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

Osteoarthritis (OA) is a common age-related degenerative joint disease, affecting 7% of the global population, more than 500 million people worldwide. Exosomes from mesenchymal stem cells (MSCs) showed promise for OA treatment, but the insufficient biological targeting weakens its efficacy and might bring side effects. Here, we report the chondrocyte-targeted exosomes synthesized via click chemistry as a novel treatment for OA. Exosomes are isolated from human umbilical cord-derived MSCs (hUC-MSCs) using multistep ultracentrifugation process, and identified by electron microscope and nanoparticle tracking analysis (NTA). Chondrocyte affinity peptide (CAP) is conjugated on the surface of exosomes using click chemistry. For tracking, nontagged exosomes and CAP-exosomes are labeled by Dil, a fluorescent dye that highlights the lipid membrane of exosomes. To verify the effects of CAP-exosomes, nontagged exosomes and CAP-exosomes are added into the culture medium of interleukin (IL)-1β-induced chondrocytes. Immunofluorescence are used to test the expression of matrix metalloproteinase (MMP)-13. CAP-exosomes, compared with nontagged exosomes, are more easily absorbed by chondrocytes. What's more, CAP-exosomes induced lower MMP-13 expression of chondrocytes when compared with nontagged exosomes (p<0.001). CAP-exosomes show chondrocyte-targeting and exert better protective effect than nontagged exosomes on chondrocyte extracellular matrix. Histological and in vivo validation are now being conducted

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

Osteoarthritis, the most common degenerative joint disease, significantly impairs life quality and labor capability of patients. Synovial inflammation, initiated by HMGB1 (High mobility group box 1)-induced activation of macrophage, precedes other pathological changes. As an upstream regulator of NF-κB (nuclear factor-kappa B) and MAPK (mitogen-activated protein kinase) signaling pathway, TAK1 (TGF-β activated kinase 1) participates in macrophage activation, while its function in osteoarthritis remains unveiled. This study aims to investigate the role of TAK1 in the pathogenesis of osteoarthritis via both in vitro and in vivo approaches. We performed immunohistochemical staining for TAK1 in synovial tissue, both in osteoarthritis patients and healthy control. Besides, immunofluorescence staining for F4/80 as macrophage marker and TAK1 were conducted as well. TAK1 expression was examined in RAW264.7 macrophages stimulated by HMGB1 via qPCR (Quantitative polymerase chain reaction) and Western blotting, and the effect of TAK1 inhibitor (5z-7 oxozeaenol) on TNF-α production was evaluated by immunofluorescence staining. Further, we explored the influence of intra-articular shRNA (short hairpin RNA) targeting TAK1 on collagenase-induced osteoarthritis in mice. Immunohistochemical staining confirmed significant elevation of TAK1 in osteoarthritic synovium, and immunofluorescence staining suggested macrophages as predominant residence of TAK1. In HMGB1-stimulated RAW264.7 macrophages, TAK1 expression was up-regulated both in mRNA and protein level. Besides, TAK1 inhibitor significantly impairs the production of TNF-α by macrophages upon HMGB1 stimulation. Moreover, intra-articular injection of lentivirus loaded with shRNA targeting TAK1 (sh-TAK1) reduced peri-articular osteophyte formation in collagenase-induced osteoarthritis in mice. TAK1 exerts a potent role in the pathogenesis of osteoarthritis by mediating the activation of macrophages

Osteoarthritis (OA) is a degenerative joint disease affecting millions worldwide. Early detection of OA and monitoring its progression is essential for effective treatment and for preventing irreversible damage. Although sensors have emerged as a promising tool for monitoring analytes in patients, their application for monitoring the state of pathology is currently restricted to specific fields (such as diabetes). In this study, we present the development of an optical sensor system for real-time monitoring of inflammation based on the measurement of nitric oxide (NO), a molecule highly produced in tissues during inflammation. Single-walled carbon nanotubes (SWCNT) were functionalized with a single-stranded DNA (ssDNA) wrapping designed using an artificial intelligence approach and tested using S-nitroso-N-acetyl penicillamine (SNAP) as a standard released-NO marker. An optical SWIR reader with LED excitation at 650 nm, 730 nm and detecting emission above 1000 nm was developed to read the fluorescence signal from the SWCNTs. Finally, the SWCNT was embedded in GelMa to prove the feasibility of monitoring the release of NO in bovine chondrocyte and osteochondral inflamed cultures (1–10 ng/ml IL1β) monitored over 48 hours. The stability of the inflammation model and NO release was indirectly validated using the Griess and DAF-FM methods. A microfabricated sensor tag was developed to explore the possibility of using ssDNA-SWCNT in an ex vivo anatomic set-up for surgical feasibility, the limit of detection, and the stability under dynamic flexion. The SWCNT sensor was sensitive to NO in both in silico and in vitro conditions during the inflammatory response from chondrocyte and osteochondral plug cultures. The fluorescence signal decreased in the inflamed group compared to control, indicating increased NO concentration. The micro-tag was suitable and stable in joints showing a readable signal at a depth of up to 6 mm under the skin. The ssDNA-SWCNT technology showed the possibility of monitoring inflammation continuously in an in vitro set-up and good stability inside the joint. However, further studies in vivo are needed to prove the possibility of monitoring disease progression and treatment efficacy in vivo. Acknowledgments: The project was co-financed by Innosuisse (grant nr. 56034.1 IP-LS)

