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.

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.

Functionalization of biomimetic nanomaterials allows to reproduce the composition of native bone, permitting better regeneration, while nanoscale surface morphologies provide cues for cell adhesion, proliferation and differentiation. Functionalization of 3D printed and bioprinted constructs, by plasma-assisted deposition of calcium phosphates-based (CaP) nanostructured coatings and by nanoparticles, respectively, will be presented. Stoichiometric and ion doped CaP- based nanocoatings, including green materials (mussel seashells and cuttlefish bone), will be introduced to guide tissue regeneration. We will show interactions between biomimetic surfaces and MSCs to address bone regeneration and SAOS-2 cells for bone tumor models. Our results show that combining AM and nanostructured biomimetic films permits to reproduce the architecture and the mechanical and compositional characteristics of bone. Stability behavior of the coatings, as well as MSCs behavior strongly depend on the starting CaP material, with more soluble CaPs and ion-doped ones showing better biological behavior. Green materials appear promising, as biomimetic films can be successfully obtained upon conversion of the marine precursors into hydroxyapatite. Last-not-least, nanoparticles-loaded scaffolds could be bioprinting without loss of cell viability, but ink characteristics depend on ion-doping as demonstrated for SAOS-2 cells over 14 days of culture. Biomimetic nanomaterials for functionalization in AM is a promising approach for bone modelling and regeneration.

Biofabrication is a popular technique to produce personalized constructs for tissue engineering. In this study we combined laponite (Lap), gellan gum (GG) with platelet-rich plasma (PRP) aiming to enhance the endothelial regeneration through the synergistic effects of their individual properties. Laponite has the ability to form porous three-dimensional networks mimicking the extracellular matrix structure, and PRP delivery of growth factors stimulates the endothelial cell proliferation and migration, offering a composite bioink for cell growth and support. The sustained release of these growth factors from the GG-laponite-PRP composite material over time provides a continuous source of stimulation for the cells, leading to more effective tissue engineering strategies for endothelial tissue regeneration. Four blend compositions comprising 1% w/v GG and 0.5 or 1% w/v Lap and 25% v/v PRP were combined with Wharton jelly mesenchymal stem cells (WJ-MSCs) and bioprinted into vessel-like structures with an inner diameter of 3 mm and a wall thickness of 1 mm. Stress/strain analysis revealed the elastomeric properties of the hydrogels with Young modulus values of 10 MPa. Increasing the Lap concentration led to a non-significant decrease of swelling ratio from 93 to 91%. Live/dead assay revealed cell viability of at least 76%, with the 0.5%Lap-GG viability exceeding 99% on day 21. Gradual increase of glycosaminoglycans accumulation and collagen production indicate promotion of ECM formation. The expression and membranous localization of PECAM-1 from day 7 and the granular intracellular localization of vWF after 2 weeks demonstrate in vitro endothelial functionality. In vivo subcutaneous implantation indicated the absence of any adverse immunological reactions. The results reveal the expression of both vWF and PECAM-1 by WJ-MSCs entrapped in all four construct compositions with significantly higher expression of vWF in the presence of PRP.

The primary aim was to assess the reliability of ultrasound in the assessment of humeral shaft fracture healing. The secondary aim was to estimate the accuracy of ultrasound assessment in predicting humeral shaft nonunion.

Twelve patients (mean age 54yrs [20–81], 58% [n=7/12] female) with a non-operatively managed humeral diaphyseal fracture were prospectively recruited and underwent ultrasound scanning at six and 12wks post-injury. Scans were reviewed by seven blinded observers to evaluate the presence of sonographic callus. Intra- and inter-observer reliability were determined using the weighted kappa and intraclass correlation coefficient (ICC). Accuracy of ultrasound assessment in nonunion prediction was estimated by comparing scans for patients that united (n=10/12) with those that developed a nonunion (n=2/12).

At both six and 12wks, sonographic callus was present in 11 patients (10 united, one developed a nonunion) and sonographic bridging callus (SBC) was present in seven patients (all united). Ultrasound assessment demonstrated substantial intra- (6wk kappa 0.75, 95% CI 0.47-1.03; 12wk kappa 0.75, 95% CI 0.46-1.04) and inter-observer reliability (6wk ICC 0.60, 95% CI 0.38-0.83; 12wk ICC 0.76, 95% CI 0.58-0.91). Absence of sonographic callus demonstrated a sensitivity of 50%, specificity 100%, positive predictive value (PPV) 100% and negative predictive value (NPV) 91% in nonunion prediction (accuracy 92%). Absence of SBC demonstrated a sensitivity of 100%, specificity 70%, PPV 40% and NPV 100% (accuracy 75%). Of three patients at risk of nonunion based on reduced radiographic callus formation (Radiographic Union Score for HUmeral fractures <8), one had SBC on 6wk ultrasound (and united) and the other two had non-bridging or absent sonographic callus (both developed a nonunion).

Ultrasound assessment of humeral shaft fracture healing was reliable and predictive of nonunion, and may be a useful tool in defining the risk of nonunion among patients with reduced radiographic callus formation.

Developments in the field of additive manufacturing have allowed significant improvements in the design and production of scaffolds with biologically relevant features to treat bone defects. Unfortunately, the workflow to generate personalized scaffolds is source of inaccuracies leading to a poor fit between the implant and patients' bone defects. In addition, scaffolds are often brittle and fragile, uneasing their handling by surgeons, with significant risks of fracture during their insertion in the defect. Consequently, we developed organo-mineral cementitious scaffolds displaying evolutive mechanical properties which are currently being evaluated to treat maxillofacial bone deformities in veterinary clinics. Treatment of dog patients was approved by ethic and welfare committees (CERVO-2022-14-V). To date, 8 puppies with cleft palate/lip deformities received the following treatment. Two weeks prior surgery, CT-scan of patient's skull was performed to allow for surgical planning and scaffold designing. Organo-mineral printable pastes were formulated by mixing an inorganic cement precursor (α-Ca3(PO4)2) to a self-reticulating hydrogel (silanized hyaluronic acid) supplemented with a viscosifier (hydroxymethylpropylcellulose). Scaffolds were produced by robocasting of these pastes. Surgical interventions included the reconstruction of soft tissues, and the insertion of the scaffold soaked with autologous bone marrow. Bone formation was monitored 3 and 6 months after reconstruction, and a biopsy at 6 months was performed for more detailed analyses. Scaffolds displayed great handling properties and were inserted within bone defects without significant issue with a relevant bone edges/scaffold contact. Osteointegration of the scaffolds was observed after 3 months, and regeneration of the defect at 6 months seemed quite promising. Preliminary results have demonstrated a potential of the set-up strategy to treat cleft lip/palate deformities in real, spontaneous clinical setting. Translation of these innovative scaffolds to orthopedics is planned for a near future.

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.

Artificial Intelligence (AI) is becoming more powerful but is barely used to counter the growth in health care burden. AI applications to increase efficiency in orthopedics are rare. We questioned if (1) we could train machine learning (ML) algorithms, based on answers from digitalized history taking questionnaires, to predict treatment of hip osteoartritis (either conservative or surgical); (2) such an algorithm could streamline clinical consultation.

Multiple ML models were trained on 600 annotated (80% training, 20% test) digital history taking questionnaires, acquired before consultation. Best performing models, based on balanced accuracy and optimized automated hyperparameter tuning, were build into our daily clinical orthopedic practice. Fifty patients with hip complaints (>45 years) were prospectively predicted and planned (partly blinded, partly unblinded) for consultation with the physician assistant (conservative) or orthopedic surgeon (operative). Tailored patient information based on the prediction was automatically sent to a smartphone app. Level of evidence: IV.

Random Forest and BernoulliNB were the most accurate ML models (0.75 balanced accuracy). Treatment prediction was correct in 45 out of 50 consultations (90%), p<0.0001 (sign and binomial test). Specialized consultations where conservatively predicted patients were seen by the physician assistant and surgical patients by the orthopedic surgeon were highly appreciated and effective.

Treatment strategy of hip osteoartritis based on answers from digital history taking questionnaires was accurately predicted before patients entered the hospital. This can make outpatient consultation scheduling more efficient and tailor pre-consultation patient education.

Chondral defects in the knee have cartilage biomechanical differences due to defect size and orientation. This study examines how the tibiofemoral contact pressure is affected by increasing full-thickness chondral defect size on the medial and lateral condyle at full extension.

Isolated full-thickness, square chondral defects increasing from 0.09cm² to 1.0cm² were created sequentially on the medial and lateral femoral condyles of six human cadaveric knees with intact ligaments and menisci. Chondral defects were created 1.0cm from the femoral notch posteriorly. The knees were fixed to a uniaxial load frame and loaded from 0N to 600N at full extension. Contact pressures between the femoral and tibial condyles were measured using pressure mapping sensors. The peak contact pressure was defined as the highest value in the 2.54mm² area around the defect. The location of the peak contact pressure was determined relative to the centre of the defect.

Peak contact pressure was significantly different between (4.30MPa) 0.09cm² and (6.91MPa) 1.0cm² defects (p=0.04) on the medial condyle. On the lateral condyle, post-hoc analysis showed differences in contact pressures between (3.63MPa) 0.09cm² and (5.81MPa) 1.0cm² defect sizes (p=0.02).

The location of the stress point shifted from being posteromedial (67% of knees) to anterolateral (83%) after reaching a 0.49cm² defect size (p < 0.01) in the medial condyle. Conversely, the location of the peak contact pressure point moved from being anterolateral (50%) to a posterolateral (67%) location in defect sizes greater than 0.49cm² (p < 0.01).

Changes in contact area redistribution and cartilage stress from 0.49cm² to 1.0cm² impact adjacent cartilage integrity. The location of the maximum stress point also varied with larger defects. This study suggests that size cutoffs exist earlier in the natural history of chondral defects, as small as 0.49cm², than previously studied, suggesting a lower threshold for intervention.

In March 2020, COVID-19 was declared a pandemic by the World Health Organization. The pandemic imposed drastic changes in our social and professional routine. Professionally at all levels our hospital tasks were changed and prioritized. Surgeons and residents were deployed on rotations to fields other than their expertise in orthopaedics. Health-care education received major changes in these challenging times, and students did face difficulties in receiving education, as well as training due to limited clinical and surgical exposure.

In response to the WHO regulations, most of the teaching centres and hospitals worldwide have adopted the web-based teaching and learning model to continue the education and training of orthopaedic residents. These results brought significant changes to the training experience in orthopaedic surgery in combination with the fact that clinical duty hours and case volume were substantially reduced.

In what concerns orthopaedic journal publications, the Covid-19 pandemic resulted in a decline in the annual publication rate for the first time in over 20 years. Although not uniform, the reduction was most likely due to multifactorial causes.

Regarding the appraisal at the end of training, at the Orthopaedic European Board Examination we were able to verify that the outcome at the written part 1 exam was good, equivalent to the outcome prior to the pandemic. However the oral viva was much worse, probably due to the fact that residents skipped much of the clinical and surgical teaching and exposure during 2020 and 2021. At the end of training, theoretical/factual knowledge was good but poor from the clinical practical experience.

Lower back pain (LBP) is a worldwide clinical problem and a prominent area for research. Numerous in vitro biomechanical studies on spine specimens have been undertaken, attempting to understand spinal response to loading and possible factors contributing to LBP. However, despite employing similar testing protocols, there are challenges in replicating in vivo conditions and significant variations in published results. The aim of this study was to use the University of Bath (UoB) spine simulator to perform tests to highlight the major limitations associated with six degree of freedom (DOF) dynamic spine testing.

A steel helical spring was used as a validation model and was potted in Wood's metal. Six porcine lumbar spinal motion segments were harvested and dissected to produce isolated spinal disc specimens. These were potted in Wood's metal, ensuring the midplane of the disc remained horizontal and then sprayed with 0.9% saline and wrapped in saline-soaked tissue and plastic wrap to prevent dehydration. A 400N axial preload was used for spinal specimens. Specimens were tested under the stiffness and flexibility protocols.

Tests were performed using the UoB custom 6-axis spine simulator with coordinate axes. Tests comprised five cycles with data acquired at 100Hz. Stiffness and flexibility matrices were evaluated from the last three motion cycles using the linear least squares method.

According to theory, inverted flexibility matrices should equal stiffness matrices. In the case of the spring, the matrices matched analytical solutions and inverted flexibility matrices were equivalent to stiffness matrices. Matrices from the spinal tests demonstrated some symmetry, with similarities between inverted flexibility- and stiffness matrices, though these were unequal overall. Matrix element values were significantly affected by displacements assumed to occur at disc centre.

Spring tests proved that for linear, elastic specimens, the spine simulator functioned as expected. However, multiple factors limit the confidence in spine test results. Centre of rotation, displacement assumptions and rigid body transformations are known to impact the results from spinal testing, and these should be addressed going forward to improve the replication of in vivo conditions.

