Intervertebral disc (IVD) degeneration occurs with aging, leading to low back pain (LBP), which is one of the leading conditions of disability worldwide. With the lack of effective treatment, decellularized extracellular matrix (dECM) – based biomaterials have been proposed for IVD regeneration. However, the impact of donor ages on tissue repair had never been explored before in the disc field. Therefore, we aimed to address this question.

For that, a decellularization protocol for bovine nucleus pulposus (NP) of different aged donors (fetus, young and old) was optimized by testing several detergents (SDS and Triton). The process efficiency was evaluated in terms of DNA and cell removal, as well as ECM preservation. Afterwards, dECMs were repopulated with bovine NP cells and cultured ex vivo. At day 7, cell behavior, ECM de novo synthesis and remodeling were evaluated [1]. Moreover, dECMs’ inflammatory response was assessed after in vivo CAM assay. Finally, inflammatory and angiogenic cytokines were analyzed in the conditioned media-derived from dECMs by using a cytokine array.

As results, an optimal decellularization protocol (SDS 0.1%, 1h), efficient at removing cells and DNA from bovine NPs, while preserving ECM cues of native tissues, was developed. After repopulation, aggrecan increased in younger NPs, while collagen 2 decreased which may be indicative of matrix remodeling [1]. After in vivo CAM assay, fetal dECMs showed the highest inflammatory response. Finally, no statistically significant changes of cytokines were detected in the matrices, despite for a trend of higher IFN-α, IFN-γ and LIF in fetal dECMs, IL-1β in young dECMs and Decorin in old dECMs.

Overall, this work uncovered the importance of tissue donor ages for tissue regenerative purpose, opening new avenues for the development of appropriate therapeutic strategies for IVD degeneration.

Acknowledgments: FCT, EUROSPINE, ON Foundation.

The most important outcome predictor of Legg-Calvé-Perthes disease (LCPD) is the shape of the healed femoral head. However, the deformity of the femoral head is currently evaluated by non-reproducible, categorical, and qualitative classifications. In this regard, recent advances in computer vision might provide the opportunity to automatically detect and delineate the outlines of bone in radiographic images for calculating a continuous measure of femoral head deformity. This study aimed to construct a pipeline for accurately detecting and delineating the proximal femur in radiographs of LCPD patients employing existing algorithms. To detect the proximal femur, the pretrained stateof-the-art object detection model, YOLOv5, was trained on 1580 manually annotated radiographs, validated on 338 radiographs, and tested on 338 radiographs. Additionally, 200 radiographs of shoulders and chests were added to the dataset to make the model more robust to false positives and increase generalizability. The convolutional neural network architecture, U-Net, was then employed to segment the detected proximal femur. The network was trained on 80 manually annotated radiographs using real-time data augmentation to increase the number of training images and enhance the generalizability of the segmentation model. The network was validated on 60 radiographs and tested on 60 radiographs. The object detection model achieved a mean Average Precision (mAP) of 0.998 using an Intersection over Union (IoU) threshold of 0.5, and a mAP of 0.712 over IoU thresholds of 0.5 to 0.95 on the test set. The segmentation model achieved an accuracy score of 0.912, a Dice Coefficient of 0.937, and a binary IoU score of 0.854 on the test set. The proposed fully automatic proximal femur detection and segmentation system provides a promising method for accurately detecting and delineating the proximal femoral bone contour in radiographic images, which is necessary for further image analysis.

Intervertebral discs (IVD) provide flexibility to the back and ensure functional distributions of the spinal loads. They are avascular, and internal diffusion-dependent metabolic transport is vital to supply nutrients to disc cells1, but interactions with personalized IVD shapes and mechanics remain poorly explored. Poromechanical finite element models of seven personalized lumbar IVD geometries, with mean heights ranging from 8 to 16 mm were coupled with a reactive oxygen, glucose and lactate transport model linked with tissue deformations and osmosis . In previous studies, reduced formulations of the divergence of the solute flux (∇ .J = ∇ . (D∇ C) = ∇ D. ∇ C +D∇ 2C) ignored the dependence of the diffusion on the deformation gradients, ∇ D. ∇C. We simulated this phenomenon to explore its significance in mechano-metabolic -transport couplings, in the different geometries, over 24h of simulated rest (8h) and physical activity (16h). ∇ D. ∇ C affected the daily variations of glucose concentrations in IVD thinner than 12 mm but with neglectable variation ranges, while not considering ∇ D. ∇ C in taller discs only slightly overestimated the glucose concentration. Most importantly, tall IVD had nearly 60% less glucose than thin IVD, with local drops below the concentration of 0.5 mM, considered to be critical for disc cells3, in the anterior nucleus pulposus. On the one hand, previous reduced formulations for mechanometabolic-transport models of the IVD seem acceptable, even for patient-specific modelling. On the other hand, tall IVD might suffer from unfortunate combinations of deformation-dependent solute diffusion and large diffusion distances, which may favor early

Acknowledgements: Catalan Government and European Commission (2020 BP 00282; ERC-2021-CoG-O-Health-101044828)

It is known that the gait dynamics of elderly substantially differs from that of young people. However, it has not been well studied how this age-related gait dynamics affects the knee biomechanics, e.g., cartilage mechanical response. In this study, we investigated how aging affects knee biomechanics in a female population using subject-specific computational models.

Two female subjects (ages of 23 and 69) with no musculoskeletal disorders were recruited. Korea National Institute for Bioethics Policy Review Board approved the study. Participants walked at a self-selected speed (SWS), 110% of SWS, and 120% of SWS on 10 m flat ground. Three-dimensional marker trajectories and ground reaction forces (Motion Analysis, USA), and lower limbs’ muscle activities were measured (EMG, Noraxon USA). Knee cartilage and menisci geometries were obtained from subjects’ magnetic resonance images (3T, GE Health Care). An EMG-assisted musculoskeletal finite element modeling workflow was used to estimate knee cartilage tissue mechanics in walking trials. Knee cartilage and menisci were modeled using a transversely isotropic poroviscoelastic material model.

Walking speed in SWS, 110%, and 120% of SWS were 1.38 m/s, 1.51 m/s, and 1.65 m/s for the young, and 1.21 m/s, 1.34 m/s and 1.46 m/s for the elderly, respectively. The maximum tensile stress in the elderly tibial cartilage was ~25%, ~33%, and ~32% lower than the young at SWS, 110%, and 120% of SWS, respectively. These preliminary results suggest that the cartilage in the elderly may not have enough stimulation even at 20% increases in walking speed, which may be one reason for tissue degeneration. To enhance these findings, further study with more subjects and different genders will investigate how age-related gait dynamics affects knee biomechanics.

Acknowledgments: Australian NHMRC Ideas Grant (APP2001734), KITECH (JE220006)

One-fourth of all ankle trauma involve injury to the syndesmotic ankle complex, which may lead to syndesmotic instability and/or posttraumatic ankle osteoarthritis in the long term if left untreated. The diagnosis of these injuries still poses a deceitful challenge, as MRI scans lack physiologic weightbearing and plain weightbearing radiographs are subject to beam rotation and lack 3D information. Weightbearing cone-beam CT (WBCT) overcomes these challenges by imaging both ankles during bipedal stance, but ongoingdebate remains whether these should be taken under weightbearing conditions and/or during application of external rotation stress. The aim of this study is study therefore to compare both conditions in the assessment of syndesmotic ankle injuries using WBCT imaging combined with 3D measurement techniques.

In this retrospective study, 21 patients with an acute ankle injury were analyzed using a WBCT. Patients with confirmed syndesmotic ligament injury on MRI were included, while fracture associated syndesmotic injuries were excluded. WBCT imaging was performed in weightbearing and combined weightbearing-external rotation. In the latter, the patient was asked to internally rotate the shin until pain (VAS>8/10) or a maximal range of motion was encountered. 3D models were developed from the CT slices, whereafter. The following 3D measurements were calculated using a custom-made Matlab® script; Anterior tibiofibular distance (AFTD), Alpha angle, posterior Tibiofibular distance (PFTD) and Talar rotation (TR) in comparison to the contralateral non-injured ankle.

The difference in neutral-stressed Alpha angle and AFTD were significant between patients with a syndesmotic ankle lesion and contralateral control (P=0.046 and P=0.039, respectively). There was no significant difference in neutral-stressed PFTD and TR angle.

Combined weightbearing-external rotation during CT scanning revealed an increased AFTD in patients with syndesmotic ligament injuries. Based on this study, application of external rotation during WBCT scans could enhance the diagnostic accuracy of subtle syndesmotic instability.

A comprehensive understanding of the self-repair abilities of menisci and their overall function in the knee joint requires three-dimensional information. However, previous investigations of the meniscal blood supply have been limited to two-dimensional imaging methods, which fail to accurately capture tissue complexity. In this study, micro-CT was used to analyse the 3D microvascular structure of the meniscus, providing a detailed visualization and precise quantification of the vascular network.

A contrast agent (μAngiofil®) was injected directly into the femoral artery of cadaver legs to provide the proper contrast enhancement. First, the entire knee joint was analysed with micro-CT, then to increase the applicable resolution the lateral and medial menisci were excised and investigated with a maximum resolution of up to 4 μm. The resulting micro-CT datasets were analysed both qualitatively and quantitatively. Key parameters of the vascular network, such as vascular volume fraction, vessel radius, vessel length density, and tortuosity, were separately determined for the lateral and medial meniscus, and their four circumferential zones defined by Cooper.

In accordance with previous literature, the quantitative micro-CT data confirm a decrease in vascular volume fraction along the meniscal zones. The highest concentration of blood vessels was measured in the meniscocapsular region 0, which is characterized by vascular segments with a significantly larger average radius. Furthermore, the highest vessel length density observed in zone 0 suggests a more rapid delivery of oxygen and nutrients compared to other regions. Vascular tortuosity was detected in all circumferential regions, indicating the occurrence of vascular remodelling in all tissue areas.

In conclusion, micro-CT is a non-invasive imaging technique that allows for the visualization of the internal structure of an object in three dimensions. These advanced 3D vascular analyses have the potential to establish new surgical approaches that rely on the healing potential of specific areas of the meniscus.

Acknowledgements: The authors acknowledge R. Hlushchuk, S. Halm, and O. Khoma from the University of Bern for their help with contrast agent perfusions.

A medializing calcaneal osteotomy (MCO) is one of the key inframalleolar osteotomies to correct progressive collapsing foot deformity (PCFD). While many studies were able to determine the hind- and midfoot alignment after PCFD correction, the subtalar joint remained obscured by superposition on plain radiography. Therefore, we aimed to perform a 3D measurement assessment of the hind- and subtalar joint alignment pre- compared to post-operatively using weightbearing CT (WBCT) imaging.

Fifteen patients with a mean age of 44,3 years (range 17-65yrs) were retrospectively analyzed in a pre-post study design. Inclusion criteria consisted of PCFD deformity correct by MCO and imaged by WBCT. Exclusion criteria were patients who had concomitant midfoot fusions or hindfoot coalitions. Image data were used to generate 3D models and compute the hindfoot - and talocalcaneal angle as well as distance maps.

Pre-operative radiographic parameters of the hindfoot and subtalar joint alignment improved significantly relative to the post-operative position (HA, MA_Sa, and MA_Co). The post-operative talus showed significant inversion, abduction, and dorsiflexion of the talus (2.79° ±1.72, 1.32° ±1.98, 2.11°±1.47) compared to the pre-operative position. The talus shifted significantly different from 0 in the posterior and superior direction (0.62mm ±0.52 and 0.35mm ±0.32). The distance between the talus and calcaneum at the sinus tarsi increased significantly (0.64mm ±0.44).

This study found pre-dominantly changes in the sagittal, axial and coronal plane alignment of the subtalar joint, which corresponded to a decompression of the sinus tarsi. These findings demonstrate the amount of alternation in the subtalar joint alignment that can be expected after MCO. However, further studies are needed to determine at what stage a calcaneal lengthening osteotomy or corrective arthrodesis is indicated to obtain a higher degree of subtalar joint alignment correction.

Bone infections due to fractures or implants are a big medical problem. In experimental medicine, many experimental models have been created on different animal species to simulate the disease condition and to do experience treatments. The aim of this paper was to present an antibacterial efficacy of using a bone allograft developed according to the Marburg system of bone bank on a model of chronic osteomyelitis induced in rabbits.

In research was used 54 rabbits. Osteomyelitis was induced in rabbits by a human strain of St. aureus ATCC 43300, in the rabbit femur. There have been created 3 groups of animals. In 1^st group used antibiotic impregnated biodegradable material “PerOssal”. In 2^nd group used antibiotic impregnated whole bone allograft. In 3^rd group used antibiotic impregnated perforated bone allograft. Evaluation of installation and evolution of the disease was done by microbiological. A separate study of microbiological data is presented here.

This study showed, in the 1^st and 3^rd groups there is a persistent decrease in CFU by 14 knocks to 120.4 in the 1^st group and to 3.5 in the 3^rd group, and in the 2^nd group, on the contrary, there is an increase in CFU to 237.33. This shows the lack of effectiveness of using a whole bone allograft.

The results showed, after 7 days there was no statistically significant difference between the groups. After 14 days the perforated bone allograft impregnated with antibiotic was better than the biodegradable material “PerOssal”.

3D Printed polyether-ether-ketone (PEEK) has gained widespread use in clinical practice due to its excellent biocompatibility, biomechanical compatibility, and personalization. However, pre-printed PEEK implants are not without their flaws, including bioinert, optimization distortion of 3D printing digital model and prosthetic mismatching. Recent advancements in mechanical processing technology have made it possible to print bone implants with PEEK fused deposition, allowing for the construction of mechanically adaptable implants. In this study, we aimed to synthesize silanized polycitrate (PCS) via thermal polymerization and in situ graft it to PEEK surface to construct an elastomer coating for 3D printed PEEK implants (PEEK-PCS). This incorporation of PCS allows the implant to exhibit adaptive space filling ability and stress dispersal. In vivo and in vitro results, PEEK-PCS exhibited exceptional osseointegration and osteogenesis properties along with macrophage M2 phenotypic polarization, inflammatory factors reducing, promotion of osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). Additionally, PEEK-PCS displays good autofluorescence properties in vitro and in vivo, with stable fluorescence for 14 days, suggesting potential bioimaging applications. The study confirms that PEEK in situ grafting with thermo-polymerized PCS elastomers is a viable approach for creating multifunctional (bone defect adaptation, bioimaging, immune regulation, and osseointegration) implants for bone tissue engineering.

The multimodal management of canal stenosis is increasing, and inhibitors of central sensitization are playing a crucial role in central sensitization processes. Pregabalin and gabapentin are antiepileptic drugs that reduce presynaptic excitability. The objective of this study was to investigate whether the use of pregabalin and gabapentin is effective in the symptomatic management of canal stenosis.

A literature search was conducted in four databases. The inclusion criteria were studies that compared pregabalin or gabapentin with a control group in lumbar canal stenosis. Randomized clinical trials and a comparative retrospective cohort study were included. The main clinical endpoints were VAS/NRS, ODI, and RDQ (Roland Morris Disability Questionnaire) at 2, 4, 8 weeks, and 3 months, adverse events, and walking distance were also collected. Data were combined using Review Manager 5.4 software.

Six studies and 392 patients were included. The mean age was 60.25. No significant differences were observed in VAS at 2, 4, and 8 weeks: (MD: 0.23; 95% CI: −0.63-1.09), (MD: −0.04; 95% CI: −0.64 to −0.57), and (MD: −0.6; 95% CI: −1.22 to 0.02). Significant differences were observed in favor of pregabalin with respect to VAS at three months: (MD: −2.97; 95% CI: −3.43 to −2.51). No significant differences were observed in ODI (MD: −3.47; 95% CI: −7.15 to −0.21). Adverse events were significantly higher in the pregabalin/gabapentin group (OR 5.88, 95%CI 1.28-27.05). Walking distance and RDQ could not be compared, although the results were controversial.

Gabapentinoids have not been shown to be superior to other drugs used in the treatment of LSS or to placebo. However, they have shown a higher incidence of adverse effects, improved results in VAS at 3 months, and a slight improvement in ambulation at 4 months in combination with NSAIDs compared to NSAIDs in monotherapy.

Poor tendon repair is an unsolved issue in clinical practice, due to complex tendon structure. Tendon stem/progenitor cells (TSPCs) play key roles in homeostasis, regeneration, and inflammation regulation in acute tendon injuries, and rely on TGF-β signaling for recruitment into degenerative tendons. In this study, we aimed to develop an in vitro model for tenogenesis adopting a dynamic culture of a fibrin 3D scaffold, bioengineered with human TSPCs collected from both healthy and tendinopathic surgery explants (Review Board prot./SCCE n.151, 29 October 2020). 3D culture was maintained for 21 days under perfusion provided by a custom-made bioreactor, in a medium supplemented with hTGF-β1 at 20 ng/mL. The data collected suggested that the 3D in vitro model well supported survival of both pathological and healthy cells, and that hTGF-β signaling, coupled to a dynamic environment, promoted differentiation events. However, pathological hTSPCs showed a different expression pattern of tendon-related genes throughout the culture and an impaired balance of pro-inflammatory and anti-inflammatory cytokines, compared to healthy hTSPCs, as indicated by qRT-PCT and immunofluorescence analyses. Additionally, the expression of both tenogenic and cytokine genes in hTSPCs was influenced by hTGF-β1, indicating that the environment assembled was suitable for studying tendon stem cells differentiation. The study offers insights into the use of 3D cultures of hTSPCs as an in vitro model for investigating their behavior during tenogenic events and opens perspectives for following the potential impact on resident stem cells during regeneration and healing events.

Adipose-derived stem cells (ADSCs) are an effective alternative for Teno-regeneration. Despite their applications in tendon engineering, the mechanisms promoting tendon healing still need to be understood. Since there is scattered information on ovine ADSCs, this research aims to investigate in vitro their teno-differentiation for potential use in preclinical tendon regeneration models.

Ovine ADSCs were isolated from the tail region according to FAT-STEM laboratories, expanded until passage six (P6), and characterized in terms of stemness, adhesion and MHC markers by Flow Cytometry (FCM) and immunocytochemistry (ICC). Cell proliferation and senescence were evaluated with MTT and Beta-galactosidase assays, respectively. P1 ADSCs’ teno-differentiation was assessed by culturing them with teno-inductive Conditioned Media (CM) or engineering them on tendon-mimetic PLGA scaffolds. ADSCs teno-differentiation was evaluated by morphological, molecular (qRT-PCR), and biochemical (WesternBlot) approaches.

ADSCs exhibited mesenchymal phenotype, positive for stemness (SOX2, NANOG, OCT4), adhesion (CD29, CD44, CD90, CD166) and MHC-I markers, while negative for hematopoietic (CD31, CD45) and MHC-II markers, showing no difference between passages. ICC staining confirmed these results, where ADSCs showed nuclear positivity for SOX2 (≅ 56%) and NANOG (≅ 67%), with high proliferation capacity without senescence until P6. Interestingly, ADSCs cultured with the teno-inductive CM did not express tenomodulin (TNMD) protein or gene. Conversely, ADSCs seeded on scaffolds teno-differentiated, acquiring a spindle shape supported by TNMD protein expression at 48h (p<0.05 vs. ADSCs 48h) with a significant increase at 14 days of culture (p<0.05 vs. ADSCs + fleece 48h).

Ovine ADSCs respond differently upon distinct teno-inductive strategies. While the molecules on the CM could not trigger a teno-differentiation in the cells, the scaffold's topological stimulus did, resulting in the best strategy to apply. More insights are requested to better understand ovine ADSCs’ tenogenic commitment before using them in vivo for tendon regeneration.

Acknowledgements: This research is part of the P4FIT project ESR5, under the H2020MSCA-ITN-EJD-P4 FIT-Grant Agreement ID:955685.

Tendon injuries are a common problem that can significantly impact an individual's quality of life. While traditional surgical methods have been used to address this issue, Extracellular Vesicles (EVs) have emerged as a promising approach to promote tendon repair and regeneration mechanisms, as they deliver specific biological signals to neighbouring cells. In this study, we extracted human Tendon Progenitor Stem cells (hTPSCs) from surgery explants and isolated their EVs from perfused and static media.

hTPSCs were isolated from tendon surgery biopsy (Review Board prot./SCCE n.151, 29/10/2020) and cultured in both static and dynamic conditions, using a perfusion bioreactor (1ml/min). When cells reached 80% confluence, they were switched into a serum-free medium for 24 hours for EVs-production. Conditioned media was ultra-centrifuged for 90min (100000g). The recovered pellet was then characterized by size and concentration (Nanosight NS300), Zeta potential (Mastersizer S), morphology (SEM and TEM) and protein quantification.

hTPSCs stemness and multipotency were confirmed through CD73, CD90, and CD105 expression and confirmation of quad-lineage (adipo-osteo-chondro-teno) differentiation. After 7 days, hTPSCs were ready for EVs-production. Ultracentrifugation revealed the presence of particles with a concentration of 7×107 particles/mL consistent across both cultures. Further characterization indicated that EVs collected from perfused conditions displayed an elevated vesicle mean size (mean 143±6.5 nm) in comparison to static conditions (mean 112±7.4 nm). Consistent with, but not in proportion with, the above protein content was measured at 20 ng/ml (dynamic) and 7 ng/mL (static) indicating a nearly 3-fold increase in concentration associated with a ~22% increase in particle size.

Proposed data showed that sub-200 diameter vesicles were successfully collected from multipotent hTPSCs starvation, and the vesicle size and protein concentration were compatible with established EV literature; furthermore, dynamic culture conditions seemed more suitable for EVs-production. Further characterization will be required to better understand, EVs-compositions and their role in tendon regenerative events.

Regenerative medicine (RM) promises to restore both the mechanical functionality and the biological composition of tissues after damage. Three-dimensional scaffolds are used in RM to host cells and let them produce proteins that are the building blocks of the native tissues. While regenerating tissues evolve over time through dynamic biomechanical and biochemical changes, current scaffolds’ generation are passive causing mechanical mismatch, suboptimal growth, and pain. Furthermore, current scaffolds ignore the complexity of the reciprocal bio-mechanics regulation, hindering the design of the next-gen scaffolds. To regenerate tissues and organs, biofabrication strategies that impart spatiotemporal control over cell-cell and cell-extracellular matrix communication, often through control over cell and material deposition and placement, are being developed. To achieve these targets, the spatiotemporal control over biological signals at the interface between cells and materials is often aimed for. Alternatively, biological activity can be triggered through the control of mechanical cues, harnessing more fundamental know-how in mechanobiology that could be combined with biofabrication strategies. Here, I present some of our most recent advancements in merging mechanobiology with biofabrication that enabled the control of cell activity, moving towards enhanced tissue regeneration as well as the possibility to create more complex 3D in vitro models to study biological processes.

Relevant in vitro models emulating tendinopathies are highly needed to study these diseases and develop better treatments. We have recently proposed a new strategy that allows the automated 3D writing of microphysiological systems (MPS) embedded into its own biomimetic fibrillar support platform based on the self-assembling of cellulose nanocrystals (CNCs). Here, we explored this CNC platform for writing humanized in vitro tendon models using tendon decellularized extracellular matrix (dECM)-based bioinks to closely recapitulate the biophysical and biochemical cues of tendon cell niche and self-induce the tenogenic differentiation of stem cells. The proposed concept was further explored to study the crosstalk between the tendon core and vascular compartment.

Porcine flexor tendons were decellularized to produce the dECM bioink hydrogel. hASCs were used as cell source and the bioink was directly printed within the CNC fluid gel. Tendon constructs were co-printed with compartmentalized microvascular structures to evaluate the cellular crosstalk with endothelial cells. The tendon-on-chip models showed high cell viability and proliferation during culture up to 21 days, and the synergy between dECM cues and printed patterns induced anisotropic cell organization similar to tendon tissues. Gene and protein analysis showed upregulation of the most important tendon related markers on tendon constructs, demonstrating that the biophysical and biochemical cues of dECM induced hASCs commitment toward tenogenic phenotype. In co-culture system, chemotaxis induced endothelial cells migration toward the tendon compartment, but without significant infiltration. Gene and protein expression results suggest that the cellular crosstalk established in this MPS with endothelial cells boosted hASCs tenogenesis, emulating tendon development stages.

Overall, the proposed system might be promising for the automated fabrication of organotypic tendon-on-chip models that will be a valuable new tool to study tendon physiology, pathology, or the effect of drugs for the treatment of tendinopathy.

Acknowledgments: EU H2020 for ERC-2017-CoG-772817; ERC-PoC-BioCHIPs-101069302; FCT/MCTES for 2022.05526.PTDC, 2020.03410.CEECIND, and PD/BD/129403/2017

The aim of this study is to print 3D polycaprolactone (PCL) scaffolds at high and low temperature (HT/LT) combined with salt leaching to induced porosity/larger pore size and improve material degradation without compromising cellular activity of printed scaffolds. PCL solutions with sodium chloride (NaCl) particles either directly printed in LT or were casted, dried, and printed in HT followed by washing in deionized water (DI) to leach out the salt. Micro-Computed tomography (Micro-CT) and scanning electron microscope (SEM) were performed for morphological analysis. The effect of the porosity on the mechanical properties and degradation was evaluated by a tensile test and etching with NaOH, respectively. To evaluate cellular responses, human bone marrow-derived mesenchymal stem/stromal cells (hBMSCs) were cultured on the scaffolds and their viability, attachment, morphology, proliferation, and osteogenic differentiation were assessed. Micro-CT and SEM analysis showed that porosity induced by the salt leaching increased with increasing the salt content in HT, however no change was observed in LT. Structure thickness reduced with elevating NaCl content. Mass loss of scaffolds dramatically increased with elevated porosity in HT. Dog bone-shaped specimens with induced porosity exhibited higher ductility and toughness but less strength and stiffness under the tension in HT whereas they showed decrease in all mechanical properties in LT. All scaffolds showed excellent cytocompatibility. Cells were able to attach on the surface of the scaffolds and grow up to 14 days. Microscopy images of the seeded scaffolds showed substantial increase in the formation of extracellular matrix (ECM) network and elongation of the cells. The study demonstrated the ability of combining 3D printing and particulate leaching together to fabricate porous PCL scaffolds. The scaffolds were successfully printed with various salt content without negatively affecting cell responses. Printing porous thermoplastic polymer could be of great importance for temporary biocompatible implants in bone tissue engineering applications.

Articular cartilage is a multi-zonal tissue that coats the epiphysis of long bones and avoids its wear during motion. An unusual friction could micro-fracture this connective membrane and progress into an osteochondral defect (OD), where the affected cartilage suffers inflammation, fibrillation, and forfeiture of its anisotropic structure.

Clinical treatment for ODs has been focused on micro-fracture techniques, where the defect area is removed and small incisions are performed in the subchondral bone, which allows the exudation of mesenchymal stem cells (hMSCs) to the abraded zone. However, hMSCs represent less than 0.01% of the total cell population and are not able to self-organise coherently, so the treatments fail in the long term. To select, support and steer hMSCs from the bone marrow into a specific differentiation stage, and recreate the cartilage anisotropic microenvironment, multilayer dual-porosity 3D-printed scaffolds were developed.