Osteoarthritis (OA) is the most common degenerative joint disorder. Its multifactorial etiology includes age, sex, joint overloading, genetic or nervous influences. In particular, the autonomic nervous system is increasingly gaining in importance. Its two branches, the sympathetic (SNS) and parasympathetic nervous system, are well-balanced under healthy conditions. OA patients seem to be prone to an autonomic imbalance and therefore, we analyzed their autonomic status. More than 200 participants including patients with early and late stage knee OA (before and 1 year after knee replacement surgery) and healthy probands (age-matched) were analyzed. Heart rate variability was measured via electrocardiogram to assess long-term sympathetic (low-frequency=LF) and parasympathetic (high-frequency=HF, pRR50) activities or general variability (RMSSD, SDRR). Serum cortisol concentrations were measured by ELISA. Perceived chronic stress (PSQ) was assessed via questionnaire. Multivariant regression was performed for data analysis. LF/HF value of early OA was slightly increased compared to healthy controls but significantly higher compared to late OA patients before (p>0.05) and after TKR (p>0.01). HF in late OA patients before TKR was significantly decreased compared to patients after TKR (p>0.001) or healthy controls (p>0.05). Healthy probands exhibited the highest SDRR values, early OA patients had slightly lower levels and late OA patients before TKR displayed significantly reduced SDRR (p>0.001). The same differences were observed in pRR50 and RMSSD. Serum cortisol concentrations and PSQ scores increased in late OA patients before TKR. At the time point of TKR, women with beta blocker medication had significantly higher age (71 ± 9 years) than those without (63 ± 12 years)(p>0.01). An autonomic dysfunction with sympathetic dominance occurs in OA patients. The fact that beta blocker medication in women delayed the need of TKR indicates that SNS inhibition might counteract OA. Future therapeutic interventions for OA should consider a systemic approach with special regard on the ANS

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

Abstract. Introduction. Precision health aims to develop personalised and proactive strategies for predicting, preventing, and treating complex diseases such as osteoarthritis (OA), a degenerative joint disease affecting over 300 million people worldwide. Due to OA heterogeneity, which makes developing effective treatments challenging, identifying patients at risk for accelerated disease progression is essential for efficient clinical trial design and new treatment target discovery and development. Objectives. This study aims to create a trustworthy and interpretable precision health tool that predicts rapid knee OA progression based on baseline patient characteristics using an advanced automated machine learning (autoML) framework, “Autoprognosis 2.0”. Methods. All available 2-year follow-up periods of 600 patients from the FNIH OA Biomarker Consortium were analysed using “Autoprognosis 2.0” in two separate approaches, with distinct definitions of clinical outcomes: multi-class predictions (categorising patients into non-progressors, pain-only progressors, radiographic-only progressors, and both pain and radiographic progressors) and binary predictions (categorising patients into non-progressors and progressors). Models were developed using a training set of 1352 instances and all available variables (including clinical, X-ray, MRI, and biochemical features), and validated through both stratified 10-fold cross-validation and hold-out validation on a testing set of 339 instances. Model performance was assessed using multiple evaluation metrics, such as AUC-ROC, AUC-PRC, F1-score, precision, and recall. Additionally, interpretability analyses were carried out to identify important predictors of rapid disease progression. Results. Our final models yielded high accuracy scores for both multi-class predictions (AUC-ROC: 0.858, 95% CI: 0.856–0.860; AUC-PRC: 0.675, 95% CI: 0.671–0.679; F1-score: 0.560, 95% CI: 0.554–0.566) and binary predictions (AUC-ROC: 0.717, 95% CI: 0.712–0.722; AUC-PRC: 0.620, 95% CI: 0.616–0.624; F1-score: 0.676, 95% CI: 0.673–0679). Important predictors of rapid disease progression included the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores and MRI features. Our models were further successfully validated using a hold-out dataset, which was previously omitted from model development and training (AUC-ROC: 0.877 for multi-class predictions; AUC-ROC: 0.746 for binary predictions). Additionally, accurate ML models were developed for predicting OA progression in a subgroup of patients aged 65 or younger (AUC-ROC: 0.862, 95% CI: 0.861–0.863 for multi-class predictions; AUC-ROC: 0.736, 95% CI: 0.734–0.738 for binary predictions). Conclusions. This study presents a reliable and interpretable precision health tool for predicting rapid knee OA progression using “Autoprognosis 2.0”. Our models provide accurate predictions and offer insights into important predictors of rapid disease progression. Furthermore, the transparency and interpretability of our methods may facilitate their acceptance by clinicians and patients, enabling effective utilisation in clinical practice. Future work should focus on refining these models by increasing the sample size, integrating additional features, and using independent datasets for external validation. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Abstract. Objectives. Current use of hard biomaterials such as cobalt-chrome alloys or ceramics to articulate against the relatively soft, compliant native cartilage surface reduces the joint contact area by up to two thirds. This gives rise to high and abnormal loading conditions which promotes degradation and erosion of the mating cartilage leading to pain, stiffness, and loss of function. Biomimetic soft lubrication strategies have been developed by grafting hydrophilic polymers onto substrates to form a gel-type surface. Surface grafted gels mimic the natural mechanisms of friction dissipation in synovial joints, showing a promising potential for use in hemiarthroplasty. This project aims to develop implant surfaces with properties tailored to match articular cartilage to retain and promote natural joint function ahead of total joint replacement. Methods. Four different types of monomers were grafted in a one-step photopolymerisation procedure onto polished PEEK substrates. The functionalised surfaces were investigated using surface wettability, FTIR, and simplified 2D-tribometry tests against glass and animal cartilage specimens to assess their lubricity and mechanical properties for hemiarthroplasty articulations. Results. Polymer functionalised surfaces under different grafting conditions were assessed for their wettability, graft density and quality. A reduction in water contact angle from 90° to < 20° was seen for functionalised highly hydrophilic PEEK surfaces. Similarly a reduction in the coefficient of friction (and subsequently shear stresses acting on cartilage) of 95% to ∼ 10. −2. was seen for functionalised PEEK surfaces slid against glass and cartilage in PBS. Conclusions. Development of this technology has the potential to vastly improve the performance of hemiarthroplasty. Providing earlier and targeted interventions for degenerative joint disease whilst preserving the function of the remaining healthy cartilage. Future work will concern using these promising hydrated functionalised surface architectures as focal cartilage deflects plugs along with long-term performance and suitability for implantation assessments using joint simulator testing