Injury of the intervertebral disc (IVD) can occur for many reasons including structural weakness due to disc degeneration. A common disc injury is herniation. A herniated nucleus can compress spinal nerves, causing pain, and nucleus depressurisation changes mechanical behaviour. Many studies have investigated in vitro IVD injuries including endplate fracture, incisions, and nucleotomy. There is, however, a lack of consensus on how the biomechanical behaviour of spinal motion segments is affected. The aim of this study was to induce defined changes to IVDs of spine specimens in vitro and apply 6 degree of freedom testing to evaluate the effect of these changes.

Six porcine lumbar spinal motion segments were harvested from organically farmed pigs. Posterior structures were removed to produce isolated spinal disc specimens. Specimens were potted in Wood's metal, ensuring the midplane of the IVD remained horizontal. After potting, specimens were sprayed with 0.9% saline, wrapped in saline-soaked tissue and plastic wrap to prevent dehydration. A 400N axial preload was equilibrated for 30 minutes before testing. Specimens were tested intact and after a partial nucleotomy removing ~0.34g of nuclear material with a curette through an annular incision.

Stiffness tests were performed using the University of Bath's custom 6-axis spine simulator with coordinate axes and displacement amplitudes. Tests comprised five cycles with data acquired at 100Hz. Stiffness matrices were evaluated from the last three motion cycles using the linear least squares method.

Stiffness matrices for intact and nucleotomy tests were compared. No significant differences in shear, axial or torsional stiffnesses were noted. Nucleotomy caused significantly higher stiffness in lateral bending and flexion-extension with increased linearity and the load-displacement behaviour in these axes displayed no neutral zone (NZ).

Induced changes were designed to replicate posterolaterally herniated discs. Unaffected shear, axial and torsional stiffnesses suggest the annulus is crucial in these axes. However, reduced ROM and NZ after nucleotomy suggests bending is most affected by herniation. Increased linearity and lack of defined NZ in these axes demonstrates herniation causes major changes to the viscoelastic behaviour of spine specimens in response to loading.

Osteosarcoma is a highly malignant primary tumor of bone tissue. The 5-year survival rate of patients with metastasis is below 20% and this scenario is unchanged in the last two decades, despite great efforts in pre-clinical and clinical research. Traditional preclinical models of osteosarcoma do not consider the whole complexity of its microenvironment, leading to poor correlation between in vitro/in vivo results and clinical outcomes. Spheroids are a promising in vitro model to mimic osteosarcoma and perform drug-screening tests, as they (i) reproduce the microarchitecture of the tumor, (ii) are characterized by hypoxic regions and necrotic core as the in vivo tumor, (iii) and recapitulate the chemo-resistance phenomena. However, to date, the spheroid model is scarcely used in osteosarcoma research.

Our aim is to develop a customized culture dish to grow and characterize spheroids and to perform advanced drug-screening tests. The resulting platform must be adapted to automated image acquisition systems, to overcome the drawbacks of commercial spheroids platforms.

To this purpose, we designed and developed a micro-patterned culture dish by casting agarose on a 3D printed mold from a CAD design. We successfully obtained viable and reproducible homotypic osteosarcoma spheroids, with two different cells lines from osteosarcoma (i.e., 143b and MG-63). Using the platform, we performed viability assays and live fluorescent stainings (e.g., Calcein AM) with low reagent consumption. Moreover, the culture dish was validated as drug screening platform, administrating Doxorubicin at different doses, and evaluating its effect on OS spheroids, in terms of morphology and viability. This platform can be considered an attractive alternative to the highly expensive commercial spheroid platforms to obtain homogeneous and reproducible spheroids in a high-throughput and cost effective mode.

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.

Fracture healing is a spatially controlled process involving crosstalk of multiple tissues. To precisely capture and understand molecular mechanism underlying impaired healing, there is a need to integrate spatially-resolved molecular analyses into preclinical fracture healing models. I will present our recent data obtained by spatial transcriptomics of musculoskeletal samples from fracture healing studies in mice. Subsequently, I will show how spatial transcriptomics can be integrated into multimodal approaches in preclinical fracture healing models. In combination with established in vivo imaging and emerging omics techniques, spatially-resolved analyses have the potential to elucidate the molecular mechanisms underlying impaired healing with optimization of treatments.

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.

To determine the clinical efficacy of vitamin-D supplementation on pain intensity and functional disability in patients with chronic lower back pain.

This prospective cohort study was conducted from 20th March 2017 to 19th March 2019. The inclusion criteria were patients of CLBP aged between 15 to 55 years. Exclusion criteria included all the patients with Disc prolapse, Spinal stenosis, Any signs of neurological involvement, Metabolic bone disease (Hypo- or Hyperparathyroidism) and Chronic kidney disease/Chronic liver disease. Patients were supplemented with 50,000 IU of oral vitamin-D3 every week for 8 weeks (induction phase) and 50,000 IU of oral vitamin-D3 once monthly for 6 months (maintenance phase). Efficacy parameters included pain intensity and functional disability measured by VAS and modified Oswestry disability questionnaire (MODQ) scores at baseline, 2, 3 and 6 months post-supplementation. Vitamin-D3 levels were measured at baseline,2,3 and 6 months.

A total of 600 patients were included in the study. The mean age of patients was 44.2 ± 11.92 years. There were 337 (56.2%) male patients while 263 (43.8%) female patients. Baseline mean vitamin-D levels were 13.32 ± 6.10 ng/mL and increased to 37.18 ± 11.72 post supplementation (P < 0.0001). There was a significant decrease in the pain score after 2nd, 3rd& 6th months (61.7 ± 4.8, 45.2 ± 4.6 & 36.9 ± 7.9, respectively) than 81.2 ± 2.4 before supplementation (P < 0.001). The modified Oswestry disability score also showed significant improvement after 2nd, 3rd & 6th months (35.5, 30.2 & 25.8, respectively) as compared to baseline 46.4 (P < 0.001). About 418 (69.7%) patients attained normal levels after 6 months.

Vitamin-D supplementation in chronic lower back pain patients may lead to improvement in pain intensity and functional ability.

Total knee and hip arthroplasty (TKA and THA) are the most commonly performed surgical procedures, the costs of which constitute a significant healthcare burden. Improving access to care for THA/TKA requires better efficiency. It is hypothesized that this may be possible through a two-stage approach that utilizes prediction of surgical time to enable optimization of operating room (OR) schedules.

Data from 499,432 elective unilateral arthroplasty procedures, including 302,490 TKAs, and 196,942 THAs, performed from 2014-2019 was extracted from the American College of Surgeons (ACS) National Surgical and Quality Improvement (NSQIP) database. A deep multilayer perceptron model was trained to predict duration of surgery (DOS) based on pre-operative clinical and biochemical patient factors. A two-stage approach, utilizing predicted DOS from a held out “test” dataset, was utilized to inform the daily OR schedule. The objective function of the optimization was the total OR utilization, with a penalty for overtime. The scheduling problem and constraints were simulated based on a high-volume elective arthroplasty centre in Canada. This approach was compared to current patient scheduling based on mean procedure DOS. Approaches were compared by performing 1000 simulated OR schedules.

The predict then optimize approach achieved an 18% increase in OR utilization over the mean regressor. The two-stage approach reduced overtime by 25-minutes per OR day, however it created a 7-minute increase in underutilization. Better objective value was seen in 85.1% of the simulations.

With deep learning prediction and mathematical optimization of patient scheduling it is possible to improve overall OR utilization compared to typical scheduling practices. Maximizing utilization of existing healthcare resources can, in limited resource environments, improve patient's access to arthritis care by increasing patient throughput, reducing surgical wait times and in the immediate future, help clear the backlog associated with the COVID-19 pandemic.

Pelvic tilt can vary over time due to aging and the possible appearance of sagittal spine disorders. Cup position in total hip arthroplasty (THA) can be influenced due to these changes. We assessed the evolution of pelvic tilt and cup position after THA and the possible appearance of complications for a minimum follow-up of ten years.

343 patients received a THA between 2006 and 2009. All were diagnosed with primary osteoarthritis and their mean age was 63.3 years (range, 56 to 80). 168 were women and 175 men. 250 had no significant lumbar pathology, 76 had significant lumbar pathology and 16 had lumbar fusion. Radiological analysis included sacro-femoral-pubic (SFP), acetabular abduction (AA) and anteversion cup (AV) angles. Measurements were done pre-operatively and at 6 weeks, and at five and ten years post-operatively. Three measurements were recorded and the mean obtained at all intervals. All radiographs were evaluated by the same author, who was not involved in the surgery.

There were nine dislocations: six were solved with closed reduction, and three required cup revision. All the mean angles changed over time; the SFP angle from 59.2º to 60º (p=0.249), the AA angle from 44.5º to 46.8º (p=0.218), and the AV angle from 14.7º to 16.2º (p=0.002). The SFP angle was lower in older patients at all intervals (p<0.001). The SFP angle changed from 63.8 to 60.4º in women and from 59.4º to 59.3º in men, from 58.6º to 59.6º (p=0.012). The SFP angle changed from 62.7º to 60.9º in patients without lumbar pathology, from 58.6º to 57.4º in patients with lumbar pathology, and from 57.0º to 56.4º in patients with a lumbar fusion (p=0.919). The SFP cup angle was higher in patients without lumbar pathology than in the other groups (p<0.001), however, it changed more than in patients with lumbar pathology or fusion at ten years after THA (p=0.04).

Posterior pelvic tilt changed with aging, influencing the cup position in patients after a THA. Changes due to lumbar pathology could influence the appearance of complications long-term.

Lumbar diseases have become a major problem affecting human health worldwide. Conservative treatment of lumbar diseases is difficult to achieve ideal results, and surgical treatment of trauma, complications, it is imperative to develop a new treatment method. This study aims to explore the regulatory mechanism of cartilage endplate ossification caused by abnormal stress, and design intervention targets for this mechanism, so as to provide theoretical reference for the prevention and treatment of lumbar degeneration.

In vivo, we constructed spinal instability model in mice. In vitro, we used a mechanical tensile machine to simulate the abnormal stress conditions of the endplate cartilage cells. Through the high-throughput sequencing, we found the enrichment of Hippo signaling pathway. As YAP is a key protein in the Hippo signaling pathway, we then created cartilaginous YAP elimination mice (Col2::YAPfl/fl). The lumbar spine model was constructed again in these mice for H&E, SOFG and immunofluorescence staining. In vitro lentivirus was used to knock out YAP, immunofluorescence staining, WB and qPCR were performed. Finally, we conducted therapeutic experiments by using YAP agonist and AAV5 carrying YAP plasmids.

We collected 8w samples from C57/BL6 mice after modeling. We found ossification of the endplate in mice similar to human disc degeneration. High-throughput sequencing of stretched cells demonstrated high enrichment of the Hippo signaling pathway. By immunofluorescence staining, it was confirmed that Col-II decreased and Col-X gradually increased in the endplate cartilage of mice. This was also confirmed at 7 days after an in vitro stretch of 5% and 12%. Meanwhile, we found that cartilaginous YAP elimination mice developed very severe endplate degeneration. However, the endplate was well protected by intraperitoneal injection of YAP agonist or AAV5-YAP endplate injection, and the results in vitro were consistent with that.

In the process of cartilaginous ossification, abnormal stress regulates Col10a1 to promote cartilage endplate ossification through Hippo signaling pathway mediated YAP, and we expect to find potential drug targets for treatment through this mechanism.

Although bone morphogenetic protein 2 (BMP-2) has been FDA-approved for spinal fusion for decades, its disadvantages of promoting osteoclast-based bone resorption and suboptimal carrier (absorbable collagen sponge) leading to premature release of the protein limit its clinical applications. Our recent study showed an excellent effect on bone regeneration when BMP-2 and zoledronic acid (ZA) were co-delivered based on a calcium sulphate/hydroxyapatite (CaS/HA) scaffold in a rat critical-size femoral defect model. Therefore, the aim of this study was to evaluate whether local application of BMP-2 and ZA released from a CaS/HA scaffold is favorable for spinal fusion. We hypothesized that CaS/HA mediated controlled co-delivery of rhBMP-2 and ZA could show an improved effect in spinal fusion over BMP-2 alone. 120, 8-week-old male Wistar rats (protocol no. 25-5131/474/38) were randomly divided into six groups in this study (CaS/HA, CaS/HA + BMP-2, CaS/HA + systemic ZA, CaS/HA + local ZA, CaS/HA + BMP-2 + systemic ZA, CaS/HA + BMP-2 + local ZA). A posterolateral spinal fusion at L4 to L5 was performed bilaterally by implanting group-dependent scaffolds. At 3 weeks and 6 weeks, 10 animals per group were euthanized for µCT, histological staining, or mechanical testing. µCT and histological results showed that the CaS/HA + BMP-2 + local ZA group significantly promoted bone regeneration than other treated groups. Biomechanical testing showed breaking force in CaS/HA + BMP + local ZA group was significantly higher than other groups at 6 weeks. In conclusion, the CaS/HA-based biomaterial functionalized with bioactive molecules rhBMP-2 and ZA enhanced bone formation and concomitant spinal fusion outcome

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

We have developed a novel technique to analyse bone, using imaging mass cytometry (IMC) without the constraints of using immunofluorescent histochemistry. IMC can measure the expression of over 40 proteins simultaneously, without autofluorescence. We analysed mitochondrial respiratory chain (RC) protein deficiencies in human bone which are thought to contribute to osteoporosis with increasing age.