Dual-porosity scaffolds were printed using prepared inks, containing specific ratios of poly-(d,l)lactide-co-caprolactone copolymer and gelatine microspheres of different diameters, which acted as sacrificial micro-pore templates and were leached after printing. The cell adhesion capability was investigated showing an increased cell number in dual-porosity scaffolds as compared to non-porous ones. To mimic the stiffness of the three cartilage zones, several patterns were designed, printed, and checked by dynamic-mechanical analysis under compression at 37 ºC. Three patterns with specific formulations were chosen as candidates to recreate the mechanical properties of the cartilage layers. Differentiation studies in the selected scaffolds showed the formation of mature cartilage by gene expression, protein deposition and biomolecular analysis. Given the obtained results, designed scaffolds were able to guide hMSC behaviour.

In conclusion, biocompatible, multilayer and dual-porosity scaffolds with cell entrapment capability were manufactured. These anisotropic scaffolds were able to recreate the physical microenvironment of the natural cartilage, which in turn stimulated cell differentiation and the formation of mature cartilage.

Acknowledgments: This work was supported by the EMAKIKER grant.

For chondral damage in younger patients, surgical best practice is microfracture, which involves drilling into the bone to liberate the bone marrow. This leads to a mechanically inferior fibrocartilage formed over the defect as opposed to the desired hyaline cartilage that properly withstands joint loading. While some devices have been developed to aid microfracture and enable its use in larger defects, fibrocartilage is still produced and there is no clear clinical improvement over microfracture alone in the long term. Our goal is to develop 3D printed devices, which surgeons can implant with a minimally invasive technique. The scaffolds should match the functional properties of cartilage and expose endogenous marrow cells to suitable mechanobiological stimuli in-situ, in order to promote healing of articular cartilage lesions before they progress to osteoarthritis, and rapidly restore joint health and mobility. Importantly, scaffolds should direct a physiological host reaction, instead of a foreign body reaction, associated with chronic inflammation and fibrous capsule formation, negatively influencing the regenerative outcome.

Our novel silica/polytetrahydrofuran/polycaprolactone hybrids were prepared by sol-gel synthesis and scaffolds were 3D printed by direct ink writing. 3D printed hybrid scaffolds with pore channels of ~250 µm mimic the compressive behaviour of cartilage. Our results show that these scaffolds support human bone marrow stem/stromal cell (hMSC) differentiation towards chondrogenesis in vitro under hypoxic conditions to produce markers integral to articular cartilage-like matrix evaluated by immunostaining and gene expression analysis. Macroscopic and microscopic evaluation of subcutaneously implanted scaffolds in mice showed that scaffolds caused a minimal resolving inflammatory response. Our findings show that 3D printed hybrid scaffolds have the potential to support cartilage regeneration.

Acknowledgements: Authors acknowledge funding provided by EPSRC grant EP/N025059/1.

Precision health aims to develop personalised and proactive strategies for predicting, preventing, and treating complex diseases such as osteoarthritis (OA). Due to OA heterogeneity, which makes developing effective treatments challenging, identifying patients at risk for accelerated disease progression is essential for efficient clinical trial design and new treatment target discovery and development.

To create a reliable and interpretable precision health tool that predicts rapid knee OA progression over a 2-year period from baseline patient characteristics using an advanced automated machine learning (autoML) framework, “Autoprognosis 2.0”.

All available 2-year follow-up periods of 600 patients from the FNIH OA Biomarker Consortium were analysed using “Autoprognosis 2.0” in two separate approaches, with distinct definitions of clinical outcomes: multi-class predictions (categorising disease progression into pain and/or radiographic progression) and binary predictions. Models were developed using a training set of 1352 instances and all available variables (including clinical, X-ray, MRI, and biochemical features), and validated through both stratified 10-fold cross-validation and hold-out validation on a testing set of 339 instances. Model performance was assessed using multiple evaluation metrics. Interpretability analyses were carried out to identify important predictors of progression.

Our final models yielded higher accuracy scores for multi-class predictions (AUC-ROC: 0.858, 95% CI: 0.856-0.860) compared to binary predictions (AUC-ROC: 0.717, 95% CI: 0.712-0.722). Important predictors of rapid disease progression included WOMAC scores and MRI features. Additionally, accurate ML models were developed for predicting OA progression in a subgroup of patients aged 65 or younger.

This study presents a reliable and interpretable precision health tool for predicting rapid knee OA progression. Our models provide accurate predictions and, importantly, allow specific predictors of rapid disease progression to be identified. Furthermore, the transparency and explainability of our methods may facilitate their acceptance by clinicians and patients, enabling effective translation to clinical practice.

Despite the clinical relevance of back pain and intervertebral disc herniation, the lack of reliable models has strained their molecular understanding. We characterized the lumbar spinal phenotype of C57BL/6 and SM/J mice during aging. Interestingly, old SM/J lumbar discs evidenced accelerated degeneration, associated with high rates of disc herniation. SM/J AF's and degenerative human's AF transcriptomic profiles showed altered immune cell, inflammation, and p53 pathways. Old SM/J mice presented increased neuronal markers in herniated discs, thicker subchondral bone, and higher sensitization to pain. Dorsal root ganglia transcriptomic studies and spinal cord analysis exhibited increased pain and neuroinflammatory markers associated with altered extracellular matrix regulation. Immune system single-cell and tissue level analysis showed distinctive T-cell and B-cell modulation and negative correlation between mechanical allodynia and INF-α, IL-1β, IL2, and IL4, respectively. This study underscores the multisystemic network behind back pain and highlights the role of genetic background and the immune system in disc herniation disease.

Acknowledgments: This study is supported by grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) R01AR055655, R01AR064733, R01AR074813 to MVR.

Intervertebral disc (IVD) degeneration is responsible for severe clinical symptoms including chronic back pain. Galectins are a family of carbohydrate-binding proteins, some of which can induce functional disease markers in IVD cells and other musculoskeletal diseases. Galectins −4 and −8 were shown to trigger disease-promoting activity in chondrocytes but their effects on IVD cells have not been investigated yet. This study elucidates the role of galectin-4 and −8 in IVD degeneration.

Immunohistochemical evidence for the presence of galectin-4 and −8 in the IVD was comparatively provided in specimens of 36 patients with spondylochondrosis, spondylolisthesis, or spinal deformity. Confocal microscopy revealed co-localization of galectin-4 and −8 in chondrocyte clusters of degenerated cartilage. The immunohistochemical presence of galectin-4 correlated with histopathological and clinical degeneration scores of patients, whereas galectin-8 did not show significant correlations. The specimens were separated into annulus fibrosus (AF), nucleus pulposus (NP) and endplate, which was confirmed histologically. Separate cell cultures of AF and NP (n=20) were established and characterized using cell type-specific markers. Potential binding sites for galectins including sialylated N-glycans and LacdiNAc structures were determined in AF and NP cells using LC/ESI-MS-MS. To assess galectin functions, cell cultures were treated with recombinant galectin-4 or −8, in comparison to IL-1β, and analyzed using RT-qPCR and In-cell Western blot. In vitro, both galectins triggered the induction of functional disease markers (CXCL8 and MMP3) on mRNA level and activated the nuclear factor-kB pathway. NP cells were significantly more responsive to galectin-8 and Il-1β than AF cells. Phosphorylation of p-65 was time-dependently induced by both galectins in both cell types to a comparable extent.

Taken together, this study provides evidence for a functional role of glycobiological processes in IVD degeneration and highlights galectin-4 and −8 as regulators of pro-inflammatory and degrative processes in AF and NP cells.

Tendons mainly consist of collagen in order to withstand high tensile forces. Compared to other, high turnover tissues, cellularity and vascularity in tendons are low. Thus, the natural healing process of tendons takes long and can be problematic. In case of injury to the enthesis, the special transition from tendon over cartilage to bone is replaced by a fibrous scar tissue, which remains an unsolved problem in rotator cuff repair.

To improve tendon healing, many different approaches have been described using scaffolds, stem cells, cytokines, blood products, gene therapy and others. Despite promising in vitro and in vivo results, translation to patient care is challenging. In clinics however, tendon auto- or allografts remain still first choice to augment tendon healing if needed.

Therefore, it is important to understand natural tendon properties and natural tendon healing first. Like in other tissues, senescence of tenocytes seems to play an important role for tendon degeneration which is interestingly not age depended. Our in vivo healing studies have shown improved and accelerated healing by adding collagen type I, which is now used in clinics, for example for augmentation of rotator cuff repair. Certain cytokines, cells and scaffolds may further improve tendon healing but are not yet used routinely, mainly due to missing clinical data, regulatory issues and costs.

In conclusion, the correct diagnosis and correct first line treatment of tendon injuries are important to avoid the necessity to biologically augment tendon healing. However, strategies to improve and accelerate tendon healing are still desirable. New treatment opportunities may arise with further advances in tendon engineering in the future.

The advent of modular implants aims to minimise morbidity associated with revision of hemiarthroplasty or total shoulder arthroplasty (TSA) to reverse shoulder arthroplasty (RSR) by allowing retention of the humeral stem. This systematic review aimed to summarise outcomes following its use and reasons why modular humeral stems may be revised.

A systematic review of Pubmed, Medline and EMBASE was performed according to PRISMA guidelines of all patients undergoing revision of a modular hemiarthroplasty or TSA to RSR. Primary implants, glenoid revisions, surgical technique and opinion based reports were excluded. Collected data included demographics, outcomes and incidence of complications.

277 patients were included, with a mean age of 69.8 years (44-91) and 119 being female. Revisions were performed an average of 30 months (6-147) after the index procedure, with the most common reason for revision being cuff failure in 57 patients. 165 patients underwent modular conversion and 112 underwent stem revision. Of those that underwent humeral stem revision, 18 had the stem too proximal, in 15 the stem was loose, 10 was due to infection and 1 stem had significant retroversion. After a mean follow up of 37.6 months (12-91), the Constant score improved from a mean of 21.8 to 48.7. Stem revision was associated with a higher complication rate (OR 3.13, 95% CI 1.82-5.39).

The increased use of modular stems has reduced stem revision, however 40% of these implants still require revision due to intra-operative findings. Further large volume comparative studies between revised and maintained humeral stems post revision of modular implants can adequately inform implant innovation to further improve the stem revision rate.

Total shoulder arthroplasty (TSA) and Reverse Total shoulder arthroplasty (RSA) are two of the most performed shoulder operations today. Traditionally postoperative rehabilitation included a period of immobilisation, protecting the joint and allowing time for soft tissue healing. This immobilisation period may significantly impact a patient's quality of life (Qol)and ability to perform activities of daily living (ADL's). This period of immobilisation could be safely avoided, accelerating return to function and improving postoperative QoL.

This systematic review examines the safety of early mobilisation compared to immobilisation after shoulder arthroplasty focusing on outcomes at one year.

The number of seven needed knots to provide secure hold of high strength sutures was previously reported. New technologies like tape sutures and sutures with a salt infused silicon core have been developed, potentially reducing the number of needed knots. Study aims: To investigate the influence of (1) throw number and (2) different ambient conditions on knot security in two different high-strength sutures, and (3) to compare their biomechanical competence.

Two sutures (SutureTape (FT); n=56 and DynaTape (DT); n=56) were assigned for knot tying. Specimens were exposed to different media during tying, namely air, saline solution, and fat. A monotonic tensile ramp was applied. For each suture and ambient condition, seven specimens with 3 to 7 throws each were tested (n=7), evaluating their slippage and ultimate force to failure. The minimum number of throws preventing suture unraveling was determined in each suture and condition.

For each suture type and condition failure occurred via rupturing in all specimens for the following minimum number of throws: FT: dry–6, wet–6, fatty-wet–6; DT: dry–6; wet–4; fatty-wet–5. No significant differences were found comparing ultimate load to rupture of the two groups with minimum number of needed throws in each media. (FT dry-6 vs. DT dry-6; p<0.07); (FT wet-6 vs. DT wet-4; p<0.20); (FT fat-6 vs. DT fat-5; p<0.58). Knot slippage of DT was significantly higher in wet and fatty conditions compared to ST p<0.001 and p<0.004.

In fatty-wet conditions DT requires 5 throws to achieve a secure knot. In wet conditions this number can be reduced to 4 throws. FT needs 6 throws to provide a stable knot in all conditions. The biomechanical competence of both sutures in terms of knot slippage and peak force are comparable.

As high incidences of tendinopathies are observed particularly in those who intensively use their tendons, we assume that pathological changes are caused, at least partially, by mechanical overload. This has led to the so-called overload hypothesis, explaining the development of tendinopathies by structural failure resulting from excessive load. At the same time, tendon loading is an important part in tendon rehabilitation. Currently, exercise treatment approaches such as eccentric training or heavy load resistance training are widely applied in tendinopathy rehabilitation, with good clinical results such as an improvement in function and a reduction in pain. Particularly those rehabilitative approaches which impose high strains on the tendon may induce an adaptation of the tendon's mechanical properties such as increased tendon stiffness. An increased tendon stiffness is often interpreted as desirable, as it may protect the tendon from overloading and thus prevent future strain injuries. However, the tendinopathic tendon is not necessarily less stiff than the tendon in the contralateral leg and an improvement in tendon stiffness is not necessarily accompanied by an improvement in tendon pain or function. In addition, metabolic factors, resulting e.g. in low-level systemic inflammation, may contribute to pathological tendon tissue changes and are not necessarily affected by an exercise program, while nutritional interventions or dietary supplements may potentially affect tendon cell metabolism. Indeed, dietary supplements have been introduced as an additional therapeutic approach in the treatment of tendinopathies in recent years, and their positive curative effects have been reported for both the general population and athletes. In the management of tendinopathies, it may thus be advisable if therapeutic approaches aim to address both tendon mechanics and tendon metabolism for better treatment effectiveness and a sustainable improvement in pain and function.

Cell-based therapies offer a promising strategy to treat tendon injuries and diseases. Both mesenchymal stromal cells (MSCs) and pluripotent stem cells (PSCs) are good candidates for such applications due to their self-renewing and differentiation capacity. However, the translation of cell-based therapies from bench to bedside can be hindered by the use of animal-derived components in ancillary materials and by the lack of standardised media and protocols for in vitro tenogenic differentiation. To address this, we have optimized animal component-free (ACF) workflows for differentiating human MSCs and PSCs to tenocyte-like cells (TLCs) respectively. MSCs isolated from bone marrow (n = 3) or adipose tissue (n = 3) were expanded using MesenCult™-ACF Plus Culture Kit for at least 2 passages, and differentiated to TLCs in 21 days using a step-wise approach. Briefly, confluent cultures were treated with an ACF tenogenic induction medium for 3 days, followed by treatment with an ACF maturation medium for 18 days. Monolayer cultures were maintained at high density without passaging for the entire duration of the protocol, and the medium was changed every 2 – 3 days. In a similar fashion, embryonic (n = 3) or induced PSCs (n = 3) were first differentiated to acquire a mesenchymal progenitor cell (MPC) phenotype in 21 days using STEMdiff™ Mesenchymal Progenitor Kit, followed by the aforementioned tenogenic protocol for an additional 21 days. In all cases, the optimized workflows using ACF formulations consistently activated a tenogenic transcriptional program, leading to the generation of elongated, spindle-shaped tenomodulin-positive (TNMD+) cells and deposition of an extracellular matrix predominantly composed of collagen type I. In summary, here we describe novel workflows that can robustly generate TLCs from MSCs and hPSC-derived MPCs for potential translational applications.

Although remnant-preserved ACL reconstruction (ACLR) restores knee joint stability and dampens the problem of acute ACL rupture-induced knee pain, an increasing number of patients still develop post-traumatic osteoarthritis (PTOA) after 10 to 15 years of ACLR. We previously found that remnant-preserved ACLR with concomitant medial and lateral meniscus repair may not prevent cartilage degeneration and weaken muscle strength, while the clinical features of PTOA are not clear. We hypothesized that remnant-preserved ACLR with concomitant medial and lateral meniscus tears is related to early cartilage damage, worse function recovery, patient-reported outcomes (PROs) and delayed duration to return to sports. The aim is to evaluate the remnant-preserved ACLR with complicated meniscal injuries in predicting which patients are at higher risk of osteoarthritic changes, worse function and limited activities after ACLR for 12 months.

Human ethical issue was approved by a committee from Xi'an Jiaotong University. 26 young and active patients (24 male, 2 female) with ACL injuries (Sherman type I and II) with concomitant medial and lateral meniscus within 2 months were included from January 2014 to March 2022. The average age of the ACLR+ meniscus repair was 26.77±1.52 (8 right, 5 left) and isolated ACLR control was 31.92±2.61 years old (7 left, 6 right). Remnant-preserved ACLR with a 5- to 6-strand hamstring tendon graft was operated on by the same sports medicine specialists. MRI CUBE-T₂ scanning with 48 channels was conducted by a professional radiologist. The volume of the ACL graft was created through 3 dimensional MRI model (Mimics 19, Ann Arbor). Anterior Cruciate Ligament OsteoArthritis Score (ACLOAS) was applied to score visible cartilage damage. IKDC 2000 score and VAS were assessed by two blinded researchers. Results were presented as mean± SEM of each group.

The cross-sectional area and 3D volume of the ACL graft were greater in the remnant-preserved ACLR+meniscus group compared with isolated ACLR (p=0.01). It showed that ACLR+ meniscus group had early signs of joint damage and delayed meniscus healing regarding ACLOAS compared to control group (p=0.045). MRI CUBE-T₂ prediction of radiographic cartilage degeneration was not obvious in both groups post remnant-preserved ACLR over 12 months (p>0.05). However, higher VAS scores, lower IKDC scores, and long-last joint swelling were reported in the ACLR+ meniscus repair group at the end of 12 months follow-up. Although remnant-preserved ACLR+ meniscus was able to maintain the restore the knee function, it showed delayed timing (>12 months) to return to play at the pre-injury stage, while no difference between the timing of returning to the normal daily routine of their ACLR knee compared to control (p=0.30). The cost of ACLR+ meniscus (average 10,520.76$) was higher than the control group (6,452.92$, p=0.018).

Remnants-preserved ACLR with concomitant injured medial and lateral meniscus repair shows a higher risk of cartilage damage, greater cost, worse functional performance, and longer time for young male patients to return to sports after 12-month follow-up compared to isolated ACLR. Further evidence and long-term follow-up are needed to better understand the association between these results and the risk of development of PTOA in this patient cohort.

Early identification of patients at risk for impaired tendon healing and corresponding novel therapeutic approaches are urgent medical needs. This study aimed to clarify the role of CD3+ T-cells during acute Achilles tendon (AT) healing. Blood and hematoma aspirate were taken from 26 patients during AT reconstruction, and additional blood samples were obtained during clinical follow-up at 6, 26 and 52 weeks after surgery. T-cell subsets were analyzed by flow cytometry using CD3, CD4, CD8, CD11a, CD57 and CD28 antibodies. Clinical follow-up included functional tests, MRI assessments, and subjective questionnaires. In vitro, the functional behavior of patient-derived tenocytes was investigated in co-cultures with autologous unpolarized CD4+ or CD8+ T-cells, or IFNy-polarized CD8+ or IL17-polarized CD4+ Tcells (n=5-6). This included alterations in gene expression (qPCR), MMP secretion (ELISA), migration rate (scratch wound healing assay) or contractility (collagen gels). Analysis revealed that elevated CD4+ T-cell levels and reduced CD8+ T-cell levels (increased CD4/CD8 ratio) in hematoma aspirate and pre-operative blood were associated with inferior clinical outcomes regarding pain and function at 26 and 52 weeks. Increased levels of CD8+ -memory T-cell subpopulations in blood 6 weeks after surgery were associated with less tendon elongation. In vitro, tenocytes showed increased MMP1/2/3 levels and collagen III/I ratio in co-culture with unpolarized and/or IL17-polarized CD4+ T-cells compared to unpolarized CD8+ T-cells. This coincided with increased IL17 receptor expression in tenocytes co-cultured with CD4+ T-cells. Exposure of tenocytes to IL17-polarized CD4+ T-cells decreased their migration rate and increased their matrix contractility, especially compared to IFNy-polarized CD8+ T-cells. The CD4+ /CD8+ T-cell ratio could serve as prognostic marker for early identification of patients with impaired AT healing potential. Local reduction of CD4+ T-cell levels or their IL17 secretion represent a potential therapeutic approach to improve AT healing and to prevent weakening of the tendon ECM.

Glutamate regulates the expression of apoptosis-related genes and triggers the apoptosis of fibroblasts in rotator cuff tendons. Subacromial bursitis is always accompanied by symptomatic rotator cuff tear (RCT). However, no study has been reported on the presence of glutamate in subacromial bursa and on its involvement of shoulder pain in patients who had RCT. The purposes of this study were to determine whether the glutamate expression in subacromial bursa is associated with the presence of RCT and with the severity of shoulder pain accompanying RCT.

Subacromial bursal tissues were harvested from patients who underwent arthroscopic rotator cuff tendon repair or glenoid labral repair with intact rotator cuff tendon. Glutamate tissue concentrations were measured, using a glutamate assay kit. Expressions of glutamate and its receptors in subacromial bursae were histologically determined. The sizes of RCT were determined by arthroscopic findings, using the DeOrio and Cofield classification. The severity of shoulder pain was determined, using visual analog scale (VAS). Any associations between glutamate concentrations and the size of RCT were evaluated, using logistic regression analysis. The correlation between glutamate concentrations and the severity of pain was determined, using the Pearson correlation coefficient. Differences with a probability <0.05 were considered statistically significant.

Glutamate concentrations showed significant differences between the torn tendon group and the intact tendon group (P = 0.009). Concentrations of glutamate significantly increased according to increases in tear size (P < 0.001). In histological studies, the expressions of glutamate and of its ionotropic and metabotropic receptors have been confirmed in subacromial bursa. Glutamate concentrations were significantly correlated with pain on VAS (Rho=0.56 and P =0.01).

The expression of glutamate in subacromial bursa is significantly associated with the presence of RCT and significantly correlated with its accompanying shoulder pain.

Acknowledgements: This research was supported by the Basic Science Research Program, through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015R1D1A3A01018955 and 2017R1D1A1B03035232).

In absence of available quantitative measures, the assessment of fracture healing based on clinical examination and X-rays remains a subjective matter. Lacking reliable information on the state of healing, rehabilitation is hardly individualized and mostly follows non evidence-based protocols building on common guidelines and personal experience. Measurement of fracture stiffness has been demonstrated as a valid outcome measure for the maturity of the repair tissue but so far has not found its way to clinical application outside the research space. However, with the recent technological advancements and trends towards digital health care, this seems about to change with new generations of instrumented implants – often unfortunately termed “smart implants” – being developed as medical devices.

The AO Fracture Monitor is a novel, active, implantable sensor system designed to provide an objective measure for the assessment of fracture healing progression (1). It consists of an implantable sensor that is attached to conventional locking plates and continuously measures implant load during physiological weight bearing. Data is recorded and processed in real-time on the implant, from where it is wirelessly transmitted to a cloud application via the patient's smartphone. Thus, the system allows for timely, remote and X-ray free provision of feedback upon the mechanical competence of the repair tissue to support therapeutic decision making and individualized aftercare.

The device has been developed according to medical device standards and underwent extensive verification and validation, including an in-vivo study in an ovine tibial osteotomy model, that confirmed the device's capability to depict the course of fracture healing as well as its long-term technical performance. Currently a multi-center clinical investigation is underway to demonstrate clinical safety of the novel implant system. Rendering the progression of bone fracture healing assessable, the AO Fracture Monitor carries potential to enhance today's postoperative care of fracture patients.

Decreasing the bulk weight without losing the biomechanical properties of commercial pure titanium (Cp-Ti) medical implants is now possible by using Laser Powder Bed Fusion (L-PBF) technology. Gyroid lattice structures that have precious mechanical and biological advantages because of similarity to trabecular bone. The aim of the study was to design and develop L-PBF process parameter optimization for manufacturing gyroid lattice Cp-Ti structures. The cleaning process was then optimized to remove the non-melted powder from the gyroid surface without mechanical loss.

Gyroid cubic designs were created with various relative densities by nTopology. L-PBF process parameter optimization was progressed using with Cp-Ti (EOS TiCP Grade2) powder in the EOS M290 machine to achieve parts that have almost full dense and dimensional accuracy. The metallography method was made for density. Dimensional accuracy at gyroid wall thicknesses was investigated between designed and manufactured via stereomicroscope, also mechanical tests were applied with real time experiment and numerical analysis (ANSYS). Mass loss and strut thickness loss were investigated for chemical etching cleaning process.

Gyroid parts had 99,5% density. High dimensional accuracy was achieved during L-PBF process parameters optimization. Final L-PBF parameters gave the highest 19% elongation and 427 MPa yield strength values at tensile test. Mechanical properties of gyroid were controlled with changing relative density. A minute chemical etching provided to remove non-melted powders.

Compression test results of gyroids at numerical and real-time analysis gave unrelated while deformation behaviors were compatible with each other. Gyroid Cp-Ti osteosynthesis mini plates will be produced with final L-PBF process parameters. MTT cytotoxicity test will be characterized for cell viability.

Acknowledgements This project is granted by TUBITAK (120N943). Feza Korkusuz MD is a member of the Turkish Academy of Sciences (TÜBA).

According to the latest report from the German Arthroplasty Registry, aseptic loosening is the primary cause of implant failure following primary hip arthroplasty. Osteolysis of the proximal femur due to the stress-shielding of the bone by the implant causes loss of fixation of the proximal femoral stem, while the distal stem remains fixed.

Removing a fixed stem is a challenging process. Current removal methods rely on manual tools such as chisels, burrs, osteotomes, drills and mills, which pose the risk of bone fracture and cortical perforation. Others such as ultrasound and laser, generate temperatures that could cause thermal injury to the surrounding tissues and bone. It is crucial to develop techniques that preserve the host bone, as its quality after implant removal affects the outcome of a revision surgery.

A gentler removal method based on the transcutaneous heating of the implant by induction is proposed. By reaching the glass transition temperature (T_G) of the periprosthetic cement, the cement is expected to soften, enabling the implant to be gently pulled out. The in-vivo environment comprises body fluids and elevated temperatures, which deteriorate the inherent mechanical properties of bone cement, including its T_G. We aimed to investigate the effect of fluid absorption on the T_G (ASTM E2716-09) and Vicat softening temperature (VST) (ISO 306) of Palacos R cement (Heraeus Medical GmbH) when dry and after storage in Ringer's solution for up to 8 weeks.

Samples stored in Ringer's solution exhibited lower T_G and VST than those stored in air. After 8 weeks, the T_G decreased from 95.2°C to 81.5°C in the Ringer's group, while the VST decreased from 104.4°C to 91.9°C. These findings will be useful in the ultimate goal of this project which is to design an induction-based system for implant removal.

Acknowledgements: Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – SFB/TRR-298-SIIRI – Project-ID 426335750

Even minor lesions in articular cartilage (AC) can cause underlying bone damage creating an osteochondral (OC) defect. OC defects can cause pain, impaired mobility and can develop to osteoarthritis (OA). OA is a disease that affects nearly 10% of the population worldwide^[1], and represents a significant economic burden to patients and society^[2]. While significant progress has been made in this field, realising an efficacious therapeutic option for unresolved OA remains elusive and is considered one of the greatest challenges in the field of orthopaedic regenerative medicine^[3]. Therefore, there is a societal need to develop new strategies for AC regeneration. In recent years there has been increased interest in the use of tissue-specific aligned porous freeze-dried extracellular matrix (ECM) scaffolds as an off-the-shelf approach for AC repair, as they allow for cell infiltration, provide biological cues to direct target-tissue repair and permit aligned tissue deposition, desired in AC repair^[4]. However, most ECM-scaffolds lack the appropriate mechanical properties to withstand the loads passing through the joint^[5]. One solution to this problem is to reinforce the ECM with a stiffer framework made of synthetic materials, such as polylactic acid (PLA)^[6]. Such framework can be 3D printed to produce anatomically accurate implants^[7], attractive in personalized medicine. However, typical 3D prints are static, their design is not optimized for soft-hard interfaces (OC interface), and they may not adapt to the cyclic loading passing through our joints, thus risking implant failure. To tackle this limitation, more compliant or dynamic designs can be printed, such as coil-shaped structures^[8]. Thus, in this study we use finite element modelling to create different designs that mimic the mechanical properties of AC and prototype them in PLA, using polyvinyl alcohol as support. The optimal design will be combined with an ECM scaffold containing a tailored microarchitecture mimicking aspects of native AC.

