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

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

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

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

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

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

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

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

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

Varus malalignment increases the susceptibility of cartilage to mechanical overloading, which stimulates catabolic metabolism to break down the extracellular matrix and lead to osteoarthritis (OA). The altered mechanical axis from the hip, knee to ankle leads to knee joint pain and ensuing cartilage wear and deterioration, which impact millions of the aged population. Stabilization of the remaining damaged cartilage, and prevention of further deterioration, could provide immense clinical utility and prolong joint function. Our previous work showed that high tibial osteotomy (HTO) could shift the mechanical stress from an imbalanced status to a neutral alignment. However, the underlying mechanisms of endogenous cartilage stabilization after HTO remain unclear. We hypothesize that cartilage-resident mesenchymal stem cells (MSCs) dampen damaged cartilage injury and promote endogenous repair in a varus malaligned knee. The goal of this study is to further examine whether HTO-mediated off-loading would affect human cartilage-resident MSCs' anabolic and catabolic metabolism. This study was approved by IACUC at Xi'an Jiaotong University. Patients with medial compartment OA (52.75±6.85 yrs, left knee 18, right knee 20) underwent open-wedge HTO by the same surgeons at one single academic sports medicine center. Clinical data was documented by the Epic HIS between the dates of April 2019 and April 2022 and radiographic images were collected with a minimum of 12 months of follow-up. Medial compartment OA with/without medial meniscus injury patients with unilateral Kellgren /Lawrence grade 3–4 was confirmed by X-ray. All incisions of the lower extremity healed well after the HTO operation without incision infection. Joint space width (JSW) was measured by uploading to ImageJ software. The Knee injury and Osteoarthritis Outcome Score (KOOS) toolkit was applied to assess the pain level. Outerbridge scores were obtained from a second-look arthroscopic examination. RNA was extracted to quantify catabolic targets and pro-inflammatory genes (QiaGen). Student's t test for two group comparisons and ANOVA analysis for differences between more than 2 groups were utilized. To understand the role of mechanical loading-induced cartilage repair, we measured the serial changes of joint space width (JSW) after HTO for assessing the state of the cartilage stabilization. Our data showed that HTO increased the JSW, decreased the VAS score and improved the KOOS score significantly. We further scored cartilage lesion severity using the Outerbridge classification under a second-look arthroscopic examination while removing the HTO plate. It showed the cartilage lesion area decreased significantly, the full thickness of cartilage increased and mechanical strength was better compared to the pre-HTO baseline. HTO dampened medial tibiofemoral cartilage degeneration and accelerate cartilage repair from Outerbridge grade 2 to 3 to Outerbridge 0 to 1 compared to untreated varus OA. It suggested that physical loading was involved in HTO-induced cartilage regeneration. Given that HTO surgery increases joint space width and creates a physical loading environment, we hypothesize that HTO could increase cartilage composition and collagen accumulation. Consistent with our observation, a group of cartilage-resident MSCs was identified. Our data further showed decreased expression of RUNX2, COL10 and increased SOX9 in MSCs at the RNA level, indicating that catabolic activities were halted during mechanical off-loading. To understand the role of cartilage-resident MSCs in cartilage repair in a biophysical environment, we investigated the differentiation potential of MSCs under 3-dimensional mechanical loading conditions. The physical loading inhibited catabolic markers (IL-1 and IL-6) and increased anabolic markers (SOX9, COL2).

Knee-preserved HTO intervention alleviates varus malalignment-related knee joint pain, improves daily and recreation function, and repairs degenerated cartilage of medial compartment OA. The off-loading effect of HTO may allow the mechanoregulation of cartilage repair through the differentiation of endogenous cartilage-derived MSCs.

Growing evidence has suggested that paracrine mechanisms of Mesenchymal stem cell (MSC) may be involved in the underlying mechanism of MSC after transplantation, and extracellular vesicles (EVs) are an important component of this paracrine role. The aim of this study was to investigate the in vitro osteogenic effects of EVs derived from undifferentiated mesenchymal stem cells and from chemically induced to differentiate into osteogenic cells for 7 days. Further, the osteoinductive potential of EVs for bone regeneration in rat calvarial defects was assessed.

We could isolate and characterize EVs from naïve and osteogenic-induced MSCs. Proteomic analysis revealed that EVs contained distinct protein profiles, with Osteo-EVs having more differentially expressed proteins with osteogenic properties. EVs were found to enhance the proliferation and migration of cultured MSC. In addition, the study found that Osteo-EVs/MEM combination scaffolds could enhance greater bone formation after 4 weeks as compared to native MEM loaded with serum-free media.

The study suggests that EVs derived from chemically osteogenic-induced MSCs for 7 days can significantly enhance both the osteogenic differentiation activity of cultured hMSCs and the osteoinductivity of MEM scaffolds. The results indicate that Osteo-MSC-secreted nanocarriers-EVs combined with MEM scaffolds can be used for repairing bone defects.

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

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

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

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

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

Nanovesicle-based therapy is increasingly being pursued as a safe, cell-free strategy to combat various immunological, musculoskeletal and neurodegenerative diseases. Small secreted extracellular vesicles (sEVs) obtained from multipotent mesenchymal stromal cells (MSCs) are of particular interest for therapeutic use since they convey anti-inflammatory, anti-scarring and neuroprotective activities to the recipient cells. Cell-derived vesicles (CDVs) produced by a proprietary extrusion process are surrounded by a lipid bilayer membrane with correct membrane topology, display biological activities similar to MSC-derived EVs and may find specific application for organ-targeted drug delivery systems. Translation of nanovesicle-based therapeutics into clinical application requires quantitative and reproducible analysis of bioactivity and stability, and the potential for GMP-compliant manufacturing. Manufacturing and regulatory considerations as well as preclinical models to support clinical translation will be discussed.

Mesenchymal stromal cells (MSC) have been proposed as an emerging cell therapy for bone tissue engineering applications. However, the healing capacity of the bone tissue is often compromised by patient's age and comorbidities, such as osteoporosis. In this context, it is important to understand the impact of donor age on the therapeutic potential of MSC. Importantly, the impact on donor age is not restricted to cells themselves but also to their microenvironment that is known to affect cell function.

The extracellular matrix (ECM) has an important role in stem cell microenvironment, being able to modulate cell proliferation, self-renewal and differentiation. Decellularized cell-derived ECM (dECM) has been explored for regenerative medicine applications due to its bioactivity and its resemblance to the in vivo microenvironment. Thus, dECM offers the opportunity not only to develop microenvironments with customizable properties for improvement of cellular functions but also as a platform to study cellular niches in health and disease. In this study, we investigated the capacity of the microenvironment to rescue the impaired proliferative and osteogenic potential of aged MSC. The goal of this work was to understand if the osteogenic capacity of MSC could be modulated by exposure to a dECM derived from cells obtained from young donors. When aged MSC were cultured on dECM derived from young MSC, their in vitro proliferative and osteogenic capacities were enhanced. Our results suggest that the microenvironment, specifically the ECM, plays a crucial role in the osteogenic differentiation capacity of MSC. dECM might be a valuable clinical strategy to overcome the age-related decline in the osteogenic potential of MSC by recapitulating a younger microenvironment, attenuating the effects of aging on the stem cell niche. Overall, this study opens new possibilities for developing clinical strategies for elderly patients with limited bone formation capacity who currently lack effective treatments.