Introduction. Articular cartilage injuries have a limited potential to heal and, over time, may lead to osteoarthritis, an inflammatory and degenerative joint disease associated with activity-related pain, swelling, and impaired mobility. Regeneration and restoration of the joint tissue functionality remain unmet challenges. Stem cell-based tissue engineering is a promising paradigm to treat cartilage degeneration. In this context, hydrogels have emerged as promising biomaterials, due to their biocompatibility, ability to mimic the tissue extracellular matrix and excellent permeability. Different stimulation strategies have been investigated to guarantee proper conditions for mesenchymal stem cell differentiation into chondrocytes, including growth factors, cell-cell interactions, and biomaterials. An interesting tool to facilitate chondrogenesis is external ultrasound stimulation. In particular, low-intensity pulsed ultrasound (LIPUS) has been demonstrated to have a role in regulating the differentiation of adipose mesenchymal stromal cells (ASCs). However, chondrogenic differentiation of ASCs has been never associated to a precisely measured ultrasound dose. In this study, we aimed to investigate whether dose-controlled LIPUS is able to influence chondrogenic differentiation of ASCs embedded in a 3D hydrogel. Materials and Methods. Human adipose mesenchymal stromal cells at 2∗10. 6. cells/mL were embedded in a hydrogel ratio 1:2 (VitroGel RGD®) and exposed to LIPUS stimulation (frequency: 1 MHz, intensity: 250 mW/cm. 2. , duty cycle: 20%, pulse repetition frequency: 1 kHz, stimulation time: 5 min) in order to assess its influence on cell differentiation. Hydrogel-loaded ASCs were cultured and differentiated for 2, 7, 10 and 28 days. At each time point cell viability (Live&Dead), metabolic activity (Alamar Blue), cytotoxicity (LDH), gene expression (COL2, aggrecan, SOX9, and COL1), histology and immunohistochemistry (COL2, aggrecan, SOX9, and COL1) were evaluated respect to a non-stimulated control. Results. Histological analysis evidenced a uniform distribution of ASCs both at the periphery and at the center of the hydrogel. Live & Dead test evidenced that the encapsulated ASCs were viable, with no signs of cytotoxicity. We found that LIPUS induced chondrogenesis of ASCs embedded in the hydrogel, as demonstrated by increased expression of COL2, aggrecan and SOX9 genes and proteins, and decreased expression of COL1 respect to the non-stimulated control. Conclusions. These results suggest that the LIPUS treatment could be a valuable tool in cartilage tissue engineering, to push the differentiation of ASCs encapsulated in a 3D hydrogel