Osteoporosis is characterised by reduced bone mineral density (BMD) and fragility fractures. Humans accumulate mitochondrial mutations and RC deficiency with age and this has been linked to the changing phenotype in advancing age and age-related disease. Mitochondrial mutations are detectable from the age of 30 onwards, coincidently the age BMD begins to decline. Mitochondria contain their own genome which accumulates somatic variants at around 10 times the rate of nuclear DNA. Once these mutations exceed a threshold, RC deficiency and cellular dysfunction occur. The PolgD257A/D257A mouse model expresses a proof-reading deficient version of PolgA, a mtDNA polymerase. These mice accumulate mutations 3-5 times higher than wild-type mice showing enhanced levels of age-related osteoporosis and RC deficiency in osteoblasts.

Bone samples were analysed from young and old patients, developing a protocol and analysis framework for IMC in bone tissue sections to analyse osteoblasts in-situ for RC deficiency.

Samples from the femoral neck of 10 older healthy volunteers aged 40 – 85 were compared with samples from young patients aged 1-19. We have identified RC complex I defect in osteoblasts from 6 of the older volunteers, complex II defects in 2 of the older volunteers, complex IV defect in just 1 older volunteer, and complex V defect in 4 of the older volunteers.

These observations are consistent with the PolgD257A/D257A mouse-model and suggest that RC deficiency, due to age-related pathogenic mitochondrial DNA mutations, may play a significant role in the pathogenesis of human age-related osteoporosis.

Nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is key in maintaining redox homeostasis and the pathogenesis of osteoarthritis (OA) involves oxidative distress. We thus investigated whether Nrf2/ARE signaling may control expression of key chondrogenic differentiation and hyaline cartilage maintenance factor SOX9.

In human C-28/I2 chondrocytes SOX9 expression was measured by RT–qPCR after shRNA-mediated knockdown of Nrf2 or its antagonist the Kelch-like erythroid cell-derived protein with cap “n” collar homology-associated protein 1 (Keap1). Putative ARE-binding sites in the proximal SOX9 promoter region were inactivated, cloned into pGL3, and co-transfected with phRL–TK for dual-luciferase assays to verify whether Nrf2 transcriptionally regulates SOX9. SOX9 promoter activity without and with Nrf2-inducer methysticin were analyzed. Sox9 expression in articular chondrocytes was correlated to cartilage thickness and degeneration in wild-type (WT) and Nrf2-knockout mice. Data were analyzed by one-way ANOVA, a Student's t-test, or Wilcoxon rank-sum test, according to the normal distribution. Statistical significance was set to p < 0.05.

While Keap1-specific RNAi increased SOX9 expression, Nrf2-specific RNAi significantly decreased it. Putative ARE sites (ARE₁, ARE₂) were identified in the SOX9 promoter region. ARE₂ mutagenesis significantly reduced SOX9 promoter activity, while truncation of ARE₁ did not. A functional ARE₂ site was thus essential for methysticin-mediated induction of SOX9 promoter activity. Knee cartilage of young Nrf2-knockout mice further revealed significantly fewer Sox9-positive chondrocytes as compared to old Nrf2-knockout animals, which further showed thinner cartilage and more severe cartilage erosion.

Our data suggest that SOX9 expression in articular cartilage is directly Nrf2-dependent and that pharmacological Nrf2 activation may hold potential to diminish age-dependent osteoarthritic changes in knee cartilage through improving protective SOX9 expression.

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).

Conventional proximal tibial osteotomy is a widely successful joint-preserving treatment for osteoarthritis; however, conventional procedures do not adequately control the posterior tibial slope (PTS). Alterations to PTS can affect knee instability, ligament tensioning, knee kinematics, muscle and joint contact forces as well as range of motion.

This study primarily aimed to provide a comprehensive investigation of the variables influencing PTS during high tibial osteotomy using a 3D surgical simulation approach. Secondly, it aimed to provide a simple means of implementing the findings in future 3D pre-operative planning and /or clinically.

The influence of two key variables: the gap opening angle and the hinge axis orientation on PTS was investigated using three independent approaches: (1) 3D computational simulation using CAD software to perform virtual osteotomy surgery and simulate the post-operative outcome. (2) Derivation of a closed-form mathematical solution using a generalised vector rotation approach (3) Clinical assessment of synthetically generated x-rays of osteoarthritis patients (n=28; REC reference: 17/HRA/0033, RD&E NHS, UK) for comparison against the theoretical/computational approaches.

The results from the computational and analytical assessments agreed precisely. For three different opening angles (6°, 9° and 12°) and 7 different hinge axis orientations (from −30° to 30°), the results obtained were identical. A simple analytical solution for the change in PTS, ΔP_s, based on the hinge axis angle, α, and the osteotomy opening angle, θ, was derived:

ΔP_s=sin^-1(sin α sin θ)

The clinical assessment demonstrated that the absolute values of PTS, and changes resulting from various osteotomies, matched the results from the two relative prediction methods.

This study has demonstrated that PTS is impacted by the hinge axis angle and the extent of the osteotomy opening angle and provided computational evidence and analytical formula for general use.

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).

Integrin α2β1 is one of the major transmembrane receptors for fibrillary collagen. In native bone we could show that the absence of this protein led to a protective effect against age-related osteoporosis. The objective of this study was to elucidate the effects of integrin α2β1 deficiency on fracture repair and its underlying mechanisms.

Standardised femoral fractures were stabilised by an intramedullary nail in 12 week old female C57Bl/6J mice (wild type and integrin α2^-/-). After 7, 14 and 28 days mice were sacrificed. Dissected femura were subjected to µCT and histological analyses. To evaluate the biomechanical properties, 28-day-healed femura were tested in a torsional testing device. Masson goldner staining, Alizarin blue, IHC and IF staining were performed on paraffin slices. Blood serum of the animals were measured by ELISA for BMP-2. Primary osteoblasts were analysed by in/on-cell western technology and qRT-PCR.

Integrin α2β1 deficient animals showed earlier transition from cartilaginous callus to mineralized callus during fracture repair. The shift from chondrocytes over hypertrophic chondrocytes to bone-forming osteoblasts was accelerated. Collagen production was increased in mutant fracture callus. Serum levels of BMP-2 were increased in healing KO mice. Isolated integrin deficient osteoblast presented an earlier expression and production of active BMP-2 during the differentiation, which led to earlier mineralisation. Biomechanical testing showed no differences between wild-type and mutant bones.

Knockout of integrin α2β1 leads to a beneficial outcome for fracture repair. Callus maturation is accelerated, leading to faster recovery, accompanied by an increased generation of extra-cellular matrix material. Biomechanical properties are not diminished by this accelerated healing. The underlying mechanism is driven by an earlier availability of BMP-2, one main effectors for bone development. Local inhibition of integrin α2β1 is therefore a promising target to accelerate fracture repair, especially in patients with retarded healing.

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).

Orthopedic Device-Related Infections (ODRIs) are a major medical challenge, particularly due to the involvement of biofilm-encased and multidrug-resistant bacteria. Current treatments, based on antibiotic administration, have proven to be ineffective. Consequently, there is a need for antibiotic-free alternatives. Antimicrobial peptides (AMPs) are a promising solution due to their broad-spectrum of activity, high efficacy at very low concentrations, and low propensity to induce resistance. We aim to develop a new AMP-based chitosan nanogel to be injected during orthopedic device implantation to prevent ODRIs. Chitosan was functionalized with norbornenes (NorChit) through the reaction with carbic anhydride and then, a cysteine-modified AMP, Dhvar5, a peptide with potent antibacterial activity, even against methicillin-resistant Staphylococcus aureus (MRSA), was covalently conjugated to NorChit (NorChit- Dhvar5), through a thiol-norbornene photoclick chemistry (UV= 365 nm). For NorChit-Dhvar5 nanogels production, the NorChit-Dhvar5 solution (0.15% w/v) and Milli-Q water were injected separately into microfluidic system. The nanogels were characterized regarding size, concentration, and shape, using Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA) and Dynamic light scattering (DLS). The nanogels antibacterial properties were assessed in Phosphate Buffer (PBS) for 6 h, against four relevant microorganisms (Pseudomonas aeruginosa, S. aureus and MRSA, and in Muller- Hinton Broth (MHB), 50% (v/v) in PBS, supplemented with human plasma (1% (v/v)), for 6 and 24 h against MRSA. The obtained NorChit-Dhvar5 nanogels, presented a round-shaped and ∼100 nm. NorChit- Dhvar5 nanogels in a concentration of 10¹⁰ nanogels/mL in PBS were capable of reducing the initial inoculum of P. aeruginosa by 99%, S. aureus by 99%, and MRSA by 90%. These results were corroborated by a 99% MRSA reduction, after 24 h in medium. Furthermore, NorChit-Dhvar5 nanogels do not demonstrate signs of cytotoxicity against MC3T3-E1 cells (a pre-osteoblast cell line) after 14 days, having high potential to prevent antibiotic-resistant infection in the context of ODRIs.

Virtual mechanical testing is a method for measuring bone healing using finite element models built from computed tomography (CT) scans. Previously, we validated a dual-zone material model for ovine fracture callus that differentiates between mineralized woven bone and soft tissue based on radiodensity.¹ The objective of this study was to translate the dual-zone material model from sheep to two important clinical scenarios: human tibial fractures in early-stage healing and late-stage nonunions.

CT scans for N = 19 tibial shaft fractures were obtained prospectively at 12 weeks post-op. A second group of N = 33 tibial nonunions with CT scans were retrospectively identified. The modeling techniques were based on our published method.² The dual-zone material model was implemented for humans by performing a cutoff sweep for both the 12-week and nonunion groups. Virtual torsional rigidity (VTR) was calculated as VTR = ML/φ [N-m²/°], where M is the moment reaction, L is the diaphyseal segment length, and φ is the angle of twist.

As the soft tissue cutoff was increased, the rigidity of the clinical fractures decreased and soft tissue located within the fracture gaps produced higher strains that are not predicted without the dual zone approach. The structural integrity of the nonunions varied, ranging from very low rigidities in atrophic cases to very high rigidities in highly calcified hypertrophic cases, even with dual-zone material modeling.

Human fracture calluses are heterogeneous, comprising of woven bone and interstitial soft tissue. Use of a dual-zone callus material model may be instrumental in identifying delayed unions during early healing when callus formation is minimal and/or predominantly fibrous with little mineralization.

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.

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.

Chronic inflammatory events have been associated to almost every chronic disease, including cardiovascular-, neurodegenerative- and autoimmune- diseases, cancer, and host-implant rejection. Given the toll of chronic inflammation in healthcare and socioeconomical costs developing strategies to resolve and control chronic states of inflammation remain a priority for the significant benefit of patients.

Macrophages (Mφ) hold a central role both in the initiation and resolution of inflammatory events, assuming different functional profiles. The outstanding features of Mφ counting with the easy access to tissues, and the extended networking make Mφ excellent candidates for precision therapy. Moreover, sophisticated macrophage-oriented systems could offer innovative immune-regulatory alternatives to effectively regulate chronic environments that traditional pharmacological agents cannot provide.

We propose magnetically assisted systems for balancing Mφ functions at the injury site. This platform combines polymers, inflammatory miRNA antagonists and magnetically responsive nanoparticles to stimulate Mφ functions towards pro-regenerative phenotypes. Strategies with magnetically assisted systems include contactless presentation of immune-modulatory molecules, cell internalization of regulatory agents for functional programming via magnetofection, and multiple payload delivery and release.

Overall, Mφ-oriented systems stimulated pro-regenerative functions of Mφ supporting magnetically assisted theranostic nanoplatforms for precision therapies, envisioning safer and more effective control over the distribution of sensitive nanotherapeutics for the treatments of chronical inflammatory conditions.

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

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

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.

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.