Acknowledgements: The authors thank FCT for funding through the project DentalBioMatrix (PTDC/BTM-MAT/3538/2020) and to the research institutions iBB (UIDB/04565/2020 and UIDP/04565/2020) and Associate Laboratory i4HB (LA/P/0140/2020).

The effects of dexamethasone (dex), during in vitro human osteogenesis, are contrasting. Indeed, dex downregulates SOX9 during osteogenic differentiation of human bone marrow mesenchymal stromal cells (HBMSCs). However, dex also promotes PPARG expression, resulting in the formation of adipocyte-like cells within the osteogenic monolayers. The regulation of both SOX9 and PPARG seems to be downstream the transactivation activity of the glucocorticoid receptor (GR), thus the effect of dex on SOX9 downregulation is indirect. This study aims at determining whether PPAR-γ regulates SOX9 expression levels, as suggested by several studies.

HBMSCs were isolated from bone marrow of patients with written informed consent. HBMSCs were cultured in different osteogenic induction media containing 10 or 100 nM dex. Undifferentiated cells were used as controls. Cells were treated either with a pharmacological PPAR-γ inhibitor T0070907 (donors n=4) or with a PPARG-targeting siRNA (donors n=2). Differentiation markers or PPAR-γ target genes were analysed by RT-qPCR. Mineral deposition was assessed by ARS staining. Two-way ANOVA followed by a Tukey's multiple comparison test compared the effects of treatments.

At day 7, T0070907 downregulated ADIPOQ and upregulated CXCL8, respectively targets of PPAR-γ-mediated transactivation and transrepression. RUNX2 and SOX9 were also significantly downregulated in absence of dex. PPARG was successfully downregulated by siRNA. ADIPOQ expression was also inhibited, while CXCL8 did not show any significant difference between siRNA treatment groups. RUNX2 was downregulated by the PPARG-siRNA treatment in presence of 100 nM dexamethasone, while SOX9 levels were not affected. ARS showed no change in the mineralization levels when PPARG expression or activity was inhibited.

Understanding how dex regulates HBMSC differentiation is of pivotal importance to refine current in vitro models. These results suggest that PPARG does not mediate SOX9 downregulation. Unexpectedly, RUNX2 expression was also unaltered or even downregulated after PPAR-γ inhibition.

Acknowledgements: AO Foundation, AO Research Institute (CH) and PRIN 2017 MUR (IT) for financial support.

Despite osteoarthritis (OA) representing a large burden for healthcare systems, there remains no effective intervention capable of regenerating the damaged cartilage in OA. Mesenchymal stromal cells (MSCs) are adult-derived, multipotent cells which are a candidate for musculoskeletal cell therapy. However, their precise mechanism of action remains poorly understood.

The effects of an intra-articular injection of human bone-marrow derived MSCs into a knee osteochondral injury model were investigated in C57Bl/6 mice. The cell therapy was retrieved at different time points and single cell RNA sequencing was performed to elucidate the transcriptomic changes relevant to driving tissue repair. Mass cytometry was also used to study changes in the mouse immune cell populations during repair.

Histological assessment reveals that MSC treatment is associated with improved tissue repair in C57Bl/6 mice. Single cell analysis of retrieved human MSCs showed spatial and temporal transcriptional heterogeneity between the repair tissue (in the epiphysis) and synovial tissue. A transcriptomic map has emerged of some of the distinct genes and pathways enriched in human MSCs isolated from different tissues following osteochondral injury. Several MSC subpopulations have been identified, including proliferative and reparative subpopulations at both 7 days and 28 days after injury. Supported by the mass cytometry results, the immunomodulatory role of MSCs was further emphasised, as MSC therapy was associated with the induction of increased numbers of regulatory T cells correlating with enhanced repair in the mouse knee.

The transcriptomes of a retrieved MSC therapy were studied for the first time. An important barrier to the translation of MSC therapies is a lack of understanding of their heterogeneity, and the consequent lack of precision in its use. MSC subpopulations with different functional roles may be implicated in the different phases of tissue repair and this work offers further insights into repair process.

Approximately 30% of general practice consultations for musculoskeletal pain are related to tendon disorders, causing substantial personal suffering and enormous related healthcare costs. Treatments are often prone to long rehabilitation times, incomplete functional recovery, and secondary complications following surgical repair. Overall, due to their hypocellular and hypovascular nature, the regenerative capacity of tendons is very poor and intrinsically a disorganized scar tissue with inferior biomechanical properties forms after injury. Therefore, advanced therapeutic modalities need to be developed to enable functional tissue regeneration within a degenerative environment, moving beyond pure mechanical repair and overcoming the natural biological limits of tendon healing.

Our recent studies have focused on developing biologically augmented treatment strategies for tendon injuries, aiming at restoring a physiological microenvironment and boosting endogenous tissue repair. Along these lines, we have demonstrated that the local application of mesenchymal stromal cell-derived small extracellular vesicles (sEVs) has the potential to improve rotator cuff tendon repair by modulating local inflammation and reduce fibrotic scarring. In another approach, we investigated if the local delivery of the tendon ECM protein SPARC, which we previously demonstrated to be essential for tendon maturation and tissue homeostasis, has the potential to enhance tendon healing. Finally, I will present results demonstrating the utility of nanoparticle-delivered, chemically modified mRNAs (cmRNA) to improve tendon repair.

Gait analysis is an indispensable tool for scientific assessment and treatment of individuals whose ability to walk is impaired. The high cost of installation and operation are a major limitation for wide-spread use in clinical routine.

Advances in Artificial Intelligence (AI) could significantly reduce the required instrumentation. A mobile phone could be all equipment necessary for 3D gait analysis. MediaPipe Pose provided by Google Research is such a Machine Learning approach for human body tracking from monocular RGB video frames that is detecting 3D-landmarks of the human body.

Aim of this study was to analyze the accuracy of gait phase detection based on the joint landmarks identified by the AI system.

Motion data from 10 healthy volunteers walking on a treadmill with a fixed speed of 4.5km/h (Callis, Sprintex, Germany) was sampled with a mobile phone (iPhone SE 2nd Generation, Apple). The video was processed with Mediapipe Pose (Version 0.9.1.0) using custom python software. Gait phases (Initial Contact - IC and Toe Off - TO) were detected from the angular velocities of the lower legs. For the determination of ground truth, the movement was simultaneously recorded with the AS-200 System (LaiTronic GmbH, Innsbruck, Austria).

The number of detected strides, the error in IC detection and stance phase duration was calculated.

In total, 1692 strides were detected from the reference system during the trials from which the AI-system identified 679 strides. The absolute mean error (AME) in IC detection was 39.3 ± 36.6 ms while the AME for stance duration was 187.6 ± 140 ms.