Acknowledgments: This project has received funding from the European Union's Horizon Europe research and innovation MSCA PF programme under grant agreement No. 101110000.

While high-performance ceramics like alumina and zirconia exhibit excellent wear resistance, they provide poor osseointegration capacity. As osseointegration is crucial for non-cemented joint prostheses, new techniques have been successfully developed for biofunctionalizing high-performance ceramic surfaces. Stable cell adhesion can be achieved by covalently bound specific peptides. In this study we investigate the effect of sterilization processes on organo-chemically functionalized surfaces.

To enhance the performance of alumina-toughened zirconia ceramics (ATZ), a 3-aminopropyldiisopropylethoxysilane (APDS) monolayer was applied and coupled with cyclo-RGD peptides (cRGD) by using bifunctional crosslinker bis(sulfosuccinimidyl)suberat (BS³). The samples were sterilized using e-beam or gamma-sterilization at 25 kGy, either before or after biofunctionalization with cRGD. Functionalization stability was investigated by contact angle measurements. The functionality of cRGD after sterilization was demonstrated using proliferation tests and cytotoxicity assays. Immunofluorescence staining (pFAK, Actin, DAPI) was conducted to evaluate the adhesion potential between the samples and human mesenchymal stem cells (hMSCs).

Functionalized samples before and after sterilization showed no significant difference regarding their contact angles. A proliferation test demonstrated that the cells on functionalized samples proliferate significantly more than on untreated samples before and after sterilization. hMSCs showed a significant higher proliferation on gamma sterilized samples compared to all other groups after 14 days. It was confirmed that the samples did not exhibit cytotoxic behavior before or after sterilization. Fluorescence microscopy demonstrated that both, cells on sterilized and on non-sterilized samples, expressed high levels of pFAK-Y397.

The investigated functionalization enables improved adhesion and proliferation of hMSCs and is stable against the investigated sterilization processes. This is of importance as the option of having a sterile product enables the start of the translation of this biofunctional coating towards preclinical and subsequently first-in-man applications.

Acknowledgments: We acknowledge the financial support of the Federal Ministry of Education and Research, BMBF (13GW0452A-C).

Articular cartilage is a relatively hypoxic tissue with a unique extracellular matrix that is enriched with cations, resulting in an elevated interstitial fluid osmolarity. Several biomechanical and physicochemical stimuli are reported to influence chondrocyte metabolism. For regenerative in vitro applications, increasing the extracellular osmolarity above plasma level to more physiological valuesinduces chondrogenic marker expression and the differentiation of chondroprogenitor cells. Calcineurin inhibitor FK506 modulates the differentiation of primary chondrocytes under such conditions and its effect on cell proliferation, extracellular matrix quality, and BMP- and TGF-β signaling will be described. Supraphysiological osmolarity compromises chondrocyte proliferation, while physosmolarity or FK506 did not. Rather, the combination of the latter increased proteoglycan and collagen expression in chondrocytesin vitro and in situ, affecting expression of TGF-β-inducible protein TGFBI and chondrogenic (SOX9, Col2) as well as terminal differentiation markers (e.g., Col10). Surprisingly, expression of particularly minor collagens (e.g., Col9, Col11) was improved. Physiological osmolarity seems to promote terminal chondrogenic differentiation of progenitor cells through sensitization of TGF-β superfamily signaling at the type I receptor. While hyperosmolarity alone facilitates TGF-β superfamily signaling, FK506 seems to enhance signaling by releasing the FKBP12 break from the type I receptor to improve collagenous marker expression. Our data help explaining seemingly contradictory earlier findings and potentially benefit future cell-based cartilage repair strategies.

Development of osteoarthritis (OA) correlates with epigenetic alteration in chondrocytes. H3K27me3 demethylase UTX is known to regulate tissue homeostasis, but its role in the homeostasis of articulating joint tissue is poorly understood. Forced UTX expression upregulated H3K27me3 enrichment at the Sox9 promoter region to inhibit key extracellular matrix (ECM) molecules, like e.g. type II collagen, aggrecan, and glycosaminoglycans in articular chondrocytes. Utx loss in vitro altered the H3K27me3-binding epigenomic landscape, which contributes to mitochondrial activity, cellular senescence, and cartilage development. Functional target genes of Utx comprise insulin-like growth factor 2 (Igf2) and polycomb repressive complex 2 (PRC2) core components Eed and Suz12. Specifically, Utx deletion promoted Tfam transcription, mitochondrial respiration, ATP production and Igf2 transcription, but inhibited Eed and Suz12 expression. Igf2 inhibition or forced Eed or Suz12 expression increased H3K27 trimethylation and H3K27me3 enrichment at the Sox9 promoter, compromising Utx loss-induced ECM overproduction. Overexpression of Utx in murine knee joints aggravated OA development, including articular cartilage damage, synovitis, osteophyte formation, and subchondral bone loss. Transgenic mice with a chondrocytespecific Utx knockout develop thicker articular cartilage as compared to wild-type controls and show fewer gonarthrotic symptoms during destabilized medial meniscus- and collagenase-induced joint injury. In summary, UTX represses chondrocytic activity and accelerates cartilage degradation during OA, while Utx loss promotes cartilage integrity through epigenetic stimulation of mitochondrial biogenesis and Igf2 transcription. This highlights a novel noncanonical role of Utx that regulates articular chondrocyte anabolism and OA development.

Chondrocytic activity is downregulated by compromised autophagy and mitochondrial dysfunction to accelerate the development of osteoarthritis (OA). Irisin is a cleaved form of fibronectin type III domain containing 5 (FNDC5) and known to regulate bone turnover and muscle homeostasis. However, little is known about the role of irisin in chondrocytes and the development of OA. This talk will shed light on FNDC5 expression by human articular chondrocytes and compare normal and osteoarthritic cells with respect to autophagosome marker LC3-II and oxidative DNA damage marker 8-hydroxydeoxyguanosine (8-OHdG). In chondrocytes in vitro, irisin improves IL-1β-mediated growth inhibition, loss of specific cartilage markers and glycosaminoglycan production. Irisin further suppressed Sirt3 and UCP- 1 to improve mitochondrial membrane potential, ATP production, and catalase. This attenuated IL-1β-mediated production of reactive oxygen species, mitochondrial fusion, mitophagy, and autophagosome formation. In a surgical murine model of destabilization of the medial meniscus (DMM) intra-articular administration of irisin alleviates symptoms like cartilage erosion and synovitis. Furthermore, gait profiles of the treated limbs improved. In chondrocytes, irisin treatment upregulates autophagy, 8-OHdG and apoptosis in cartilage of DMM limbs. Loss of FNDC5 in chondrocytes correlates with human knee OA and irisin repressed inflammation-mediated oxidative stress and deficient extracellular matrix synthesis through retaining mitochondrial biogenesis and autophagy. The talk sheds new light on the chondroprotective actions of this myokine and highlights the remedial effects of irisin during progression of OA.

Osteoporosis (OP) and osteoarthritis (OA) are leading causes of musculoskeletal dysfunction in elderly, with chondrocyte senescence, inflammation, oxidative stress, subcellular organelle dysfunction, and genomic instability as prominent features. Age-related intestinal disorders and gut dysbiosis contribute to host tissue inflammation and oxidative stress by affecting host immune responses and cell metabolism. Not surprisingly, the development of OP and OA correlate with dysregulations of the gut microflora in rodents and humans. Intestinal microorganisms produce metabolites, including short-chain fatty acids, bile acids, trimethylamine N-oxide, and liposaccharides, affecting mitochondrial function, metabolism, biogenesis, autophagy, and redox reactions in chondrocytes to regulate joint homeostasis. Modulating the abundance of specific gut bacteria, like Lactobacillus and Bifidobacterium, by probiotics or fecal microbiota transplantation appears to suppress age-induced chronic inflammation and oxidative damage in musculoskeletal tissue and holds potential to slow down OP development. The talk will highlight treatment options with probiotics or metabolites for modulating the progression of OA and OP.

Within the field of disc degeneration-related low back pain, the spine community has been increasingly acknowledging the regenerative potential of extracellular vesicles (EVs). EVs are small lipid bilayer-delimited particles naturally released by cells, involved in intercellular signaling. They do so by interacting with recipient cells and releasing their biological cargo (e.g., mRNA, miRNA, DNA, protein, lipid)

EVs derived from mesenchymal stromal cells and, more recently, also EVs from notochordal cells, the cells residing within the core of the juvenile human disc, are being actively studied. In general, they have been proposed to mitigate inflammation/catabolic processes, reduce apoptosis, stimulate proliferation and even improve the matrix producing capacity of the treated cells. Within this context, appropriate characterization of EVs is essential to increase the level of evidence that the reported effects are indeed EV-associated. To analyze the purity and biochemical composition of EV preparations the International Society for Extracellular Vesicles (ISEV) has prepared guidelines recommending the analysis of multiple (EV) markers, as well as proteins co-isolated/recovered with EVs. Alongside, to prove that the effects are EV-associated and not due to co-isolated factors from the tissue or cells used to derive the EVs, appropriate technical controls need to be taken along (during cell/tissue culture). As such the question arises: “what is the evidence so far?”

While from a fundamental perspective EVs are very appealing, the use of natural EVs in clinical applications is challenging. It comes with drawbacks, including biologic variability, yield, cumbersome isolation, and challenging upscaling and storage to achieve industrial levels. To date there is no FDA-approved EV-based therapy for disc-related lower back pain.

Nonetheless, EV-based therapeutic approaches have unique advantages over the use of (pluripotent) stem cell-based therapies, such as a high biologic, but low immunogenic and tumorigenic potential.

Acknowledgements: This talk is based on experiences from part of the project NC-CHOICE [no. 19251] of the research talent programme VICI financed by the Dutch Research Council (NWO) and the iPSpine project that receives funding from the European Union's Horizon 2020 research and innovation program under grant agreement no. 825925.

Messenger RNA (mRNA) is a new class of drug that can be used to express a therapeutic protein and, in contrast to DNA, is safer and inexpensive. Among its advantages, mRNA will immediately begin to express its encoded protein in the cell cytoplasm. The protein will be expressed for a period of time, after which the mRNA is degraded. There is no risk of genetic damage, one of the concerns with plasmid DNA (pDNA) used in traditional gene therapy approaches. Nevertheless, mRNA application in tissue regeneration and regenerative medicine remains limited. In this case, mRNA must overcome its main hurdles: immunogenicity, lack of stability, and intracellular delivery. Research has been done to overcome these limitations, and the future of mRNA seems promising for tissue repair^1,2. This keynote talk will address questions including: What are the opportunities for mRNA to improve outcomes in musculoskeletal tissue repair, in particular bone and cartilage? What are the key factors and challenges to expediting this technology to patient treatment (beyond COVID-19 vaccination)?

Acknowledgements: E.R.B thanks the cmRNAbone project funded by the European Union's Horizon 2020 research and innovation program under the grant agreement no. 874790 and the NIH R01 AR074395 from NIAMS for funding her mRNA work.

Articulating cartilage experiences a multitude of biophysical cues. Due to its primary function in distributing load with near frictionless articulation, it is clear that a major stimulus for cartilage homeostasis and regeneration is the mechanical load it experiences on a daily basis. While these effects are considered when performing in vivo studies, in vitro studies are still largely performed under static conditions. Therefore, an increasing complexity of in vitro culture models is required, with the ultimate aim to recreate the articulating joint as accurately as possible. We have for many years utilized a complex multiaxial load bioreactor capable of applying tightly regulated compression and shear loading protocols. Using this bioreactor, we have been able to demonstrate the mechanical induction of human bone marrow stromal cell (BMSC) chondrogenesis in the absence of exogenous growth factors. Building on previous bioreactor studies that demonstrated the mechanical activation of endogenous TGFβ, and subsequent chondrogenesis of human bone marrow derived MSCs, we have been further increasing the complexity of in vitro models. For example, the addition of high molecular weight hyaluronic acid, a component of synovial fluid, culture medium leads to reduced hypertrophy and increased glycosaminoglycan deposition. The ultimate aim of all of these endeavors is to identify promising materials and therapies during in vitro/ ex vivo studies, therefore reducing the numbers or candidates that are finally tested using in vivo studies. This 3R approach can improve the opportunities for success while leading to more ethically acceptable product development pathways.

Photobiomodulation (PBM), the use of light for regenerative purposes, has a long history with first documentations several thousand years ago in ancient Egypt and a Nobel Price on this topic at the beginning of last century (by Niels Finsen). Nowadays, it is in clinical use for indications such as wound healing, pain relief and anti-inflammatory treatment. Given the rising numbers of in vitro studies, there is increasing evidence for the underlying mechanisms such as wavelength dependent reactive oxygen production and adenosine triphosphate generation. In cartilage regeneration, the use of PBM is controversially discussed with divergent results in clinics and insufficient in vitro studies. As non-invasive therapy, PMB is, though, of particular importance, since a general regenerative stimulus would be of great benefit in the otherwise only surgically accessible tissues. We therefore investigated the influence of different wavelengths - blue (475 nm), green (516 nm) or red (635 nm) of a low-level laser (LLL) - on the chondrogenic differentiation of chondrocytes and adipose derived stromal cells of different human donors and applied the light in different settings (2D, 3D) with cells in a proliferative or differentiating stage. All assessed parameters (spheroid growth, histology, matrix quantification and gene expression) revealed an influence of LLL on chondrogenesis in a donor-, wavelength- and culture-model-dependent manner. Especially encouraging was the finding, that cells with poor chondrogenic potential could be improved by one single 2D treatment. Amongst the three wave lengths, red light was the most promising one with the most positive impact. Although in vivo data are still missing, these in vitro results provide evidence for a proper biofunctional effect of LLL.

Osteoarthritis (OA) is the most prevalent degenerative joint disease that is a leading cause of disability worldwide. Existing therapies of OA only address the symptoms. Liraglutide is a well-known anti-diabetic medication that is used to treat type 2 diabetes and obesity. In inflammatory and post-traumatic OA animal models, liraglutide has demonstrated anti-inflammatory, pain-relieving, and cartilage-regenerating effects1 . The objective of this study is to investigate liraglutide's ability to reduce inflammation and promote anabolism in human OA chondrocytes in vitro. Pellets formed with human OA chondrocytes were cultured with a chondrogenic medium for one week to form cartilage tissue. Afterward, pellets were cultured for another 2 weeks with a chondropermissive medium. The OA group was treated with IL-1β to mimic an inflammatory OA condition. The drug group was treated with 0.5 or 10 µM liraglutide. On days 0, 1, and 14, pellets were collected. Conditioned medium was collected over the 2 weeks culture period. The gene and protein expression levels of regenerative and inflammatory biomarkers were evaluated and histological analyzes were performed. Results showed that the nitric oxide release of the OA + 0.5 µM liraglutide and OA + 10 µM liraglutide groups were lower than the OA group. The DNA content of the OA + 0.5 µM liraglutide and OA + 10 µM liraglutide groups were higher than the OA group on day 14. The RT-qPCR results showed that the anabolism (ACAN, COMP, and COL2) markers were higher expressed in the OA + 0.5 µM liraglutide and OA + 10 µM liraglutide groups when compared with the OA group. The inflammation (CCL-2 and IL-8) markers and catabolism markers (MMP-1, MMP-3, ADAMTS4, and ADAMTS5) had lower expression levels in the OA + liraglutide groups compared to the OA group. The histomorphometric analysis (Figure 1) supported the RT-qPCR results. The results indicate that liraglutide has anabolic and anti-inflammatory effects on human OA chondrocyte pellets.

Acknowledgments: This project has received funding from the Eurostars-2 joint program with co-funding from the European Union Horizon 2020 research and innovation program. The funding agencies supporting this work are (in alphabetical order of participating countries): France: BPI France; Germany: Project Management Agency (DLR), which acts on behalf of the Federal Ministry of Education and Research (BMBF); The Netherlands: Netherlands Enterprise Agency (RVO); Switzerland: Innosuisse (the Swiss Innovation Agency).

For any figures and tables, please contact the authors directly.

Knee swelling is common after injury or surgery, resulting in pain, restricted range of movement and limited mobility. Accurately measuring knee swelling is critical to assess recovery. However, current measurement methods are either unreliable or expensive [1,2]. Therefore, a new measurement method is developed. This wearable (the ‘smart brace’) has shown the ability to distinguish a swollen knee from a not swollen knee using multi-frequency-bio impedance analysis (MF-BIA) [3].

This study aimed to determine the accuracy of this smart brace. The study involved 25 usable measurements on patients treated for unilateral knee osteoartritis with a 5mL injection of Lidocaïne + DepoMedrol (1:4). MF-BIA measurements were taken before and after the injection, both on the treated and untreated knee. The smart brace accurately measured the effect of the injection by a decrease in resistance of up to 2.6% at 100kHz (p<0.01), where commonly used gel electrodes were unable to measure the relative difference. Remarkably, both the smart brace and gel electrodes showed a time component in the MF-BIA measurements.

To further investigate this time component, 10 participants were asked to lie down for 30 minutes, with measurements taken every 3 minutes using both gel electrodes and the smart brace on both legs. The relative change between each time step was calculated to determine changes over time. The results showed presence of a physiological aspect (settling of knee fluids), and for the brace also a mechanical aspect (skin-electrode interface) [4]. The mechanical aspect mainly interfered with reactance values.

Overall, the smart brace is a feasible method for quantitatively measuring knee swelling as a relative change over time. However, the skin-electrode interface should be improved for reliable measurements at different moments in time. The findings suggest that the smart brace could be a promising tool for monitoring knee swelling during rehabilitation.

Osteoarthritis (OA) and diabetis mellitus type 2 (DMT2) are pathogenetically linked. Complement dysregulation contributes to OA and could be involved in DMT2. The inflammatory anaphylatoxin C5a is released during complement activation. This study aims to understand the specific responses of chondrocytes isolated from diabetic and non-diabetic rats exposed to C5a and/or the proinflammatory cytokine TNFα in vitro dependent on the glucose supply. Articular chondrocytes of adult Zucker Diabetic Fatty (ZDF) rats (homozygous: fa/fa, diabetic, heterozygous: fa/+, lean controls) were exposed to 10 ng/mL TNFα and 25 ng/mL C5a alone or in combination, both, under normo- (NG, 1 g/L glucose) and hyperglycemic (HG, 4.5 g/L glucose) conditions (4 or 24 h). Chondrocyte survival, metabolic activity and gene expression of collagen type 2, suppressors of cytokine signaling (SOCS)1, −3 and anti-oxidative hemoxygenase-1 (HMOX1) were assessed. The complement regulatory protein CD46 and cell nuclei sizes were analyzed. Chondrocyte vitality remained unaffected by the treatment. Metabolic activity was impaired in chondrocytes of non-diabetic rats under HG conditions. Collagen type 2 transcription was suppressed by TNFα under HG condition in chondrocytes from nondiabetic donors and under both conditions in those of DMT2 rats (24 h)

Except for DMT2 chondrocytes under HG (4 h), HMOX1 was generally induced by TNFα +/- C5a (NG, HG). C5a elevated HMOX1 only in chondrocytes of controls. The SOCS1/3 genes were increased by TNFα (NG, diabetic, non diabetic, 4 and 24 h). This could also be observed in chondrocytes of diabetic, but not of lean rats (24 h, HG). At 4 h, C5a induced SOCS1 only in non diabetic chondrocytes (NG, HG). Cytoprotective CD46 protein was suppressed by TNFα under NG condition. Nuclear volumes of chondrocyte were lower in chondrocytes from DMT2 rats compared to those from controls. The differential response suggests that chondrocytes are irreversibly compromised by DMT2.

Achnowledgement: The authors are grateful for the support by the “Stiftung Edoprothetik (S 04/21)”

The application of immune regenerative strategies to deal with unsolved pathologies, such as tendinopathies, is getting attention in the field of tissue engineering exploiting the innate immunomodulatory potential of stem cells [1]. In this context, Amniotic Epithelial Cells (AECs) represent an innovative immune regenerative strategy due to their teno-inductive and immunomodulatory properties [2], and because of their high paracrine activity, become a potential stem cell source for a cell-free treatment to overcome the limitations of traditional cell-based therapies. Nevertheless, these immunomodulatory mechanisms on AECs are still not fully known to date. In these studies, we explored standardized protocols [3] to better comprehend the different phenotypic behavior between epithelial AECs (eAECs) and mesenchymal AECs (mAECs), and to further produce an enhanced immunomodulatory AECs-derived secretome by exposing cells to different stimuli. Hence, in order to fulfill these aims, eAECs and mAECs at third passage were silenced for CIITA and Nrf2, respectively, to understand the role of these molecules in an inflammatory response. Furthermore, AECs at first passage were seeded under normal or GO-coated coverslips to study the effect of GO on AECs, and further exposed to LPS and/or IL17 priming to increase the anti-inflammatory paracrine activity. The obtained results demonstrated how CIITA and Nrf2 control the immune response of eAECs and mAECs, respectively, under standard or immune-activated conditions (LPS priming). Additionally, GO exposition led to a faster activation of the Epithelial-Mesenchymal transition (EMT) through the TGFβ/SMAD signaling pathway with a change in the anti-inflammatory properties. Finally, the combinatory inflammatory stimuli of LPS+IL17 enhanced the paracrine activity and immunomodulatory properties of AECs. Therefore, AECs-derived secretome has emerged as a potential treatment option for inflammatory disorders such as tendinopathies.

Acknowledgement: This research is part of the P4FIT project ESR1, funded under the H2020-ITN-EJD-Marie-Skłodowska-Curie grant agreement 955685.

Digital Ventilated Cages (DVC) offer an innovative technology to obtain accurate movement data from a single mouse over time [1]. Thus, they could be used to determine the occurrence of a tendon damage event as well as inform on tissue regeneration [2,3]. Therefore, using the mouse model of tendon experimental damage, in this study it has been tested whether the recovery of tissue microarchitecture and of extracellular matrix (ECM) correlates with the motion data collected through this technology.

Mice models were used to induce acute injury in Achilles tendons (ATs), while healthy ones were used as control. During the healing process, the mice were housed in DVC cages (Tecniplast) to monitor animal welfare and to study biomechanics assessing movement activity, an indicator of the recovery of tendon tissue functionality. After 28 days, the AT were harvested and assessed for their histological and immunohistochemical properties to obtain a total histological score (TSH) that was then correlated to the movement data.

DVC cages showed the capacity to distinguish activity patterns in groups from the two different conditions. The data collected showed that the mice with access to the mouse wheel had a higher activity as compared to the blocked wheel group, which suggests that the extra movement during tendon healing improved motion ability. The histological results showed a clear difference between different analyzed groups. The bilateral free wheel group showed the best histological recovery, offering the highest TSH score, thus confirming the results of the DVC cages and the correlation between movement activity and structural recovery.

Data obtained showed a correlation between TSH and the DVC cages, displaying structural and movement differences between the tested groups. This successful correlation allows the usage of DVC type cages as a non-invasive method to predict tissue regeneration and recovery.

Acknowledgements: This research is part of the P4FIT project ESR13, funded by the H2020-ITN-EJD MSCA grant agreement No.955685.

Stem cell therapy for the intervertebral disc (IVD) is highly debated but holds great promises. From previous studies, it is known that notochordal cells are highly regenerative and may stimulate other differentiated cells to produce more matrix. Lately, a particular tissue-specific progenitor cell population has been identified in the centre of the intervertebral disc (IVD. The current hope is that these nucleus pulposus progenitor cells (NPPC) could play a particular role in IVD regeneration.

Current evidence confirms the presence of these cells in murine, canine, bovine and in the human fetal/surgical samples. Noteworthy, one of the main markers to identify these cells, i.e., Tie2, is a typical marker for endothelial cells. Thus, it is not very clear what their origin and their role might be in the context of developmental biology. In human surgical specimens, their presence is, even more, obscured depending on the donor's age and the condition of the IVD and other yet unknown factors.

Here, I revisit the recent literature on regenerative cells identified for the IVD in the past decades. Current evidence how these NPPC can be isolated and detected in various species and tissues will be recapitulated. Future directions will be provided on how these progenitor cells could be used for regenerative medicine and tissue engineering.

Back pain is a leading cause of disability worldwide and it is primarily considered to be triggered by intervertebral disc (IVD) degeneration (IVDD). Current treatments may improve pain and mobility, but carry high costs and fail to address IVD repair or regeneration. As no effective therapeutic approach has been proposed to restore inflamed and degenerated IVDs, there is the urgent need to clarify the key pathomechanism of IVDD, the involvement of inflammation, particularly complement activation in matrix catabolism, and how to target them towards tissue repair/regeneration. Mesenchymal stem cell (MSC)-based therapies have become the focus of several regenerative IVD studies. Although patients in clinical trials reported less pain after cell therapy, the long-term success of cell engraftment is unclear due to the hostile IVD environment. The mechanism-of-action of MSCs is mostly dependent on the secreted soluble factors. Moreover, priming of MSC with interleukin (IL)-1β modulates the secretome content, improving its anti-inflammatory and regenerative effect on IVDD organ culture models. MSC-derived extracellular vesicles (EVs) have also been shown to modulate human IVD cells towards a healthy IVD phenotype in vitro. However, the mechanisms involved in the effect of secretome and EVs, particularly with regard to immunomodulation and matrix metabolism, are not fully understood. Our work investigates the effects of secretome and EVs secreted by IL-1β-primed MSCs to impair IVD matrix degradation and/or improve matrix formation in IVDD.

Mechanical loading is important to maintain the homeostasis of the intervertebral disc (IVD) under physiological conditions but can also accelerate cell death and tissue breakdown in a degenerative state. Bioreactor loaded whole organ cultures are instrumental for investigating the effects of the mechanical environment on the IVD integrity and for preclinical testing of new therapies under simulated physiological conditions. Thereby the loading parameters that determine the beneficial or detrimental reactions largely depend on the IVD model and its preparation. Within this symposium we are discussing the use of bovine caudal IVD culture models to reproduce tissue inflammation or matrix degradation with or without bioreactor controlled mechanical loading. Furthermore, the outcome parameters that define the degenerative state of the whole IVD model will be outlined. Besides the disc height, matrix integrity, cell viability and phenotype expression, the tissue secretome can provide indications about potential interactions of the IVD with other cell types such as neurons. Finally, a novel multiaxial bioreactor setup capable of mimicking the six degrees-of-freedom loading environment of IVDs will be introduced that further advances the relevance of preclinical ex-vivo testing.

The relationship of degeneration to symptoms has been questioned. MRI detects apparently similar disc degeneration and degenerative changes in subjects both with and without back pain. We aimed to overcome these problems by re-annotating MRIs from asymptomatic and symptomatic groups onto the same grading system.