Achilles tendon mechanical properties depend on a complex hierarchical design, with collagen being the smallest load-bearing unit. At the nanoscale, collagen molecules are organized into fibrils, which at the microscale are assembled into fibers, followed by larger structures such as sub-tendons or fascicles. Degree of in vivo loading affects the collagen content, and organization and consequently the tissue's mechanical response. We aim to unravel how composition, structural organization, and mechanical response are affected by degree of in vivo loading at each length scale. The presentation will outline the results to date about to the use of high-resolution synchrotron-based tissue characterisation methods on several length scales in combination with in situ mechanical tests. We use a rat model, where the tendons are subjected to varying loading in vivo. To characterize the tissue microstructure, phase-contrast enhanced synchrotron micro-tomography is performed. The 3D fiber organization in fully loaded tendons is highly aligned, whereas the fibers in unloaded tendons are significantly more heterogeneously arranged and crimped. To characterize the collagen fibril response, Small Angle X-ray Scattering is performed. Two types of fibril organizations are found; a single population oriented towards the main load direction and two fibril subpopulations with clearly distinct orientations. Scattering during loading showed that the fibrils in unloaded tendons did not strain as much in fully loaded. In situ loading concurrently with high resolution synchrotron experiments show the complex tendon response to in situ load and its relation to in vivo loading and tendon hierarchical structure. Unloading seems to alter the organization of the fibrils and fibers, e.g. increased crimping and more pronounced sub-tendon twists.

Acknowledgements: Funding from Knut and Alice Wallenberg Foundation and European Research Council (101002516). Paul Scherrer Institut, Switzerland for beamtime at cSAXS and TOMCAT.

Despite promising results in attempting intervertebral disc regeneration, intradiscal cell transplantation is affected by several drawbacks, including poor viability in the harsh disc environment, low cost-effectiveness, and immunogenic/tumorigenic concerns. Recently, the development of cell-free approaches is gaining increasing interest in the field, with a particular regard towards extracellular vesicles (EVs). Nucleus pulposus cell (NPC) progenitors characterized by Tie2 expression have shown a higher chondrogenic differentiation potential compared to MSCs. The aim of this study was to investigate the putative regenerative effects of EVs isolated from Tie2-overexpressing NPC progenitors on degenerative NPCs.

NPCs were isolated from young donors and underwent an optimized culture protocol to maximize Tie2 expression (NPCs^Tie2+) or a standard protocol (NPCs^STD). Following EV characterization, NPC isolated from patients affected by intervertebral disc degeneration (IDD) were treated with either NPCs^Tie2+-EVs or NPCs^STD-EVs. Cell proliferation and viability were assessed with the CCK-8 assay. Cell apoptosis and necrosis were evaluated with the Annexin V/PI assay. Cell senescence was investigated with b-galactosidase staining. EV uptake was assessed with PKH26 staining of EVs under confocal microscopy.

Treatment with EVs isolated from young NPC donors significantly increased degenerative NPC viability, especially in samples treated with NPCs^Tie2+-EVs. Likewise, NPCs^Tie2+-EVs significantly reduced cell senescence and did not show to exert necrotic nor apoptotic effects on recipient cells. Furthermore, EV uptake was successfully observed in all treated cells.

NPCs^Tie2+-EVs demonstrated to significantly enhance degenerative NPC viability, senescence and apoptosis. The use of committed progenitors naturally residing the in the nucleus pulposus may optimize EV regenerative properties and constitute the basis for a new therapy for IDD.

Osteoarthritis (OA) affects the whole joint and leads to chronic pain. The sympathetic nervous system (SNS) seems to be involved in OA pathogenesis, as indicated by in vitro studies as well as by our latest work demonstrating that sympathectomy in mice results in increased subchondral bone volume in the OA knee joint. We assume that chronic stress may lead to opposite effects, such as an increased bone loss in OA due to an elevated sympathetic tone. Therefore, we analyzed experimental OA progression in mice exposed to chronic stress. OA was induced in male C57BL/6J mice by surgical destabilization of the medial meniscus (DMM) and Sham as well as non-operated mice served as controls. Half of these groups were exposed to chronic unpredictable mild stress (CUMS). After 12 weeks, chronic stress efficiency was assessed using behavioral tests. In addition to measuring body weight and length, changes in subchondral bone were analyzed by μCT. Dynamic Weight Bearing system was used to monitor OA-related pain. Histological scoring will be conducted to investigate the severity cartilage degeneration and synovial inflammation. CUMS resulted in increased anxiety and significant decrease in body weight gain in all CUMS groups compared to non-CUMS groups. CUMS also increased serum corticosterone in healthy mice, with even higher levels in CUMS mice after DMM surgery. CUMS had no significant effect on subchondral bone, but subarticular bone mineral density and trabecular thickness were increased. Moreover, CUMS resulted in significant potentiation of DMM-associated pain. Our results suggest that the autonomic imbalance with increased sympathetic nervous activity induced by chronic stress exacerbates the severity of OA pain perception. We expect significantly increased cartilage degeneration as well as more severe synovial inflammation in CUMS DMM mice compared to DMM mice.

Mesenchymal stem cells-derived extracellular vesicles (MSC-EVs) have great promise in the field of orthopaedic nanomedicine due to their regenerative, as well as immunomodulatory and anti-inflammatory properties. Researchers are interested in harnessing these biologically sourced nanovesicles as powerful therapeutic tools with intrinsic bioactivity to help treat various orthopaedic diseases and defects. Recently, a new class of EV mimetics has emerged known as nanoghosts (NGs). These vesicles are derived from the plasma membrane of ghost cells, thus inheriting the surface functionalities and characteristics of the parent cell while at the same time allowing for a more standardized and reproducible production and significantly greater yield when compared to EVs. This study aims to investigate and compare the osteoinductive potential of MSC-EVs and MSC-NGs in vitro as novel tools in the field of bone tissue engineering and nanomedicine. To carry out this investigation, MSC-EVs were isolated from serum-free MSC conditioned media through differential ultracentrifugation. The remaining cells were treated with hypotonic buffer to produce MSC-ghosts that were then homogenized and serially extruded through 400 and 200 nm polycarbonate membranes to form the MSC-NGs. The concentration, size distribution, zeta potential, and protein content of the isolated nanoparticles were assessed. Afterwards, MSCs were treated with either MSC-EVs or MSC-NGs under osteogenic conditions, and their differentiation was assessed through secreted ALP assay, qPCR, and Alizarin Red mineralization staining. Isolation of MSC-EVs and MSC-NGs was successful, with relatively similar mean diameter size and colloidal stability. No effect on MSC viability and metabolic activity was observed with either treatment. Both MSC-EV and MSC-NG groups had enhanced osteogenic outcomes compared to the control; however, a trend was observed that suggests MSC-NGs as better osteoinductive mediators compared to MSC-EVs.

Acknowledgements: The authors would like to acknowledge Canada Research Chair – Tier 1 in Regenerative Medicine and Nanomedicine, CHRP, and McGill's Faculty of Dental Medicine and Oral Health Sciences for their financial support.

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Based on Ilizarov's law of tension-stress principle, distraction histogenesis technique has been widely applied in orthopaedic surgery for decades. Derived from this technique, cranial bone transport technique was mainly used for treating cranial deformities and calvarial defects. Recent studies reported that there are dense short vascular connections between skull marrow and meninges for immune cells trafficking, highlighting complex and tight association between skull and brain. Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia without effective therapy. Meningeal lymphatics have been recognized as an important mediator in neurological diseases. The augmentation of meningeal lymphatic drainage might be a promising therapeutic target for AD. Our proof-of-concept study has indicated that cranial bone transport can promote ischemic stroke recovery via modulating meningeal lymphatic drainage function, providing a rationale for treating AD using cranial bone maneuver (CBM). This study aims to investigate the effects of CBM on AD and to further explore the potential mechanisms.

Transgenic 5xFAD mice model was used in this study. After osteotomy, a bone flap was used to perform CBM without damaging the dura. Open filed test, novel object recognition test and Barn's maze test were used to evaluate neurological functions of 5xFAD mice after CBM treatment. Congo red and immunofluorescence staining were used to evaluate amyloid depositions and Aβ plaques in different brain regions. Lymphangiogenesis and the level of VEGF-C were examined after CBM treatment. OVA-A647 was intra-cisterna-magna injected to evaluate meningeal lymphatic drainage function after CBM treatment.

CBM significantly improved memory functions and reduced amyloid depositions and Aβ plaques in the hippocampus of 5xFAD mice. A significant increase of meningeal lymphatic vessels in superior sagittal sinus and transverse sinus, and the upregulation of VEGF-C in meninges were observed in 5xFAD mice treated with CBM. Moreover, CBM remarkably enhanced meningeal lymphatic drainage function in 5xFAD mice (n=5-16 mice/group for all studies).

CBM may promote meningeal lymphangiogenesis and lymphatic drainage function through VEGF-C-VEGFR3 pathway, and further reduce amyloid depositions and Aβ plaques and alleviate memory deficits in AD.

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In a clinical setting, there is a need for simple gait kinematic measurements to facilitate objective unobtrusive patient monitoring. The objective of this study is to determine if a learned classification model's output can be used to monitor a person's recovery status post-TKA.

The gait kinematics of 20 asymptomatic and 17 people with TKA were measured using a full-body Xsens model¹. The experimental group was measured at 6 weeks, 3, 6, and 12 months post-surgery. Joint angles of the ankle, knee, hip, and spine per stride (10 strides) were extracted from the Xsens software (MVN Awinda studio 4.4)¹.

Statistical features for each subject at each evaluation moment were derived from the kinematic time-series data. We normalised the features using standard scaling². We trained a logistic regression (LR) model using L1-regularisation on the 6 weeks post-surgery data2–4.

After training, we applied the trained LR- model to the normalised features computed for the subsequent timepoints. The model returns a score between 0 (100% confident the person is an asymptomatic control) and 1 (100% confident this person is a patient). The decision boundary is set at 0.5.

The classification accuracy of our LR-model was 94.58%. Our population's probability of belonging to the patient class decreases over time. At 12 months post-TKA, 38% of our patients were classified as asymptomatic.

Imaging can provide valuable information about the function of tissues and organs. The capacity for detecting and measuring imaging biomarkers of biological activities, allows for a better understanding of the pathophysiology of any process in the human body, including the musculoskeletal system. This is of particular importance in oncologic, metabolic and rheumatologic diseases, but not limited to these.

In the domain of the musculoskeletal system, functional imaging also means to be able to address biomechanical evaluations.

Weight-bearing imaging and dynamic studies have a prominent role. All imaging techniques (X-rays, CT, MR, ultrasound) are in demand, and offer different applications, specific equipment and novel methods for addressing this.

Functional imaging is also essential to drive minimally invasive treatments – i.e. interventional radiology, and new treatment approaches move together with the advances on imaging guidance methods.

On both the diagnostic and the interventional side, the increasing availability of dedicated equipment and the development of specific imaging methods and protocols greatly helps the transition from research to clinical practice.

Menisci are crucial structures for knee homeostasis: they provide increase of congruence between the articular surfaces of the distal femur and tibial plateau, bear loading, shock absorption, lubrication, and proprioception. After a meniscal lesion, the golden rule, now, is to save as much meniscus as possible: only the meniscus tissue which is identified as unrepairable should be excised and meniscal sutures find more and more indications. Several different methods have been proposed to improve meniscal healing. They include very basic techniques, such as needling, abrasion, trephination and gluing, or more complex methods, such as synovial flaps, meniscal wrapping, or the application of fibrin clots. Basic research of meniscal substitutes has also become very active in the last decades. The features needed for a meniscal scaffold are: promotion of cell migration, it should be biomimetic and biocompatible, it should resist forces applied and transmitted by the knee, it should slowly biodegrade and should be easy to handle and implant. Several materials have been tested, that can be divided into synthetic and biological. The first have the advantage to be manufactured with the desired shapes and sizes and with precise porosity dimension and biomechanical characteristics. To date, the most common polymers are polylactic acid (PGA); poly-(L)-lactic acid (PLLA); poly- (lactic-co-glycolic acid) (PLGA); polyurethane (PU); polyester carbon and polycaprolactone (PCL). The possible complications, more common in synthetic than natural polymers are poor cell adhesion and the possibility of developing a foreign body reaction or aseptic inflammation, leading to alter the joint architecture and consequently to worsen the functional outcomes. The biological materials that have been used over time are the periosteal tissue, the perichondrium, the small intestine submucosa (SIS), acellular porcine meniscal tissue, bacterial cellulose. Although these have a very high biocompatibility, some components are not suitable for tissue engineering as their conformation and mechanical properties cannot be modified. Collagen or proteoglycans are excellent candidates for meniscal engineering, as they maintain a high biocompatibility, they allow for the modification of the porosity texture and size and the adaptation to the patient meniscus shape. On the other hand, they have poor biomechanical characteristics and a more rapid degradation rate, compared to others, which could interfere with the complete replacement by the host tissue. An interesting alternative is represented by hydrogel scaffolds. Their semi-liquid nature allows for the generation of scaffolds with very precise geometries obtained from diagnostic images (i.e. MRI).