Landmark detection is a challenging task for the AI-system as can clearly be seen be the rate of only 40% detected strides. As mentioned by Fadillioglu et al., error in TO-detection is higher than in IC-detection.

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

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

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

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

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

In a consecutive retrospective analysis of 190 patients treated with the Masquelet technique at the BG Klinikum Hamburg from January 2012 to January 2022, defect-specific features such as the extent and morphology of the defect were recorded, and their influence on the time to reach full weight-bearing of the affected limb was investigated.

A total of 217 defects were treated in 190 patients using the Masquelet technique. 70% of all defects were located in the tibia, followed by 22% in the femur and only about 7% in the upper extremity. The average length of all defects was 58 mm (+/−31 mm), with the largest defect measuring 180 mm and the smallest measuring 20 mm. 89% of the patients achieved full weight-bearing at the end of therapy. The average time from initiation of therapy to reaching safe full weight-bearing was 589 days. There was a significant correlation between defect length and time to reach full weight-bearing (p = 0.0134). These results could serve as a basis for creating a score for prognostics and evaluation of bone healing after treatment with the Masquelet technique. Additionally, the results could help guide indications for secondary stabilization using internal fixation.

The management of comminuted metaphyseal fractures is a technical challenge and satisfactory outcomes of such fixations often remain elusive. The small articular fragments and bone loss often make it difficult for standard fixation implants for proper fixation. We developed a novel technique to achieve anatomical reduction in multiple cases of comminuted metaphyseal fractures at different sites by employing the cantilever mechanism with the help of multiple thin Kirschner wires augmented by standard fixation implants.

We performed a retrospective study of 10 patients with different metaphyseal fractures complicated by comminution and loss of bone stock. All patients were treated with the help of cantilever mechanism using multiple Kirschner wires augmented by compression plates. All the patients were operated by the same surgeon between November 2020 to March 2021 and followed up till March 2023. Surgical outcomes were evaluated according to the clinical and radiological criteria.

A total of 10 patients were included in the study. Since we only included patients with highly unstable and comminuted fractures which were difficult to fix with traditional methods, the number of patients in the study were less. All 10 patients showed satisfactory clinical and radiological union at the end of the study with good range of motion. One of the patient in the study had post-operative wound complication which was managed conservatively with regular dressings and oral antibiotics.

Comminuted metaphyseal fractures might differ in pattern and presentation with every patient and there can be no standard treatment for all. The cantilever technique of fracture fixation is based on the principle of cantilever mechanism used in bridges and helps achieve good anatomical reduction and fixation. It provides a decent alternative when standard modes of fixation don't give desired result owing to comminuted nature of fractures and deficiency of bone stock.

Primary bone tumors are rare, complex and highly heterogeneous. Its diagnostic and treatment are a challenge for the multidisciplinary team. Developments on tumor biomarkers, immunohistochemistry, histology, molecular, bioinformatics, and genetics are fundamental for an early diagnosis and identification of prognostic factors. The personalized medicine allows an effective patient tailored treatment. The bone biopsy is essential for diagnosis. Treatment may include systemic therapy and local therapy. Frequently, a limb salvage surgery includes wide resection and reconstruction with endoprosthesis, biological or composites. The risk for local recurrence and distant metastases depends on the primary tumor and treatment response.

Cancer patients are living longer and bone metastases are increasing. Bone is the third most frequently location for distant lesions. Bone metastases are associated to pain, pathological fractures, functional impairment, and neurological deficits. It impacts survival and patient quality of life. The treatment of metastatic disease is a challenge due to its complexity and heterogeneity, vascularization, reduced size and limited access. It requires a multidisciplinary treatment and depending on different factors it is palliative or curative-like treatment. For multiple bone metastases it is important to relief pain and increases function in order to provide the best quality of life and expect to prolong survival. Advances in nanotechnology, bioinformatics, and genomics, will increase biomarkers for early detection, prognosis, and targeted treatment effectiveness. We are taking the leap forward in precision medicine and personalized care.

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

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

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

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

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

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

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

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

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

For many years, marker-based systems have been used for motion analysis. However, the emergence of new technologies, such as 4D scanners provide exciting new opportunities for motion analysis. In 4D scanners, the subjects are measured as a dense mesh, which enables the use of shape analysis techniques. In this talk, we will explore how the combination of the rising new motion analysis methods and shape modelling may change the way we think about movement and its analysis.

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

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

Syndesmotic ankle lesions involve disruption of the osseous tibiofibular mortise configuration as well as ligamentous structures stabilizing the ankle joint. Incomplete diagnosis and maltreatment of these injuries is frequent, resulting in chronic pain and progressive instability thus promoting development of ankle osteoarthritis in the long term. Although the pathogenesis is not fully understood, abnormal mechanics has been implicated as a principal determinant of ankle joint degeneration after syndesmotic ankle lesions. Therefore, the focus of this presentation will be on our recent development of a computationally efficient algorithm to calculate the contact pressure distribution in patients with a syndesmotic ankle lesion, enabling us to stratify the risk of OA development in the long term and thereby guiding patient treatment.

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

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

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

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

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

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

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

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

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

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

Matrix metalloproteinase enzymes (MMPs) play a crucial role in the remodeling of articular cartilage, contributing also to osteoarthritis (OA) progression. The pericellular matrix (PCM) is a specialized space surrounding each chondrocyte, containing collagen type VI and perlecan. It acts as a transducer of biomechanical and biochemical signals for the chondrocyte. This study investigates the impact of MMP-2, -3, and -7 on the integrity and biomechanical characteristics of the PCM.

Human articular cartilage explants (n=10 patients, ethical-nr.:674/2016BO2) were incubated with activated MMP-2, -3, or -7 as well as combinations of these enzymes. The structural degradative effect on the PCM was assessed by immunolabelling of the PCM's main components: collagen type VI and perlecan. Biomechanical properties of the PCM in form of the elastic moduli (EM) were determined by means of atomic force microscopy (AFM), using a spherical cantilever tip (2.5µm).

MMPs disrupted the PCM-integrity, resulting in altered collagen type VI and perlecan structure and dispersed pericellular arrangement. A total of 3600 AFM-measurements revealed that incubation with single MMPs resulted in decreased PCM stiffness (p<0.001) when compared to the untreated group. The overall EM were reduced by ∼36% for all the 3 individual enzymes. The enzyme combinations altered the biomechanical properties at a comparable level (∼36%, p<0.001), except for MMP-2/-7 (p=0.202).

MMP-induced changes in the PCM composition have a significant impact on the biomechanical properties of the PCM, similar to those observed in early OA. Each individual MMP was shown to be highly capable of selectively degrading the PCM microenvironment. The combination of MMP-2 and -7 showed a lower potency in reducing the PCM stiffness, suggesting a possible interplay between the two enzymes. Our study showed that MMP-2, -3, and -7 play a direct role in the functional and structural remodeling of the PCM.