Introduction and Objective. Osteoarthritis of the knee joint is common in old age population in every part of world. Pain is the major source of disability in patients with osteoarthritis of the knee joint. Subchondral bone marrow is richly innervated with nociceptive pain fibers and may be a source of pain in patients with symptomatic degenerative joint disease. Current therapy for managing bone marrow oedema is core decompression (CD), combining core decompression and injection of hydroxyapatite cement or autologus chondrocyte supplementtion. But all of this work has been done in femoral head and authors documented good result with minimal complication. There are various studies in literature suggesting treatment to repair BME by restoring support and relieving abnormal stresses with accepted internal fixation and bone stimulating surgical techniques in relieving knee OA pain. In this study, we present efficacy of knee arthroscopy with adjunctive core decompression and supplementation with structural scaffold to improve self-rated visual analog scale (VAS) pain scores, rate of conversion to arthroplasty, and patient satisfaction levels. Materials and Methods. The study included patients aged between 40 and 75 years old, with pain in the knee for at least six months, associated with high-signal MRI lesion on T2 sequences, on the tibia or femur. Trephine was used as the bone decompression instrument. Trephine has a diameter of 8–10 mm and operation with trephine requires that a cortical incision window be made prior to decompression treatment, thus necessitating strict disinfection. This procedure was done under spinal anesthesia. After diagnostic arthroscopy, decompression was done under C –ARM in desired area on MRI. After decompression, defect was filled with Poly ester urea's scaffold impregnated with BMAC. Results. Patients were assessed using the visual analog pain scale and the KOOS score, one week before surgery and one, three, six, 12, and 24 weeks after the procedure. MRI images were analyzed Lesions were mapped and measured in the axial, coronal, and sagittal views to plan the injection site and the trajectory of the cannula used for the procedure. Radiographs using anteroposterior, profile, and Rosenberg views of the knee and lower limb were performed to classify the lesion according to the Kellgren-Lawrence classification and to assess lower limb alignment. Evaluation using the KOOS showed a mean total score in the preoperative period of 38.44 points and of 60.7, 59.08, 56.92, 64.40, and 71.36 points at one, three, six, 12, and 24 weeks after surgery, respectively. In the VAS assessment, mean was 7.8 points preoperatively and 2.8, 2.6, 2.5, 1.3, and 0.5 points in the same periods. Conclusions. Hence it can be Concluded that this new innovative technique has provided significant improvements in the parameters of pain and functional capacity in the short-term assessment

Introduction and Objective. Osteoarthritis (OA) is the most common inflammatory and degenerative joint disease. Mesenchymal Stromal Cells (MSCs), with their chondro-protective and immune-regulatory properties, have been considered as a new approach to treat OA. Considering the risk of cell leakage outside the articular space and the poor survival rate after intra-articular (IA) injection, we hypothesized that cell encapsulation in cytoprotective hydrogels could overcome these limitations and provide cells with a suitable 3D microenvironment supporting their biological activity. We previously generated micromolded alginate particles (diameter 150 μm) and demonstrated the long-term viability of microencapsulated MSCs isolated from human adipose tissue (hASCs). Encapsulated cells maintained their in vitro ability to sense and respond to a pro-inflammatory environment (IFN-γ/TNF-α or synovial fluids from OA patients) by secreting PGE. 2. , IDO, HGF and TGF-β. In this study, we evaluated the anti-OA efficacy of these microencapsulated hASCs in a post-traumatic OA model in rabbits. Materials and Methods. OA was surgically induced by anterior cruciate ligament transection (ACLT)-mediated destabilization of the right knee in rabbits (n=24). Eight weeks after surgery, destabilized joints were injected (IA, 26G needle) with 200 μL of either PBS, blank microparticles, non-encapsulated or microencapsulated cells (5×10. 5. cells). Six weeks after injection, rabbits were euthanized and all destabilized (right) and sham-operated (left contralateral) joints were dissected and analyzed for OA severity. Tibial subchondral bone histomorphometric parameters were measured by quantitative micro-computed tomography (micro-CT). Histological sections of samples were analyzed after Safranin-O staining and quantitatively assessed according to a modified Osteoarthritis Research Society International (OARSI) scoring system. Immunohistochemical detection of NITEGE was performed to assess the extracellular matrix degradation. Results. Micro-CT analysis of destabilized joints confirmed that the rabbit ACLT significantly affected the tibial subchondral bone architecture as early as eight weeks, as revealed by significant changes of the subchondral bone parameters of operated joints compared to the sham operated joints. In particular, destabilized joints exhibited a Bone Volume/Tissue Volume ratio (BV/TV) ranging from 53.4% to 56.6%, compared to a mean BV/TV of 65.4% for sham operated joints. All destabilized joints also exhibited a significantly increased modified OARSI score, ranging from 7.4±0.4 for those injected with encapsulated cells to 8.9±0.2 for those injected with PBS, as compared to 4.8±0.4 for sham-operated joints. Of interest, we identified a slight, while not significant, reduction of the severity of OA lesions after injection of microencapsulated cells using the modified OARSI scoring. Finally, semi-quantitative analysis of NITEGE immunostaining revealed a significant increase in all destabilized joints that were injected with PBS or blank microparticles, in comparison with sham ones. On the contrary, NITEGE immunostaining in destabilized joints that were injected with non-encapsulated or encapsulated hASC revealed a significant reduced NITEGE immunostaining, indicating a decreased matrix degradation. Conclusions. Our data suggest that the microencapsulated hASCs exerted their anti-OA properties after IA injection in rabbit knees, as evidenced by the tendency toward a reduced modified OARSI score, and most importantly a significant reduction in NITEGE immunostaining associated matrix degradation. Further studies are now warranted to investigate the anti-OA efficacy of microencapsulated hASCs in the long-term