Promising results have been reported with alginate and polyvinyl alcohol (PVA). Furthermore, hydrogel scaffolds can be enriched with growth factors, platelet-rich plasma (PRP) and Bone Marrow Aspirate Concentrate (BMAC). In recent years, several researchers have developed meniscal scaffolds combining different biomaterials, to optimize the mechanical and biological characteristics of each polymer. For example, biological polymers such as chitosan, collagen and gelatin allow for excellent cellular interactions, on the contrary synthetic polymers guarantee better biomechanical properties and greater reliability in the degradation time. Three-dimensional (3D) printing is a very interesting method for meniscus repair because it allows for a patient-specific customization of the scaffolds. The optimal scaffold should be characterized by many biophysical and biochemical properties as well as bioactivity to ensure an ECM-like microenvironment for cell survival and differentiation and restoration of the anatomical and mechanical properties of the native meniscus. The new technological advances in recent years, such as 3D bioprinting and mesenchymal stem cells management will probably lead to an acceleration in the design, development, and validation of new and effective meniscal substitutes.

Mechanical failure of spine posterior fixation in the lumbar region Is suspected to occur more frequently when the sagittal balance is not properly restored. While failures at the proximal extremity have been studied in the literature, the lumbar distal junctional pathology has received less attention. The aim of this work was to investigate if the spinopelvic parameters, which characterize the sagittal balance, could predict the mechanical failure of the posterior fixation in the distal lumbar region.

All the spine surgeries performed in 2017-2019 at Rizzoli Institute were retrospectively analysed to extract all cases of lumbar distal junctional pathology. All the revision surgeries performed due to the pedicle screws pull-out, or the breakage of rods or screws, or the vertebral fracture, or the degenerative disc disease, in the distal extremity, were included in the junctional (JUNCT) group. A total of 83 cases were identified as JUNCT group. All the 241 fixation surgeries which to date have not failed were included in the control (CONTROL) group. Clinical data were extracted from both groups, and the main spinopelvic parameters were assessed from sagittal standing preoperative (pre-op) and postoperative (post-op) radiographs with the software Surgimap (Nemaris). In particular, pelvic incidence (PI), sagittal vertical axis (SVA), pelvic tilt (PT), T1 pelvic angle (TPA), sacral slope (SS) and lumbar lordosis (LL) have been measured.

In JUNCT, the main failure cause was the screws pull-out (45%). Spine fixation with 7 or more levels were the most common in JUNCT (52%) in contrast to CONTROL (14%). In CONTROL, PT, TPA, SS and PI-LL were inside the recommended ranges of good sagittal balance. For these parameters, statistically significant differences were observed between pre-op and post-op (p<0.0001, p=0.01, p<0.0001, p=0.004, respectively, Wilcoxon test). In JUNCT, the spinopelvic parameters were out of the ranges of the good sagittal balance and the worsening of the balance was confirmed by the increase in PT, TPA, SVA, PI-LL and by the decrease of LL (p=0.002, p=0.003, p<0.0001, p=0.001, p=0.001, respectively, paired t-test) before the revision surgery. TPA (p=0.003, Kolmogorov-Smirnov test) and SS (p=0.03, unpaired t-test) differed significantly in pre-op between JUNCT and CONTROL. In post-op, PI-LL was significantly different between JUNCT and CONTROL (p=0.04, unpaired t-test). The regression model of PT vs PI was significantly different between JUNCT and CONTROL in pre-op (p=0.01, Z-test).

These results showed that failure is most common in long fused segments, likely due to long lever arms leading to implant failure. If the sagittal balance is not properly restored, after the surgery the balance is expected to worsen, eventually leading to failure: this effect was confirmed by the worsening of all the spinopelvic parameters before the revision surgery in JUNCT. Conversely, a good sagittal balance seems to avoid a revision surgery, as it is visible is CONTROL. The mismatch PI-LL after the fixation seems to confirm a good sagittal balance and predict a good correction. The linear regression of PT vs PI suggests that the spine deformity and pelvic conformation could be a predictor for the failure after a fixation.

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Hamstring tendons (HT) represent a widely used autograft for ACL reconstruction. Harvesting, processing and pretensioning procedures together with the time out of the joint could theoretically hamper tendon cells (TCs) viability. The authors hypothesize that HT cells are not impaired at the end of the surgical procedures and their tenogenic phenotype may be strongly improved by exposure to PEMF.

Biomedical imaging is essential in the diagnosis of musculoskeletal pathologies and postoperative evaluations. In this context, Cone-Beam technology-based Computed Tomography (CBCT) can make important contributions in orthopaedics. CBCT relies on divergent cone X-rays on the whole field of view and a rotating source-detector element to generate three-dimensional (3D) volumes. For the lower limb, they can allow acquisitions under real loading conditions, taking the name Weight-Bearing CBCT (WB-CBCT). Assessments at the foot, ankle, knee, and at the upper limb, can benefit from it in situations where loading is critical to understanding the interactions between anatomical structures. The present study reports 4 recent applications using WB-CBCT in an orthopaedic centre.

Patient scans by WB-CBCT were collected for examinations of the lower limb in monopodal standing position. An initial volumetric reconstruction is obtained, and the DICOM file is segmented to obtain 3D bone models. A reference frame is then established on each bone model by virtual landmark palpation or principal component analysis. Based on the variance of the model point cloud, this analysis automatically calculates longitudinal, vertical and mid-lateral axes. Using the defined references, absolute or relative orientations of the bones can be calculated in 3D.

In 19 diabetic patients, 3D reconstructed bone models of the foot under load were combined with plantar pressure measurement. Significant correlations were found between bone orientations, heights above the ground, and pressure values, revealing anatomic areas potentially prone to ulceration. In 4 patients enrolled for total ankle arthroplasty, preoperative 3D reconstructions were used for prosthetic design customization, allowing prosthesis-bone mismatch to be minimized. 20 knees with femoral ligament reconstruction were acquired with WB-CBCT and standard CT (in unloading). Bone reconstructions were used to assess congruency angle and patellar tilt and TT-TG. The values obtained show differences between loading and unloading, questioning what has been observed so far. Twenty flat feet were scanned before and after Grice surgery. WB-CBCT allowed characterization of the deformity and bone realignment after surgery, demonstrating the complexity and multi-planarity of the pathology.

These applications show how a more complete and realistic 3D geometric characterization of the of lower limb bones is now possible in loading using WB-CBCT. This allows for more accurate diagnoses, surgical planning, and postoperative evaluations, even by automatisms. Other applications are in progress.

Introduction:

Most of the published papers on AI based diagnosis have focused on the algorithm's diagnostic performance in a ‘binary’ setting (i.e. disease vs no disease). However, no study evaluated the actual value for the clinicians of an AI based approach in diagnostic. Detection of Traumatic thoracolumbar (TL) fractures is challenging on planar radiographs, resulting in significant rates of missed diagnoses (30-60%), thus constituting a field in which a performance improvement is needed. Aim of this study is therefore to evaluate the value provided by AI generated saliency maps (SM), i.e. the maps that highlight the AI identified region of interests.

Scoliosis correction surgery is one of the longest and most complex procedures of all orthopedic surgery. The complication rate is therefore not negligible and is particularly high when the surgery is performed in patients with neuromuscular or connective tissue disease or complex genetic syndromes. In fact, these patients have various comorbidities and organ deficits (respiratory capacity, swallowing / nutrition, heart function, etc.), which can compromise the outcome of the surgery. In these cases, an accurate assessment and preparation for surgery is essential, also making use of external consultants. To make this phase simpler, more effective and homogeneous, a multidisciplinary path of peri-operative optimization is being developed in our Institute, which also includes the possibility of post-operative hospitalization for rehabilitation and recovery. The goal is to improve the basic functional status as much as possible, in order to ensure faster functional recovery and minimize the incidence of peri-operative complications, to be assessed by clinical audit. The path model and the preliminary results on the first patients managed according to the new modality are presented here.

The multidisciplinary path involves the execution of the following assessments / interventions: • Pediatric visit with particular attention to the state of the upper airways and the evaluation of chronic or frequent inflammatory states • Cardiological Consultation with Echocardiogram. • Respiratory Function Tests, Blood Gas Analysis and Pneumological Consultation to evaluate indications for preoperative respiratory physiotherapy cycles, Non-Invasive Ventilation (NIV) cycles, Cough Machine. Possible Polysomnography. • Nutrition consultancy to assess the need for nutritional preparation in order to improve muscle trophism. • Consultation of the speech therapist in cases of dysphagia for liquids and / or solids. • Electroencephalogram and Neurological Consultation in epileptic patients. • Physiological consultation in patients already being treated with a cough machine and / or NIV. • Availability of postoperative hospitalization in the rehabilitation center (with skills in respiratory and neurological rehabilitation) for the most complex cases. When all the appropriate assessments have been completed, the anesthetist in charge at our Institute examines the clinical documentation and establishes whether the path can be considered complete and whether the patient is ready for surgery. At the end of the surgery, the patient is admitted to the Post-operative Intensive Care Unit of the Institute. If necessary, a new program of postoperative rehabilitation (respiratory, neuromotor, etc.) is programmed in a specialist reference center.

To date, two patients have been referred to the preoperative optimization path: one with Ullrich Congenital Muscular Dystrophy, and one with 6q25 Microdeletion Syndrome. In the first case, the surgery was performed successfully, and the patient was discharged at home. In the second case, after completing the optimization process, the surgery was postponed due to the finding of urethral malformation with the impossibility of bladder catheterization, which made it necessary to proceed with urological surgery first.

The preliminary case series presented here is still very limited and does not allow evaluations on the impact of the program on the clinical practice and the complication rate. However, these first experiences made it possible to demonstrate the feasibility of this complex multidisciplinary path in which a network of specialists takes part.

Low back pain (LBP) is the leading cause of disability worldwide. Recently, treatment of the intervertebral disc (IVD) with stem cells has been used for the treatment of degenerate discs (IDD) which cause at least the 40% of LBP cases. Despite pain reduction, follow-up in clinical studies have not shown an improvement in the structural integrity of IVD. A valid alternative could be the use of progenitor disc cells (notocordal cells, NC) or of their precursors. Mesendoderm progenitor cells (MEPC) have the ability to replicate and differentiate toward NC. In this preliminary study we evaluated in a preclinical large animal IDD model the viability and NC differentiation of MEPC derived from induced pluripotent stem cells (iPSC).

MEPC, derived from iPSC and developed during the iPSpine project (# 825925), were thawed and plated on laminin for 24h and labeled with PKH26.

Two adult sheep were subjected to nucleotomy of five lumbar discs for the induction of IDD. After 5 weeks, 3 of the 5 degenerate discs were treated with MEPC at 3 different doses (low, medium and high). One sheep was sacrificed after 7 days and the other after 30 days from the treatment injection procedure. Clinical parameters were collected to evaluate the safety of treatment. Discs were paraffin embedded and analysed using histological techniques. Survival (PKH26), proliferation (PCNA), notocordal cell differentiation (Brachyury, Cytokeratin 8/18/19, Sox9, Foxa2) and endodermal differentiation (Sox17) were evaluated.

After the injection of the cells, both sheep lost about 20% of body weight. The analysis showed that only in discs treated with the highest dose the PKH26 stained cells resulted alive after 30 days from the procedure. These cells turn out to be:

This preliminary study shows that MEPC, derived from iPSC and injected into ovine discs degenerated by nucleotomy, are able to survive 30 days from treatment and differentiate within the disc predominantly towards the notocordal phenotype.

Abstract

Cranial cruciate ligament (CrCL) disease/rupture is a highly prevalent orthopaedic disease in dogs and common cause of pain, lameness, and secondary joint osteoarthritis (OA). Previous experiments investigating the role of glutamate receptors (GluR) in arthritic degeneration and pain revealed that OA biomarkers assessing early bone turnover and inflammation, including osteoprotegerin (OPG) and the receptor activator of nuclear factor kappa-B ligand (RANKL) are more likely to be influenced by glutamate signalling. Moreover, interleukin-6 (IL-6) has a complex and potentially bi directional (beneficial and detrimental) effect, and it is a critical mediator of arthritic pain, OA progression and joint destruction.

Abstract

There is no consensus on how to evaluate and grade pin site infection. A precise, objective and reliable pin site infectious score is warranted. The literature was reviewed for pin site infection classification systems, The Modified Gordon Score (MGS) grade 0-6 was used. The aim was to test the reliability of The Modified Gordon Infection Score. The observed agreement and inter-rater reliability were investigated between nurse and doctors.