Acknowledgements: This work was supported by the Faculty of Medicine of the University of Tübingen (grant number.: 2650-0-0).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Tendinopathy is a disease associated with pain and tendon degeneration, leading to a decreased range of motion and an increased risk of tendon rupture. The etiology of this frequent disease is still unknown. In other musculoskeletal tissues like cartilage and intervertebral discs, transient receptor potential channels (TRP- channels) were shown to play a major role in the progression of degeneration. Due to their responsiveness to a wide range of stimuli like temperature, pH, osmolarity and mechanical load, they are potentially relevant factors in tendon degeneration as well. We therefore hypothesize that TRP- channels are expressed in tendon cells and respond to degeneration inducing stimuli.

By immunohistochemistry, qRT-PCR and western blot analyses, we found three TRP channel members, belonging to the vanilloid (TRPV), and ankyrin (TRPA) subfamily, respectively, to be expressed in healthy human tendon tissue as well as in rodent tendon, with expression being located to cells within the dense tendon proper, as well as to endotenon resident cells. In vitro-inflammatory and ex vivo-mechanical stimulation led to a significant upregulation of TRPA1 expression in tendon cells, which correlates well with the fact that TRPA1 is considered as mechanosensitive channel being sensitized by inflammatory mediators.

This is the first description of TRP- channels in human and rodent tendon. As these channels are pharmacologically targetable by both agonists and antagonists, they may represent a promising target for novel treatments of tendinopathy.

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

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

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

Geometric deep learning is a relatively new field that combines the principles of deep learning with techniques from geometry and topology to analyze data with complex structures, such as graphs and manifolds. In orthopedic research, geometric deep learning has been applied to a variety of tasks, including the analysis of imaging data to detect and classify abnormalities, the prediction of patient outcomes following surgical interventions, and the identification of risk factors for degenerative joint disease. This review aims to summarize the current state of the field and highlight the key findings and applications of geometric deep learning in orthopedic research. The review also discusses the potential benefits and limitations of these approaches and identifies areas for future research. Overall, the use of geometric deep learning in orthopedic research has the potential to greatly advance our understanding of the musculoskeletal system and improve patient care.

Wearable inertial sensors can detect abnormal gait associated with knee or hip osteoarthritis (OA). However, few studies have compared sensor-derived gait parameters between patients with hip and knee OA or evaluated the efficacy of sensors suitable for remote monitoring in distinguishing between the two. Hence, our study seeks to examine the differences in accelerations captured by low-frequency wearable sensors in patients with knee and hip OA and classify their gait patterns.

We included patients with unilateral hip and knee OA. Gait analysis was conducted using an accelerometer ipsilateral with the affected joint on the lateral distal thighs. Statistical parametric mapping (SPM) was used to compare acceleration signals. The k-Nearest Neighbor (k-NN) algorithm was trained on 80% of the signals' Fourier coefficients and validated on the remaining 20% using 10-fold cross-validation to classify the gait patterns into hip and knee OA.

We included 42 hip OA patients (19 females, age 70 [63–78], BMI of 28.3 [24.8–30.9]) and 59 knee OA patients (31 females, age 68 [62–74], BMI of 29.7 [26.3–32.6]). The SPM results indicated that one cluster (12–20%) along the vertical axis had accelerations exceeding the critical threshold of 2.956 (p=0.024). For the anteroposterior axis, three clusters were observed exceeding the threshold of 3.031 at 5–19% (p = 0.0001), 39–54% (p=0.00005), and 88–96% (p = 0.01). Regarding the mediolateral axis, four clusters were identified exceeding the threshold of 2.875 at 0–9% (p = 0.02), 14–20% (p=0.04), 28–68% (p < 0.00001), and 84–100% (p = 0.004). The k-NN model achieved an AUC of 0.79, an accuracy of 80%, and a precision of 85%.

In conclusion, the Fourier coefficients of the signals recorded by wearable sensors can effectively discriminate the gait patterns of knee and hip OA. In addition, the most remarkable differences in the time domain were observed along the mediolateral axis.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

An overview about 3D printing technology in orthopaedic applications will be given based on examples. The process from early prototypes to certified implants coming from serial production will be demonstrated also considering relevant surrounding conditions. Today's focus is mostly on orthopaedic implants, but there is a high potential for new implant-related surgical instrument solutions taking into account up-coming clinical demands and user needs accessible by actual 3D printing technologies.

Production of porous titanium bone implants is a highly promising research and application area due to providing high osseointegration and achieving the desired mechanical properties. Production of controlled porosity in titanium implants is possible with laser powder bed fusion (L- PBF) technology. The main topics of this presentation includes the L-PBF process parameter optimization to manufacture thin walls of porous titanium structures with almost full density and good mechanical properties as well as good dimensional accuracy. Moreover, the cleaning and coating process of these structures to further increase osseointegration and then in-vitro biocompatibility will be covered.

Tissue repair is believed to rely on tissue-resident progenitor cell populations proliferating, migrating, and undergoing differentiation at the site of injury. During these processes, the crosstalk between mesenchymal stromal/stem cells (MSCs) and macrophages has been shown to play a pivotal role. However, the influence of extracellular matrix (ECM) remodelling in this crosstalk, remains elusive.

Human MSCs cultured on tissue culture plastic (TCP) and encased within fibrin in vitro were treated with/without TNFα and IFNγ. Human monocytes were cocultured with untreated/pretreated MSCs on TCP or within fibrin. After seven days, the conditioned media (CM) were collected. Human chondrocytes were exposed to CM in a migration assay. The impact of TGFβ was assessed by adding an inhibitor (TGFβRi). Cell activity was assessed using RT-qPCR and XL-protein-profiler-array.

Previously, we demonstrated that culturing human MSCs within 3D-environments significantly enhances their immunoregulatory activity in response to pro-inflammatory stimuli. In this study, monocytes were co-cultured with MSCs within fibrin, acquiring a distinct M2-like repair macrophage phenotype in contrast to TCP co-cultures. MSC/macrophage CM characterization using a protein array demonstrated differences in release of several factors, including chemokines, growth factors and ECM components. Chondrocyte migration was significantly reduced in CM from untreated MSC/monocytes co-cultures in fibrin compared to CM of untreated MSCs/monocytes on TCP. This impact on migration was not seen with chondrocytes cultured in CM of monocytes co-cultured with pretreated MSCs in fibrin. The CM of monocytes co-cultured with pretreated MSCs in fibrin up-regulates COL2A1 and SOX9 compared to TCP. Chondrogenesis and migration were TGFβ dependent.

MSC/macrophage crosstalk and responsiveness to cytokines are influenced by the ECM environment, which subsequently impacts tissue-resident cell migration and chondrogenesis. The direct effects of ECM on MSC/macrophage secretory phenotype is complemented by the dynamic ECM binding and release of growth factors such as TGFβ.