In a healthy joint, mechanical loading increases matrix synthesis and maintains cell phenotype, while reducing catabolic activities. It activates several pathways, most of them yet largely unknown, with integrins, TGF-β, canonical (Erk 1/2) and stress-activated (JNK) MAPK playing a key role. Degenerative joint diseases are characterized by Wnt upregulation and by the presence of proteolytic fibronectin fragments (FB-fs). Despite they are known to impair some of the aforementioned pathways, little is known on their modulatory effect on cartilage mechanoresponsiveness. This study aims at investigating the effect of mechanical loading in healthy and in vitro diseased cartilage models using pro-hypertrophic Wnt agonist CHIR99021 and the pro-catabolic FB-fs 30 kDa. Human primary chondrocytes from OA patients have been grown in alginate hydrogels for one week, prior to be incubated for 4 days with 3μM CHIR99021 or 1 μM FB-fs. Human cartilage explants isolated from OA patients have incubated 4 days with 3 μM CHIR99021 or 1 μM FB-fs. Both groups have then been mechanically stimulated (unconfined compression, 10% displacement, 1.5 hours, 1 Hz), using a BioDynamic bioreactor 5270 from TA Instruments. Expression of collagen type I, II and X, aggrecan, ALK-1, ALK-5, αV, α5 and β1 integrins, TGF-β1 have been assessed by Real Time-PCR and normalized with the expression of S29. Percentage of phosphorylated Smad2, Smad1 and JNK were determined through western blot. TGF-β1 content was quantified by sandwich ELISA; MMP-13 and GAG by western blot and DMMB assay, respectively. At least three biological replicates were used. ANOVA test was used for parametric analysis; Kruskal-Wallis and Mann-Whitney post hoc test for non-parametric. Preliminary data show that compression increased collagen II expression in control, but not in CHIR99021 and FB-fs pre-treated group (Fig. 1A-B). This was associated with downregulation of β1-integrin expression, which is the main collagen receptor and further regulates collagen II expression, suggesting inhibition of Erk1/2 pathway. A trend of increase expression of collagen type X after mechanical loading was observed in CHIR and FB-fs group. ALK-1 and ALK-5 showed a trend toward stronger upregulation in CHIR99021 group after compression, suggesting the activation of both Smad1/5/8 and Smad 2/3 pathways. To further investigate pathways leading to these different mechano-responses, the phosphorylation levels of Smad1 and Smad2, Erk1/2 and JNK proteins are currently being studied. Preliminary results show that Smad2, Smad1 and JNK protein levels increased in all groups after mechanical loading, independently of an increase in TGF-β1 expression or content. Compression further increased phosphorylation of Smad2, but not of Smad1, in all groups

Osteoarthritis (OA) is a progressive and degenerative joint disease resulting in changes to articular cartilage. In focal early OA defects, autologous chondrocyte implantation (ACI) has a 2-fold failure rate due to poor graft integration and presence of inflammatory factors (e.g. Interleukin-1β). Bone marrow derived mesenchymal stem cells (MSCs) are an alternative cell source for cell-based treatments due to their chondrogenic capacity, though in vivo implantation leads to bone formation. In vivo, chondrocytes reside under an oxygen tension between 2–7% oxygen or physioxia. Physioxia enhances MSC chondrogenesis with reduced hypertrophic marker (collagen X and MMP13) expression compared to hyperoxic conditions (20% oxygen). This study sought to understand whether implantation of physioxic preconditioned MSCs improves cartilage regeneration in an early OA defect model compared to hyperoxic MSCs. Bone marrow extracted from New Zealand white rabbits (male: 5–6 months old; n = 6) was split equally for expansion under 2% (physioxia) or 20% (hyperoxia) oxygen. Chondrogenic pellets (2 × 105 cells/pellet) formed at passage 1 were cultured in the presence of TGF-β1 under their expansion conditions and measured for their wet weight and GAG content after 21 days. During bone marrow extraction, a dental drill (2.5mm diameter) was applied to medial femoral condyle on both the right and left knee and left untreated for 6 weeks. Following this period, physioxia and hyperoxia preconditioned MSCs were seeded into a hyaluronic acid (TETEC) hydrogel. Fibrous tissue was scraped and then MSC-hydrogel was injected into the right (hyperoxic MSCs) and left (physioxia MSCs) knee. Additional control rabbits with drilled defects had fibrous tissue scrapped and then left untreated without MSC-hydrogel treatment for the duration of the experiment. Rabbits were sacrificed at 6 (n = 3) and 12 (n = 3) weeks post-treatment, condyles harvested, decalcified in 10% EDTA and sectioned using a cryostat. Region of interest was identified; sections stained with Safranin-O/Fast green and evaluated for cartilage regeneration using the Sellers scoring system by three blinded observers. Physioxic culture of rabbit MSCs showed significantly shorter doubling time and greater cell numbers compared to hyperoxic culture (∗p < 0.05). Furthermore, physioxia enhanced MSC chondrogenesis via significant increases in pellet wet weight and GAG content (∗p < 0.05). Implantation of physioxic preconditioned MSCs showed significantly improved cartilage regeneration (Mean Sellers score = 7 ± 3; ∗p < 0.05) compared to hyperoxic MSCs (Sellers score = 12 ± 2) and empty defects (Sellers score = 17 ± 3). Physioxia enhances in vitro rabbit MSC chondrogenesis. Subsequent in vivo implantation of physioxia preconditioned MSCs improved cartilage regeneration in an early OA defect model compared to hyperoxic MSCs. Future studies will investigate the mechanisms for enhanced in vivo regeneration using physioxia preconditioned MSCs