We analysed disc degeneration in pre-existing large MRI datasets. Their MRIs were all originally annotated on different scales. We re-annotated all MRIs independent of their initial grading system, using a verified, rapid automated MRI annotation system (SpineNet) which reported degeneration on the Pfirrmann (1-5) scale, and other degenerative features (herniation, endplate defects, marrow signs, spinal stenosis) as binary present/absent. We compared prevalence of degenerative features between symptomatics and asymptomatics.

Pfirrmann degeneration grades in relation to age and spinal level were very similar for the two independent groups of symptomatics over all ages and spinal levels. Severe degenerative changes were significantly more prevalent in discs of symptomatics than asymptomatics in the caudal but not the rostral lumbar discs in subjects < 60 years. We found high co-existence of degenerative features in both populations. Degeneration was minimal in around 30% of symptomatics < 50 years.

We confirmed age and disc level are significant in determining imaging differences between asymptomatic and symptomatic populations and should not be ignored. Automated analysis, by rapidly combining and comparing data from existing groups with MRIs and information on LBP, provides a way in which epidemiological and ‘big data’ analysis could be advanced without the expense of collecting new groups.

Bottom-up tissue engineering (TE) strategies employing microscale living materials as building blocks provide a promising avenue for generating intricate 3D constructs resembling native tissues. These microtissue units exhibit high cell densities and a diverse extracellular matrix (ECM) composition, enhancing their biological relevance. By thoughtfully integrating different cell types, the establishment of vital cell-cell and cell-matrix interactions can be promoted, enabling the recreation of biomimetic micro-niches and the replication of complex morphogenetic processes. Notably, by co-assembling blood vessel-forming endothelial cells with supportive stromal cells, microtissues with stable capillary beds, referred to as vascular units (VUs), can be generated. Through a modular TE approach, these VUs can be further combined with other microtissues and biomaterials to construct large-scale vascularized tissues from the bottom up. Integration of VUs with technologies such as 3D bioprinting and microfluidics allows for the creation of structurally intricate and perfusable constructs. In this presentation, we will showcase examples of VUs and explore their applications in regenerative medicine and tissue modeling.

Acknowledgements: This work was supported by project EndoSWITCH (PTDC/BTM-ORG/5154/2020) funded by FCT (Portuguese Foundation for Science and Technology).

Aging impairs the regenerative capacity of musculoskeletal tissues and is associated with poor healing outcomes. PolgA^D257A/D257A (PolgA) mice present a premature aging phenotype due to the accumulation of mitochondrial DNA (mtDNA) point mutations at rates 3 – 5 fold higher compared to wild type mice. Consequently, PolgA mice exhibit the premature onset of clinically-relevant musculoskeletal aging characteristics including frailty, osteo-sarcopenia, and kyphosis. I will present our recent findings on the use of PolgA mice to investigate the effects of aging on the regenerative capacity of bone. In particular, I will focus on the mechano-sensitivity of the regenerative process in aged bone environments and the opportunities it presents for clinical translation of mechanical intervention therapies.

Despite the major advances in osteosynthesis after trauma, there remains a small proportion of patients (<10%) who exhibit delayed healing and/or eventual progression to non-union. While known risk factors exist, e.g. advanced age or diabetes, the exact molecular mechanism underlying the impaired healing is largely unknown and identifying which specific patient will develop healing complications is still not possible in clinical practice. The talk will cover our novel multimodal approaches in small animals, which have the potential to precisely capture and understand biological changes during fracture healing on an individual basis. Via combining emerging omics technologies with our recently developed femur defect loading equipment in mice, we provide a platform to precisely link mechanical and molecular analyses during fracture healing.

The Masquelet technique is a variable method for treating critical-sized bone defects, but there is a need to develop a technique for promoting bone regeneration. In recent studies of bone fracture healing promotion, macrophage-mesenchymal stem cell (MSC) cross-talk has drawn attention. This study aimed to investigate macrophage expression in the induced membrane (IM) of the Masquelet technique using a mouse critical-sized bone defect model.

The study involved a 3-mm bone defect created in the femur of mice and fixed with a mouse locking plate. The Masquelet (M) group, in which a spacer was inserted, and the Control (C) group, in which the defect was left intact, were established. Additionally, a spacer was inserted under the fascia of the back (B group) to form a membrane due to the foreign body reaction. Tissues were collected at 1, 2, and 4 weeks after surgery (n=5 in each group), and immunostaining (CD68, CD163: M1, M2 macrophage markers) and RT-qPCR were performed to investigate macrophage localization and expression in the tissues.

The study found that CD68-positive cells were present in the IM of the M group at all weeks, and RT-qPCR showed the highest CD68 expression at 1 week. In addition, there was similar localization and expression of CD163. The C group showed lower expression of CD68 and CD163 than the M group at all weeks. The B group exhibited CD68-positive cells in the fibrous capsule and CD163-positive cells in the connective tissue outside the capsule, with lower expression of both markers compared to the M group at all weeks.

Macrophage expression in IM in M group had different characteristics compared to C group and B group. These results suggest that the IM differs from the fibrous capsules due to the foreign body reaction, and the macrophage-MSC cross-talk may be involved in Masquelet technique.

Orthopaedic soft tissues, such as tendons, ligaments, and articular cartilage, rely on their unique collagen fiber architectures for proper functionality. When these structures are disrupted in disease or fail to regenerate in engineered tissues, the tissues transform into dysfunctional fibrous tissues. Unfortunately, collagen synthesis in regenerating tissues is often slow, and in some cases, collagen fibers do not regenerate naturally after injury, limiting repair options. One of the research focuses of my team is to develop functional fiber replacements that can promote in vivo repair of musculoskeletal tissues throughout the body. In this presentation, I will discuss our recent advancements in electrowriting 3D printing of natural polymers for creating functional fiber replacements. This manufacturing process utilizes electrical signals to control the flow of polymeric materials through an extrusion nozzle, enabling precise deposition of polymeric fibers with sizes that cannot be achieved using conventional extrusion printing methods. Furthermore, it allows for the formation of fiber organizations that surpass the capabilities of conventional electrospinning processes. During the presentation, I will showcase examples of electrowritten microfiber scaffolds using various naturally-derived polymers, such as gelatin (a denatured form of collagen) and silk fibroin. I will discuss the functional properties of silk-based scaffolds and highlight how they exhibit restored β-sheet and α-helix structures [1]. This restoration results in an elastic response of up to 20% deformation and the ability to withstand cyclic loading without plastic deformation. Additionally, I will present our latest results on the compatibility of this technique with patterning cell-laden fiber structures [2]. This novel biofabrication process allows for the printing of biomimetic microscale architectures with high cell viability, and offers a promising approach to understanding how shear and elongation forces influence cell development of hierarchical (collagen) fibers.

Acknowledgements: The author would like to thank the Reprint project (OCENW.XS5.161) and the program “Materials Driven Regeneration” (024.003.013) by the Netherlands Organization for Scientific Research for the financial support.

The anterior cruciate ligament (ACL) is the connective tissue located at the end of long bones providing stability to the knee joint. After tear or rupture clinical reconstruction of the tissue remains a challenge due to the particular mechanical properties required for proper functioning of the tissue. The outstanding mechanical properties of the ACL are characterized by a viscoelastic behavior responsible of the dissipation of the loads that are transmitted to the bone. These mechanical properties are the result of a very specialized graded extracellular matrix that transitions smoothly between the heterotypic cells, stiffness and composition of the ACL and the adjacent bone. Thus, mimicking the zonal biochemical composition, cellular phenotype and organization are key to reset the proper functioning of the ACL.

We have previously shown how the biochemical composition presented to cells in electrospun scaffolds results in haptokinesis, reverting contact-guidance effects.^[1] Here, we demonstrate that contact guidance can also be disrupted by structural parameters in aligned wavy scaffolds. The presentation of a wavy fiber arrangement affected the cell organization and the deposition of a specific ECM characteristic of fibrocartilage. Cells cultured in wavy scaffolds grew in aggregates, deposited an abundant ECM rich in fibronectin and collagen II, and expressed higher amounts of collagen II, X and tenomodulin as compared to aligned scaffolds. In-vivo implantation in rabbits of triphasic scaffolds accounting for aligned-wavy-aligned zones showed a high cellular infiltration and the formation of an oriented ECM, as compared to traditional aligned scaffolds.^[2]

A major obstacle in biofabrication is replicating the organization of the extracellular matrix and cellular patterns found in anisotropic tissues within bioengineered constructs. While magnetically-assisted 3D bioprinting techniques have the potential to create scaffolds that mimic natural biological structures, they currently lack the ability to accurately control the dispersion of magnetic substances within the bioinks without compromising the fidelity of the intended composite. To overcome this dichotomy, the concepts of magnetically- and matrix-assisted 3D bioprinting are combined here. This method preserves the resolution of printed structures by keeping low viscosity bioinks uncrosslinked during printing, which allows for the arrangement of magnetically-responsive microfibers without compromising the structural integrity of the design. Solidification is induced after the microfibers are arranged in the desired pattern. Furthermore, the precise design of these magnetic microfillers permits the utilization of low levels of inorganic materials and weak magnetic field strengths, which reduces the potential risks that may be associated with their use. The effectiveness of this approach is evaluated in the context of tendon tissue engineering, and the results demonstrate that combining the tendons like anisotropic fibrous microstructure with remote magneto-mechanical stimulation during in vitro maturation provides both biochemical and biophysical cues that effectively guide human adipose-derived stem cells towards a tenogenic phenotype In summary, the developed strategy allows the fabrication of anisotropic high-resolution magnetic composites with remote stimulation functionalities, opening new horizons for tissue engineering applications.

Acknowledgments: ERC Grant CoG MagTendon nr 772817, BioChips PoC project nr 10106930, (PD/BD/129403/2017), (CEECIND/01375/2017), (2020.03410.CEECIND), (2022.05526.PTDC), (ED481B2019/025).

Cartilage lesions often undergo irreversible progression due to low self-repair capability of this tissue. Tissue engineered approaches based in extrusion bioprinting of constructs loaded with stem cell spheroids may offer valuable alternatives for the treatment of cartilage lesions. Human mesenchymal stromal cell (hMSC) spheroids can be chondrogenically differentiated faster and more efficiently than single cells. This approach allows obtaining larger tissues in a rapid, controlled and reproducible way. However, it is challenging to control tissue architecture, construct stability, and cell viability during maturation. In this study we aimed at the development of a reproducible bioprinting process followed by post-bioprinting chondrogenic differentiation procedure using large quantities of hMSC spheroids encapsulated in a xanthan gum-alginate hydrogel. Multi-layered constructs were bioprinted, ionically crosslinked, and chondrogenically differentiated for 28 days. The expression of glycosaminoglycan, collagen II and IV were observed. After 56 days in culture, the bioprinted constructs were still stable and show satisfactory cell metabolic activity with profuse extracellular matrix production. These results showed a promising procedure to obtain 3D cartilage-like constructs that could be potential use as stable chondral tissue implants for future therapies.

Acknowledgments: The National Council for Scientific and Technological Development (CNPq, Brazil – Grants # 314 724/2021-4, 307 829/2018-9, 430 860/2018-8, 142 050/2018-0 and 465 656/2014-5), the Coordination for the Improvement of Higher Educational Personnel (CAPES, Brazil – PrInt 88 887.364849/2019-00 and PrInt 88 887.310405/2018-00), the Fund for Support to Teaching, Research and Extension from the University of Campinas (FAEPEX/UNICAMP, Brazil – Grants # 2921/18, 2324/21), and the European Union's Horizon 2020 JointPromise project – Precision manufacturing of microengineered complex joint implants, under grant agreement 874 837 are acknowledged for the financial support of this study.

Years ago, we identified the need of a dedicated group and conference for advanced therapies with musculoskeletal indications. We saw a disconnect between high-level science and the criticality of actual medical need, thus creating a gap between research and industry – a gap that needed to be bridged.

To achieve this goal, a vehicle to connect and amplify the expertise of key opinion leaders in advanced therapies in orthopaedics was needed. With that purpose in mind and after years of preparation, the “Advanced Therapies in Orthopaedics Foundation” (ATiO) was established with the aim to create a network consisting of all important stake holders in the field, ranging from clinics & research, to corporates, finance and regulators – an Alliance for Advanced Therapies in Orthopaedics to form the future.

After initial hesitance and failures, with growing knowledge about advanced products and their characteristics, increasingly more medtech and also pharma companies enter the advanced therapies market. However, due to the specifics of the biology and regulation of advanced therapy products, a lot of new know-how is necessary to be successful in this highly promising field.

The HIPGEN study funded under EU Horizon 2020 (Grant 7792939) has the aim to investigate the potential of the first regenerative cell therapy for the improvement of recovery after muscle injury in hip fracture patients. For this aim we intramuscularly injected placental derived mesenchymal stromal cells during hip fracture arthroplasty. Despite not having reached the primary endpoint, which was the Short Physical Performance Battery, we could observe an increase in abductor muscle strength and a faster return to balance looking at symmetry in insole measurements during follow up.

Bone regeneration is a complex but very well organized process in which the immune system has a decisive role. The adaptive immune system and its experience level (percentage of effector and memory T cells) has been proven to influence the healing cascade especially in the early healing phases. This opens the possibility of an early intervention to enhance bone healing during the primary clinical treatment. Patients stratified for possible delayed bone healing could benefit from immunomodulatory treatment approaches. In pre-clinical studies cells and signaling molecules have been identified that could represent promising candidates to help patients in need.

Osteoarthritis (OA) is the most common joint disease, affecting approximately 16% of the adult population worldwide. The chronic inflammation in the joint leads to the breakdown of cartilage, which leads to permanent pain and limitations in everyday life at an early stage of the disease. To date, there is no therapy that can interrupt the inflammatory state or reverse cartilage damage. The PROTO consortium (funded by the EU Horizon Europe program, Grant 101095635) aims to prevent the development of OA by correcting a pathological biomechanical pattern by a digital training intervention and to treat early stage OA with an innovative allogeneic cell therapy.

Osteotomies in the musculoskeletal system are joint preserving procedures to correct the alignment of the patient. In the lower limb, most of the pre-operative planning is performed on full leg weightbearing radiographs. However, these images contain a 2-dimensional projection of a 3-dimensional deformity, lack a clear visualization of the joint surface and are prone to rotational errors during patient positioning. Weightbearing CT imaging has demonstrated to overcome these shortcomings during the first applications of this device at level of the foot and ankle. Recent advances allow to scan the entire lower limb and novel applications at the level of the knee and hip are on the rise. Here, we will demonstrated the current techniques and 3-dimensional measurements used in supra- and inframalleolar osteotomies around the ankle. Several of these techniques will be transposed to other parts in the lower limb to spark future studies in this field.

3D accurate measurements of the skeletal structures of the foot, in physiological and impaired subjects, are now possible using Cone-Beam CT (CBCT) under real-world loading conditions. In detail, this feature allows a more realistic representation of the relative bone-bone interactions of the foot as they occur under patient-specific body weight conditions. In this context, varus/valgus of the hindfoot under altered conditions or the thinning of plantar tissues that occurs with advancing age are among the most complex and interesting to represent, and numerous measurement proposals have been proposed. This study aims to analyze and compare these measurements from CBCT in weight-bearing scans in a clinical population. Sixteen feet of diabetic patients and ten feet with severe adult flatfoot acquired before/after corrective surgery underwent CBCT scans (Carestream, USA) while standing on the leg of interest. Corresponding 3D shapes of each bone of the shank and hindfoot were reconstructed (Materialise, Belgium). Six different techniques found in the literature were used to calculate the varus/valgus deformity, i.e., the inclination of the hindfoot in the frontal plane of the shank, and the distance between the ground and the metatarsal heads was calculated along with different solutions for the identification of possible calcifications. Starting with an accurate 3D reconstruction of the skeletal structures of the foot, a wide range of measurements representing the same angle of hindfoot alignment were found, some of them very different from each other. Interesting correlations were found between metatarsal height and subject age, significant in diabetic feet for the fourth and fifth metatarsal bones. Finally, CBCT allows 3D assessment of foot deformities under loaded conditions. The observed traditional measurement differences and new measurement solutions suggest that clinicians should consider carefully the anatomical and functional concepts underlying measurement techniques when drawing clinical and surgical conclusions.

Applications of weightbearing computed tomography (WBCT) imaging in the foot and ankle have emerged over the past decade. However, the potential diagnostic benefits are scattered across the literature, and a concise overview is currently lacking. Therefore, we aimed to systematically review all reported diagnostic applications per anatomical region in the foot and ankle. A systematic literature search was performed in the electronic databases PubMed, EMBASE, Cochrane Library, and Web of Science. Search terms consisted of “weightbearing/standing CT and ankle, hind-, mid- or forefoot”. English language studies analyzing the diagnostic applications of WBCT were included. Studies were excluded if they simulated weightbearing CT, described normal subjects, included cadaveric samples or samples were case reports. The modified Methodological Index for Non-Randomized Studies (MINORS) was applied for quality assessment. The added value was defined as the review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines and registered in the Prospero database (CRD42019106980). A total of 48 studies (prospective N=8, retrospective N=36, cohort study N=1, diagnostic N=2, prognostic comparative study N=1) were found to be eligible for review. The following diagnostic applications were identified per anatomical area in the foot: ankle (osteoarthritis N=5, ligament injury N=6); hindfoot (deformity N=9); midfoot (Lisfranc injury N=2, flatfoot deformity N=13, osteoarthritis N=1); forefoot (hallux valgus N=12). The identified studies contained diagnostic applications that could not be used on plain radiographs. The mean MINORS equaled 10.1 on a total of 16 (range: 8 to 12). Diagnostic applications of weightbearing CT imaging are most frequently studied in hindfoot deformity, but other area's areas are on the rise. Post-processing of images was identified as the main added value compared to WBRX. However, the findings should be interpreted with caution as the average quality score was moderate. Therefore, future prospective studies are warranted to consolidate the role of WBCT in diagnostic and therapeutic algorithms.

Varus ankle osteoarthritis (OA) is typically associated with peritalar instability, which may result in altered subtalar joint position. This study aimed to determine the extent to which total ankle replacement (TAR) in varus ankle OA can restore the subtalar position alignment using 3-dimensional semi-automated measurements on WBCT. Fourteen patients (15 ankles, mean age 61) who underwent TAR for varus ankle OA were retrospectively analyzed using semi- automated measurements of the hindfoot based on pre-and postoperative weightbearing WBCT (WBCT) imaging. Eight 3-dimensional angular measurements were obtained to quantify the ankle and subtalar joint alignment. Twenty healthy individuals were served as a control groups and were used for reliability assessments. All ankle and hindfoot angles improved between preoperative and a minimum of 1 year (mean 2.1 years) postoperative and were statistically significant in 6 out of 8 angles (P<0.05). Values The post-op angles were in a similar range to as those of healthy controls were achieved in all measurements and did not demonstrated statistical difference (P>0.05). Our findings indicate that talus repositioning after TAR within the ankle mortise improves restores the subtalar position joint alignment within normal values. These data inform foot and ankle surgeons on the amount of correction at the level of the subtalar joint that can be expected after TAR. This may contribute to improved biomechanics of the hindfoot complex. However, future studies are required to implement these findings in surgical algorithms for TAR in prescence of hindfoot deformity.

Acute syndesmotic ankle injuries continue to impose a diagnostic dilemma and it remains unclear whether weighbearing or external rotation should be exerted rotation during the imaging process. Therefore, we aimed to implement both axial load (weightbearing) and external rotation in the assessment of a clinical cohort of patients with syndesmotic ankle injuries syndesmotic using weightbearing CT imaging. In this retrospective comparative cohort study, patients with an acute syndesmotic ankle injury were analyzed using a WBCT (N= 20; Mean age= 31,64 years; SD= 14,07. Inclusion criteria were an MRI confirmed syndesmotic ankle injury imaged by a bilateral WBCT of the ankle during weightbearing and combined weightbearing-external rotation. Exclusion criteria consisted of fracture associated syndesmotic ankle injuries. Three-dimensional (3D) models were generated from the CT slices. Tibiofibular displacement and Talar Rotation was quantified using automated3D measurements (Anterior TibioFibular Distance (ATFD), Alpha Angle, Posterior TibioFibular Distance (PTFD) and Talar Rotation (TR) Angle) in comparison to a cohort of non-injured ankles.

The term macromolecular crowding is used to describe equilibria and kinetics of biochemical reactions and biological processes that occur via mutual volume exclusion of macromolecules in a highly crowded structureless medium. In vivo, the extracellular space is heavily crowded by a diverse range of macromolecules and thus, biological processes occur rapidly, whilst in vitro, in the absence of macromolecules, the same processes occur very slowly, if they are initiated at all (1-3). This talk will discuss the concept of macromolecular crowding, alone or in combination with other in vitro microenvironment modulators, in tendon engineering context.

Acknowledgements: This work has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme, grant agreement No. 866126. This publication has emanated from research supported by grants from Science Foundation Ireland (SFI) under grant number 19/FFP/6982.

Critical size bone defects deriving from large bone loss are an unmet clinical challenge1. To account for disadvantages with clinical treatments, researchers focus on designing biological substitutes, which mimic endogenous healing through osteogenic differentiation promotion. Some studies have however suggested that this notion fails to consider the full complexity of native bone with respect to the interplay between osteoclast and osteoblasts, thus leading to the regeneration of less functional tissue2. The objective of this research is to assess the ability of our laboratory's previously developed 6-Bromoindirubin-3’-Oxime (BIO) incorporated guanosine diphosphate crosslinked chitosan scaffold in promoting multilineage differentiation of myoblastic C2C12 cells and monocytes into osteoblasts and osteoclasts1, 3, 4. BIO addition has been previously demonstrated to promote osteogenic differentiation in cell cultures5, but implementation of a co-culture model here is expected to encourage crosstalk thus further supporting differentiation, as well as the secretion of regulatory molecules and cytokines2.

Biocompatibility testing of both cell types is performed using AlamarBlue for metabolic activity, and nucleic acid staining for distribution. Osteoblastic differentiation is assessed through quantification of ALP and osteopontin secretion, as well as osteocalcin and mineralization staining. Differentiation into osteoclasts is verified using SEM and TEM, qPCR, and TRAP staining.

Cellular viability of C2C12 cells and monocytes was maintained when cultured separately in scaffolds with and without BIO for 21 days. Both scaffold variations showed a characteristic increase in ALP secretion from day 1 to 7, indicating early differentiation but BIO-incorporated sponges yielded higher values compared to controls. SEM and TEM imaging confirmed initial aggregation and fusion of monocytes on the scaffold's surface, but BIO addition appeared to result in smoother cell surfaces indicating a change in morphology. Late-stage differentiation assessment and co-culture work in the scaffold are ongoing, but initial results show promise in the material's ability to support multilineage differentiation.

Acknowledgements: The authors would like to acknowledge the financial support of the Collaborative Health Research Program (CHRP) through CIHR and NSERC, as well as Canada Research Chair – Tier 1 in Regenerative Medicine and Nanomedicine, and the FRQ-S.

Calcium phosphates-based (CaPs) nanocoatings on metallic prosthesis are widely studied in orthopedics and dentistry because they mimic the mineral component of native human bone and favor the osseointegration process. Despite the fact that different calcium phosphates have different properties (composition, crystallinity, and ion release), only stoichiometric hydroxyapatite (HA) films have been analyzed in deep. Here, we have realized films of different CaPs (HA, beta-tricalcium phosphate (β-TCP) and brushite (DCPD)) onto Ti6Al4V microrough substrates by Ionized Jet Deposition (IJD). We have implemented the heating of substrates at 400°C during deposition to see the effect on coating properties.

Different film features are evaluated: morphology and topography (FEG-SEM, AFM), physical-chemical composition (FT-IR and EDS), dissolution profile and adhesion to substrate (scratch test), with a focus on how the different CaPs and temperature changed the coating features. After coating optimization, we have studied the in vitro BM-MSC behavior, in term of viability and early adhesion.

We have obtained good transfer of fidelity in composition from target to coating for all CaPs, with nanostructured films formed by globular aggregates (~300 nm diameter), with homogeneous and uniform coverage of the substrate surface, without cracks. The heating during deposition has increased the adhesion of the films to the substrate, with higher stability in medium immersion and wettability, features that can improve the biological behavior of cells. All CaP coatings have showed excellent biocompatibility, with DCPD coating that promote higher cells viability at 14 days respect to HA and β- TCP films. About the early cell adhesion, the BM-MSC have showed switch from a globular to an elongated morphology at 6 hours in all coatings respect to the uncoated titanium, sign of better adhesion.

From these results, the fabrication of different CaP nanocoatings with IJD can be a promising for applications in orthopedics and dentistry.

Tendons and tendon-to-bone entheses don't usually regenerate after injury, and the hierarchical organization of such tissues makes them challenging sites of study for tissue engineers. In this study, we have tried a novel approach using miRNA and a bioactive bioink to stimulate the regeneration of the enthesis. microRNAs (miRNAs) are short, non-coding sequences of RNA that act as post-transcriptional regulators of gene and protein expression [1]. Mimics or inhibitors of specific miRNAs can be used to restore lost functions at the cell level or improve healing at the tissue level [2,3]. We characterized the healing of a rat patellar enthesis and found that miRNA-16-5p was upregulated in the fibrotic portion of the injured tissue 10 days after the injury. Based on the reported interactions of miRNA-16-5p with the TGF-β pathway via targeting of SMAD3, we aimed to explore the effects of miRNA-16-5p mimics on the tenogenic differentiation of adipose-derived stem cells (ASCs) encapsulated in a bioactive bioink [4,5]. Bioinks with different properties are used for the 3D printing of biomimetic constructs. By integrating cells, materials, and bioactive molecules it is possible to tailor the regenerative capacity of the ink to meet the particular requirements of the tissue to engineer [5]. Here we have encapsulated ASCs in a gelatin-methacryloyl (GelMa) bioink that incorporates miR-16-5p mimics and magnetically responsive microfibers (MRFs). When the bioink is crosslinked in the presence of a magnetic field, the MRFs align unidirectionally to create an anisotropic construct with the ability to promote the tenogenic differentiation of the encapsulated ASCs. Additionally, the obtained GelMA hydrogels retained the encapsulated miRNA probes, which permitted the effective 3D transfection of the ASC and therefore, the regulation of gene expression, allowing to investigate the effects of the miR-16-5p mimics on the tenogenic differentiation of the ASCs in a biomimetic scenario.

Electrospinning is an advantageous technique for cartilage tissue engineering (CTE) applications due to its ability to produce nanofibers recapitulating the size and alignment of the collagen fibers present within the articular cartilage superficial zone. Moreover, coaxial electrospinning allows the fabrication of core-shell fibers able to encapsulate and release bioactive molecules in a sustained manner. Kartogenin (KTG) is a small heterocyclic molecule, which was demonstrated to promote the chondrogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells(hBMSCs)[1].

In this work, we developed and evaluated the biological performance of core-shell poly(glycerol sebacate)(PGS)/poly(caprolactone)(PCL) aligned nanofibers (core:PGS/shell:PCL) mimicking the native articular cartilage extracellular matrix(ECM) and able to promote the sustained release of the chondroinductive drug KTG[2].