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

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

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

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

Acknowledgments: FCT, EUROSPINE, ON Foundation.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Osteosarcoma is common in children and adolescents with high mortality due to rapid progression. Therapeutic approaches for osteosarcoma are limited and may cause side effects. Cannabinoid ligands exert antiproliferative, apoptotic effect in cancer cells via CB1/2 or TRPV1 receptors. In this study, we hypothesized that synthetic specific CB2R agonist CB65 might have an antiproliferative and apoptotic effect on osteosarcoma cell lines in vitro. If so, this agent might be a chemotherapeutic candidate for osteosarcoma, with prolonged release, increased stability and bioavailability when loaded into a liposomal system. We first determined CB2 receptor expression in MG63 and Saos-2 osteosarcoma cells by qRT- PCR and FCM. CB65 reduced proliferation in osteosarcoma cells by WST-1 and RTCA. IC50 for MG63 and Saos-2 cells were calculated as 1.11×10-11 and 4.95×10-11 M, respectively. The antiproliferative effect of CB65 on osteosarcoma cells was inhibited by CB2 antagonist AM630. IC50 of CB65 induced late apoptosis of MG63 and Saos-2 cells at 24 and 48 hours, respectively by FCM. CB65 was loaded into the liposomal system by thin film hydration method and particle size, polydispersity index, and zeta potentials were 141.7±0.6 nm, 0.451±0.026, and -10.9±0.3 mV, respectively. The CB65-loaded liposomal formulation reduced MG63 and Saos-2 cell proliferation by RTCA. IC50 of CB65 and CB65-loaded liposomal formulation induced late apoptosis of MG63 and Saos-2 cells at 24 and 48 hours, respectively, by FCM. Scratch width was higher in CB65 and CB65-loaded liposome-treated cells compared to control. In this study, the real-time antiproliferative and apoptotic effect of synthetic specific CB2 agonist CB65 in osteosarcoma cell lines was demonstrated for the first time, and the real time therapeutic window was determined. The CB65-loaded liposomal formulation presents a potential treatment option that can be translated to clinic following its validation within animal models and production under GMP conditions.

Tryfonidou leads the Horizon 2020 consortium (iPSpine; 2019–2023) bringing a transdisciplinary team of 21 partners together to address the challenges and bottlenecks of iPS-based advanced therapies towards their transition to the clinic. Here, chronic back pain due to intervertebral disc degeneration is employed as a show case. The project develops the iPS-technology and designed smart biomaterials to carry, protect and instruct the iPS cells within the degenerate disc environment. This work will be presented including ongoing activities focus on translating the developed methodology and tools towards clinically relevant animal models.

The consortium optimized the protocol for the differentiated iPS-notochordal-like cells (iPS-NLCs) and shortlisted two biomaterials shortlisted based on their physicochemical, cytotoxicity, biomechanical and biocompatibility testing. Both were shown to be safe and have been tested with the progenitors of iPS-NLCs. An advanced platform (e.g., the dynamic loading bioreactor for disc tissue) was used to evaluate their performance: the biomaterials supported the iPS-NLC progenitors after injection into the degenerate disc and seem to also support their maturation towards NLCs. Furthermore, we confirmed the capacity of these cells to survive inside degenerated discs at 30 days upon injection in sheep, whereafter we continued with their evaluation at 3 months post-injection. We achieved full evaluation of the sheep spines, including biomechanical analysis using the portable spine biomechanics tester prior analysis at the macro- and microscopic, and biochemical level.

Osteoporosis is a progressive, chronic disease of bone metabolism, characterized by decreased bone mass and mineral density, predisposing individuals to an increased risk of fractures. The use of animal models, which is the gold standard for the screening of anti-osteoporosis drugs, raises numerous ethical concerns and is highly debated because the composition and structure of animal bones is very different from human bones. In addition, there is currently a poor translation of pre-clinical efficacy in animal models to human trials, meaning that there is a need for an alternative method of screening and evaluating new therapeutics for metabolic bone disorders, in vitro.

The aim of this project is to develop a 3D Bone-On-A-Chip that summarizes the spatial orientation and mutual influences of the key cellular components of bone tissue, in a citrate and hydroxyapatite-enriched 3D matrix, acting as a 3D model of osteoporosis. To this purpose, a polydimethylsiloxane microfluidic device was developed by CAD modelling, stereolithography and replica molding. The device is composed by two layers: (i) a bottom layer for a 3D culture of osteocytes embedded in an osteomimetic collagen-enriched matrigel matrix with citrate-doped hydroxyapatite nanocrystals, and (ii) a upper layer for a 2D perfused co-culture of osteoblasts and osteoclasts seeded on a microporous PET membrane.

Cell vitality was evaluated via live/dead assay. Bone deposition and bone resorption was analysed respectively with ALP, Alizarin RED and TRACP staining. Osteocytes dendrite expression was evaluated via immunofluorescence. Subsequently, the model was validated as drug screening platform inducing osteocytes apoptosis and administrating standard anti-osteoporotic drugs.

This device has the potential to substitute or minimize animal models in pre-clinical studies of osteoporosis, contributing to pave the way for a more precise and punctual personalized treatment.

Intervertebral disc (IVD) degeneration (IDD) involves imbalance between the anabolic and the catabolic processes that regulate the extracellular matrix of its tissues. These processes are complex, and improved integration of knowledge is needed. Accordingly, we present a nucleus pulposus cell (NPC) regulatory network model (RNM) that integrates critical biochemical interactions in IVD regulation and can replicate experimental results. The RNM was built from a curated corpus of 130 specialized journal articles. Proteins were represented as nodes that interact through activation and inhibition edges. Semi-quantitative steady states (SS) of node activations were calculated. Then, a full factorial sensitivity analysis (SA) identified which out of the RNM 15 cytokines, and 4 growth factors affected most the structural proteins and degrading enzymes. The RNM was further evaluated against metabolic events measured in non-healthy human NP explant cultures, after 2 days of 1ng/ml IL-1B catabolic induction. The RNM represented successfully an anabolic basal SS, as expected in normal IVD. IL-1B was able to increase catabolic markers and angiogenic factors and decrease matrix proteins. Such activity was confirmed by the explant culture measurements. The SA identified TGF-β and IL1RA as the two most powerful rescue mediators. Accordingly, TGFβ signaling-based IDD treatments have been proposed and IL-1RA gene therapy diminished the expression of proteases. It resulted challenging to simulate rescue strategies by IL-10, but interestingly, IL-1B could not induce IL-10 expression in the explant cultures. Our RNM was confronted to independent in vitro measurements and stands for a unique model, to integrate soluble protein signaling and explore IDD.

Acknowledgements: European Commission (Disc4All-ITN-ETN-955735)

The Global Burden of Disease Study 2019 showed a 33.4% increase in fractures and a 65.3% increase in Years lived with disability (YLD) since 1990. Although the overall rate of fracture related infection (FRI) is low, it increases to 30% in complex fractures. In addition, the implantation of foreign materials, such as fracture stabilizing implants, decreases the number of bacteria needed to cause an infection. Then, when infections do occur, they are difficult to treat and often require multiple surgeries to heal. The bacteria can persist in the canaliculi of the bony tissue, in cells, in a biofilm on material or necrotic bone or in abscess communities. In the last decades, different approaches have been pursued to modify biomaterials as well as implant surface and to develop antimicrobial surfaces or local drug release strategies. This talk will give an introduction to the problem of bony and implant associated infections and presents the development and preclinical (as well as clinical) studies of two approaches for local drug delivery.