Total knee arthroplasty is a well-established treatment for degenerative joint disease, on the other hand metal ion release of cobalt or chromium and particle formation can trigger intolerance reactions. Biotribological examinations can help to assess the metal ion release in different settings. The purpose of this study was the evaluation of inter-laboratory differences in the metal ion concentration analysis. Samples were generated in a 3+1 station knee wear simulator (EndoLab GmbH, Thansau, Germany) with a medium size Columbus Knee System with or without AS multilayer coating. The wear simulation was performed under highly demanding activity (HDA) profile and samples were taken after 0.5, 5.0, 5.5. and 8.0 million cycles. The samples were blinded and sent to three different laboratories and the content of chromium, cobalt, molybdenum, nickel, and zirconium was assessed by inductively coupled plasma mass spectrometry (ICP-MS). The AS multilayer coating clearly reduced the release of chromium, cobalt and molybdenum. Mean levels were: Chromium 9329.78µg/l ± 985.44 vs 503.75µg/l ± 54.19, cobalt 10419.00µg/l ± 15.517.53 vs 2.60µg/l ± 1.35, molybdenum 2496.33µg/l ± 102.62 vs 2.46µg/l ± 2.31. Interestingly we found especially for nickel and zirconium big inter-laboratory differences in the metal assessment. There were up to 10-fold higher values in comparison of one laboratory to another. The data demonstrate that results of metal ion assessment should be evaluated by interlaboratory comparison and should be critically interpreted

Introduction. Osteoarthritis (OA) of the knee, a prevalently degenerative joint disorder provoked by articular cartilage loss, accounts for the leading cause of total knee arthroplasty. Autophagy is an indispensable intracellular event that maintains chondrocyte survival and metabolism. MicroRNAs are non-coding small RNAs participating in tissue morphogenesis, remodeling, and homeostasis. This study was undertaken to investigate the effect of microRNA-128 (miR-128) knockdown on the development of OA knees. Materials/Methods. Knee joints in rats were subjected to anterior cruciate ligament transection (ACLT) for inducing OA. Articular cartilage, synovium, and subchondral bone microarchitecture were assessed by OARSI scoring system, histomorphometry, and μCT imaging. Chondrocyte autophagy in terms of the expression of autophagic markers Atg4, Atg12, microtubule-associated protein 1 light chain 3 (LC3), and autophagosome formation was verified. Expression of microRNA, mRNA and signaling transduction were quantified with in situ hybridization, RT- quantitative PCR, and immunoblotting. Results. Chondrocytes in the affected knees showed weak expression of autophagic markers Atg4, Atg12, and LC3-II abundances in conjunction with significant increases in OARSI scores and a 2.5-fold elevation in miR-128 expression. The gain of miR-128 signaling in intact joints through intra-articular injection of miR-128 precursor resulted in 1.8–2.1-fold elevations in serum cartilage breakdown products CTX-II and COMP concentrations. miR-128 overexpression caused the joints to show evident chondrocyte apoptosis as evidenced by TUNEL staining concomitant with severe cartilage damage. Of note, antisense oligonucleotide knockdown of miR-128 (miR-128-AS) enabled the affected knee joints to show minor responses to the ACLT escalation of autophagy dysfunction in chondrocytes, cartilage breakdown histopathology, and OARSI scores. Administration with miR-128-AS also attenuated the ACLT-induced synovial membrane thickening, hyper-angiogenesis, and hypercellularity, which subsequently alleviated osteophyte accumulation, subchondral plate destruction, and trabecular microstructure loss. Conclusion. miR-128 signaling impairs chondrocyte autophagy, which ramps up chondrocyte apoptosis and OA knee development. This study highlights an emerging miR-128 knockdown strategy that sustains cartilage microarchitecture integrity and thereby delays OA knee pathogenesis