The produced coaxial aligned PGS/PCL scaffolds were characterized in terms of their structure and fiber diameter, chemical composition, thermal properties, mechanical performance under tensile testing and in vitro degradation kinetics, in comparison to monoaxial PCL aligned fibers and respective non-aligned controls. KTG was incorporated into the core PGS solution to generate core-shell PGS-KTG/PCL nanofibers and its release kinetics was studied by HPLC analysis. KTG-loaded electrospun aligned scaffolds capacity to promote hBMSCs chondrogenic differentiation was evaluated by assessing cell proliferation, typical cartilage-ECM production (sulfated glycosaminiglycans(sGAG)) and chondrogenic marker genes expression in comparison to non-loaded controls. All the scaffolds fabricated showed average fiber diameters within the nanometer-scale and the core-shell structure of the fibers was clearly confirmed by TEM. The coaxial PGS-KTG/PCL nanofibers evidenced a more sustained drug release over 21 days. Remarkably, in the absence of the chondrogenic cytokine TGF-β3, KTG-loaded nanofibers promoted significantly the proliferation and chondrogenic differentiation of hBMSCs, as suggested by the increased cell numbers, higher sGAG amounts and up-regulation of the chondrogenic genes COL2A1, Sox9, ACAN and PRG4 expression. Overall, our results highlight the potential of core-shell PGS-KTG/PCL aligned nanofibers for the development of novel MSC-based CTE strategies.

Acknowledgements: The authors thank FCT for funding through the project InSilico4OCReg (PTDC/EME-SIS/0838/2021) and to institutions iBB (UID/BIO/04565/2020) and Associate Laboratory I4HB (LA/P/0140/2020).

Vascular inflammation and activation of myofibroblasts are significant contributors to the progression of fibrosis, which can severely impair tissue function. In various tissues, including tendons, Transforming growth factor beta 1 (TGF-β1) has been identified as a critical driver of adhesion and scar formation. Nevertheless, the mechanisms that underlie fibrotic peritendinous adhesions are still not well comprehended, and human microphysiological systems to help identify effective therapies remain scarce. To address this issue, we developed a novel human Tendon-on-a-Chip (hToC), comprised of an endothelialized vascular compartment harboring circulating monocytes and separated by a 5 μm/100 nm dual-scale ultrathin porous membrane from a type I/III collagen hydrogel with primary tendon fibroblasts and tissue-resident macrophages, all under defined serum-free conditions. The hToC models the crosstalk of the various cells in the system leading to the induction of inflammatory and fibrotic pathways including the activation of mTOR signaling. Consistent with phenotypes observed in vivo in mouse models and clinical human samples, we observed myofibroblast differentiation and senescence, tissue contraction, excessive extracellular matrix deposition, and monocytes’ transmigration and macrophages’ secretion of inflammatory cytokines, which were dependent on the presence of the endothelial barrier. This model offers novel insights on the role of vasculature in the pathophysiology of adhesions, which were previously underappreciated. Moreover, in testing whether the hToC could be used to evaluate efficacy of therapeutics, we were able to capture donor-specific variability in the response to Rapamycin treatment, which reduced myofibroblast activation regardless. Thus, our findings demonstrate the value of the hToC as a human microphysiological system for investigating the pathophysiology of fibrotic conditions in the context of peritendinous injury and similar fibrotic conditions, providing an alternative to animal testing.

Titanium alloys are one of the most used for orthopaedic implants and the fabrication of them by 3D printing technology is a raising technology, which could effectively resolve existing challenges. Surface modification of Ti surfaces is often necessary to improve biocorrosion resistance, especially in inflammatory conditions. Such modification can be made by coatings based on hydrogels, like alginate (Alg) - a naturally occurring anionic polymer. The properties of the hydrogel can be further enhanced with calcium phosphates like octacalcium phosphate (OCP) as a precursor of biologically formed hydroxyapatite. Formed Alg-OCP matrices have a high potential in wound healing, delivery of bioactive agents etc. but their effect on 3D printed Ti alloys performance was not well known.

In this work, Alg-OCP coated 3D printed samples were studied with electrochemical measurements and revealed significant variations of corrosion resistance vs. composition of the coating. The potentiodynamic polarization test showed that the Alg-OCP-coated samples had lower corrosion current density than simple Alg-coated samples. Electrochemical impedance spectroscopy indicated that OCP incorporated hydrogels had also a high value of the Bode modulus and phase angle. Hence Alg-OCP hydrogels could be highly beneficial in protecting 3D printed Ti alloys especially when the host conditions for the implant placement are inflammatory.

AcThis work was supported by the European Union Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Actions GA860462 (PREMUROSA). The authors also acknowledge the access to the infrastructure and expertise of the BBCE – Baltic Biomaterials Centre of Excellence (European Union Horizon 2020 programme under GA857287).

In the native articular cartilage microenvironment, chondrocytes are constantly subjected to dynamic physical stimuli that maintains tissue homeostasis. They produce extra cellular matrix (ECM) components such as collagens (type II mainly, 50-75%), proteoglycans (10-30%) and other type of proteins¹ . While collagen offers a large resistance in tension, proteoglycans are the responsible of the viscoelastic response under compression due to the negative charge they confer to the ECM allowing it to entrap a large amount of interstitial fluid. In pathologic states (e.g. osteoarthritis), this ECM is degenerated and the negative charge becomes unbalanced, losing the chondroprotective properties and resulting on an overloaded chondrocytes that further degenerate the matrix.

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) has been used to generate acoustic (pressure) waves that create bubbles that collapse with cells, inducing a stimulus that can modulate cell response². This mechanical stimulation promotes the expression of type II collagen, type X collagen, aggrecan and TGF-β, appearing as a great strategy to regenerate cartilage. However, current strategies make use of extrinsic forces to stimulate cartilage formation overlooking the physico-chemical properties of the degenerated cartilage, resulting in an excessive load-transfer to chondrocytes and the consequent hypertrophy and degeneration.

Here, interpenetrated networks (IPNs) with different compositions were created using methacrylated gelatin (GelMA), to mimic the collagen, and alginate functionalized with tyramine (Alg-tyr) to mimic glycosaminoglycans and to introduce a negative charge in the model. Within the matrix chondrocytes where encapsulated and stimulated under different conditions to identify the ultrasound parameters that enhance tissue formation. Samples with and without stimulation were compared analysing the expression and deposition of collagen II, aggrecan, collagen X and TGF-β. The results suggested that the chondrogenic marker expression of the samples stimulated for 10 minutes per day for 28 days, was two times higher overall in all of the cases, which was correlated to the tissue formation detected.

Acknowledgments: The authors would like to thank the Basque Government for the “Predoctoral Training Program for Non-Doctoral Research Staff 2021-2022” (Grant ref.: PRE_2021_1_0403). This work was supported by the RETOS grant PID2020-114901RA-I00 of the Ministry of Science and Innovation (MICINN).

Cartilage lacks the ability to self-repair when damaged, which can lead to the development of degenerative joint disease. Despite intensive research in the field of cartilage tissue engineering, there is still no regenerative treatment that consistently promotes the development of hyaline cartilage. Extracellular matrix (ECM) derived hydrogels have shown to support cell adhesion, growth and differentiation [1,2]. In this study, porcine articular cartilage was decellularized, solubilised and subsequently modified into a photo-crosslinkable methacrylated cartilage ECM hydrogel. Bone marrow derived mesenchymal stem/stromal cells (MSCs) were encapsulated into both methacrylated ECM hydrogels (ECM-MA) and gelatin methacryloyl (GelMA) as control hydrogel, and their chondrogenic potential was assessed using biochemical assays and histological analysis. We found that successful decellularization of the cartilage tissue could be achieved while preserving key ECM components, including collagen and glycosaminoglycans. A live-dead assay demonstrated good viability of MSCs withing both GelMA and ECM-MA hydrogels on day 7. Large increases in sGAG accumulation was observed after 21 days of culture in chondrogenic media in both groups. Histological analysis revealed the presence of a more fibrocartilage tissue in the GelMA group, while cells embedded within the ECM-MA showed a round and chondrocytic-like morphology. Both groups stained positively for proteoglycans and collagen, with limited evidence of calcium deposition following Alizarin Red staining. These results show that ECM-MA hydrogels support a hyaline cartilage phenotype and robust cartilaginous matrix production. Future studies will focus on the printability of ECM-MA hydrogels to enable their use as bioinks for the biofabrication of functional tissues.

We have developed plasmonic fibrin-based hydrogels that incorporate gold nanoparticles which transduce incident near-infrared (NIR) light into heat. Human adenovirus serotype type-5 vectors encoding a firefly luciferase (fLuc) coding sequence driven by a heat-inducible promoter were incorporated into the hydrogels. Transmission electronic microscopic analysis revealed that the adenoviral vectors were associated to the fibrin fibers. In vitro experiments in which human cells were cultured with plasmonic hydrogels showed that the adenoviral vectors can diffuse from the hydrogels, transduce the cells, and stimulate heat-induced transgene expression upon NIR irradiation. The hydrogels were implanted in 4.2 mm drill hole defects generated in the humerus of male rabbits. Three days after implantation, the defects were NIR-irradiated. Six h later, the animals were euthanized and samples from the bone defect zone were processed for immunohistochemical analyses using a specific fLuc antibody. The results showed strong expression of fLuc in tissues surrounding the implants of NIR-irradiated rabbits, while non- irradiated animals exhibited negligible expression. We next aimed to use the temperature increase to induce the production of transgenic bone morphogenetic protein 6 (BMP-6), using safe gene switches that can provide tighter control of in vivo transgene expression than heat-inducible promoters. These switches are only activated by heat in the presence of rapamycin and maintain a high level of targeted transgene expression for several days after heat activation. Adenoviral vectors encoding the safe switches that control the expression of BMP-6 were incorporated to the composites. The resulting NIR-responsive hydrogels were implanted in the bone defects generated in rabbits and used as a platform to transduce host cells, generate local hyperthermia and stimulate BMP-6 production.

Acknowledgements: This research was supported by grants RTI2018-095159-B-I00 and PID2021-126325OB-I00 (MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe”), by grant P2022/BMD- 7406 (Regional Government of Madrid). M.A.L-J. is the recipient of predoctoral fellowship PRE2019-090430 (MCIN/AEI/10.13039/501100011033).

Bone regeneration is pivotal for the healing of fractures. In case this process is disturbed a non-union can occur. This can be induced by environmental factors such as smoking, overloading etc. Co-morbidities such as diabetes, osteoporosis etc. may be more intrinsic factors besides other disturbances in the process. Those pathways negatively influence the bone regeneration process. Several intrinsic signal transduction pathways (WNT, BMP etc.) can be affected. Furthermore, on the transcriptional level, important mRNA expression can be obstructed by deregulated miRNA levels. For instance, several miRNAs have been shown to be upregulated during osteoporotic fractures. They are detrimental for osteogenesis as they block bone formation and accelerate bone resorption. Modulating those miRNAs may revert the physiological homeostasis. Indeed, physiological fracture healing has a typical miRNA signature. Besides using molecular pathways for possible treatment of non-union fractures, providing osteogenic cells is another solution. In 5 clinical cases with non-union fractures with defects larger than 10 cm, successful administration of a 3D printed PCL-TCP scaffold with autologous bone marrow aspirate concentrate and a modulator of the pathogenetic pathway has been achieved. All patients recovered well and showed a complete union of their fractures within one year after start of the regenerative treatment.

Thus, non-union fractures are a diverse entity. Nevertheless, there seem to be common pathogenetic disturbances. Those can be counteracted at several levels from molecular to cell. Compositions of those may be the best option for future therapies. They can also be used in a more personalized fashion in case more specific measurements such as miRNA signature and stem cell activity are applied.

Secondary bone healing is impacted by the extent of interfragmentary motion at the fracture site. It provides mechanical stimulus that is required for the formation of fracture callus. In clinical settings, interfragmentary motion is induced by physiological loading of the broken bone – for example, by weight-bearing. However, there is no consensus about when mechanical stimuli should be applied to achieve fast and robust healing response. Therefore, this study aims to identify the effect of the immediate and delayed application of mechanical stimuli on secondary bone healing.

A partial tibial osteotomy was created in twelve Swiss White Alpine sheep and stabilized using an active external fixator that induced well-controlled interfragmentary motion in form of a strain gradient. Animals were randomly assigned into two groups which mimicked early (immediate group) and late (delayed group) weight-bearing. The immediate group received daily stimulation (1000 cycles/day) from the first day post-op and the delayed group from the 22nd day post-op. Healing progression was evaluated by measurements of the stiffness of the repair tissue during mechanical stimulation and by quantifying callus area on weekly radiographs. At the end of the five weeks period, callus volume was measured on the post-mortem high-resolution computer tomography (HRCT) scan.

Stiffness of the repair tissue (p<0.05) and callus progression (p<0.01) on weekly radiographs were significantly larger for the immediate group compared to the delayed group. The callus volume measured on the HRCT was nearly 3.2 times larger for the immediate group than for the delayed group (p<0.01).

This study demonstrates that the absence of immediate mechanical stimuli delays callus formation, and that mechanical stimulation already applied in the early post-op phase promotes bone healing.

The aim of this scoping review is to understand the extent and type of evidence in relation to the use of guided growth for correcting rotational deformities of long bones. Guided growth is routinely used to correct angular deformities in long bones in children. It has also been proven to be a viable method to correct rotational deformities, but the concept is not yet fully examined. Databases searched include Medline, Embase, Cochrane Library, Web of Science and Google Scholar.

All identified citations were uploaded into Rayyan.ai and screened by at least two reviewers. The search resulted in 3569 hits. 14 studies were included: 1 review, 3 clinical trials and 10 pre-clinical trials. Clinical trials: a total of 21 children (32 femurs and 5 tibiae) were included. Surgical methods were 2 canulated screws connected by cable, PediPlates obliquely oriented, and separated Hinge Plates connected by FiberTape. Rotation was achieved in all but 1 child. Adverse effects reported include limb length discrepancy (LLD), knee stiffness and rebound of rotation after removal of tethers. 2 pre-clinical studies were ex-vivo studies, 1 using 8-plates on Sawbones and 1 using a novel z-shaped plates on human cadaver femurs. There were 5 lapine studies (2 using femoral plates, 2 using tibial plates and 1 using an external device on tibia), 1 ovine (external device on tibia), 1 bovine (screws and cable on metacarp) and a case-report on a dog that had an external device spanning from femur to tibia. Rotation was achieved in all studies. Adverse effects reported include implant extrusions, LLD, articular deformities, joint stiffness and rebound. All included studies conclude that guided growth is a viable treatment for rotational deformities of long bones, but there is great variation in models and surgical methods used, and in reported adverse effects.

Various approaches have been implemented to enhance bone regeneration, including the utilization of autologous platelet-rich plasma and bone morphogenetic protein-2. The objective of this study was to evaluate the impact of Marburg Bone Bank-derived bone grafts in conjunction with platelet-rich plasma (PRP), recombinant human bone morphogenetic protein-2 (rhBMP-2), and zoledronic acid (ZA) on osteogenesis within rabbit bone defects.

Healing after bone fracture is assessed by frequent radiographs, which expose patients to radiation and lacks behind biological healing. This study aimed to investigate whether the electrical impedance using electrical impedance spectroscopy correlated to quantitative scores of bone healing obtained from micro-CT and mechanical bending test.

Eighteen rabbits were subjected to tibial fracture that was stabilized with external fixator. Two electrodes were positioned, one electrode placed within the medullary cavity and the other on the lateral cortex, both three millimeters from the fracture site. Impedance was measured daily across the fracture site at a frequency range of 5 Hz to 1 MHz. The animals were divided into three groups with different follow-up time: 1, 3 and 6 weeks for micro-CT (Bone volume/tissue volume (BV/TV, %)) and mechanical testing (maximum stress (MPa), failure energy (kJ/cm3), young modulus (Mpa)).

There was a statistically significant correlation between last measured impedance at 5 Hz frequency immediately prior to euthanasia and BV/TV of callus (−0.68, 95%CI: (−0.87; −0.31)). Considering the mechanical testing with three-point bending, no significant correlation was found between last measured impedance at 5 Hz frequency immediately prior to euthanasia and maximum stress (−0.35, 95%CI: (−0.70; 0.14)), failure energy (−0.23, 95%CI: (−0.63; 0.26)), or young modulus (−0.28, 95%CI: (−0.66; 0.22)).

The significant negative correlation between impedance and BV/TV might indicate that impedances correlate with the relative bone volume in the callus site. The lack of correlation between impedance and mechanical parameters when at the same time observing a correlation between impedance and days since operation (0-42 days), might indicate that the impedance can measure biological changes at an earlier time point than rough mechanical testing.

Bone tissue is known to possess an intrinsic regeneration potential. However, in cases of major injury, trauma, and disease, bone loss is present, and the regeneration potential of the tissue is often impaired. The process of bone regeneration relies on a complex interaction of molecules. MicroRNAs (miRNA) are small, non-coding RNAs that inhibit messenger RNAs (mRNA). One miRNA can inhibit several mRNAs and one mRNA can be inhibited by several miRNAs. Functionally, miRNAs regulate the entire proteome via the local inhibition of translation. In fact, miRNA modulation has been shown to be involved in several musculoskeletal diseases¹. In those pathologies, they modulate the transcriptional activity of mRNAs important for differentiation, tissue-specific activity, extracellular matrix production, etc. Because of their function in inhibiting translation, miRNAs are being researched in many diseases and are already being used for interventional treatment². Bone tissue and its related conditions have been widely investigated up to this day^1,3. This talk will focus on the relevancy of miRNAs to bone tissue, its homeostasis, and disease. After, examples will be given of how miRNAs can be used in bone regeneration and diseases such as osteoporosis and osteosarcoma. The use of miRNAs in both, detection and therapy will be discussed.

Osteoclasts (OCs) are multinucleated cells that play a pivotal role in skeletal development and bone remodeling. Abnormal activation of OCs contributes to the development of bone-related diseases, such as osteoporosis, bone metastasis and osteoarthritis. Restoring the normal function of OCs is crucial for bone homeostasis. Recently, RNA therapeutics emerged as a new field of research for osteoarticular diseases.

The aim of this study is to use non-coding RNAs (ncRNAs) to molecularly engineer OCs and modulate their function. Specifically, we investigated the role of the microRNAs (namely miR-16) and long ncRNAs (namely DLEU1) in OCs differentiation and fusion.

DLEU1/DLEU2 region, located at chromosome 13q14, also encodes miR-15 and miR-16. Our results show that levels of these ncRNA transcripts are differently expressed at distinct stages of the OCs differentiation. Specifically, silencing of DLEU1 by small interfering RNAs (siDLEU1) and overexpression of miR-16 by synthetic miRNA mimics (miR-16-mimics) led to a significant reduction in the number of OCs formed per field (OC/field), both at day 5 and 9 of the differentiation stage. Importantly, time-lapse analysis, used to track OCs behavior, revealed a significant decrease in fusion events after transfection with siDLEU1 or miR-16-mimics and an alteration in the fusion mode and partners. Next, we investigated the migration profile of these OCs, and the results show that only miR-16-mimics-OCs, but not siDLEU-OCs, have a lower percentage of immobile cells and an increase in cells with mobile regime, compared with controls. No differences in cell shape were found. Moreover, mass-spectrometry quantitative proteomic analysis revealed independent effects of siDLEU1 and miR-16-mimics at the protein levels. Importantly, DLEU1 and miR-16 act by distinct processes and pathways.

Collectively, our findings support the ncRNAs DLEU1 and miR-16 as therapeutic targets to modulate early stages of OCs differentiation and, consequently, to impair OC fusion, advancing ncRNA-therapeutics for bone-related diseases.

Acknowledgements: Authors would like to thank to AO CMF / AO Foundation (AOCMFS-21-23A). SRM and MIA are supported by FCT (SFRH/BD/147229/2019 and BiotechHealth Program; CEECINST/00091/2018/CP1500/CT0011, respectively).

Bone homeostasis is a highly regulated process involving pathways in bone as WNT, FGF or BMP, but also requiring support from surrounding tissues as vessels and nerves. In bone diseases, the bone-vessel-nerve triad is impacted. Recently, new players appeared as regulators of bone homeostasis: microRNAs (miRNA). Five miRNAs associated with osteoporotic fractures are already known, among which miR-125b is decreasing bone formation by downregulating human mesenchymal stem cells (hMSCs) differentiation. Other miRNAs, as miR-214 (in cluster with miR-199a), are secreted by osteoclasts to regulate osteoblasts and inhibit bone formation. This forms a very complex regulatory network.

hMSCs and osteoblasts (n=3) were transfected with mimic/antagomiR of miR-125b, miR-199a-5p or miR-214, or with a scrambled miRNA (negative control) in osteogenic differentiation calcium-enriched medium (Ca++). Mineralization was assessed by Alizarin Red/CPC staining, miRNA expression by qPCR and protein by western blotting.

Exposure of hMSCs or osteoblasts to Ca++ increased mineralization compared to basal medium. hMSCs transfected with miR-125b mimic in Ca++ presented less mineralization compared to scramble. This correlated with decreased levels of BMPR2 and RUNX2. hMSCs transfected with miR-125b inhibitor presented higher mineralization. Interestingly, hMSCs transfected with miR-214 mimic in Ca++ presented no mineralization while miR-214 inhibitor increased mineralization. No differences were observed in hMSCs transfected with miR-199a-5p modulators. On the contrary, osteoblasts transfected with miR-199a-5p mimic present less mineralization than scrambled-transfected and same was observed for miR-214 and miR-125b mimics.

We highlight that miR-125b and miR-214 decrease mineralization of hMSCs in calcium-enriched medium. We noticed that miR-199a-5p is able to regulate mineralization in osteoblasts but not in hMSCs suggesting that this effect is cell-specific. Interestingly, the cluster miR-199a/214 is known as modulator of vascular function and could thus contribute to bone remodeling via different ways. With this work we slightly open the door to possible therapeutic approaches for bone diseases.

Bone morphogenetic proteins (BMPs) have been widely investigated for treating non-healing fractures. They participate in bone reconstruction by inducing osteoblast differentiation, and osteoid matrix production.¹ The human recombinant protein of BMP-7 was among the first growth factors approved for clinical use. Despite achieving comparable results to autologous bone grafting, severe side effects have been associated with its use.² Furthermore, BMP-7 was removed from the market.³ These complications are related to the high doses used (1.5-40 miligrams per surgery)² compared to the physiological concentration of BMP in fracture healing (in the nanogram to picogram per milliliter range).⁴ In this study, we use transcript therapy to deliver chemically modified mRNA (cmRNA) encoding BMP-7. Compared to direct use of proteins, transcript therapy allows the sustained synthesis of proteins with native conformation and true post-translational modifications using doses comparable to the physiological ones.⁵ Moreover, cmRNA technology overcomes the safety and affordability limitations of standard gene therapy i.e. pDNA.⁶ BMP-7 cmRNA was delivered using Lipofectamine™ MessengerMAX™ to human mesenchymal stromal cells (hMSCs). We assessed protein expression and osteogenic capacity of hMSCs in monolayer culture and in a house-made, collagen hydroxyapatite scaffold. Using fluorescently-labelled cmRNA we observed an even distribution after loading complexes into the scaffold and a complete release after 3 days. For both monolayer and 3D culture, BMP-7 production peaked at 24 hours post-transfection, however cells transfected in scaffolds showed a sustained expression. BMP-7 transfected hMSCs yielded significantly higher ALP activity and Alizarin red staining at later timepoints compared to the untransfected group. Interestingly, BMP-7 cmRNA treatment triggered expression of osteogenic genes like OSX, RUNX-2 and OPN, which was also reflected in immunostainings. This work highlights the relevance of cmRNA technology that may overcome the shortcomings of protein delivery while circumventing issues of traditional pDNA-based gene therapy for bone regeneration.

Acknowledgement: This work has been performed as part of the cmRNAbone project and has received funding from the European Union's Horizon 2020 research and innovation programme under the Grant Agreement No 874790.

MicroRNA (miR) delivery to regulate chronic inflammation hold extraordinary promise, with new therapeutic possibilities emanating from their ability to fine-tune multiple target gene regulation pathways which is an important factor in controlling aberrant inflammatory reactions in complex multifactorial disease. However, several hurdles have prevented advancements in miR-based therapies. These include off-target effects of miRs, limited trafficking, and inefficient delivery. We propose a magnetically guided nanocarrier to transport therapeutically relevant miRs to assist self- resolving inflammation processes at injury sites and reduce the impact of chronic inflammation- related diseases such as tendinopathies. The high prevalence, significant socio-economic burden and increasing recognition of dysregulated immune mediated pathways in tendon disease provide a compelling rationale for exploring inflammation-targeting strategies as novel treatments in this condition. By combining cationic polymers, miR species (e.g., miR 29a, miR155 antagonist), and magnetic nanoparticles in the form of magnetoplexes with highly efficient magnetofection procedures, we developed inexpensive, easy-to-fabricate, and biocompatible systems with competent miR-binding and fast cellular uptake into different types of human cells, namely macrophages and tendon-derived cells. The system was shown to be cell-compatible and to successfully modulate the expression and production of inflammatory markers in tendon cells, with evidence of functional pro-healing changes in immune cell phenotypes. Hence, magnetoplexes represent a simple, safe, and non-viral nanoplatform that enables contactless miR delivery and high- precision control to reprogram cell profiles toward improved pro-regenerative environments.

Acknowledgements: ERC CoG MagTendon No.772817; FCT Doctoral Grant SFRD/BD/144816/2019, and TERM

RES Hub (Norte-01-0145-FEDER-022190).

Despite extensive research aimed at improving surgical outcomes of enthesis injuries, re-tears remain a common problem, as the repairs often lead to fibrovascular scar as opposed to a zonal enthesis. Zonal enthesis formation involves anchoring collagen fibers, synthesizing proteoglycan-rich fibrocartilage, and mineralizing this fibrocartilage [1]. During development, the hedgehog signaling pathway promotes the formation and maturation of fibrocartilage within the zonal tendon-to-bone enthesis [1-4]. However, whether this pathway has a similar role in adult zonal tendon-to-bone repair is not known. Therefore, we developed a murine anterior cruciate ligament (ACL) reconstruction model [5] to better understand the zonal tendon-to-bone repair process and perturb key developmental regulators to determine the extent to which they can promote successful repair in the adult. In doing so, we activated the hedgehog signaling pathway both genetically using transgenic mice and pharmacologically via agonist injections. We demonstrated that both treatments improved the formation of zonal attachments and tunnel integration strength [6]. These improved outcomes were due in part to hedgehog signaling's positive role in proliferation of the bone marrow stromal cell (bMSC) progenitor pool and subsequent fibrocartilage production of bMSC progeny cells that form the attachments. These results suggest that, similar to growth and development, hedgehog signaling promotes the production and maturation of fibrocartilage during tendon-to-bone integration in adults. Lastly, we developed localized drug delivery systems to further improve the treatment of these debilitating injuries in future translational studies.

Acknowledgements: This work was supported by NIH R01AR076381, R21AR078429, R00AR067283, F31AR079840, T32AR007132, and P30AR069619, in addition to the McCabe Fund Pilot Award at the University of Pennsylvania.

Platelet Rich Plasma (PRP), either rich (L-PRP) or poor (P-PRP) of leukocytes, is frequently used as an anti-inflammatory and regenerative tool in osteoarthritis (OA). PRP contains proteins but not genes as it is derived from megakaryocytes. Proteomics but not metabolomics of PRP was recently studied. Metabolomics is a field of ‘omics’ research involved in comprehensive portrayal of the small molecules, metabolites, in the metabolome. These small molecules can be endogenous metabolites or exogenous compounds found in an organism (1). Our aim was to determine the difference between L-PRP and P-PRP.