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

In orthopedic surgery, implant infections are a serious issue and difficult to treat. The aim of this study was to use superparamagnetic nanoporous silica nanoparticles (MNPSNP) as candidates for directed drug delivery. Currently, short blood circulation half-life due to interactions with the host's immune system hinder nanoparticles in general from being clinically used. PEGylation is an approach to reduce these interactions and to enhance blood circulation time. The effect of PEGylation of the used ⁶⁸Ga-labelled MNPSNP on the distribution and implant accumulation was examined by PET/CT imaging and gamma counting in an implant mouse model.

Female Balb/c mice (n=24) received a magnetic implant subcutaneously on the left and a titanium implant on the right hind leg. On day one, 12 of these mice received an additional clodronate®-injection for macrophage depletion. On the second postoperative day, mice were anaesthetized and MNPSNP (native or PEGylated) injected intravenously, followed by a dynamic PET-scan over 60 minutes, a CT- and a static PET-scan at 120 min. As control, 12 mice received only ⁶⁸Ga-MNPSNP (native or PEGylated). Gamma counting of inner organs, urine, blood and implant area was performed as further final analysis. Although PEGylation of the nanoparticles already resulted in lower liver uptakes, both variants of ⁶⁸Ga-labeled MNPSNP accumulated in liver and spleen. Combination of PEGylation with clodronate®-injection led to a highly significant effect whereas clodronate®-injection alone could not reveal significant differences. In gamma counting, a significantly higher %I.D./g was found for the tissue surrounding the magnetic implants compared to the titanium control, although in a low range. PEGylation and/or clodronate®-injection revealed no significant differences regarding nanoparticle accumulation at the implantation site. PEGylation increases circulation time, but MNPSNP accumulation at the implant site was still insufficient for treatment of infections. Additional efforts have to further increase circulation time and local accumulation.

Acknowledgements: This work is funded by the German Research Foundation (DFG, project number 280642759).

Total hip arthroplasty (THA) outcome in patients with osteonecrosis of the femoral head ONFH) are excellent, however, there is controversy when compared with those in patients with osteoarthritis (OA). Reduced mineralization capacity of osteoblasts of the proximal femur in patients with ONFH could affect implant fixation.

We asked if THA fixation in patients with ONFH is worse than in those with OA.

We carried out a prospective comparative case (OA)-control (ONFH) study of patients undergoing THA at our hospital between 2017 and 2019. The minimum follow-up was 2 years. Inclusion criteria were patients with uncemented THA, younger than 70 years old, a Dorr femoral type C and idiopathic ONFH. We compared the clinical (Merlé D'Aubigné-Postel score) and radiological results related with implant positioning and fixation. Engh criteria and subsidence were assessed at the immediate postoperative, 12 weeks, 6 months, 12 months and yearly. Osteoblastic activity was determined by mineralization assay on primary cultures of osteoblasts isolated from trabecular bone samples collected from the intertrochanteric area obtained during surgery.

Group 1 (ONFH) included 18 patients and group 2 (OA), 22. Average age was 55.9 years old in group 1 and 61.3 in group 2. (p=0.08). There were no differences related with sex, Dorr femoral type or femoral filling. The mean clinical outcome score was 17.1 in group 1 and 16.5 in group 2 (p=0.03). There were no cases of dislocation, infection, or revision surgery in this series. There were 5 cases (28%) of femoral stem subsidence greater than 3mm within 6 first months in group 1 and 1 case (4.5%) in group 2 (p=0.05).

Although there were no significant differences related to clinical results, bone fixation was slower, and a greater subsidence was observed in patients with ONFH. Greater femoral stem subsidence was associated with a lower capacity for mineral nodule formation in cultured osteoblasts. The surgical technique could influence THA outcome in patients with reduced mineralization capacity of osteoblasts.

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

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

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

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

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

The ability of the body to constantly maintain metabolism homeostasis while fulling the heightened energy and macromolecule demand is crucial to ensure successful tissue healing outcomes. Studies investigating the local metabolic environment during healing are scarce to date. Here, using Type 2 Diabetes (T2D) as a study model, we investigate the impact of metabolism dysregulation on scaffold-guided large-volume bone regeneration. Our study treated wild-type or T2D rats with 5 mm critical-sized femoral defects with 3D-printed polycaprolactone (PCL) scaffolds with 70% porosity. Metabolomics was leveraged for a holistic view of metabolism alteration as healing progress and correlated to regenerated bone tissue volume and quality assessed using micro-computed tomography (µ-CT), histology, and immunohistology. Semi-targeted metabolomics analysis indicated dysregulation in the glycolysis and TCA cycle – the main energy production pathways, in T2D compared to healthy animals. The abundance of metabolites substrates, i.e., amino acids – for protein/ extracellular matrix synthesis was also affected in T2D. Tissue-level metabolites observations aligned with morphological observation with less newly formed bone observed in T2D than wild-type rats. This study enlightens the metabolism landscape during scaffold-guided large-volume bone regeneration in wild-type vs. T2D to further guide the personalization of the scaffold to drive successful regeneration.

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

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

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

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

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

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

Mesenchymal stem cells (MSCs) have been studied for the treatment of Osteoarthritis (OA), a potential mechanism of MSC therapies has been attributed to paracrine activity, in which extracellular vesicles (EVs) may play a major role. It is suggested that MSCs from younger donor compete with adult MSC in their EV production capabilities. Therefore, MSCs generated from induced pluripotent mesenchymal stem cells (iMSC) appear to provide a promising source. In this study, MSCs and iMSC during long term-expansion using a serum free clinical grade condition, were characterized for surface expression pattern, proliferation and differentiation capacity, and senescence rate. Culture media were collected continuously during cell expansion, and EVs were isolated. Nanoparticle tracking analysis (NTA), transmission electron microscopy, western blots, and flow cytometry were used to identify EVs. We evaluated the biological effects of MSC and iMSC-derived EVs on human chondrocytes treated with IL-1α, to mimic the OA environment.

In both cell types, from early to late passages, the amount of EVs detected by NTA increased significantly, EVs collected during cells expansion, retained tetraspanins (CD9, CD63 and CD81) expression. The anti-inflammatory activity of MSC-EVs was evaluated in vitro using OA chondrocytes, the expression of IL-6, IL-8 and COX-2 was significantly reduced after the treatment with hMSC-derived EVs isolated at early passage. The miRNA content of EVs was also investigated, we identify miRNA that are involved in specific biological function.

At the same time, we defined the best culture conditions to maintain iMSC and define the best time window in which to isolate EVs with highest biological activity.

In conclusion, a clinical grade serum-free medium was found to be suitable for the isolation and expansion of MSCs and iMSC with increased EVs production for therapeutic applications.

Acknowledgments: This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 874671

Mesenchymal stem cells (MSC) have been used for bone regenerative applications as an alternative approach to bone grafting. Selecting the appropriate source of MSC is vital for the success of this therapeutic approach. MSC can be obtained from various tissues, but the most used sources of MSC are Bone marrow (BMSC), followed by adipose tissue (ASC). A donor-matched comparison of these two sources of MSC ensures robust and reliable results.