Osteoarthritis (OA) is the most common degenerative joint disease causing joint immobility and chronic pain. Treatment is mainly based on alleviating pain and reducing disease progression. During OA progression the chondrocyte undergoes a hypertrophic switch in which extracellular matrix (ECM) -degrading enzymes are released, actively degrading the ECM. However, cell biological based therapies to slow down or reverse this katabolic phenotype are still to be developed. Bone morphogenetic protein 7 (BMP-7) has been shown to have OA disease-modifying properties. BMP-7 suppresses the chondrocyte hypertrophic and katabolic phenotype and may be the first biological treatment to target the chondrocyte phenotype in OA. However, intra-articular use of BMP-7 is at risk in the proteolytic and hydrolytic joint-environment. Weekly intra-articular injections are necessary to maintain biological activity, a frequency unacceptable for clinical use. Additionally, production of GMP-grade BMP-7 is challenging and expensive. To enable its clinical use, we sought for BMP-7 mimicking peptides better compatible with the joint-environment while still biologically active and which potentially can be incorporated in a drug-delivery system. We hypothesized that human BMP-7 derived peptides are able to mimic the disease modifying properties of the full-length human BMP-7 protein on the OA chondrocyte phenotype. A BMP-7 peptide library was synthesized consisting of overlapping 20-mer peptides with 18 amino-acids overlap between sequential peptides. OA human articular chondrocytes (HACs) were isolated from OA cartilage from total knee arthroplasty (n=18 donors). HACs were exposed to BMP-7 (1 nM) or BMP-7 library peptides at different concentrations (1, 10, 100 or 1000 nM). Gene-expression levels of important chondrogenic-, hypertrophic-, cartilage degrading- and inflammatory mediators were determined by RT-qPCR. GAG and ALP activity were determined using a colorimetric assay and PGE levels were measured by EIA. During the BMP-7 peptide library screening human BMP-7 derived peptides were screened for their full-length human BMP-7 mimicking properties at different concentrations (1, 10, 100 or 1000nM) on a pool of human chondrocytes. Gene expression as well as GAG, ALP and PGE2 level analysis revealed two distinct peptide regions in the BMP-7 protein based on their pro-chondrogenic and anti-OA phenotype actions on human OA chondrocytes. The two most promising peptides were further analysed for their OA chondrocyte disease modifying properties in the presence of OA synovial fluid, showing similar OA phenotype suppressive activity. Conclusively, we successfully identified two peptide regions in the BMP-7 protein with in vitro OA suppressive actions. Further biochemical fine-tuning of the peptides, and in vivo evaluation, will potentially result in the first peptide-based experimental OA treatment, addressing the hypertrophic and katabolic chondrocyte phenotype in OA

Total knee arthroplasty is a well established treatment for degenerative joint disease with good clinical results. However, complications may occur due to a biological response to polyethylene wear particles, leading to osteolysis and aseptic loosening, as well as local and systemic hypersensitivity reactions triggered by metal ions and particles such as chromium, cobalt and molybdenum. Moreover, there is an increasing demand on the performance of these implants, as this treatment is also performed in heavier, younger and middle-aged adults who have a significant physical activity and higher life expectancy. The purpose of the following study was to compare the wear characteristics and performance of a zirconium nitride (ZrN) coated knee implant, designed for patients with metal ion hypersensitivity, against the clinically established cobalt-chromium (CoCr) version under a high demanding activities wear simulation. Medium size AS Columbus® DD (Aesculap AG, Tuttlingen, Germany) femoral and tibial components with a ZrN surface were tested in comparison with the cobalt-chromium version Columbus® DD. For both groups, ultra-high-molecular weight polyethylene (UHMWPE) gliding surfaces (size T3, high 10 mm) were used. Wear simulation was performed on a load controlled 4 station knee wear simulator (EndoLab GmbH, Thansau, Germany) capable of reproducing loads and movement of daily activities measured in vivo (Bergmann et al, 2014) on 8 patients and normalized to a patient weight of 100 kg (Schwiesau et al, 2014). The load profiles were applied for 5 million cycles in a combination of 40% stairs up, 40% stairs down, 10% level walking, 8% chair raising and 2% deep squatting. Test serum was changed every 0.5 million cycles and all the components were cleaned and analyzed according to ISO 14243-2:2009(E). The gliding surfaces were evaluated for gravimetric wear and wear patterns, femur components analyzed for scratches and the test medium analyzed for metal ion concentration (cobalt, chromium, molybdenum and zirconium) using ICP-MS according to ISO 17294-2. The present study showed a wear rate reduction for the ZrN group (1.01 ± 0.29 mg/million) in comparison with the CoCr group (2.40 ± 1.18 mg/million cycles). The articulation surface of the ZrN coated femurs remained polished after the testing period, whereas the uncoated femurs showed wear scratches. Furthermore, the metal ion release from the ZrN coated implants was reduced orders of magnitude in comparison with the CoCr implants through the entire test. These results demonstrate the efficiency of ZrN coated knee implants to reduce wear as well as to prevent metal ion release in the knee joint