MGS was performed in the outpatient clinic at Aalborg University Hospital, Denmark on 1472 pin sites in 119 patients by one nurse and one of three orthopaedic surgeons blinded to each other's judgement. The data was stored in a Red Cap Database for further statistical analysis. The observed agreement between the nurse and the 3 orthopaedic surgeons was evaluated with a one-way random-effect model with interclass correlation with absolute agreement. Furthermore the observed agreement for each of the 3 surgeons with the nurse was calculated.

The distribution of MGS infection grade in the 1472 pin sites was: Grade 0; n=1372, Grade 1; n=32, Grade 2; n=39, Grade 3; n=24, Grade 4; n=5, Grade 5; n=0, Grade 6; n=0.

The observed agreement between the nurse and the surgeons was calculated as 98%. The ICC estimated between nurse and the surgeons was 0,8943 (ICC >0,85 = reliable). The grading was done by three different doctors with an agreement with the nurse as follows. Rater1 (n=416) =99,5 %, Rater2 (n=1440) =97,4%, Rater3 (n=1440) =96,6%.

A limitation to this study is that the dataset represents mostly clean pin sites with MGS 0. Only 100 pin sites had signs of superficial infection MGS 1-4 none above 4. We found that the MGS infection score is highly reliable for low grade infections but we cannot conclude on reliability in severe infections.

To detect early signs of infection infrared thermography has been suggested to provide quantitative information. Our vision is to invent a pin site infection thermographic surveillance tool for patients at home. A preliminary step to this goal is the aim of this study, to automate the process of locating the pin and detecting the pin sites in thermal images efficiently, exactly, and reliably for extracting pin site temperatures.

A total of 1708 pin sites was investigated with Thermography and augmented by 9 different methods in to totally 10.409 images. The dataset was divided into a training set (n=8325), a validation set (n=1040), and a test set (n=1044) of images. The Pin Detection Model (PDM) was developed as follows: A You Only Look Once (YOLOv5) based object detection model with a Complete Detection Intersection over Union (CDIoU), it was pre-trained and finetuned by the through transfer learning. The basic performance of the YOLOv5 with CDIoU model was compared with other conventional models (FCOS and YOLOv4) for deep and transition learning to improve performance and precision. Maximum Temperature Extraction (MTE) Based on Region of Interest (ROI) for all pin sites was generated by the model. Inference of MTE using PDM with infected and un-infected datasets was investigated.

An automatic tool that can identify and annotate pin sites on conventional images using bounding boxes was established. The bounding box was transferred to the infrared image. The PMD algorithm was built on YOLOv5 with CDIoU and has a precision of 0.976. The model offers the pin site detection in 1.8 milliseconds. The thermal data from ROI at the pin site was automatically extracted.

These results enable automatic pin site annotation on thermography. The model tracks the correlation between temperature and infection from the detected pin sites and demonstrates it is a promising tool for automatic pin site detection and maximum temperature extraction for further infection studies. Our work for automatic pin site annotation on thermography paves the way for future research on infection assessment using thermography.

Abstract

Fragility ankles fractures in the geriatric population are challenging to manage, due to fracture instability, soft tissue compromise, patient co-morbidities. Traditional management options include open reduction internal fixation, or conservative treatment, both of which are fraught with high complication rates. We aimed to present functional outcomes of elderly patients with fragility ankle fractures treated with tibiotalocalcaneal nails.

171 patients received a tibiotalocalcaneal nail over a six-year period, but only twenty met the inclusion criteria of being over sixty and having poor bone stock, verified by radiological evidence of osteopenia or history of fragility fractures. Primary outcome was mortality risk from co-morbidities, according to the Charlson co-morbidity index (CCI), and patients’ post-operative mobility status compared to pre-operative mobility. Secondary outcomes include intra-operative and post-operative complications, six-month mortality rate, time to mobilisation and union.

The mean age was 77.82 years old, five of whom are type 2 diabetics. The average CCI was 5.05. Thirteen patients returned to their pre-operative mobility state. Patients with low CCI are more likely to return to pre-operative mobility status (p=0.16; OR=4.00).

Average time to bone union and mobilisation were 92.5 days and 7.63 days, respectively. Mean post-operative AOFAS ankle-hindfoot and Olerud-Molander scores were 53.0 (range 17-88) and 50.9 (range 20-85), respectively. There were four cases of broken distal locking screws, and four cases of superficial infection. Patients with high CCI were more likely to acquire superficial infections (p=0.264, OR=3.857). There were no deep infections, periprosthetic fractures, nail breakages, non-unions.

TTC nailing is an effective treatment methodology for low-demand geriatric patients with fragility ankle fractures. This technique leads to low complication rates and early mobilisation. It is not a life-changing procedure, with many able to return to their pre-operative mobility status, which is important for preventing the loss of socioeconomic independence.

Abstract

Mechanical loading of joints with osteoarthritis (OA) results in pain-related functional impairment, altered joint mechanics and physiological nociceptor interactions leading to an experience of pain. However, the current tools to measure this are largely patient reported subjective impressions of a nociceptive impact. A direct measure of nociception may offer a more objective indicator. Specifically, movement-induced physiological responses to nociception may offer a useful way to monitor knee OA. In this study, we gathered preliminary data on healthy volunteers to analyse whether integrated biomechanical and physiological sensor datasets could display linked and quantifiable information to a nociceptive stimulus.

Following ethical approval, 15 healthy volunteers completed 5 movement and stationary activities in 2 conditions; a control setting and then repeated with an applied quantified thermal pain stimulus to their right knee. An inertial measurement unit (IMU) and an electromyography (EMG) lower body marker set were tested and integrated with ground reaction force (GRF) data collection. Galvanic skin response electrodes for skin temperature and conductivity and photoplethysmography (PPG) sensors were manually timestamped to the integrated system.

Pilot data showed EMG, GRF and IMU fluctuations within 0.5 seconds of each other in response to a thermal trigger. Preliminary analysis on the 15 participants tested has shown skin conductance, PPG, EMG, GRFs, joint angles and kinematics with varying increases and fluctuations during the thermal condition in comparison to the control condition.

Preliminary results suggest physiological and biomechanical data outputs can be linked and identified in response to a defined nociceptive stimulus. Study data is currently founded on healthy volunteers as a proof-of-concept. Further exploratory statistical and sensor readout pattern analysis, alongside early and late-stage OA patient data collection, can provide the information for potential development of wearable nociceptive sensors to measure disease progression and treatment effectiveness.

A major cause of morbidity in lower limb amputees is phantom limb pain (PLP) and residual limb pain (RLP). This study aimed to determine if surgical interposition of nerve endings into adjacent muscle bellies at the time of major lower limb amputation can decrease the incidence and severity of PLP and RLP.

Data was retrospectively collected from January 2015 to January 2021, including eight patients that underwent nerve interposition (NI) and 36 that received standard treatment. Primary outcomes included the 11-point Numerical Rating Scale (NRS) for pain severity, and Patient-Reported Outcomes Measurement Information System (PROMIS) pain intensity, behaviour, and interference. Secondary outcome included Neuro-QoL Lower Extremity Function assessing mobility. Cumulative scores were transformed to standardised t scores.

Across all primary and secondary outcomes, NI patients had lower PLP and RLP. Mean ‘worst pain’ score was 3.5 out of 10 for PLP in the NI cohort, compared to 4.89 in the control cohort (p=0.298), and 2.6 out of 10 for RLP in the NI cohort, compared to 4.44 in the control cohort (p=0.035). Mean ‘best pain’ and ‘current pain’ scores were also superior in the NI cohort for PLP (p=0.003, p=0.022), and RLP (p=0.018, p=0.134).

Mean PROMIS t scores were lower for the NI cohort for RLP (40.1 vs 49.4 for pain intensity; p=0.014, 44.4 vs 48.2 for pain interference; p=0.085, 42.5 vs 49.9 for pain behaviour; p=0.025). Mean PROMIS t scores were also lower for the NI cohort for PLP (42.5 vs 52.7 for pain intensity; p=0.018); 45.0 vs 51.5 for pain interference; p=0.015, 46.3 vs 51.1 for pain behaviour; p=0.569). Mean Neuro-QoL t score was lower in NI cohort (45.4 vs 41.9;p=0.03).

Surgical interposition of nerve endings during lower limb amputation is a simple yet effective way of minimising PLP and RLP, improving patients’ subsequent quality of life. Additional comparisons with targeted muscle reinnervation should be performed to determine the optimal treatment option.

Evidence supporting the use of virtual reality (VR) training in orthopaedic procedures is rapidly growing. However, the impact of the timing of delivery of this training is yet to be tested. We aimed to investigate whether spaced VR training is more effective than massed VR training.

24 medical students with no hip arthroplasty experience were randomised to learning the direct anterior approach total hip arthroplasty using the same VR simulation, training either once-weekly or once-daily for four sessions. Participants underwent a baseline physical world assessment on a saw bone pelvis. The VR program recorded procedural errors, time, assistive prompts required and hand path length across four sessions. The VR and physical world assessments were repeated at one-week, one-month, and 3 months after the last training session.

Baseline characteristics between the groups were comparable (p > 0.05). The daily group demonstrated faster skills acquisition, reducing the median ± IQR number of procedural errors from 68 ± 67.05 (session one) to 7 ± 9.75 (session four), compared to the weekly group's improvement from 63 ± 27 (session one) to 13 ± 15.75 (session four), p < 0.001. The weekly group error count plateaued remaining at 14 ± 6.75 at one-week, 16.50 ± 16.25 at one-month and 26.45 ± 22 at 3-months, p < 0.05. However, the daily group showed poorer retention with error counts rising to 16 ± 12.25 at one-week, 17.50 ± 23 at one-month and 41.45 ± 26 at 3-months, p<0.01. A similar effect was noted for the number of assistive prompts required, procedural time and hand path length. In the real-world assessment, both groups significantly improved their acetabular component positioning accuracy, and these improvements were equally maintained (p<0.01).

Daily VR training facilitates faster skills acquisition; however weekly practice has superior skills retention.

The purpose of the study was to compare the mechanical properties, oxidation and wear resistance of a vitamin E blended and moderately crosslinked polyethylene for total knee arthroplasty (MXE) in comparison with clinically established polyethylene materials.

The following polyethylene materials were tested: CPE (30 kGy e-beam sterilized), XLPE (75 kGy gamma crosslinked @ 100°C), ViXLPE (0.1 % vitamin E blended, 80 kGy e-beam crosslinked @ 100°C), and MXE (0.1 % vitamin E blended polyethylene, 30 kGy gamma sterilized). For the different tests, the polyethylene materials were either unaged or artificially aged for two or six weeks according to ASTM F2003-02.

The oxidation index was measured based on ASTM F2102 at a 1 mm depth. Small punch testing was performed based on ASTM F2977. Mechanical properties were measured on unaged materials according to ASTM D638.

Wear simulation was performed on a load controlled 3 + 1 station knee wear simulator (EndoLab GmbH, Thansau, Germany) capable of reproducing loads and movement of highly demanding activities (HDA) as well as ISO 14243-1 load profiles. The load profiles were applied for 5 million cycles (mc) or delamination of the polyethylene components. Medium size AS e.motion^® PS Pro (Aesculap AG, Tuttlingen, Germany) femoral and tibial components with a ZrN-multilayer surface, as well as Columbus^® CR cobalt-chrome alloy femoral and tibial components were tested. Particle analysis was performed on the serum samples of the ISO 14243-1 wear simulations based on ISO 17853:2011 and ASTM F1877.

The analysis of the mechanical properties show that moderately crosslinked polyethylene (MXE) might be a superior material for total knee arthroplasty applications [Schwiesau et al. 2021]. The addition of vitamin E in a moderately crosslinked polyethylene prevented its oxidation, kept its mechanical characteristics, and maintained a low wear, even under a HDA knee wear simulation.

The objectives of the study were to investigate demographic, injury and surgery/treatment-associated factors that could influence clinical outcome, following Autologous Chondrocyte Implantation (ACI) in a large, “real-world”, 20 year longitudinally collected clinical data set.

Multilevel modelling was conducted using R and 363 ACI procedures were suitable for model inclusion. All longitudinal post-operative Lysholm scores collected after ACI treatment and before a second procedure (such as knee arthroplasty but excluding minor procedures such as arthroscopy) were included. Any patients requiring a bone graft at the time of ACI were excluded. Potential predictors of ACI outcome explored were age at the time of ACI, gender, smoker status, pre-operative Lysholm score, time from surgery, defect location, number of defects, patch type, previous operations, undergoing parallel procedure(s) at the time of ACI, cell count prior to implantation and cell passage number.