Despite the similarities in morphology and immunophenotype of donor-matched ASC and BMSC, differences existed in their proliferation and in vitro differentiation potential, particularly osteogenic differentiation that was superior for BMSC, compared to ASC. However, these differences were substantially influenced by donor variations. In vivo, although the upregulated expression of osteogenesis-related genes in both ASC and BMSC, more bone was regenerated in the calvarial defects treated with BMSC compared to ASC, especially during the initial period of healing. According to these findings, compared to ASC, BMSC may result in faster regeneration and healing, when used for bone regenerative applications.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Fractures and related complications are a common challenge in the field of skeletal tissue engineering. Vitamin D and calcium are the only broadly available medications for fracture healing, while zinc has been recognized as a nutritional supplement for healthy bones. Here, we aimed to use polaprezinc, an anti-ulcer drug and a chelate form of zinc and L-carnosine, as a supplement for fracture healing. Polaprezinc induced upregulation of osteogenesis-related genes and enhanced the osteogenic potential of human bone marrow-derived mesenchymal stem cells and osteoclast differentiation potential of mouse bone marrow-derived monocytes. In mouse experimental models with bone fractures, oral administration of polaprezinc accelerated fracture healing and maintained a high number of both osteoblasts and osteoclasts in the fracture areas. Collectively, polaprezinc promotes the fracture healing process efficiently by enhancing the activity of both osteoblasts and osteoclasts. Therefore, we suggest that drug repositioning of polaprezinc would be helpful for patients with fractures.

Biphasic calcium phosphate (BCP) with a characteristic needle-shaped submicron surface topography (MagnetOs) has attracted much attention due to its unique bone-forming ability which is essential for repairing critical-size bone defects such as those found in the posterolateral spine. Previous in vitro and ex-vivo data performed by van Dijk LA and Yuan H demonstrated that these specific surface characteristics drive a favorable response from the innate immune system.

This study aimed to evaluate and compare the in vivo performance of three commercially-available synthetic bone grafts, (1) i-FACTOR Putty^®, (2) OssDsign^® Catalyst Putty and (3) FIBERGRAFT^® BG Matrix, with that of a novel synthetic bone graft in a clinically-relevant instrumented sheep posterolateral lumbar spine fusion (PLF) model. The novel synthetic bone graft comprised of BCP granules with a needle-shaped submicron surface topography (MagnetOs) embedded in a highly porous and fibrillar collagen matrix (MagnetOs Flex Matrix).

Four synthetic bone grafts were implanted as standalone in an instrumented sheep PLF model for 12 weeks (n=3 bilateral levels per group; levels L2/3 & L4/5), after which spinal fusion was determined by manual palpation, radiograph and µCT imaging (based on the Lenke scale), range-of-motion mechanical testing, and histological and histomorphological evaluation.

Radiographic fusion assessment determined bilateral robust bone bridging (Lenke scale A) in 3/3 levels for MagnetOs Flex Matrix compared to 1/3 for all other groups. For µCT, bilateral fusion (Lenke scale A) was found in 2/3 levels for MagnetOs Flex Matrix, compared to 0/3 for i-FACTOR Putty^®, 1/3 for OssDsign^® Catalyst Putty and 0/3 for FIBERGRAFT^® BG Matrix. Fusion assessment for MagnetOs Flex Matrix was further substantiated by histology which revealed significant graft resorption complemented by abundant bone tissue and continuous bony bridging between vertebral transverse processes resulting in bilateral spinal fusion in 3/3 implants.

These results show that MagnetOs Flex Matrix achieved better fusion rates compared to three commercially-available synthetic bone grafts when used as a standalone in a clinically-relevant instrumented sheep PLF model.

The use of intraoperative navigation and robotic surgery for minimally invasive lumbar fusion has been increasing over the past decade. The aim of this study is to evaluate postoperative clinical outcomes, intraoperative parameters, and accuracy of pedicle screw insertion guided by intraoperative navigation in patients undergoing lumbar interbody fusion for spondylolisthesis. Patients who underwent posterior lumbar fusion interbody using intraoperative 3D navigation since December 2021 were included. Visual Analogue Scale (VAS), Oswestry Disability Index (ODI), and Short Form Health Survey-36 (SF-36) were assessed preoperatively and postoperatively at 1, 3, and 6 months. Screw placement accuracy, measured by Gertzbein and Robbins classification, and facet joint infringement, measured by Yson classification, were assessed by intraoperative Cone Beam CT scans performed at the end of instrumentation. Finally, operation time, intraoperative blood loss, hospital stay, and screw insertion time were evaluated. This study involved 50 patients with a mean age of 63.7 years. VAS decreased from 65.8±23 to 20±22 (p<.01). ODI decreased from 35.4%±15 to 11.8%±14 (p<.01). An increase of SF-36 from 51.5±14 to 76±13 (p<.01) was demonstrated. The accuracy of “perfect” and “clinically acceptable” pedicle screw fixation was 89.5% and 98.4%, respectively. Regarding facet violation, 96.8% of the screws were at grade 0. Finally, the average screw insertion time was 4.3±2 min, hospital stay was 4.2±0.8 days, operation time was 205±53 min, and blood loss was 169±107 ml. Finally, a statistically significant correlation of operation time with hospital stay, blood loss and placement time per screw was found. We demonstrated excellent results for accuracy of pedicle screw fixation and violation of facet joints. VAS, ODI and SF-36 showed statistically significant improvements from the control at one month after surgery.

Navigation with intraoperative 3D images represents an effective system to improve operative performance in the surgical treatment of spondylolisthesis.

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

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

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

Dupuytren's disease (DD) is a fibroproliferative soft tissue disease affecting the palmar fascia of the hand causing permanent and irreversible flexion contracture. Aberrant fibrosis is likely to manifest through a combination of extrinsic, intrinsic, and environmental factors, including genetics and epigenetics. However, the role of epigenetics in soft tissue fibrosis in diseases such as DD is not well established. Therefore, we conducted a comprehensive multi-omic study investigating the epigenetic profiles that influence gene expression in DD pathology. Using control (patients undergoing carpal tunnel release) and diseased fibroblasts (patients undergoing Dupuytren's fasciectomy), we conducted ATAC-seq to assess differential chromatin accessibility between control and diseased fibroblasts. Additionally, ChIP-seq mapped common histone modifications (histone H4; H3K4me3, H3K9me3, H3K27me3, H4K16Ac, H4K20Me3) associated with fibrosis. Furthermore, we extracted RNA from control and DD tissue and performed bulk RNA-seq.

ATAC-seq analysis identified 2470 accessible genomic loci significantly more accessible in diseased fibroblasts compared to control. Comparison between diseased and control cells identified numerous significantly different peaks in histone modifications (H4K20me3, H3K27me3, H3K9me3) associated with gene repression in control cells but not in diseased cells. Pathway analysis demonstrated a substantial overlap in genes being de-repressed across these histone modifications (Figure 1). Both, ATAC-seq and ChIP-seq analysis indicated pathways such as cell adhesion, differentiation, and extracellular matrix organisation were dysregulated as a result of epigenetic changes. Moreover, de novo motif enrichment analysis identified transcription factors that possibly contributed to the differential gene expression between control and diseased tissue, including HIC1, NFATC1 and TEAD2. RNA-seq analysis found that these transcription factors were upregulated in DD tissue compared to control tissue.