A cross-sectional clinical study was designed in six recreational male athletes between the ages of 18 and 35 years. 3 mL P-PRP and 3 mL -LPRP was prepared from 60 mL of venous blood after treating with 9 mL of sodium citrate and centrifugation at 2.700 rpm for 10 min. Half of the prepared PRP's were frozen at −20°C for a week. Fresh and frozen samples were analyzed at the Q-TOF LC/MS device after thawing to room temperature.

Untargeted metabolomic results revealed that the metabolomic profile of the L-PRP and P-PRP were significantly different from each other. A total of 33.438 peaks were found. Statistically significant (p<0.05) peaks were uploaded to the MetaboAnalyst 5.0 platform. Exogenous out of 2.308 metabolites were eliminated and metabolites found significant for our study were subjected to pathway analysis. Steroid biosynthesis, sphingolipid metabolism and metabolism of lipid pathways were affected. In the L-PRP samples, Nicotinamide riboside (FC: 2.2), MHPG (FC: 3.0), estrone sulfate (FC: 7.5), thiamine diphosphate (FC: 2.0), leukotriene E4 (FC: 7.5), PC(18:1 (9Z)e/2:0) (FC: 9.8) and Ap4A (FC: 2.1) were higher compared to P-PRP. C24 sulfatide (FC: −11.8), 3-hexaprenyl-4,5-dihydroxybenzoic acid (FC: −2.8) metabolites were furthermore lower in P-PRP. Clinical outcomes of PRP application should consider these metabolic pathways in future studies (2).

Intervertebral disc (IVD) degeneration is a pathological process often associated with chronic back pain and considered a leading cause of disability worldwide¹. During degeneration, progressive structural and biochemical changes occur, leading to blood vessel and nerve ingrowth and promoting discogenic pain². In the last decades, several cytokines have been applied to IVD cells in vitro to investigate the degenerative cascade. Particularly, IL-10 and IL-4 have been predicted as important anabolic factors in the IVD according to a regulatory network model based in silico approach³. Thus, we aim to investigate the potential presence and anabolic effect of IL-10 and IL-4 in human NP cells (in vitro) and explants (ex vivo) under hypoxia (5% O2) after a catabolic induction.

Primary human NP cells were expanded, encapsulated in 1.2% alginate beads (4 × 106 cells/ml) and cultured for two weeks in 3D for phenotype recovery while human NP explants were cultured for five days. Afterwards, both alginate and explant cultures were i) cultured for two days and subsequently treated with 10 ng/ml IL-10 or IL-4 (single treatments) or ii) stimulated with 0.1 ng/ml IL-1β for two days and subsequently treated with 10 ng/ml IL-10 or IL-4 (combined treatments).

The presence of IL-4 receptor, IL-4 and IL-10 was confirmed in human intact NP tissue (Fig 1). Additionally, IL-4 single and combined treatments induced a significant increase of proinflammatory protein secretion in vitro (Fig. 2A-C) and ex vivo (Fig. 2D and E). In contrast, no significant differences were observed in the secretome between IL-10 single and combined treatments compared to control group.

Overall, IL-4 containing treatments promote human NP cell and explant catabolism in contrast to previously reported IL-4 anti-inflammatory performance⁴. Thus, a possible pleiotropic effect of IL-4 could occur depending on the IVD culture and environmental condition.

Acknowledgements: This project was supported by the Marie Skłodowska Curie International Training Network “disc4all” under the grant agreement #955735.

For any figures and tables, please contact the authors directly.

Obesity is correlated with the development of osteoporotic diseases. Gut microbiota-derived metabolite trimethylamine-n-oxide (TMAO) accelerates obesity-mediated tissue deterioration. This study was aimed to investigate what role TMAO may play in osteoporosis development during obesity.

Mice were fed with high-fat diet (HFD; 60 kcal% fat) or chow diet (CD; 10 kcal% fat) or 0.2% TMAO in drinking water for 6 months. Body adiposis and bone microstructure were investigated using μCT imaging. Gut microbiome and serum metabolome were characterized using 16S rRNA sequencing and liquid chromatography-tandem mass spectrometry. Osteogenic differentiation of bone-marrow mesenchymal cells was quantified using RT-PCR and von Kossa staining. Cellular senescence was evaluated by key senescence markers p16, p21, p53, and senescence association β-galactosidase staining.

HFD-fed mice developed hyperglycemia, body adiposis and osteoporosis signs, including low bone mineral density, sparse trabecular microarchitecture, and decreased biomechanical strength. HFD consumption induced gut microbiota dysbiosis, which revealed a high Firmicutes/Bacteroidetes ratio and decreased α-diversity and abundances of beneficial microorganisms Akkermansiaceae, Lactobacillaceae, and Bifidobacteriaceae. Serum metabolome uncovered increased serum L-carnitine and TMAO levels in HFD-fed mice. Of note, transplantation of fecal microbiota from CD-fed mice compromised HFD consumption-induced TMAO overproduction and attenuated loss in bone mass, trabecular microstructure, and bone formation rate. TMAO treatment inhibited trabecular and cortical bone mass and biomechanical characteristics; and repressed osteogenic differentiation capacity of bone-marrow mesenchymal cells. Mechanistically, TMAO accelerated mitochondrial dysfunction and senescence program, interrupted mineralized matrix production in osteoblasts.

Gut microbial metabolite TMAO induced osteoblast dysfunction, accelerating the development of obesity-induced skeletal deterioration. This study, for the first time, conveys a productive insight into the catabolic role of gut microflora metabolite TMAO in regulating osteoblast activity and bone tissue integrity during obesity.

The interleukin-6/gp130-associated Janus Kinases/STAT3 axis is known to play an important role in mediating inflammatory signals, resulting in production of matrix metalloproteinase-3 (MMP-3). The hip joints with rapidly destructive coxopathy (RDC) demonstrate rapid chondrolysis, probably by increased production of MMP-3 observed in the early stage of RDC. In the recent study, no apparent activation of STAT3 has been shown in the synovial tissues obtained from the osteoarthritic joint at operation. However, no data are currently available on STAT3 activation in the synovial tissues in the early stage of RDC. This study aimed to elucidate STAT3 activation in the synovial tissues in the early stage of RDC. Synovial tissues within 7 months from the disease onset were obtained from four RDC patients with femoral head destruction and high serum levels of MMP-3. RDC synovial tissues showed the synovial lining hyperplasia with an increase of CD68-positive macrophages and CD3-positive T lymphocytes. STAT3 phosphorylation was found in the synovial tissues by immunohistochemistry using anti-phospho-STAT3 antibody. The majority of phospho-STAT3-positive cells were the synovial lining cells and exhibited negative expression of macrophage or T cell marker. Treatment with tofacitinib, a Janus Kinase inhibitor, resulted in a decrease in phospho-STAT3-positive cells, especially with high intensity, indicating effective suppression of STAT3 activation in RDC synovial tissues. Inhibitory effect of tofacitinib could act through the Janus Kinase/STAT3 axis in the synovial tissues in the early stage of RDC. Therefore, STAT3 may be a potential therapeutic target for prevention of joint structural damage in RDC.

Acknowledgements: This study was supported by Katakami Foundation for Clinical Research.

Mincing cartilage with commercially available shavers is increasingly used for treating focal cartilage defects. This study aimed to compare the impact of mincing bovine articular cartilage using different shaver blades on chondrocyte viability.

Bovine articular cartilage was harvested using a scalpel or three different shaver blades (2.5 mm, 3.5 mm, or 4.2 mm) from a commercially available shaver. The cartilage obtained with a scalpel was minced into fragments smaller than 1 mm³. All four conditions were cultivated in a culture medium for seven days. After Day 1 and Day 7, metabolic activity, RNA isolation, and gene expression of anabolic (COL2A1, ACAN) and catabolic genes (MMP1, MMP13), Live/Dead staining and visualization using confocal microscopy, and flow cytometric characterization of minced cartilage chondrocytes were measured.

The study found that mincing cartilage with shavers significantly reduced metabolic activity after one and seven days compared to scalpel mincing (p<0.001). Gene expression of anabolic genes was reduced, while catabolic genes were increased after day 7 in all shaver conditions. The MMP13/COL2A1 ratio was also increased in all shaver conditions. Confocal microscopy revealed a thin line of dead cells at the lesion site with viable cells below for the scalpel mincing and a higher number of dead cells diffusely distributed in the shaver conditions. After seven days, there was a significant decrease in viable cells in the shaver conditions compared to scalpel mincing (p<0.05). Flow cytometric characterization revealed fewer intact cells and proportionally more dead cells in all shaver conditions compared to the scalpel mincing.

Mincing bovine articular cartilage with commercially available shavers reduces the viability of chondrocytes compared to scalpel mincing. This indicates that mincing cartilage with a shaver should be considered a matrix rather than a cell therapy. Further experimental and clinical studies are required to standardize the mincing process with a shaver.

Acknowledgements: This study received unrestricted funding from KARL STORZ SE & Co. KG.

Elective orthopaedic procedures, and particularly total hip arthroplasty (THA), in octogenarians and nonagenarians patients are burdened of several implications. Besides the comorbidities and the anesthesiological issues, legal and ethical implications are present. Some literature data show the clinical improvement of THA in elderly patient but the psychological aspects are not yet evaluated. Aim of this study is to evaluate the clinical aspects and the psychological impact in daily living in octogenarians and nonagenarians patients addressing THA.

We conducted a retrospective evaluation of 81 THA in 81 patients of age more than 85 years with a minimum follow-up of 6 months. Clinical aspects were evaluated using the Hip disability and Osteoarthritis Outcome Score (HOOS). The psychological issues were evaluated with the Short Form 12 (SF-12) using both the Physical Component Summary (PCS) and the Mental Component Summary (MCS). From the starter cohort of 81 patients, 8 patients were died for causes unrelated to surgery, 13 were lost to follow-up, 1 patient was revised for periprosthetic fracture; 59 patients composed the final cohort. Mean HOOS rased from 18,07 ± 17,81 to 92,36 ± 5,74 with statistically significant distribution both in the global score than in all of the different subscales. The PCS raised from 26,81 ± 10,81 to 51,86 ± 4,45 and The MCS raised from 34,84 ± 10,81 to 56,70 ± 5,04, but none of them showed a statistically significant distribution. THA in octogenarians and nonagenarians patients could be a safe procedure with positive results for clinical and psychological aspects.

Dislocation post THA confers a higher risk of re-dislocation (Kotwal et al, 2009). The dual mobility (DM) cup design (1974) was aimed at improving the stability by increasing the femoral head to neck ratio (Cuthbert et al., 2019) combining the ideas of low friction arthroplasty with increased jump distance associated with a big head arthroplasty.

Understand the dislocation rates, rates of aseptic loosening, infection rate and revision rates between the 2 types of constructs to provide current and up-to date evidence.

Medline, pubmed, embase and Cochrane databases were used based on PRISMA guidelines. RevMan software was used for the meta-analysis. Studies (English literature) which used DM construct with atleast 6 months follow-up used as intervention and non DM construct as control were included. 2 independent reviewers conducted the review with a third reviewer in case of difference in opinion regarding eligibility. Primary outcome was dislocation rate and secondary outcome was rate of revision.

564 articles identified out of which 44 articles were screened for full texts and eventually 4 systematic review articles found eligible for the study. Thus, study became a review of systematic reviews. From the 4 systematic reviews, another 35 studies were identified for data extraction and 13 papers were used for meta-analysis. Systematic reviews evaluated, projected an average follow up of 6-8 years with significantly lower dislocation rates for DM cups. The total number of patients undergoing DM cup primary THA were 30,559 with an average age 71 years while the control group consisted of 218,834 patients with an average age of 69 years. DM group had lower rate of dislocation (p < 0.00001), total lower rate of cup revision (p < 0.00001, higher incidence of fracture (p>0.05).

DM THA is a viable alternative for conventional THA. The long-term results of DM cups in primary THA need to be further evaluated using high quality prospective studies and RCTs.

Several studies have evaluated the risk of peroneal nerve (PN) injuries in all-inside lateral meniscal repair using standard knee magnetic resonance imaging (MRI) with the 30 degrees flexed knee position which is different from the knee position during actual arthroscopic lateral meniscal repair. The point of concern is “Can the risk of PN injury using standard knee MRIs be accurately determined”.

To evaluate and compare the risk of PN injury in all-inside lateral meniscal repair in relation to both borders of the popliteus tendon (PT) using MRIs of the two knee positions in the same patients.

Using axial MRI studies with standard knee MRIs and figure-of-4 with joint fluid dilatation actual arthroscopic lateral meniscal repair position MRIs, direct lines were drawn simulating a straight all-inside meniscal repair device from the anteromedial and anterolateral portals to the medial and lateral borders of the PT. The distance from the tip of each line to the PN was measured. If a line touched or passed the PN, a potential risk of iatrogenic injury was noted and a new line was drawn from the same portal to the border of the PN. The danger area was measured from the first line to the new direct line along the joint capsule.

In 28 adult patients, the closest distances from each line to the PN in standard knee MRI images were significantly shorter than arthroscopic position MRI images (all p-values < 0.05). All danger areas assessed in the actual arthroscopic position MRIs were included within the danger areas as assessed by the standard knee MRIs.

We found that the standard knee MRIs can be used to determine the risk of peroneal nerve injury in arthroscopic lateral meniscal repair, although the risks are slightly overestimated.

The need to accurately forecast the injury burden has never been higher. With an aging, ever expanding trauma population and less than half of the beds available compared to 1990, the National Health Service (NHS) is stretched to breaking point^1,2.

We utilised a dataset of 22,585 trauma patients across the four countries of the United Kingdom (UK) admitted to 83 hospitals between 22/08/22 – 16/10/22 to determine whether it is possible to predict the proportionality of injuries treated operatively within orthopaedic departments based on their number of Neck of Femur fracture (NOF) patients.

More operations were performed for elderly hip fractures alone than for the combined totals of the next four most common fractures: ankle, distal radius, tibial shaft and forearm (6387 vs 5922). Conversely, 10 out of the 13 fracture types were not encountered by at least one hospital and 93% of hospitals encountered less than 2 fractures of a certain type.

60% trauma is treated within Trauma Units (TUs) however, per unit, Major Trauma Centres (MTCs) treat approximately 43% more patients.

After excluding NOF, lower limb fractures accounted for approximately 57% of fractures in all countries and ankle and distal radius fracture combined comprised more than 50% in 74% of regions.

The number of hip fractures seen on average by an individual unit remains relatively consistent as does the regional variation of any given fracture; resultantly, it is possible to predict injury proportionality based off a unit's hip fracture numbers. This powerful tool could transform both resource allocation and recruitment.

Stiffness is reported in up to 16% of patients after total knee replacement (TKR)¹. Treatment of stiffness after TKR remains a challenge. Manipulation under anaesthesia (MUA) accounts for between 6%-36% of readmissions following TKR^2,3. The outcomes of MUA remain variable/unpredictable. Post-operative CPM is used as an adjuvant to MUA, potentially offering improved ROM, however, remains the subject of debate. We report a retrospective study comparing MUA with and without post-operative CPM.

In our institution patients undergoing MUA to receive CPM post-operatively. Owing to the COVID-19 pandemic hospital admissions were limited. During this period MUA procedures were undertaken without CPM. Two cohorts were included: 1) MUA + post-operative CPM 2) Daycase MUA. Patients’ demographics, pre-manipulation ROM, post-MUA ROM, and ROM at final follow-up were recorded.

Between 2017-2022 126 patients underwent MUA and were admitted for CPM and 42 had daycase MUA. The median Age was 66.5 and 64% were female. 57% had extension deficit (>5^o), 70% had flexion deficit (< 90^o), and 37% had both. The mean Pre-operative ROM was 72.3^o(SD:18.3^o) vs. 68.5^o(19.0^o), ROM at MUA was 95.5^o(SD:20.7^o) vs 108.3^o(SD:14.1^o) [p< 0.01], and at final follow-up 87.4^o(SD:21.9^o) vs. 92.1^o(SD:18.2^o) for daycase and CPM groups respectively. At final follow-up for the daycase and CPM groups respectively 10% vs. 7% improved, 29% vs. 13% maintained, and 57% vs. 79% regressed from the ROM achieved at MUA. The mean percentage of ROM gained at MUA maintained at final follow-up was 92%(SD:17) and 85%(SD:14)[p=0.03] for daycase and CPM groups respectively.

There was no significant difference in ROM achieved at final follow-up despite the significantly greater improvement in ROM achieved at MUA for the CPM group. The CPM group lost a greater ROM after MUA (15% vs. 8%). We conclude that post-operative CPM does not improve ROM achieved after MUA.

Tourniquet is a commonly used tool in orthopaedic practice. Incidence of complications is low but if any develops, it is devastating. Transient nerve damage, ischemia or skin burns are the possible tourniquet related complications. There is big variation in practice regarding the limb occlusion pressure.

51 procedures in 50 patients were reviewed retrospectively in our district general hospital. We looked at quality of documentation guided by the BOAST standard (The Safe Use of Intraoperative Tourniquets, published in October 2021). Limb occlusion pressure and ischemic time were analysed. Intra-operative and post-operative notes were reviewed to assess quality of documentation and post-operative complications.

Although limb occlusion pressure was above the recommended range in more than 75% of cases, there were no significant complications observed. Two cases only developed transient neuropraxia in common peroneal nerve and median nerve following tibial plateau ORIF and trapeziectomy simultaneously. Tibial ORIF fixation case had prolonged ischemic time (more than 120 minutes) and the limb occlusion pressure for the hand case was above the recommended range. Both have recovered within few days with no long-term consequences. Minimum documentation threshold was not met with regarding tourniquet site condition, method of skin isolation and padding, and exsanguination method.

This relatively new standard with no previous similar guidance needs time until it is followed by the health care professionals especially when there is no high incidence of complications related to the use of the tourniquet. However, it is crucial to increase the theatre staff awareness of such standards. This will prevent devastating complications specifically in vulnerable patients. Adjustments to theatre checklist have been suggested to improved documentation. Additionally, local teaching sessions will be delivered to theatre personnel aiming at improving our compliance to this standard.

Bone defects and fractures, caused by injury, trauma or tumour resection require hospital treatment and temporary loss of mobility, representing an important burden for societies and health systems worldwide. Autografts are the gold standard for promoting new bone formation, but these may provide insufficient material and lead to donor site morbidity and pain. We previously showed that Fibrinogen (Fg) scaffolds promote bone regeneration in vivo (1), and that modifying them with 10mM of Magnesium (Mg) ions modulates macrophage response in vitro and in vivo (2). Also, we showed that Extracellular Vesicles (EV) secreted by Dendritic Cells (DC) recruit Mesenchymal Stem/Stromal Cells (MSC)(3).

Herein, we aim to functionalize FgMg scaffolds with DC-EV, to promote recruitment and osteogenic differentiation of MSC.

Scaffolds were produced by freeze-drying (2). Ethical permission was sought for all studies. Primary human peripheral blood monocyte-derived DC were cultured, their secreted EV were isolated by differential (ultra)-centrifugation and characterised by transmission electron microscopy and nanoparticle tracking analysis (3). Bone marrow MSC were used to determine the impact of EV-functionalized scaffolds through migration assays and their osteogenic differentiation was assessed by Alizarin Red staining.

Fg and FgMg scaffolds functionalized with EV were characterized. Fg and FgMg scaffolds functionalized with DC-secreted EV were more efficient at recruiting MSC than scaffolds alone. MSC cultured on FgMg scaffolds showed significantly increased calcium deposits, in comparison with those cultured on Fg scaffolds.

Fg scaffold modification by Mg promotes MSC osteogenic differentiation, while their functionalization with DC-secreted EV acts to promote MSC recruitment. This renders the FgMg-EV functionalized scaffolds an attractive material to promote new bone formation.

Acknowledgments: Work funded by Orthoregeneration Network (ON Pilot Grant Spine 2021, EVS4Fusion). MCT supported by ERASMUS+ program.

The optimal treatment strategy for post-traumatic long bone non-unions is subject of an ongoing discussion. At the Maastricht University Medical Center (MUMC+) the induced membrane technique is used to treat post-traumatic long bone non-unions. This technique uses a multimodal treatment algorithm involving bone marrow aspirate concentrate (BMAC), the reamer-irrigator-aspirator (RIA) and P-15 bioactive peptide (iFactor, Cerapedics). Bioactive glass (S53P4 BAG, Bonalive) is added when infection is suspected. This study aims to objectify the effect of this treatment algorithm on the health-related quality of life (HRQoL) of patients with post-traumatic long bone non-unions. We hypothesized that HRQoL would improve after treatment.

From January 2020 to March 2023, consecutive patients who were referred to a multidisciplinary (trauma, orthopaedic and plastic surgery) non-union clinic at the MUMC+, The Netherlands, were evaluated using the Non-Union Scoring System (NUSS). The EQ-5D-5L questionnaire and the Lower Extremity Functional Scale (LEFS) were employed to obtain HRQoL outcomes both prior to and subsequent to surgery, with a follow-up at 6, 18 and 35 weeks.

Seventy-six patients were assessed at baseline (T0), with a mean NUSS of 40 (± 13 SD). Thirty-eight patients had their first follow-up, six weeks after surgery (T1). Thirty-one patients had a second follow-up at 18 weeks (T2), and twenty patients had the third follow-up at 35 weeks (T3). The EQ-5D index mean at baseline was 0.480, followed by an index of 0.618 at T1, 0.636 at T2, and 0.702 at T3. A significant difference was found in the HRQoL score between T0 and T1, as well as T2 and T3 (p<0.001; p=0.011). The mean LEFS significantly increased from 26 before intervention to 34, 39, and 43 after treatment (p<0.001; p=0.033; p=0.016).

This study demonstrated a significant improvement in the health-related quality of life of patients with post-traumatic long bone non-unions after the standardized treatment algorithm following the induced membrane technique.

Tendon injury is debilitating and recalcitrant. With limited knowledge of disease aitiology we have are lacking in effective treatments for this prevalent musculoskeletal complaint.

This presentation will outline our findings over the past few years in which we have demonstrated the importance of the interfascicular matrix (IFM) niche in maintaining healthy tendon function and driving disease progression^1,2. It will also continue to describe our progress in developing both in vivo and in vitro models to interrogate disease progression.

We have developed and validated a rat Achilles tendon overload model, in order to explore the impact of loading on IFM and fascicle structure, and the resulting cell response. Data highlights that structural disruption and inflammatory response both initiate in the IFM region, and can be seen in the absence of demonstrable changes to animal gait, indicating a sub-injury response in the tendon which we hypothesis may drive increased matrix turnover and repair³.

We are now looking to interrogate the pathways driving this inflammatory behaviour in an organ-chip model, exploring the interplay between IFM cells and cells within fascicles. We have demonstrated phenotypic distinction of cells from the two niche environments, localized the progenitor phenotype to the IFM region and demonstrated significant mechanosensitivity in the IFM cell population⁴. We are currently building appropriate niche environments to maintain cell phenotype in our in vitro models, to explore the metabolic changes associated with disease progression.

Acknowledgements: This body of work has received funding from: BBSRC (BB/K008412 /1); Versus Arthritis (project grant 20262); Horserace Betting Levy Board (T5); Dunhill Medical Charity (project grant RPGF1802\23); MRC (MR/T015462/1).

Recently, technologies to culture one or more cell types in three dimensions have attracted a great deal of attention in tissue engineering. Particularly, the improved viability, self-renewal capacity, and differentiation potential have been reported for stem cell spheroids. However, it is crucial to modulate spheroid functions with instructive signals to use multi-cellular spheroids in tissue engineering. We have been developing ECM-mimicking fibrous materials decorated with cell-instructive cues, which were incorporated within 3D stem cell spheroids to fine-tune their functions as modular building blocks for bottom-up tissue-engineering applications. In particular, we created composite spheroids of human adipose-derived stem cells (hADSCs) incorporating nanofibers coated with instructive signal of either transforming growth factor-β3 or bone morphogenetic growth factor-2 for chondrogenesis or osteogenesis of stem cells, respectively. The bilayer structure of osteochondral tissue was subsequently mimicked by cultivating each type of spheroid inside 3D-printed construct. The in vitro chondrogenic or osteogenic differentiation of hADSCs within the biphasic construct under general media was locally regulated by each inductive component. More importantly, hADSCs from each spheroid proliferated and sprouted to form the integrated tissue with interface of bone and cartilage tissue. This approach may be applied to engineer complex tissue with hierarchically organized structure.

Recently, a new generation of superior clavicle plates was developed featuring the variable-angle locking technology for enhanced screw positioning and optimized plate-to-bone fit design. On the other hand, mini-fragment plates used in dual plating mode have demonstrated promising clinical results. However, these two bone-implant constructs have not been investigated biomechanically in a human cadaveric model. Therefore, the aim of the current study was to compare the biomechanical competence of single superior plating using the new generation plate versus dual plating with low-profile mini-fragment plates.

Sixteen paired human cadaveric clavicles were assigned pairwise to two groups for instrumentation with either a 2.7 mm Variable Angle Locking Compression Plate placed superiorly (Group 1), or with one 2.5 mm anterior plate combined with one 2.0 mm superior matrix mandible plate (Group 2). An unstable clavicle shaft fracture AO/OTA15.2C was simulated by means of a 5 mm osteotomy gap. All specimens were cyclically tested to failure under craniocaudal cantilever bending, superimposed with bidirectional torsion around the shaft axis and monitored via motion tracking.

Initial stiffness was significantly higher in Group 2 (9.28±4.40 N/mm) compared to Group 1 (3.68±1.08 N/mm), p=0.003. The amplitudes of interfragmentary motions in terms of craniocaudal and shear displacement, fracture gap opening and torsion were significantly bigger over the course of 12500 cycles in Group 1 compared to Group 2; p≤0.038. Cycles to 2 mm shear displacement were significantly lower in Group 1 (22792±4346) compared to Group 2 (27437±1877), p=0.047.

From a biomechanical perspective, low-profile 2.5/2.0 dual plates demonstrated significantly higher initial stiffness, less interfragmentary movements, and higher resistance to failure compared to 2.7 single superior variable-angle locking plates and can therefore be considered as a useful alternative for diaphyseal clavicle fracture fixation especially in unstable fracture configurations.

To characterize the microstructural organization of collagen fibers in human medial menisci and the response to mechanical loading in relation to age. We combine high resolution imaging with mechanical compression to visualize the altered response of the tissue at the microscale. Menisci distribute the load in the knee and are predominantly composed of water and specifically hierarchically arranged collagen fibers. Structural and compositional changes are known to occur in the meniscus during aging and development of osteoarthritis. However, how microstructural changes due to degeneration affect mechanical performance is still largely unknown [1].

Fresh frozen 4 mm Ø plugs of human medial menisci (n=15, men, 20-85 years) with no macroscopic damage nor known diseases from the MENIX biobank at Skåne University Hospital were imaged by phase contrast synchrotron tomography at the TOMCAT beamline (Paul Scherrer Institute, CH). A rheometer was implemented into the beamline to perform in-situ stress relaxation (2 steps 15% and 30% strain) during imaging (21 keV, 2.75μm pixel size). 40s scans were acquired before and after loading, while 14 fast tomographs (5s acquisitions) were taken during relaxation. The fiber 3D orientations and structural changes during loading were determined using a structure tensor approach (adapting a script from [1]). The 3D collagen fiber orientation in menisci revealed alternating layers of fibers. Two main areas are shown: surfaces and bulk. The surface layers are a mesh of randomly oriented fibers. Within the bulk 2-3 layers of fibers are visible that alternate about 30° to each other. Structural degeneration with age is visible and is currently being quantified. During stress-relaxation all menisci show a similar behavior, with samples from older donors being characterized by larger standard deviation Furthermore, the behavior of the different layers of fibers is tracked during relaxation showing how fibers with different orientation respond to the applied loading.