The best fit model demonstrated that for every yearly increase in age at the time of surgery, Lysholm scores decreased by 0.2 at 1-year post-surgery. Additionally, for every point increase in pre-operative Lysholm score, post-operative Lysholm score at 1 year increased by 0.5. The number of cells implanted also impacted on Lysholm score at 1-year post-op with every point increase in log cell number resulting in a 5.3 lower score. In addition, those patients with a defect on the lateral femoral condyle (LFC), had on average Lysholm scores that were 6.3 points higher one year after surgery compared to medial femoral condyle (MFC) defects. Defect grade and location was shown to affect long term Lysholm scores, those with grade 3 and patella defects having on average higher scores compared to patients with grade 4 or trochlea defects.

Some of the predictors identified agree with previous reports, particularly that increased age, poorer pre-operative function and worse defect grades predicted poorer outcomes. Other findings were more novel, such as that a lower cell number implanted and that LFC defects were predicted to have higher Lysholm scores at 1 year and that patella lesions are associated with improved long-term outcomes cf. trochlea lesions.

Stem cell therapy is an effective means to address the repair of large segmental bone defects. However, the intense inflammatory response triggered by the implants severely impairs stem cell differentiation and tissue regeneration. High-dose transforming growth factor β1 (TGF-β1), the most locally expressed cytokine in implants, inhibits osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and promotes tissue fibrosis, severely compromising the efficacy of stem cell therapy. Small molecule inhibitors of TGF-β1 can be used to ameliorate the osteogenic disorders caused by high concentrations of TGF-β1, but systemic inhibition of TGF-β1 function will cause strong adverse effects. How to find safe and reliable molecular targets to antagonize TGF-β1 remains to be elucidated. Orphan nuclear receptor Nr4a1, an endogenous inhibitory molecule of TGF-β1, suppresses tissue fibrosis, but its role in BMSC osteogenesis is unclear. We found that TGF-β1 inhibited Nr4a1 expression through HDAC4. Overexpression of Nr4a1 in BMSCs reversed osteogenic differentiation inhibited by high levels of TGF- β1. Mechanistically, RNA sequencing showed that Nr4a1 activated the ECM-receptor interaction and Hippo signaling pathway, which in turn promoted BMSC osteogenesis. In bone defect repair and fracture healing models, transplantation of Nr4a1-overexpressing BMSCs into C57BL/6J mice or treatment with the Nr4a1 agonist Csn-B significantly ameliorated inflammation-induced bone regeneration disorders. In summary, our findings confirm the endogenous inhibitory effect of Nr4a1 on TGF- β1 and uncover the effectiveness of Nr4a1 agonists as a therapeutic tool to improve bone regeneration, which provides a new solution strategy for the treatment of clinical bone defects and inflammatory skeletal diseases.

Osteochondral injuries are a recognised factor in the development of osteoarthritis (OA). Mesenchymal stromal cells (MSCs) represent a promising biological therapeutic option as an OA-modifying treatment, and they also secrete factors that may have an anti-catabolic effect and/or encourage endogenous repair. We aim to study the effects of (i) intra-articular injection of human bone-marrow-derived MSCs and (ii) their secretome on recovery in a murine knee osteochondral injury model.

The MSC secretome was generated by stimulating human bone-marrow-derived MSCs with tumour necrosis factor alpha (TNFα). Mice (n=48) were injected with i) MSC secretome, ii) MSCs or iii) cell culture medium (control). Pain was assessed by activity monitoring, and cartilage repair, subchondral bone volume and synovial inflammation were evaluated using histology and microCT.

Both MSC- and MSC-secretome-injected mice showed significant pain reduction at day 7 when compared to control mice, but only the MSC-injected mice maintained a significant improvement over the controls at day 28. Cartilage repair was significantly improved in MSC-injected mice. No significant effects were observed with regards to synovial inflammation or subchondral bone volume.

The MSC secretome demonstrates regenerative effects but this does not appear to be as sustained as a MSC cell therapy. Further studies are required to investigate if this can be overcome using different dosing regiments for injection of the MSC secretome. As we further understand the regenerative properties of the MSC secretome, we may be able to enhance the clinical translatability of these therapies. Direct intra-articular injection of MSCs for the treatment of OA also appears promising as a potential future strategy for OA management.

Acknowledgements: MS is supported by a grant from the Wellcome Trust (PhD Programme for Clinicians)

Many age-related diseases affect our skeletal system, but bone health-targeting drug development strategies still largely rely on 2D in vitro screenings. We aimed at developing a scaffold-free progenitor cell-based 3D biomineralization model for more physiological high-throughput screenings.

MC3T3-E1 pre-osteoblast spheroids were cultured in V-shaped plates for 28 days in alpha-MEM (10% FCS, 1% L-Gln, 1X NEAA) with 1% pen/strep, changed every two days, and differentiation was induced by 10mM b-glycerophosphate and 50µg/ml ascorbic-acid. Osteogenic cell differentiation was assessed through profiling mRNA expression of selected osteogenic markers by efficiency corrected normalized 2^DDCq RT-qPCR. Biomineralization in spheroids was evaluated by histochemistry (Alizarin Red/von Kossa staining), Alkaline phosphatase (Alp) activity, Fourier transform infrared spectroscopy (FTIR) analyses, micro-CT analyses, and scanning electron microscopy on critical point-dried samples. GraphPad Prism 9 analyses comprised Shapiro-Wilk and Brown-Forsythe tests as well as 2-way ANOVA with Tukey post-hoc and non-parametric Kruskal-Wallis with Dunn post-hoc tests.

During mineralization, as opposed to non-mineralizing conditions, characteristic mRNA expression profiles of selected early and late osteoblast differentiation markers (e.g., RunX, Alp, Col1a1, Bglap) were observed between day 0 and 28 of culture; Alp was strongly upregulated (p<0.001) from day 7 on, followed by its enzymatic activity (p<0.001). Bglap and Col1a1 expression peaked on (p<0.001) and from day 14 on (p<0.05), respectively. IHC revealed osteocalcin staining in the spheroid core regions at day 14, while type I collagen staining of the cores was most prominent from day 21 on. Alizarin Red and Von Kossa confirmed central and radially outwards expanding mineralization patterns between day 14 and day 28, which was accompanied by a steady increase in extracellular calcium deposition over time (p<0.001). Micro-CT analyses allowed quantitative appreciation of the overall increase in mineral density over time (day21, p<0.05; d28, p<0.001), while SEM-EDX and FTIR ultimately confirmed a bone-like hydroxyapatite mineral deposition in 3D.

A novel and thoroughly characterized versatile bone-like 3D biomineralization in vitro model was established, which allows for studying effects of pharmacological interventions on bone mineralization ex vivo under physiomimetic conditions. Ongoing studies currently aim at elucidating in how far it specifically recapitulates intramembranous ossification.

Surgical debridement for medial epicondylitis (ME) is indicated for patients with refractory ME. The clinical efficacy of simple debridement has not been studied sufficiently. Moreover, authors experienced surgical outcome of ME was not as good as lateral epicondylitis. In this regard, authors have combined the atelocollagen injection in the debridement surgery of ME. The purpose of study was to compare clinical outcomes between simple debridement and debridement combined with atelocollagen injection in the ME.

Twenty-five patients with refractory ME and underwent surgical debridement were included in the study. Group A (n=13) was treated with isolated debridement surgery, and group B (n=12) was treated with debridement combined with 1.0 mL of type I atelocollagen. Pain and functional improvements were assessed using visual analogue scale, Mayo Elbow Performance Score (MEPS) and quick Disabilities of the Arm, Shoulder and Hand (DASH) scale respectively before surgery, at 3, 6 months after surgery and at the final follow-up.

Demographic data did not show significant difference between two groups before surgical procedures. Both groups showed improvement in pain and functional score postoperatively. However, at the 3 months after surgery, group B showed significantly better improvement as compared to group A(VAS 3.1 / 2.0, MEPS 71/82 qDASH 29/23). At the 6 months after surgery and final follow-up, both groups did not show any difference.

Surgical debridement combined with atelocollagen is effective treatment option in refractory ME and showed better short-term outcomes compared to isolated surgery.

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.

Miniscrew implants (MSIs) are widely used to provide absolute anchorage for the orthodontic treatment. However, the application of MSIs is limited by the relatively high failure rate (22.86%). In this study, we wished to investigate the effects of amorphous and crystalline biomimetic calcium phosphate coating on the surfaces of MSIs with or without the incorporated BSA for the osteointegration process with an aim to facilitate the early loading of MSIs.

Amorphous and crystalline coatings were prepared on titanium mini-pin implants. Characterizations of coatings were examined by Scanning electron microscopy (SEM), Confocal laser-scanning dual-channel-fluorescence microscopy (CLSM) and Fourier-transform infrared spectroscopy (FTIR). The loading and release kinetics of bovine serum albumin (BSA) were evaluated by Enzyme linked immunosorbent assay (ELISA). Activity of alkaline phosphate (ALP) was measured by using the primary osteoblasts. In vivo, a model of metaphyseal tibial implantation in rats was used (n=6 rats per group). We had 6 different groups: no coating no BSA, no coating but with surface adsorption of BSA and incorporation of BSA in the biomimetic coating in the amorphous and crystalline coatings. Time points were 3 days, 1, 2 and 4 weeks. Histological and histomorphometric analysis were performed and the bone to implant contact (BIC) of each group was compared.

In vitro, the incorporation of BSA changed the crystalline coating from sharp plates into curly plates, and the crystalline coating showed slow-release profile. The incorporation of BSA in crystalline coating significantly decreased the activity of ALP in vitro. In vivo study, the earliest significant increase of BIC appeared in crystalline coating group at one week.

The crystalline coating can serve as a carrier and slow release system for the bioactive agent and accelerate osteoconductivity at early stage in vivo. The presence of BSA is not favorable for the early establishment of osteointegration.

Hip precautions are currently practiced in three-quarters of trauma hospitals in the UK, despite national recommendations from the ‘Blue Book’ not stating it as a requirement. Valuable therapist time is utilised alongside the need for specialised equipment, which can potentially delay discharge whilst it is being arranged. Objective of this study was to explore the current practice of the use of hip precautions on discharge following hemiarthroplasty for hip fractures. To also explore whether they are necessary and to identify areas for improvement to benefit patient care overall.

Online survey distributed to various Trauma and Orthopaedic Departments across the UK. Survey was available over a 4-month period, collecting 55 responses overall.

Majority of responses were from trauma and orthopaedic consultants who were aware of the ‘Blue Book’ recommendations. The majority of trusts who responded did not practice hip precautions and did not feel this increased the risk of dislocations on discharge. Recommendations included integration of hip precautions in the post-op advice in coordination with the physiotherapist and information leaflets on discharge regarding hip precautions. Hip precautions were not commonly practiced, for reasons including patient compliance and the inherently stable procedure of a hemiarthroplasty compared to a THR, reducing the need for hip precautions.

Hip precautions are not widely regarded as a useful practice for post-hip hemiarthroplasty, viewed as utilising more resources and increasing costs and risk due to increased hospital stay. Thus, this potentially delays discharge overall. A consistent approach should be implemented in treating patients post-hip hemiarthroplasty.

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

Prosthetic Joint Infection (PJI) is a devastating complication that can occur after total joint replacement surgery. With increasing antimicrobial resistance, there is a need for non-antibiotic approaches to treat and prevent PJI. Doping calcium phosphates with antimicrobial ions shows promise for these purposes. This systematic review aims to search and summarise the evidence-base for the potential of calcium phosphates doped with different antimicrobial ions.

A systematic review was conducted on PubMed, Embase, Web-Of-Science, Cochrane Library and Emcare of in vitro and animal studies on the antimicrobial activity of (co)substituted calcium phosphates according to PRIMSA guidelines.. The research protocol, listing search terms and in/exclusion criteria, was registered a priori at https://doi.org/10.7910/DVN/HEP18U. Data was extracted regarding ions, micro-organisms and antimicrobial activity.

The search retrieved 1017 hits of which 148 papers were included. The substitution of 33 different ions was reported. Silver (n= 46), zinc (n=39), copper (n=18) and magnesium (n=14) were the most commonly doped ions. 36 different micro-organisms were studied of which E. coli (n=109), S. aureus (n=99), and C. albicans (n=22) were the most common. 6 different outcomes were reported, most commonly the K-ratio (n=53), the log CFU (n=41) and the bacterial inhibition zone (n=39). A validated outcome for the evaluation of biofilm prevention was lacking.

There was considerable heterogeneity in studied ions, micro-organisms and reported outcomes. A lack of clearly defined reporting guidelines in the field of antimicrobial materials has led to the use of clinically irrelevant micro-organisms and a general lack of consistency of the methods used and the reported results. Currently, there is no universally accepted measure for the effectiveness required from biomaterials for treatment and prevention of PJI.

Cartilage degeneration and loss are key events in the initiation and progression of osteoarthritis (OA). Changes to chondrocyte volume and morphology (in the form of cytoplasmic processes) and thus cell phenotype are implicated, as they lead to the production of a mechanically-weakened extracellular matrix. The chondrocyte cytoskeleton is intimately linked to cell volume and morphology and hence we have investigated alterations to levels and distribution of chondrocyte F-actin that occur during early OA.