The current epigenetic study provides insights into the aberrant fibrotic processes associated with soft tissue diseases such as DD and indicates that epigenetic-targeted therapies may be an interesting viable treatment option in future.

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

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

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

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

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

Currently implemented accuracy metrics in open-source libraries for segmentation by supervised machine learning are typically one-dimensional scores [1]. While extremely relevant to evaluate applicability in clinics, anatomical location of segmentation errors is often neglected.

This study aims to include the three-dimensional (3D) spatial information in the development of a novel framework for segmentation accuracy evaluation and comparison between different methods.

Predicted and ground truth (manually segmented) segmentation masks are meshed into 3D surfaces. A template mesh of the same anatomical structure is then registered to all ground truth 3D surfaces. This ensures all surface points on the ground truth meshes to be in the same anatomically homologous order. Next, point-wise surface deviations between the registered ground truth mesh and the meshed segmentation prediction are calculated and allow for color plotting of point-wise descriptive statistics. Statistical parametric mapping includes point-wise false discovery rate (FDR) adjusted p-values (also referred to as q-values).

The framework reads volumetric image data containing the segmentation masks of both ground truth and segmentation prediction. 3D color plots containing descriptive statistics (mean absolute value, maximal value,…) on point-wise segmentation errors are rendered. As an example, we compared segmentation results of nnUNet [2], UNet++ [3] and UNETR [4] by visualizing the mean absolute error (surface deviation from ground truth) as a color plot on the 3D model of bone and cartilage of the mean distal femur.

A novel framework to evaluate segmentation accuracy is presented. Output includes anatomical information on the segmentation errors, as well as point-wise comparative statistics on different segmentation algorithms. Clearly, this allows for a better informed decision-making process when selecting the best algorithm for a specific clinical application.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Extracellular matrix (ECM) mechanical cues guide healing in tendons. Yet, the molecular mechanisms orchestrating the healing processes remain elusive. Appropriate tissue tension is essential for tendon homeostasis and tissue health. By mapping the attainment of tensional homeostasis, we aim to understand how ECM tension regulates healing. We hypothesize that diseased tendon returns to homeostasis only after the cells reach a mechanically gated exit from wound healing.

We engineered a 3D mechano-culture system to create tendon-like constructs by embedding patient-derived tendon cells into a collagen I hydrogel. Casting the hydrogel between posts anchored in silicone allowed adjusting the post stiffness. Under this static mechanical stimulation, cells remodel the (unorganized) collagen representing wound healing mechanisms. We quantified tissue-level forces using post deflection measurements. Secreted ECM was visualized by metabolic labelling with non-canonical amino acids, click chemistry and confocal microscopy. We blocked cell-mediated actin-myosin contractility using a ROCK inhibitor (Y27632) to explore the involvement of the Rho/ROCK pathway in tension regulation.

Tissue tension forces reached the same homeostatic level at day 21 independent of post compliance (p = 0.9456). While minimal matrix was synthesized in early phases of tissue formation (d3-d5), cell-deposited ECM was present in later stages (d7-d9). More ECM was deposited by tendon constructs cultured on compliant (1Nm) compared to rigid posts (p = 0.0017). Matrix synthesized by constructs cultured on compliant posts was less aligned (greater fiber dispersion, p = 0.0021). ROCK inhibition significantly decreased tissue-level tensional forces (p < 0.0001).

Our results indicate that tendon cells balance matrix remodeling and synthesis during tissue repair to reach an intrinsically defined “mechanostat setpoint” guiding tension-mediated exit from wound healing towards homeostasis. We are identifying specific molecular mechanosensors governing tension-regulated healing in tendon and investigate the Rho/ROCK system as their possible downstream pathway.

Osteoarthritis (OA) is the leading cause of pain and disability worldwide and is characterized by the degenerative changes of articular cartilage. Joint loading is required for cartilage maintenance; however, hyper-physiologic loading is a risk factor for OA. Mechanosensitive ion channels Piezo1 and Piezo2 synergistically transduce hyper-physiologic compression of chondrocytes, leading to chondrocyte death and onset of OA. This injury response is inhibited by Piezo channel loss of function, however the mechanistic role of Piezo channels in vivo is unknown. We examined the hypothesis that deletion of Piezo in chondrocytes will protect mice from joint damage and pain-related behaviors following a surgical destabilization of the medial meniscus (DMM), investigating a key mechanistic and mechanobiological role of these channels in the pathogenesis of OA.

Aggrecan-Cre Piezo1 and Piezo1/2 knockout mice ((Agc)1-CRE^ERT2;Piezo1^fl/flPiezo2^fl/fl) were generated and given a 5-day Tamoxifen regimen at 12-weeks of age (n=6–12/group/sex). Cre-negative mice served as controls. At 16-weeks, mice received DMM surgery on the left knee. 12-weeks following DMM prior to sacrifice, activity and hyperalgesia were measured using spontaneous running wheels and a small animal algometer. Structural changes in bone, cartilage, and synovium were characterized using microCT, histology, and Modified Mankin Score criteria.

Knockout of Piezo1/2 channels was chondroprotective in both sexes following DMM surgery as demonstrated by reduced Modified Mankin Score compared to control animals. Piezo1 KO was chondroprotective in only female mice, indicating a sexually dimorphic response. Piezo1 and Piezo1/2 KO was protective against pain in male mice, while females displayed no differences compared to controls. No changes were observed in bone morphology.

Chondrocyte-specific Piezo1/2 knockout protects the knee joint from structural damage, hyperalgesia and functional deficits in a surgical model of PTOA in male and female mice, illustrating the importance of Piezo channels in response to injury in vivo. Future work aims to interrogate potential sexually dimorphic responses to cartilage damage and investigating Piezo2 KO mice.

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Abstract

Approximately 20% of primary and revision Total Knee Arthroplasty (TKA) patients require multiple revisions, which are associated with poor survivorship, with worsening outcomes for subsequent revisions. For revision surgery, either endoprosthetic replacements or metaphyseal sleeves can be used for the repair, however, in cases of severe defects that are deemed “too severe” for reconstruction, endoprosthetic replacement of the affected area is recommended. However, endoprosthetic replacements have been associated with high complication rates (high incidence rates of prosthetic joint infection), while metaphyseal sleeves have a more acceptable complication profile and are therefore preferred. Despite this, no guidance exists as to the maximal limit of bone loss, which is acceptable for the use of metaphyseal sleeves to ensure sufficient axial and rotational stability. Therefore, this study assessed the effect of increasing bone loss on the primary stability of the metaphyseal sleeve in the proximal tibia to determine the maximal bone loss that retains axial and rotational stability comparable to a no defect control.

Abstract

Abstract