Acknowledgments: We thank PSI for the beamtime at the TOMCAT beamline X02DA, and funding from Swedish Research Council (2019-00953), under the frame of ERA PerMed, and the Novo Nordisk Foundation through MathKOA (NNF21OC0065373).

The concept of guided growth was proposed by Andry in 1741. In the last decades the concept has been widely used as implants has been introduced that can modulate the growth of the bone and pediatric longitudinal and angular deformities is widely treated by this technique. However, there is there is a huge variation in techniques and implants used and high-quality clinical trials is still lacking. Recently implants correcting rotational bony deformities have been proposed and clinical case series have been published.

The current status of guided growth will be presented in this narrative review and preliminary experiences with rotational guided growth will be shared. Is guided growth to be considered a safe treatment at this time point?

Bone defects can result from different incidents such as acute trauma, infection or tumor resection. While in most instances bone healing can be achieved given the tissue's innate ability of self-repair, for critical-sized defects spontaneous regeneration is less likely to occur, therefore requiring surgical intervention. Current clinical procedures have failed to adequately address this issue. For this reason, bone tissue engineering (BTE) strategies involving the use of synthetic grafts for replacing damaged bone and promoting the tissue's regeneration are being investigated. The electrical stimulation (ES) of bone defects using direct current has yielded very promising results, with neo tissue formation being achieved in the target sites in vivo. Electroactive implantable scaffolds comprised by conductive biomaterials could be used to assist this kind of therapy by either directing the ES specifically to the damaged site or promoting the integration of electrodes within the bone tissue as a coating. In this study, we developed novel conductive heat-treated polyacrylonitrile/poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PAN/PEDOT:PSS) nanofibers via electrospinning capable of mimicking key native features of the bone tissue's extracellular matrix (ECM) and providing a platform for the delivery of exogenous ES. The developed scaffolds were doped with sulfuric acid and mineralized in Simulated Body Fluid to mimic the inorganic phase of bone ECM. As expected, the doped PAN/PEDOT:PSS nanofibers exhibited electroconductive properties and were able to preserve their fibrous structure. The addition of PEDOT:PSS was found to improve the bioactivity of the scaffolds, with a more significant in vitro mineralization being obtained. By seeding the scaffolds with MG-63 osteoblasts and human mesenchymal stem/stromal cells, an increased cell proliferation was observed for the mineralized PAN/PEDOT:PSS nanofibers, which also registered an increased expression of key osteogenic markers (e.g Osteopontin). Our findings appear to corroborate the promising potential of the generated nanofibers for future ES-based BTE applications.

Acknowledgements: The authors thank FCT for funding through the projects InSilico4OCReg (PTDC/EME-SIS/0838/2021), BioMaterARISES (EXPL/CTM-CTM/0995/2021) and OptiBioScaffold (PTDC/EME-SIS/32554/2017, POCI-01- 0145-FEDER- 32554), the PhD scholarship (2022.10572.BD) and through institutional funding to iBB (UIDB/04565/2020 and UIDP/04565/2020), Associate Laboratory i4HB (LA/P/0140/2020) and IT (UIDB/50008/2020).

Medial opening wedge high tibial osteotomy (MOWHTO) is the workhorse procedure for correcting varus malalignment of the knee. There have been recent developments in the synthetic options to fill the osteotomy gap. The current gold standard for filling this osteotomy gap is autologous bone graft which is associated with donor site morbidity. We would like to introduce and describe the process of utilizing the novel Osteopore® 3D printed, honeycomb structured, Polycaprolactone and β-Tricalcium Phosphate wedge for filling the gap in MOWHTO. In the advent of additive manufacturing and the quest for more biocompatible materials, the usage of the Osteopore® bone wedge in MOWHTO is a promising technique that may improve the biomechanical stability as well the healing of the osteotomy gap.

A novel EP4 selective agonist (KMN-159) was developed [1] and has been proven that it can act as an osteopromotive factor to repair critical-size femoral bone defects in rats at a dose-dependent manner [2]. Based on its osteopromotive properties, we hypothesized that KMN-159 could also aid in bone formation for spinal fusion. Therefore, the aim of this study was to investigate its spinal fusion effect in a dorsolateral spinal fusion model in rats. This study was performed on 192, 10-week-old male Wistar rats. The rats were randomized into 8 groups (n = 12 per group): 1) SHAM (negative control), 2) MCM (scaffold only), 3) MCM + 20 µg BMP-2 (positive control), 4-8) MCM + 0.2, 2, 20, 200 or 2000 µg KMN-159. A posterolateral intertransverse process spinal fusion at L4 to L5 was performed bilaterally by implanting group dependent scaffolds (see above) or left empty in the SHAM group (protocol no. 25-5131/474/38). Animals were euthanized after 3 weeks and 6 weeks for µCT and biomechanical testing analysis. The results showed that KMN-159 promoted new bone formation in a dose-dependent manner at 3 weeks and 6 weeks as verified by µCT. The biomechanical testing showed that the dose of 20, 200 and 2000 µg KMN-159 groups obtained comparable strength with BMP-2 group, which higher than SHAM, MCM and lower doses of 0.2 and 2 µg KMN-159 groups. In conclusion, KMN-159 could be a potential replacement of BMP-2 as a novel osteopromotive factor for spinal fusion.

Acknowledgements: We are grateful to Ulrike Heide, Anna-Maria Placht (assistance with surgeries) as well as Suzanne Manthey & Annett Wenke (histology).

Pelvic bone defect in patients with severe congenital dysplasia of the hip (CDH) lead to abnormalities in lumbar spine and lower limb alignment that can determine total hip arthroplasty (THA) patients' outcome. These variables may be different in uni- or bilateral CDH.

We compared the clinical outcome and the spinopelvic and lower limb radiological changes over time in patients undergoing THA due to uni- or bilateral CHD at a minimum follow-up of five years.

Sixty-four patients (77 hips) undergoing THA due to severe CDH between 2006 and 2015 were analyzed: Group 1 consisted of 51 patients with unilateral CDH, and group 2, 113 patients (26 hips) with bilateral CDH. There were 32 females in group 1 and 18 in group 2 (p=0.6). The mean age was 41.6 years in group 1 and 53.6 in group 2 (p<0.001). We compared the hip, spine and knee clinical outcomes. The radiological analysis included the postoperative hip reconstruction, and the evolution of the coronal and sagittal spinopelvic parameters assessing the pelvic obliquity (PO) and the sacro-femoro-pubic (SFP) angles, and the knee mechanical axis evaluating the tibio-femoral angle (TFA).

At latest follow-up, the mean Harris Hip Score was 88.6 in group 1 and 90.7 in group 2 (p=0.025). Postoperative leg length discrepancy of more than 5 mm was more frequent in group 1 (p=0.028). Postoperative lumbar back pain was reported in 23.4% of the cases and knee pain in 20.8%, however, there were no differences between groups. One supracondylar femoral osteotomy and one total knee arthroplasty were required. The radiological reconstruction of the hip was similar in both groups. The PO angle improved more in group 1 (p=0.01) from the preoperative to 6-weeks postoperative and was constant at 5 years. The SFP angle improved in both groups but there were no differences between groups (p=0.5). 30 patients in group 1 showed a TFA less than 10º and 17 in group 2 (p=0.7).

Although the clinical outcome was better in terms of hip function in patients with bilateral CDH than those with unilateral CDH, the improvement in low back and knee pain was similar. Patients with unilateral dysplasia showed a better correction of the PO after THA. All spinopelvic and knee alignment parameters were corrected and maintained over time in most cases five years after THA.

Intervertebral disc (IVD) degeneration is the most frequent cause of Low Back Pain (LBP) affecting nearly 80% of the population [1]. Current treatments fail to restore a functional IVD or to provide a long-term solution, so, there is an urgent need for novel therapeutic strategies. We have defined the IVD extracellular matrix (ECM) profile, showing that the pro-regenerative molecules Collagen type XII and XIV, are uniquely expressed during fetal stages [2]. Now we propose the first fetal injectable biomaterial to regenerate the IVD.

Fetal decellularized IVD scaffolds were recellularized with adult IVD cells and further implanted in vivo to evaluate their anti-angiogenic potential. Young decellularized IVD scaffolds were used as controls. Finally, a large scale protocol to produce a stable, biocompatible and easily injectable fetal IVD-based hydrogel was developed.

Fetal scaffolds were more effective at promoting Aggrecan and Collagen type II expression by IVD cells. In a Chorioallantoid membrane assay, only fetal matrices showed an anti-angiogenic potential. The same was observed in vivo when the angiogenesis was induced by human NP cells. In this context, human NP cells were more effective in GAG synthesis within a fetal microenvironment. Vaccum-assisted perfusion decellularized IVDs were obtained, with high DNA removal and sGAG retention. Hydrogel pre-solution passed through 21-30G needles. IVD cells seeded on the hydrogels initially decreased metabolic activity, but increased up to 70% at day 7, while LDH assay revealed cytotoxicity always below 30%.

This study will open new avenues for the establishment of a disruptive treatment for IVD degeneration with a positive impact on the angiogenesis associated with LBP, and on the improvement of patients’ quality of life.

Acknowledgements: Financial support was obtained from EUROSPINE, ON Foundation and FCT (Fundação para a Ciência e a Tecnologia).

Intervertebral disc (IVD) degeneration is inadequately understood due to the lack of in vitro systems that fully mimic the mechanical and biological complexity of this organ. We have recently made an advancement by developing a bioreactor able to simulate physiological, multiaxial IVD loading and maintain the biological environment in ex vivo IVD models [1].

To validate this new bioreactor system, we simulated natural spine movement by loading 12 bovine IVDs under a combination of static compression (0.1 MPa), cyclic flexion/extension (±3˚, ±6˚ or 0-6˚) and cyclic torsion (±2˚, ±4˚ or 0-4˚) for more than 10’000 (0.2 Hz) or 100’000 (1 Hz) cycles over 14 days. A higher number of cycles increased the release of glycosaminoglycans and nitric oxide, as an inflammation marker, whereas fewer cycles maintained these two factors at physiological levels. All applied protocols upregulated the expression of MMP13 in the outermost annulus fibrosus (AF), indicating a collagen degradation response. This was supported by fissures observed in the AF after a longer loading duration. Increasing loading cycles induced high cell death in the nucleus pulposus and inner AF, while with fewer cycles, high cell viability was maintained in all IVD regions, irrespective of the magnitude of rotation.

Less frequent multiaxial loading maintains IVD homeostasis while more frequent loading initiates an IVD degenerative profile. Specifically, the morphological and molecular changes were localized in the AF, which can be associated with combined flexion/extension and torsion. More loading cycles induced region-specific cell death and a higher release of extracellular matrix molecules from the innermost IVD regions, likely associated with longer exposure to static compression. Altogether, we demonstrated the advantages of the multiaxial bioreactor to study region-specific response in the IVD, which will allow a more profound investigation of IVD degeneration under different combinations of motions.

Growth factors produced by inflammatory cells and mesenchymal progenitors are required for proper bone regeneration. Signaling pathways activated downstream of these proteins work in concert and synergistically to drive osteoblast and/or chondrocyte differentiation. While dysregulation can result in abnormal healing, activating these pathways in the correct spatiotemporal context can enhance healing. Bone morphogenetic protein (BMP) signaling is well-recognized as being required for bone regeneration, and BMP is used clinically to enhance bone healing. However, it is imperative to develop new therapeutics that can be used alone or in conjunction with BMP to drive even more robust healing. Notch signaling is another highly conserved signaling pathway involved in tissue development and regeneration. Our work has explored Notch signaling during osteoblastogenesis and bone healing using both in vitro studies with human primary mesenchymal progenitor cells and in vivo studies with genetically modified mouse models. Notch signaling is required and sufficient for osteoblast differentiation, and is required for proper bone regeneration. Indeed, intact Notch signaling through the Jagged-1 ligand is required for BMP induced bone formation. On-going work continues to explore the intersection between BMP and Notch signaling, and determining cell types that express Notch receptors and Notch ligands during bone healing. Our long-term objective is to develop Notch signaling as a clinical therapy to repair bone.

Numerous papers present in-vivo knee kinematics data following total knee arthroplasty (TKA) from fluoroscopic testing. Comparing data is challenging given the large number of factors that potentially affect the reported kinematics. This paper aims at understanding the effect of following three different factors: implant geometry, performed activity and analysis method.

A total of 30 patients who underwent TKA were included in this study. This group was subdivided in three equal groups: each group receiving a different type of posterior stabilized total knee prosthesis. During single-plane fluoroscopic analysis, each patient performed three activities: open chain flexion extension, closed chain squatting and chair-rising. The 2D fluoroscopic data were subsequently converted to 3D implant positions and used to evaluate the tibiofemoral contact points and landmark-based kinematic parameters.

Significantly different anteroposterior translations and internal-external rotations were observed between the considered implants. In the lateral compartment, these differences only appeared after post-cam engagement. Comparing the activities, a significant more posterior position was observed for both the medial and lateral compartment in the closed chain activities during mid-flexion. A strong and significant correlation was found between the contact-points and landmarks-based analyses method. However, large individual variations were also observed, yielding a difference of up to 25% in anteroposterior position between both methods.

In conclusion, all three evaluated factors significantly affect the obtained tibiofemoral kinematics. The individual implant design significantly affects the anteroposterior tibiofemoral position, internal-external rotation and timing of post-cam engagement. Both kinematics and post-cam engagement additionally depend on the activity investigated, with a more posterior position and associated higher patella lever arm for the closed chain activities. Attention should also be paid to the considered analysis method and associated kinematics definition: analyzing the tibiofemoral contact points potentially yields significantly different results compared to a landmark-based approach.

Tibial periprosthetic fracture is an important complication of the Oxford Unicompartmental Knee Replacement (OUKR). Primary fixation of cementless OUKR tibial components relies on the interference-fit of the ‘keel’ and a slot in the proximal tibia. Clinically used double blade keel saws (DKS) create slots with two grooves, generating stress concentrations where fractures may initiate. This study aimed to investigate slot factors that may influence incidence of tibial periprosthetic fractures.

Slots were made in PCF20 polyurethane foam using the DKS plus/minus adjuvant rasping, single blade keel saw (SKS), and rasp-only. Round and square slots were machined with milling cutters. Compact tensile tests were conducted per ASTM E399 to determine tensile load to fracture (TLTF) and results were validated using bovine tibia. Cementless OUKR components were implanted into slots in custom polyurethane blocks and compressed to failure to determine anatomical load to fracture (ALTF). A custom MATLAB program calculated slot roundness from cross-sectional images.

Round slots had higher TLTF (29.5N, SD=2.7) than square (25.2N, SD=1.7, p<0.05) and DKS slots (23.3N, SD=2.7, p<0.0001). Fractures occurred at the round slot apices, square slot corners, and deepest DKS slot grooves. ALTF was not significantly different between square and round slots. Adjuvant rasping made DKS slots significantly rounder, resulting in significantly higher TLTF, but rasping did not increase ALTF. ALTF was significantly higher for SKS (850N, SD=133, p<0.01) and rasp-only (912N, SD=100, p<0.001) slots compared to standard DKS slots (703N, SD=81).

Round keel slots minimise stress concentrations and increase TLTF but do not increase ALTF. The SKS and rasp-only slots retain material at slot ends and have significantly higher ALTF. Future studies should assess saw blades that retain material and round slot ends to evaluate if their use may significantly reduce the incidence of tibial periprosthetic fracture.

Fractures of the prosthetic components after total knee arthroplasty (TKA) are rare but dangerous complications, sometimes difficult to diagnose and to manage. Aim of this study is to evaluate the incidence of component breakage and its treatment in our single institution's experience. We retrospectively review our institution registry. From 605 revision knee arthroplasties since 2000 to 2018, we found 8 cases of component breakage, of these 3 belonged to UKA, and 5 belonged to TKA. The UKA fractures were all on the metal tibial component; while 4 TKA fractures were ascribed to the liner (2 Posterior-Stabilized designs and 2 constrained designs) and only one case was on the femoral component. For every patient a revision procedure was performed, in two cases a tibial tubercle osteotomy was performed, while in one case (where the fracture was of the post cam) an arthroscopy was performed prior to the arthrotomy. All of the UKA fractures were treated with a standard revision implant. As regard the TKA, 2 liner fractures were treated with the only liner exchange, while the other 2 liner fractures and the fracture of the metallic component were treated with total knee revision. No intra- and post-operative complications were found. Component breakage after TKA is a serious complication. Its treatment, always surgical, can hide pitfalls, especially if the timing is not correct; indeed apart from the revision of one or more components, the surgeons must address any issues of management of bone defect and ligamentous stability.

After knee replacement, therapy resistant, chronic synovitis is common and leads to effusion and pain.

A cohort of 55 patients with 57 knee replacements and chronic synovitis underwent radiosynoviorthesis. In summary, 101 joints were treated using 182±9 MBq of 90Y-citrate. The number of radiosynoviorthesis ranged from 1 to 4 (53%, 21%, 23%, and 4%). Every patient received a 99mTc-MDP scintigraphy before and three months after every radiosynoviorthesis. Follow-up ranged from 5.7 to 86.7 months. For qualitative analysis, an four steps scoring was used (0 = no response or worsening, 1 = slight, 2 = good, 3 = excellent response). For quantification, the uptake was determined within the 99mTc-MDP scintigraphy soft tissue phase before and after therapy.

At the end of long-term follow-up 27% of patients have an excellent, 24% good, 30% slight and 20% no response. The duration of response was 7.5±8.3 months (maximum 27 months). In repeated treatment, the effect after the first therapy was lesser than in patients who received a single treatment in total. However, three months after the last radiosynoviorthesis, patients with repeated treatment showed a similar effectiveness than single treated patients. At the end of long-term follow-up, patients with repeated radiosynoviorthesis had a higher effectiveness at similar duration response. In the 99mTc-MDP scan 65% of patients showed a reduction of uptake. When comparing subjective and objective response 78% of patients showed a concordance in both, symptoms and scintigraphy. Pilot histological analysis revealed that the synovitis is triggered by small plastic particles.

Radiosynoviorthesis is effective in patients with knee replacement and chronic synovitis. It shows good subjective and objective response rates and long response duration. Repeated treatment leads to a stronger long-time response. The chronic synovitis is caused by plastic particles, which result from the abrasion of the polymeric inlay of endoprothesis.

In osteoarthritis, chondrocytes acquire a hypertrophic phenotype that contributes to matrix degradation. Inflammation is proposed as trigger for the shift to a hypertrophic phenotype. Using in vitro culture of human chondrocytes and cartilage explants we could not find evidence for a role of inflammatory signalling activation. We found, however, that tissue repair macrophages may contribute to the onset of hypertrophy (doi: 10.1177/19476035211021907) Intra-articularly injected triamcinolone acetonide to inhibit inflammation in a murine model of collagenase-induced osteoarthritis, increased synovial macrophage numbers and osteophytosis, confirming the role of macrophages in chondrocyte hypertrophy occurring in osteophyte formation (doi: 10.1111/bph.15780).

In search of targets to inhibit chondrocyte hypertrophy, we combined existing microarray data of different cartilage layers of murine growth plate and murine articular cartilage after induction of collagenase-induced osteoarthritis. We identified common differentially expressed genes and selected those known to be associated to inflammation. This revealed EPHA2, a tyrosine kinase receptor, as a new target. Using in silico, in vitro and in vivo models we demonstrated that inhibition of EPHA2 might be a promising treatment for osteoarthritis.

Recently, single cell RNA-seq. has revealed detailed information about different populations of chondrocytes in articular cartilage during osteoarthritis. We re-analysed a published scRNA-seq data set of healthy and osteoarthritic cartilage to obtain the differentially expressed genes in the population of hypertrophic chondrocytes compared to the other chondrocytes, applied pathway analyses and then used drug databases to search for upstream inhibitors of these pathways. This drug repurposing approach led to the selection of 6 drugs that were screened and tested using several in vitro models with human chondrocytes and cartilage explants.

In this lecture I will present this sequence of studies to highlight different approaches and models that can be used in the quest for a disease modifying drug for osteoarthritis.

The regenerative capacity of hyaline cartilage is greatly limited. To prevent the onset of osteoarthritis, cartilage defects have to be properly treated. Cartilage, tissue engineered by mean of bioactive glass (BG) scaffolds presents a promising approach. Until now, conventional BGs have been used mostly for bone regeneration, as they are able to form a hydroxyapatite (HA) layer and are therefore, less suited for cartilage reconstruction. The aim of this study is to compare two BGs based on a novel BG composition tailored specifically for cartilage (CAR12N) and patented by us with conventional BG (BG1393) with a similar topology. The highly porous scaffolds consisting of 100% BG (CAR12N, CAR12N with low Ca2+/Mg2+ and BG1393) were characterized and dynamically seeded with primary porcine articular chondrocytes (pACs) or primary human mesenchymal stem cells (hMSCs) for up to 21 days. Subsequently, cell viability, DNA and glycosaminoglycan contents, cartilage-specific gene and protein expression were evaluated. The manufacturing process led to a comparable high (over 80%) porosity in all scaffold variants. Ion release and pH profiles confirmed bioactivity for them. After both, 7 and 21 days, more than 60% of the total surfaces of all three glass scaffold variants was densely colonized by cells with a vitality rate of more than 80%. The GAG content was significantly higher in BG1393 colonized with pACs. In general, the GAG content was higher in pAC colonized scaffolds in comparison to those seeded with hMSCs. The gene expression of cartilage-specific collagen type II, aggrecan, SOX9 and FOXO1 could be detected in all scaffold variants, irrespectively whether seeded with pACs or hMSCs. Cartilage-specific ECM components could also be detected at the protein level. In conclusion, all three BGs allow the maintenance of the chondrogenic phenotype or chondrogenic differentiation of hMSCs and thus, they present a high potential for cartilage regeneration.

Decellularization techniques have advanced to reduce the risk of immune rejection in transplantation. Validation of these protocols typically relies on Crapo's criteria¹, which include the absence of visible nuclei and low DNA content. In our study, five decellularization protocols were compared to determine the optimal approach for human fascia lata (HFL) samples. However, our findings raised questions as to why recipients can still develop immunity despite meeting validation criteria.

HFL samples were decellularized using four protocols with SDS-Triton X100-DNase (D1 to D4-HFL) and one protocol using solvent-detergent-based baths (D5-HFL). The decellularized samples (D-HFL) were compared to native samples (N-HFL) using histology, and DNA content was measured. The human leukocyte antigen (HLA) content within the matrix was assessed using western blot analysis. Both D-HFL and N-HFL samples, along with negative control patches, were implanted in the backs of 28 Wistar rats. Anti-human IgG serum levels were evaluated after one month.

H&E and Hoechst staining revealed the absence of residual cells in all decellularization protocols. DNA content was consistently below the critical threshold (p<0.05). All implanted D-HFL samples resulted in significantly lower anti-human IgG levels compared to N-HFL (p<0.01). However, 2.5 out of 4 rats developed immunity after being implanted with D1 to D4-HFL, with varying levels of anti-human IgG. Only rats implanted with D5-HFL showed undetectable levels of IgG and were considered non-immunized. Western blot analysis indicated that only D5-HFL had a residual HLA content below 1%.

The literature on decellularization has primarily relied on Crapo's criteria, which do not consider the role of HLA mismatch in acute immune rejection. Our results suggest that a residual HLA content below 1% should also be considered to prevent immunization, even if other validation criteria are met. Further research is needed to evaluate the impact of residual HLA levels on human allotransplantation outcomes.

To design slow resorption patient-specific bone graft whose properties of bone regeneration are increased by its geometry and composition and to assess it in in-vitro and in-vivo models.

A graft composed by hydroxyapatite (HA) and β-TCP was designed as a cylinder with 3D gyroid porosities and 7 mm medullary space based on swine's anatomy. It was produced using a stereolithography 3D-printing machine (V6000, Prodways).

Sterile bone grafts impregnated with or without a 10µg/mL porcine BMP-2 (pBMP-2) solution were implanted into porcine femurs in a bone loss model. Bone defect was bi-weekly evaluated by X-ray during 3 months. After sacrifice, microscanner and non-decalcified histology analysis were conducted on biopsies.

Finally, osteoblasts were cultured inside the bone graft or in monolayer underneath the bone graft. Cell viability, proliferation, and gene expression were assessed after 7 and 14 days of cell culture (n=3 patients).

3D scaffolds were successfully manufactured with a composition of 80% HA and 20% β-TCP ±5% with indentation compressive strength of 4.14 MPa and bending strength of 11.8MPa.

In vivo study showed that bone regeneration was highly improved in presence of pBMP-2. Micro-CT shows a filling of the gyroid sinuses of the implant (Figure 1).

In vitro, the presence of BMP2 did not influence the viability of the osteoblasts and the mortality remained below 3%. After 7 days, the presence of BMP2 in the scaffold significantly increased by 85 and 65% the COL1A1 expression and by 8 and 33-fold the TNAP expression by osteoblasts in the monolayer or in the scaffold, respectively. This BMP2 effect was transient in monolayer and did not modify gene expression at day 14.

BMP2-impregnated bone graft is a promising patient-personalized 3D-printed solution for bone defect regeneration, by promoting neighboring host cells recruitment and solid new bone formation.

For any figures and tables, please contact the authors directly.

In this work, we combined tissue engineering and gene therapy technologies to develop a therapeutic platform for bone regeneration. We have developed photothermal fibrin-based hydrogels that incorporate degradable CuS nanoparticles (CuSNP) which transduce incident near-infrared (NIR) light into heat. A heat-activated and rapamycin-dependent transgene expression system was incorporated into mesenchymal stem cells to conditionally control the production of bone morphogenetic protein 2 (BMP-2). Genetically engineered cells were entrapped in the photothermal hydrogels. In the presence of rapamycin, photoinduced mild hyperthermia induced the release of BMP-2 from the NIR responsive cell constructs. Transcriptome analysis of BMP-2 expressing cells showed a signature of induced genes related to stem cell proliferation and angiogenesis. We next generated 4 mm diameter calvarial defects in the left parietal bone of immunocompetent mice. The defects were filled with NIR-responsive hydrogels entrapping cells that expressed BMP-2 under the control of the gene circuit. After one and eight days, rapamycin was administered intraperitoneally followed by irradiation with an NIR laser. Ten weeks after implantation, the animals were euthanized and samples from the bone defect zone were processed for histological analysis using Masson's trichrome staining and for immunohistochemistry analyses using specific CD31 and CD105 antibodies. Samples from mice that were only administered rapamycin or vehicle or that were only NIR-irradiated showed the persistence of fibrous tissue bridging the defect. In animals that were treated with rapamycin, NIR irradiation of implants resulted in the formation of new mineralized tissue with a high degree of vascularization, thus indicating the therapeutic potential of the approach.

Acknowledgements: This research was supported by grants RTI2018-095159-B-I00 and PID2021-126325OB-I00 (MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe”), by grant P2022/BMD- 7406 (Regional Government of Madrid). M.A.L-J. is the recipient of predoctoral fellowship PRE2019-090430 (MCIN/AEI/10.13039/501100011033).