Osteoarthritis (OA), the most common chronic degenerative joint disease, is characterized by inflammation, degradation of the articular cartilage and subchondral bone lesions, causing pain and decreased functionality. NF-κB pathway is involved in OA and, in most cases, its activation depends on the phosphorylation and degradation of IκBα, the NF-κB endogenous inhibitor that sequesters NF-κB in the cytosol. Under inflammatory stimuli, IκBα is degraded by the IKK signalosome and NF-κB moves into the nucleus, inducing the transcription of inflammatory mediator genes and catabolic enzymes. The IKK signalosome includes IKKβ and IKKα kinases, the latter shown to be pivotal in the OA extracellular matrix derangement. The current OA therapies are not curative and nowadays, the preclinical research is evaluating new structure-modifying pharmacological treatments, able to prevent or delay cartilage degradation. N-acetyl phenylalanine derivative (NAPA), is a derivative of glucosamine, a constituent of the glycosaminoglycans of cartilage and a chondroprotective agent. Previous in vitro studies showed the ability of NAPA to increase cartilage components and to reduce inflammatory cytokines, inhibiting IKKα kinase activity and its nuclear migration. The present study aims to further clarify the effect of NAPA in counteracting OA progression, in an in vivo mouse model after destabilization of the medial meniscus (DMM). Mice were divided into 3 groups:. -. DMM group: DMM surgery without NAPA;. -. DMM+NAPA group: DMM surgery with NAPA treatment;. -. NO DMM group: no DMM surgery. DMM surgery was performed in the right knee, according to Glasson SS [2], while the left knee did not undergo any surgery. Four weeks after surgery (mild-to-moderate OA), some animals received one intra-articular injection of NAPA (2.5 mM) and after 2 weeks, the animals were pharmacologically euthanized. The mice of the 1. st. group were euthanized 4 weeks after DMM and those of the 3. rd. group after 6 weeks from their arrival in the animal facility. At the end of experimental times, both knee joints of the animals were analyzed through histology, histomorphometry, immunohistochemistry and subchondral bone microhardness. The injection of NAPA significantly improved cartilage structure, increased cartilage thickness (p<0.0005), reduced Chambers and Mankin scores (p<0.005), fibrillation index (p<0.005) and decreased MMP13 (p<0.05) and ADAMTS5, MMP10, and IKKα (p<0.0005) staining. The microhardness measurements did not shown statistically significant differences between groups. This study demonstrated the chondroprotective activities exerted by NAPA in vivo. NAPA markedly improved the physical structure of articular cartilage and reduced the amount of catabolic enzymes, and therefore of extracellular matrix remodeling. The reduction in OA grading and catabolic enzymes paralleled the reduction of IKKα expression. This further hints at a pivotal role of IKKα in OA development by regulating MMP activity through the control of procollagenase (MMP10) expression. We believe that the preliminary preclinical data, here presented, contribute to improve the knowledge on the development of disease modifying drugs since we showed the ability of NAPA of reverting the surgically induced OA in the widely accepted DMM model

A novel scoring system for the grading of osteoarthritis has been developed. Scoring systems for the measurement of Osteoarthritis (OA) are essential for the understanding of the osteoarthritic process. OA is a mutifactorial degenerative joint disease affecting not only hyaline cartilage but also the surrounding tissues and particularly the subchondral bone. It as questionable as to why the articular cartilage remains the sole component used for histopathological assessment. The intimate relationship between the subchondral bone and overlying cartilage provide major difficulty in their independent measurement. A new scoring system has been developed to incorporate the subchondral bone into the assessment process and relating it to the structure of the overlying hyaline cartilage, which together permit a more accurate description of the degree of degenerate change. The new scoring system was developed from the analysis of 26 operative specimens from tibial plateau (TP) from patients who underwent total knee replacement (TKR). Multiple osteochondral plugs were taken from weight-bearing regions of the whole TP. The specimens were fixed and decalcified before being sectioned and stained with Masson's trichrome. Using a standard imaging system (Photoshop) the areas of bone and hyaline cartilage were identified and measured. Further parameters 1) cartilage thickness 2) tidemark integrity, 3) surface integrity 4) cartilage morphology were measured using a numeric measurement scale. The scoring system indicated a relationship between the area of subchondral bone and the hyaline cartilage degeneration. The overall sum of scores was also successful in distinguishing between the milder and more severe samples of OA. More comprehensive inter and intra observer variability needs to be tested in order validate the system. Quantifying changes to the subchondral bone may also serve beneficial to clinicians, as it is possible that monitoring these changes clinically could lead to early identification of OA