The femoral heads (FH) from hip joints (N=16) were obtained with ethical permission and patient consent following femoral neck fracture. Cartilage was assessed as grade 0 (non-degenerate) and grade 1 (superficial fibrillation) using OARSI criteria. In situ chondrocyte volume and F-actin distribution were assessed using the fluorescent indicators (5-chloromethyl fluorescein diacetate (CMFDA)) and phalloidin, and imaged and quantified by confocal microscopy, Imaris^TM and ImageJ software.

There were no differences between the volume or total F-actin levels of in situ chondrocytes within the superficial zone of grade 0 (n=164 cells) compared to grade 1 (n=145) cartilage (P>0.05). However, a more detailed analysis of phalloidin labelling was performed, which demonstrated significant increases in both intense punctuate (IP) or intense areas (IA) (P<0.0001; P=0.0175 respectively). A preliminary analysis of IP and IA F-actin labelling suggested that while the former did not appear to be associated with changes to chondrocyte morphology, most of the cytoplasmic processes were associated with the presence of IA at the starting point of the protrusion.

These results demonstrate marked changes to F-actin distribution in chondrocytes in the very early stages of cartilage degeneration as occurs in OA. These subtle changes are probably an early indication of a change to the chondrocyte phenotype and thus worthy of further study as they may lead to deleterious alterations to matrix metabolism and ultimately cartilage weakening.

The Nottingham Hip Fracture Score (NHFS) was developed in 2007 as a predictor of 30-day mortality after hip fracture surgery following a neck of femur fracture. The National Hip Fracture Database is the standard used which calculated their own score using national data.

The NHF score for 30-day mortality was calculated for 50 patients presenting with a fractured neck femur injury between January 2020 to March 2020. A score <5 was classified as low risk and >/=5 as high risk. Aim was to assess the accuracy in calculating the Nottingham Hip Fracture Score against the National Hip Fracture Database. To explore whether it should it be routinely included during initial assessment to aid clinical management?

There was an increase in the number of mortalities observed in patients who belonged to the high-risk group (>=5) compared to the low risk group. COVID-19 positive patients had worse outcomes with average 30-day mortality of 6.78 compared to the average of 6.06. GEH NHF score per month showed significant accuracy against the NHFD scores.

The identification of high-risk groups from their NHF score can allow for targeted optimisations and elucidation of risk factors easily gathered at the point of hospitalisation. The NHFS is a valuable tool and useful predictor to stratify the risk of 30-day mortality and 1-year mortality after hip fracture surgery. Inclusion of the score should be considered as mandatory Trust policy for neck of femur fracture patients to aid clinical management and improve patient safety overall.

Proximal humeral shaft fractures are commonly treated with long straight plates or intramedullary nails. Helical plates might overcome the downsides of these techniques as they are able to avoid the radial nerve distally. The aim of this study was to investigate in an artificial bone model: (1) the biomechanical competence of different plate designs and (2) to compare them against the alternative treatment option of intramedullary nails.

Twenty-four artificial humeri were assigned in 4 groups and instrumented as follows: group1 (straight 10-hole-PHILOS), group2 (MULTILOCK-nail), group3 (45°-helical-PHILOS) and group4 (90°-helical-PHILOS). An unstable proximal humeral shaft fracture was simulated. Specimens were tested under quasi-static loading in axial compression, internal/external rotation and bending in 4 directions monitored by optical motion tracking.

Axial displacement (mm) was significantly lower in group2 (0.1±0.1) compared to all other groups (1: 3.7±0.6; 3: 3.8±0.8; 4: 3.5±0.4), p<0.001. Varus stiffness in group2 (0.8±0.1) was significantly higher compared to groups1+3, p≤0.013 (1: 0.7±0.1; 3: 0.7±0.1; 4: 0.8±0.1). Varus bending (°) was significantly lower in group2 compared to all other groups (p<0.001) and group4 to group1, p=0.022. Flexion stiffness in group1 was significantly higher compared to groups2+4 (p≤0,03) and group4 to group1, p≤0,029 (1: 0.8±0.1; 2: 0.7±0.1; 3: 0.7±0.1; 4: 0.6±0.1). Flexion bending (°) in group4 was higher compared to all other groups (p≤0.024) and lower in group2 compared to groups1+4, p≤0.024. Torsional stiffness remained non significantly different, p≥0.086. Torsional deformation in group2 was significantly higher compared to all other groups, p≤0.017. Shear displacement remained non significantly different, p≥0.112.

From a biomechanical perspective, helical plating with 45° and 90° may be considered as a valid alternative fixation technique to standard straight plating of proximal third humeral fractures. Intramedullary nails demonstrated higher axial and bending stiffness as well as lower fracture gap movements during axial loading compared to all plate designs. However, despite similar torsional stiffness they were associated with higher torsional movements during internal/external rotation as compared to all investigated plate designs.

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.

Solid organ transplant (SOT) recipients present an increased medical risk; however, few studies analyze the outcomes of these patients undergoing hip fracture surgery. This study aimes to determine the incidence of hip fracture in SOT patients and to compare the outcomes of SOT patients with matched non-SOT controls after hip fracture fixation.

A retrospective review identified 20 SOT patients with hip fracture at a single center from 2016 to 2021 and were matched (1:1) with a cohort of 20 patients with hip fracture without SOT. Patient outcomes, mortality/survival and clinical outcomes were compared between two groups.

The incidence of hip fracture in SOT patients was 20/1787, 1.1%. There were significant differences in mortality rate (73.3% SOT group vs. 26.7% non-SOT group; p<0.05). There were no differences in survival time (p=0.746). There were no differences in time to surgery (5.0 days SOT group vs. 3.1 days non-SOT group; p=0.109), however, there were significant differences in the hospital length of stay (14 days SOT group vs. 8.6 days non-SOT group; p=0.018). There were no differences regarding the complication rate between the two groups (9/20, 45% vs. 6/20, 30% in the SOT and non-SOT groups, respectively).

SOT patients with associated hip fracture required longer hospital length of stay than non-SOT patients. SOT patients did not show greater clinical complications; however, they presented higher mortality rate compared to non-SOT patients.

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.

COVID-19 was declared a pandemic by the World Health Organization (WHO) on 11 March 2020. The initial response to the pandemic included the cessation of routine services including elective orthopaedic surgery. There was apprehension among both surgeons and patients about restarting elective surgical services. The high mortality rate in perioperative patients who contract COVID-19 was of particular concern. The aim of this study was to identify the perioperative viral transmission rate in orthopaedic patients at our institution following the restart of elective surgery between August 2020 and November 2020 after the first wave of Covid in the UK.

All patients who had their elective Orthopaedic surgeries at our institution from 1st August 2020 to 30th November 2020 were checked whether they were Covid positive or experienced COVID symptoms within 2 weeks after the operation. All patients were advised a 14-day period of comprehensive social distancing, 3 days of self-isolation and had a negative COVID-19 test within 72 hours of surgery and underwent surgery at a COVID free site. The patients were contacted and the hospital database was searched to identify those patients who were Covid positive or had Covid symptoms after the surgery. Baseline patient characteristics were recorded including age, gender, procedure, the subspeciality and admission type. Patients who underwent emergency procedures and trauma operations were excluded.

Out of the 499 patients, 315 were contacted over telephone and hospital database was searched for the rest of the patients. We found that none of the patients were positive for COVID or had symptoms of COVID within two weeks of surgery. 5 patients were COVID positive with symptoms few months after the procedure and all of them recovered. There were 144 inpatient admissions and 353 day cases.

The development of a COVID-free pathway for elective orthopaedic patients results in very low viral transmission rates. Findings of our study confirms that COVID-free elective pathway is an efficient process, and this could be implemented in future elective Orthopaedic surgeries during COVID times. Elective surgery can be safely resumed using dedicated pathways and procedures -Surgeons, hospital staff and patients should remain vigilant.

In the last decades, the use of artificial intelligence (AI) has been increasingly investigated in intervertebral disc degeneration (IDD) and chronic low back pain (LBP) research. To date, several AI-based cutting-edge technologies, such as computer vision, computer-assisted diagnosis, decision support system and natural language processing have been utilized to optimize LBP prevention, diagnosis, and treatment. This talk will provide an outline on contemporary AI applications to IDD and LBP research, with a particular attention towards actual knowledge gaps and promising innovative tools.

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.

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).

The purpose of the this survey study was twofold: 1) to examine different aspects of satisfaction with post-operative care in injured workers who have undergone rotator cuff surgery and 2) to examine the relationship between receiving a newly implemented summary report and the overall satisfaction with surgery and recovery.

The clinical communication summary report was given to injured workers following their review assessment to share with the family doctor or other health care providers. The form indicated a need for further assessments or investigations and return to work recommendations. The study involved using a satisfaction survey that examined different aspects of follow-up visit and workers’ opinion about their understanding of the nature of surgery, their progress, clinical management, and usefulness of the newly implemented summery report.

Eighty patients completed the questionnaire (mean age: 54 (8), 62(78%) males, of whom 26 (34%) had a rotator cuff decompression and 31 (40%) had a rotator cuff repair with 20 (26%) having both procedures and three missing data. There were no statistically significant relationships between the patient demographics (age, sex or type of surgery) and satisfaction. However, there was a significant correlation between how patients perceived the summary report in terms of helpfulness and the overall satisfaction with surgery (FTE<0.0004, p=0.001) and the satisfaction with recovery (FTE<0.0001, p=0.001). This may indicate that improvement in worker's understanding of their treatment recommendations and restrictions is associated with higher levels of overall satisfaction in this population.

Our results indicate a positive linear relationship between expressing a high satisfaction and the helpfulness of the summary report. As part of improving care, adding a summary report may facilitate sharing information with the injured workers, their care providers and their workplace.

Aseptic inflammation is the main factor causing aseptic loosening of artificial joints. Studies have shown that inflammatory cells can activate STING (stimulator of interferon genes, STING) after being stressed. This study aims to explore the specific mechanism of STING in aseptic loosening of artificial joints, and provide new strategies for disease prevention.

Titanium particles with a diameter of 1.2-10 μm were prepared to stimulate macrophages (RAW 264.7) to simulate the periprosthetic microenvironment. A lentiviral vector targeting the STING gene was designed and transfected into macrophages to construct a cell line targeting STING knockdown. The expression and secretion levels of TNF-α were detected by qPCR and ELISA, the activation levels of inflammatory pathways (NF-κB, IRF3, etc.) were detected by western blot, and the nucleus translocation of P65 and IRF3 was observed by cellular immunofluorescence.

After titanium particles stimulated macrophages, qPCR and ELISA showed that the transcription and secretion levels of TNF-α were significantly increased. Western blot showed that titanium particle stimulation could increase the phosphorylation levels of NF-κB and IRF3 pathways. While knockdown of STING can significantly reduce titanium particle-induced TNF production, attenuate the activation levels of NF-κB and IRF3 pathways as well as the nucleus translocation of P65 and IRF3.

Conclusions: STING positively regulates the level of inflammation in macrophages induced by titanium particles, and targeted inhibition of STING can reduce inflammation, which may delay the progression of aseptic loosening of artificial joints.

Osteoarthritis is a common articular cartilage disorder and causes a significant global disease burden. Articular cartilage has a limited capacity of repair and there is increasing interest in the use of cell-based therapies to facilitate repair including the use of Mesenchymal Stromal Cells (MSCs). There is some evidence in the literature that suggests that advancing age is associated with declining MSC function, including reduced proliferation and differentiation potential, and greater cellular apoptosis. In our study, we first performed a systematic review of the literature to determine the effects of chronological age on the in vitro properties of MSCs, and then performed a laboratory study to investigate these properties.

We initially conducted a PRISMA systematic review of the literature to review the evidence base for the effects of chronological age on the in vitro properties of MSCs including cell numbers, expansion, cell surface characterization and differentiation potential. This was followed by laboratory based experiments to assess these properties. Tissue from patients undergoing total knee replacement surgery was used to isolate MSCs from the bone fragments using a method developed in our laboratory. The growth kinetics was determined by calculating the population doublings per day. Following expansion in culture, MSCs at P2 were characterised for a panel of cell surface markers using flow cytometry. The cells were positive for CD73, CD90 and CD105, and negative for CD34 and CD45. The differentiation potential of the MSCs was assessed through tri-lineage differentiation assays. Clear differences between the younger and older patients were indicated.

Chronological age-related changes in MSC function have important implications on the use of these cells in clinical applications for an ageing population. The results from this study will be used to plan further work looking at the effects of chronological age on cellular senescence and identify pathways that could be targeted to potentially reverse any age-related changes.