Tendinopathy is the most common form of chronic tendon disorders, accounting for up 30% of all musculoskeletal clinic visits [1]. In tendon disease, the largely avascular tendon tissue often becomes hypervascularized and fibrotic [2]. As blood vessel growth and angiogenic signaling molecules are often induced by the lack of adequate nutrients and oxygen, hypoxic signaling is speculated to be a root cause of tendon neovascularization and tendinopathy [3,4,5]. However, how the vascular switch is initiated in tendons, and how vascularization contributes to tendon pathology remains unknown. In this talk, we provide evidence that HIF-1α is implicated in tendon disease and HIF-1α stabilization in human tendon cells induces vascular recruitment of endothelial cells via VEGFa secretion. More interesting, HIF-1α stabilization in tendon cells in vivo, seems to recapitulate all main features of fibrotic human tendon disease, including vascular ingrowth, matrix disorganization, changes in tissue mechanics, cell proliferation and innervation. Surprisingly, in vivo knock-out of VEGFa rescued angiogenesis in the tendon core but it did not affect tendon mechanical properties and tissue pathophysiological changes, suggesting that blood vessels ingrowth might not be a primary cause but a consequence of HIF-1α activation.

Tendon injuries present a major clinical challenge, as they necessitate surgical intervention and are prone to fibrotic progression. Despite advances in physical therapy and surgical technique, tendons fail to return to full native functioning, underlining the need for a biological therapeutic to improve tendon healing. Myofibroblasts are activated fibroblasts that participate in the proliferative and remodeling phases of wound healing, and while these matrix-producing cells are essential for proper healing, they are also linked to fibrotic initiation. A subset of tenocytes has been shown to give rise to the myofibroblast fate, and potentially contribute to fibrotic tendon healing. A viable anti-fibrotic therapy in other tissues has been reprogramming the fibroblast-myofibroblast differentiation route, avoiding a more pro-fibrotic myofibroblast phenotype. Thus, defining the molecular programs that underlie both physiological and pathological tendon healing is critical for the development of potential pharmacologic treatments. Towards that end, we have taken advantage of spatial transcriptomics, using the tenocyte marker Scleraxis as a tool, and have outlined three major spatiotemporally distinct tenocyte differentiation trajectories (synthetic, proliferative, and reactive) following acute tendon injury in mouse FDL. We have further outlined key transcriptional controls that may be manipulated to alter the differentiation process and influence the resulting myofibroblast phenotype, thereby promoting regenerative tendon healing.

Tendons are characterised by an inferior healing capacity when compared to other tissues, ultimately resulting in the formation of a pathologically altered extracellular matrix structure. Although our understanding of the underlying causes for the development and progression of tendinopathies remains incomplete, mounting evidence indicates a coordinated interplay between tendon-resident cells and the ECM is critical. Our recent results demonstrate that the matricellular protein SPARC (Secreted protein acidic and rich in cysteine) is essential for regulating tendon tissue homeostasis and maturation by modulating the tissue mechanical properties and aiding in collagen fibrillogenesis [1,2]. Consequently, we speculate that SPARC may also be relevant for tendon healing.

In a rat patellar tendon window defect model, we investigated whether the administration of recombinant SPARC protein can modulate tendon healing. Besides the increased mRNA expression of collagen type 1 and the downregulation of collagen type 3, a robust increase in the expression of pro-regenerative fibroblast markers in the repair tissue after a single treatment with rSPARC protein was observed. Additionally, pro-fibrotic markers were significantly decreased by the administration of rSPARC. Determination of structural characteristics was also assessed, indicating that the ECM structure can be improved by the application of rSPARC protein. Therefore, we believe that SPARC plays an important role for tendon healing and the application of recombinant SPARC to tendon defects has great potential to improve functional tendon repair.

In-vitro models of disease are valuable tools for studying disease and analysing response to therapeutics. Recently, advances in patient-derived organoid (PDO) models have been shown to faithfully recapitulate structure, function, and therapeutic response for a wide range of tissues. Frozen shoulder is a rare example of a chronic inflammatory fibrotic disease which is self-limiting, unlike many other soft tissue fibrotic disorders. As no in-vitro 3D models or in-vivo animal models exist for frozen shoulder, establishing an organoid model which recapitulates core diseases features may give insight into fibrosis resolution. Consequently, using biocompatible hydrogels, primary capsular fibroblasts, monocyte-derived macrophages and HUVEC cells, we generated stable PDO cultures which exhibited key disease phenotypes, including vascularization, increased stiffness, and an expanded lining layer over 21 days of culture. Through further investigation of cell-matrix and cell-cell interactions in the organoid model, we intend to unpack the differences between resolving and non-resolving fibrotic disease and uncover clinically relevant therapeutic targets for fibrosis.

Infections are among the most diffused complications of the implantation of medical devices. In orthopedics, they pose severe societal and economic burden and interfere with the capability of the implants to integrate in the host bone, significantly increasing failure risk. Infection is particularly severe in the case of comorbidities and especially bone tumors, since oncologic patients are fragile, have higher infection rate and impaired osteoregenerative capabilities. For this reason, prevention of infection is to be preferred over treatment.

This is even more important in the case of spine surgery, since spine is among the main site for tumor metastases and because incidence of post operative surgical-site infections is significant (up to 15-20%) and surgical options are limited by the need of avoiding damaging the spinal cord.

Functionalization of the implant surfaces, so as to address infection and, possibly, co- adjuvate anti-tumor treatments, appears as a breakthrough innovation. Unmet clinical needs in infection and tumors is presented, with a specific focus on the spine, then, new perspectives are highlighted for their treatment.

Over the last decades, biodegradable metals emerged as promising materials for various biomedical implant applications, aiming to reduce the use of permanent metallic implants and, therefore, to avoid additional surgeries for implant removal. However, among the important issue to be solved is their fast corrosion - too high to match the healing rate of the bone tissue. The most effective way to improve this characteristic is to coat biodegradable metals with substituted calcium phosphates. Tricalcium phosphate (β-TCP) is a resorbable bioceramic widely used as synthetic bone graft. In order to modulate and enhance its biological performance, the substitution of Ca2+ by various metal ions, such as strontium (Sr2+), magnesium (Mg2+), iron (Fe2+) etc., can be carried out. Among them, copper (Cu2+), manganese (Mn2+), zinc (Zn2+) etc. could add antimicrobial properties against implant-related infections. Double substitutions of TCP containing couples of Cu2+/Sr2+ or Mn2+/Sr2+ ions are considered to be the most perspective based on the results of our study. We established that single phase Ca3−2x(MˊMˊˊ)x(PO4)2 solid solutions are formed only at x ≤ 0.286, where Mˊ and Mˊˊ—divalent metal ions, such as Zn2+, Mg2+, Cu2+, Mn2+, and that in case of double substitutions, the incorporation of Sr2+ ions allows one to extend the limit of solid solution due to the enlargement of the unit cell structure. We also reported that antimicrobial properties depend on the substitution ion occupation of Ca2+ crystal sites in the β-TCP structure. The combination of two different ions in the Ca5 position, on one side, and in the Ca1, Ca2, Ca3, and Ca4 positions, on another side, significantly boosts antimicrobial properties. In the present work, zinc-lithium (Zn-Li) biodegradable alloys were coated with double substituted Mn2+/Sr2+ β-TCP and double substituted Cu2+/ Sr2+ β-TCP, with the scope to promote osteoinductive effect (due to the Sr2+ presence) and to impart antimicrobial properties (thanks to Cu2+ or Mn2+ ions). The Pulsed Laser Deposition (PLD) method was applied as the coating's preparation technique. It was shown that films deposited using PLD present good adhesion strength and hardness and are characterized by a nanostructured background with random microparticles on the surface. For coatings characterization, Fourier Transform Infrared Spectroscopy, X-ray Diffraction, and Scanning Electron Microscopy coupled with Energy Dispersive X-ray and X-ray Photoelectron Spectroscopy were applied. The microbiology tests on the prepared coated Zn-Li alloys were performed with the Gram-positive (Staphylococcus aureus, Enterococcus faecalis) and Gram-negative (Salmonella typhimurium, Escherichia coli) bacteria strains and Candida albicans fungus. The antimicrobial activity tests showed that Mn2+/Sr2+ β-TCP -coated and Cu2+/Sr2+ β-TCP coated Zn-Li alloys were able to inhibit the growth of all five microorganisms. The prepared coatings are promising in improving the degradation behavior and biological properties of Zn-Li alloys, and further studies are necessary before a possible clinical translation.

Decreasing the chance of local relapse or infection after surgical excision of bone metastases is a main goals in orthopedic oncology. Indeed, bone metastases have high incidence rate (up to 75%) and important cross-relations with infection and bone regeneration. Even in patients with advanced cancer, bone gaps resulting from tumor excision must be filled with bone substitutes. Functionalization of these substitutes with antitumor and antibacterial compounds could constitute a promising approach to overcome infection and tumor at one same time. Here, for the first time, we propose the use of nanostructured zinc-bone apatite coatings having antitumor and antimicrobial efficacy. The coatings are obtained by Ionized Jet Deposition from composite targets of zinc and bovine-derived bone apatite. Antibacterial and antibiofilm efficacy of the coatings is demonstrated in vitro against S. Aureus and E. Coli. Anti-tumor efficacy is investigated against MDA- MB-231 cells and biocompatibility is assessed on L929 and MSCs.

A microfluidic based approach is used to select the optimal concentration of zinc to be used to obtain antitumor efficacy and avoid cytotoxicity, exploiting a custom gradient generator microfluidic device, specifically designed for the experiments. Then, coatings capable of releasing the desired amount of active compounds are manufactured. Films morphology, composition and ion-release are studies by FEG- SEM/EDS, XRD and ICP. Efficacy and biocompatibility of the coatings are verified by investigating MDA, MSCs and L929 viability and morphology by Alamar Blue, Live/Dead Assay and FEG-SEM at different timepoints. Statistical analysis is performed by SPSS/PC + Statistics TM 25.0 software, one-way ANOVA and post-hoc Sheffe? test. Data are reported as Mean ± standard Deviation at a significance level of p <0.05.

Results and Discussion. Coatings have a nanostructured surface morphology and a composition mimicking the target. They permit sustained zinc release for over 14 days in medium. Thanks to these characteristics, they show high antibacterial ability (inhibition of bacteria viability and adhesion to substrate) against both the gram + and gram – strain.

The gradient generator microfluidic device permits a fine selection of the concentration of zinc to be used, with many potential perspectives for the design of biomaterials. For the first time, we show that zinc and zinc-based coatings have a selective efficacy against MDA cells. Upon mixing with bone apatite, the efficacy is maintained and cytotoxicity is avoided. For the first time, new antibacterial metal-based films are proposed for addressing bone metastases and infection at one same time. At the same time, a new approach is proposed for the design of the coatings, based on a microfluidic approach. We demonstrated the efficacy of Zn against the MDA-MB-231 cells, characterized for their ability to form bone metastases in vivo, and the possibility to use nanostructured metallic coatings against bone tumors. At the same time, we show that the gradient-generator approach is promising for the design of antitumor biomaterials. Efficacy of Zn films must be verified in vivo, but the dual-efficacy coatings appear promising for orthopedic applications.

Prosthetic joint infections represent complications connected to the implantation of biomedical devices, they have high incidence, interfere with osseointegration, and lead to a high societal burden. The microbial biofilm, which is a complex structure of microbial cells firmly attached to a surface, is one of the main issues causing infections. Biofilm- forming bacteria are acquiring more and more resistances to common clinical treatments due to the abuse of antibiotics administration. Therefore, there is increasing need to develop alternative methods exerting antibacterial activities against multidrug-resistant biofilm-forming bacteria. In this context, metal-based coatings with antimicrobial activities have been investigated and are currently used in the clinical practice. However, traditional coatings exhibit some drawbacks related to the insufficient adhesion to the substrate, scarce uniformity and scarce control over the toxic metal release reducing their efficacy. Here, we propose the use of antimicrobial silver-based nanostructured thin films to discourage bacterial infections. Coatings are obtained by Ionized Jet Deposition, a plasma-assisted technique that permits to manufacture films of submicrometric thickness having a nanostructured surface texture, allow tuning silver release, and avoid delamination. To mitigate interference with osseointegration, here silver composites with bone apatite and hydroxyapatite were explored. The antibacterial efficacy of silver films was tested in vitro against gram- positive and gram-negative species to determine the optimal coatings characteristics by assessing reduction of bacterial viability, adhesion to substrate, and biofilm formation. Efficacy was tested in an in vivo rabbit model, using a multidrug-resistant strain of Staphylococcus aureus showing significant reduction of the bacterial load on the silver prosthesis both when coated with the metal only (>99% reduction) and when in combination with bone apatite (>86% reduction). These studies indicate that IJD films are highly tunable and can be a promising route to overcome the main challenges in orthopedic prostheses.

Bone healing outcome is highly dependent on the initial mechanical fracture environment [1]. In vivo, direct bone healing requires absolute stability and an interfragmentary strain (IFS) below 2% [2]. In the majority of cases, however, endochondral ossification is engaged where frequency and amplitude of IFS are key factors. Still, at the cellular level, the influence of those parameters remains unknown. Understanding the regulation of naïve hMSC differentiation is essential for developing effective bone healing strategies.

Human bone-marrow-derived MSC (KEK-ZH-NR: 2010–0444/0) were embedded in 8% gelatin methacryol. Samples (5mm Ø x 4mm) were subjected to 0, 10 and 30% compressive strain (5sec compression, 2hrs pause sequence for 14 days) using a multi-well uniaxial bioreactor (RISystem) and in presence of chondro-permissive medium (CP, DMEM HG, 1% NEAA, 10 µM ITS, 50 µg/mL ascorbic acid, and 100 mM Dex). Cell differentiation was assessed by qRT-PCR and histo-/immunohistology staining. Experiments were repeated 5 times with cells from 5 donors in duplicate. ANOVA with Tukey post-hoc correction or Kurskal-Wallis test with Dunn's correction was used.

Data showed a strong upregulation of hypertrophic related genes COMP, MMP13 and Type 10 collagen upon stimulation when compared to chondrogenic SOX9, ACAN, Type 2 collagen or to osteoblastic related genes Type 1 Collagen, Runx2. When compared to chondrogenic control medium, cells in CP with or without stimulation showed low proteoglycan synthesis as shown by Safranine-O-green staining. In addition, the cells were significantly larger in 10% and 30% strain compared to control medium with 0% strain. Type 1 and 10 collagens immunostaining showed stronger Coll 10 expression in the samples subjected to strain compared to control.

Uniaxial deformation seems to mainly promote hypertrophic-like chondrocyte differentiation of MSC. Osteogenic or potentially late hypertrophic related genes are also induced by strain.

Acknowledgments: Funded by the AO Foundation, StrainBot sponsored by RISystemAG & PERRENS 101 GmbH

In cartilage tissue engineering (TE),new solutions are needed to effectively drive chondrogenic differentiation of mesenchymal stromal cells in both normal and inflammatory milieu. Ultrasound waves represent an interesting tool to facilitate chondrogenesis. In particular, low intensity pulsed ultrasound (LIPUS)has been shown to regulate the differentiation of adipose mesenchymal stromal cells. Hydrogels are promising biomaterials capable of encapsulating MSCs by providing an instructive biomimetic environment, graphene oxide (GO) has emerged as a promising nanomaterial for cartilage TE due to its chondroinductive properties when embedded in polymeric formulations, and piezoelectric nanomaterials, such as barium titanate nanoparticles (BTNPs),can be exploited as nanoscale transducers capable of inducing cell growth/differentiation. The aim of this study was to investigate the effect of dose-controlled LIPUS in counteracting inflammation and positively committing chondrogenesis of ASCs embedded in a 3D piezoelectric hydrogel.

ASCs at 2*10⁶ cells/mL were embedded in a 3D VitroGel RGD^® hydrogel without nanoparticles (Control) or doped with 25 µg/ml of GO nanoflakes and 50 µg/ml BTNPs.The hydrogels were exposed to basal or inflammatory milieu (+IL1β 10ng/ml)and then to LIPUS stimulation every 2 days for 10 days of culture. Hydrogels were chondrogenic differentiated and analyzed after 2,10 and 28 days. At each time point cell viability, cytotoxicity, gene expression and immunohistochemistry (COL2, aggrecan, SOX9, COL1)and inflammatory cytokines were evaluated.

Ultrasound stimulation significantly induced chondrogenic differentiation of ASCs loaded into 3D piezoelectric hydrogels under basal conditions: COL2, aggrecan and SOX9 were significantly overexpressed, while the fibrotic marker COL1 decreased compared to control samples. LIPUS also has potent anti-inflammatory effects by reducing IL6 and IL8 and maintaining its ability to boost chondrogenesis.

These results suggest that the combination of LIPUS and piezoelectric hydrogels promotes the differentiation of ASCs encapsulated in a 3D hydrogel by reducing the inflammatory milieu, thus representing a promising tool in the field of cartilage TE.

Acknowledgements: This work received funding from the European Union's Horizon 2020 research and innovation program, grant agreement No 814413, project ADMAIORA (AdvanceD nanocomposite MAterIals for in situ treatment and ultRAsound-mediated management of osteoarthritis).

Bone is a dynamic tissue that undergoes continuous mechanical forces. Mechanical stimuli applied on scaffolds resembling a part of the human bone tissue affects the osteogenesis [1]. Poly(3,4-ethylenedioxythiophene) (PEDOT) is a piezoelectric material that responds to mechanical stimulation producing an electrical signal, which in turn promotes the osteogenic differentiation of bone-forming cells by opening voltage-gated calcium channels [2]. In this study we examined the biological behavior of pre-osteoblastic cells seeded onto lyophilized piezoelectric PEDOT-containing scaffolds applying uniaxial compression.

Two different concentrations of PEDOT (0.10 and 0.15% w/v) were combined with a 5% w/v poly(vinyl alcohol) (PVA) and 5% w/v gelatin, casted into wells, freeze dried and crosslinked with 2% v/v (3-glycidyloxypropyl)trimethoxysilane and 0.025% w/v glutaraldehyde. The scaffolds were physicochemically characterized by FTIR, measurement of the elastic modulus, swelling ratio and degradation rate. The cell-loaded scaffolds were subjected to uniaxial compression with a frequency of 1 Hz and a strain of 10% for 1 h every second day for 21 days. The loading parameters were selected to resemble the in vivo loading situation [3]. Cell viability and morphology on the PEDOT/PVA/gelatin scaffolds was determined. The alkaline phosphatase (ALP) activity, the collagen and calcium production were determined.

The elastic modulus of PEDOT/PVA/gelatin scaffolds ranged between 1 and 5 MPa. The degradation rate indicates a mass loss of 15% after 21 days. The cell viability assessment displays excellent biocompatibility, while SEM images display well-spread cells. The ALP activity at days 3, 7 and 18 as well as the calcium production are higher in the dynamic culture compared to the static one. Moreover, energy dispersive spectroscopy analysis revealed the presence of calcium phosphate in the extracellular matrix after 14 days. The results demonstrate that PEDOT/PVA/gelatin scaffolds promote the adhesion, proliferation, and osteogenic differentiation of pre-osteoblastic cells under mechanical stimulation, thus favoring bone regeneration.

The growing number of non-union fractures in an aging population has increased the clinical demand for tissue-engineered bone. Electrical stimulation (ES) has been described as a promising strategy for bone regeneration treatments in several clinical studies. However the underlying mechanism by which ES augments bone formation is still poorly understood and its use in bone tissue engineering (BTE) strategies is currently underexplored. Additive manufacturing (AM) technologies (Fused Deposition Modeling/3D Printing) have been widely used in BTE due to their ability to fabricate scaffolds with a high control over their structural and mechanical properties in a reproducible and scalable manner. Thus, in this work, we combined AM methods with conductive biomaterials and ES to enhance the osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells (hBMSCs) envisaging improved BTE strategies.

First, we started by developing AM-based electro-bioreactor devices containing medical-grade electrodes (stainless steel and Ti6Al4V) to apply ES to monolayer 2D cultures and 3D cell-seeded scaffolds. Computer modeling(Finite Element Analysis-FEA) was employed to predict the magnitude/distribution of electrical fields within the ES devices and along the different conductive scaffolds. Prior to scaffold culture, 5 different ES protocols were tested in terms of their ability to promote hBMSCs proliferation and osteogenic differentiation in 2D cultures. The best performance ES protocol was then used in two different AM-based BTE strategies: 1) Two different conductive scaffolds (conductive poly lactic acid (PLA) and titanium) were seeded with hBMSCs and cultured for 21 days under osteogenic medium conditions with and without ES and their biological performance was evaluated in comparison to non-conductive standard PLA scaffolds; 2) Different PEDOT:PSS-based coating solutions were screened to obtain PEDOT:PSS/Gelatin-coated 3D polycaprolactone (PCL) scaffolds with a high(11 S.cm^-1) and stable electroconductivity. When cultured under ES, PEDOT:PSS/Gelatin-PCL scaffolds enhanced significantly hBMSCs osteogenic differentiation and mineralization(calcium deposition), highlighting their potential for BTE applications.

Acknowledgements: Funding received from FCT through projects InSilico4OCReg (PTDC/EME-SIS/0838/2021), OptiBioScaffold (PTDC/EME-SIS/4446/2020) and BioMaterARISES (EXPL/CTM-CTM/0995/2021), and to the institutions iBB (UIDB/04565/2020), CDRSP (UIDB/04044/2020) and Associate Laboratory i4HB (LA/P/0140/2020).

Anatomically, bone consists of building blocks called osteons, which in turn comprise a central canal that contains nerves and blood vessels. This indicates that bone is a highly innervated and vascularized tissue. The function of vascularization in bone (development) is well-established: providing oxygen and nutrients that are necessary for the formation, maintenance, and healing. As a result, in the field of bone tissue engineering many research efforts take vascularization into account, focusing on engineering vascularized bone. In contrast, while bone anatomy indicates that the role of innervation in bone is equally important, the role of innervation in bone tissue engineering has often been disregarded.

For many years, the role of innervation in bone was mostly clear in physiology, where innervation of a skeleton is responsible for sensing pain and other sensory stimuli. Unraveling its role on a cellular level is far more complex, yet more recent research efforts have unveiled that innervation has an influence on osteoblast and osteoclast activity. Such innervation activities have an important role in the regulation of bone homeostasis, stimulating bone formation and inhibiting resorption. Furthermore, due to their anatomical proximity, skeletal nerves and blood vessels interact and influence each other, which is also demonstrated by pathways cross-over and joint responses to stimuli.

Besides those closely connected sytems, the immune system plays also a pivotal role in bone regeneration. Certain cytokines are important to attract osteogenic cells and (partially) inhibit bone resorption. Several leukocytes also play a role in the bone regeneration process.

Overall, bone interacts with several systems. Aberrations in those systems affect the bone and are important to understand in the context of bone regeneration. This crosstalk has become more evident and is taken more into consideration. This leads to more complex tissue regeneration, but may recapitulate better physiological situations.

Bone regeneration is an area of acute medical need, but its clinical success is hampered by the need to ensure rapid vascularization of osteogenic grafts. Vascular Endothelial Growth Factor (VEGF) is the master regulator of vascular growth and during bone development angiogenesis and osteogenesis are physiologically coupled through so-called angiocrine factors produced by blood vessels. However, how to exploit this process for therapeutic bone regeneration remains a challenge (1).

Here we will describe recent work aiming at understanding the cross-talk between vascular growth and osteogenesis under conditions relevant for therapeutic bone regeneration. To this end we take advantage of a unique platform to generate controlled signalling microenvironments, by the covalent decoration of fibrin matrices with tunable doses and combinations of engineered growth factors. The combination of human osteoprogenitors and hydroxyapatite in these engineered fibrin matrices provides a controlled model to investigate how specific molecular signals regulate vascular invasion and bone formation in vivo. In particular, we found that:

In conclusion, vascularization of osteogenic grafts is not simply necessary in order to enable progenitor survival. Rather, blood vessels can actively stimulate bone regeneration in engineered grafts through specific molecular signals that can be harnessed for therapeutic purposes.

Acknowledgements: This work was supported in part by the European Union Horizon 2020 Program (Grant agreement 874790 – cmRNAbone).

For any figures and tables, please contact the authors directly.

Monomeric C reactive protein (mCRP) presents important proinflammatory effects in endothelial cells, leukocytes, or chondrocytes. However, CRP in its pentameric form exhibits weak anti-inflammatory activity. It is used as a biomarker to follow severity and progression in infectious or inflammatory diseases, such as intervertebral disc degeneration (IVDD). This work assesses for the first time the mCRP effects in human intervertebral disc cells, trying to verify the pathophysiological relevance and mechanism of action of mCRP in the etiology and progression of IVD degeneration.

We demonstrated that mCRP induces the expression of multiple proinflammatory and catabolic factors, like nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and lipocalin 2 (LCN2), in human annulus fibrosus (AF) and nucleus pulposus (NP) cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signaling of mCRP.

Our results indicate that the effect of mCRP is persistent and sustained, regardless of the proinflammatory environment, as it was similar in healthy and degenerative human primary AF cells. This is the first article that demonstrates the localization of mCRP in intravertebral disc cells of the AF and NP and that provides evidence for the functional activity of mCRP in healthy and degenerative human AF and NP disc cells.

Disease modifying approaches are commonly applied in OA patients. An aging society with better life expectancies is increasing in Europe and the globe. Orthobiologics cover intraarticular hyaluronan injections and also cellular therapies. Cellular therapies range from platelet rich plasma (PRP) applications to exosomes. Short term follow-up of limited number of patients revealed favorable results in clinical cellular therapies. Most of these studies evaluated decrease of pain and increase in function. Recent basic science studies focused on the action mechanism of orthobiologic therapies however patient perspective is less studied. Our research team has recently performed a qualitative study on the patient perspective of hyaluronan injection of the knee joint. Findings of that study will be shared and future patient knowledge based options on orthobiologics will be discussed.

The biological understanding for the disease progression osteoarthritis (OA) has uncovered specific biomarkers from either synovial fluid, articular chondrocytes or synoviocytes that can be used to diagnose the disease. Examples of these biomarkers include interleukin-1β (IL-1β) or collagen II fragments (1, 2). In parallel, isolation of chondrocytes or bone marrow derived mesenchymal stromal cells (MSCs) has yielded cell-based strategies that have shown long- term beneficial effects in a specific cohort of patients, specifically in traumatic cartilage lesions (2). This latter finding shows that patient stratification of OA is an important tool to both match patients for a specific treatment and to develop novel therapies, especially disease modifying drugs. In order to create disease stage specific therapies, the use of next generation analysis tools such as RNAseq and metabolomics, has the potential to decipher specific cellular and molecular endotypes. Alongside greater understanding of the clinical phenotype (e.g. imaging, pain, co- morbidities), therapies can be designed to alleviate the symptoms of OA at specific points of the disease in patients. This talk will outline the current biological understanding of OA and discuss how patient stratification could assist in the design of innovative therapies for the disease.

Acknowledgements: This presentation was supported by the COST action, CA21110 – Building an open European Network on Osteoarthritis Research (NetwOArk)

Translational models for OA have used a variety of small (mouse, rat) and large (sheep, pig) animal models to evaluate the efficacy of a specific therapy. Clinical trials based on the results of these animal models have yielded mixed results with respect to the treatment of the disease. Due to greater stringency in EU regulations in the use of animal models for research, ex vivo models of OA (e.g. cartilage explants, bioreactors) are being developed to mimic human joint motion as well as the inflammatory milieu (e.g. IL-1β) that can be used to understand efficacy of therapy in a physiological environment. The development of these models can enable therapies to undergo clinical trials in patients without the necessity for long-term animal studies. This presentation will describe the state of the art in this field and will discuss whether there is potential to speed up translation from bench to bedside in the future.