Introduction. The ACTIVE(Advanced Cartilage Treatment with Injectable-hydrogel Validation of the Effect) study investigates safety and performance of a novel dextran-tyramine hydrogel implant for treatment of small cartilage defects in the knee (0.5-2.0cm2). The hydrogel is composed of a mixture of natural polymer conjugates that are mixed intra-operatively and which cross-link in situ through a mild enzymatic reaction, providing a cell-free scaffold for cartilage repair. Method. The ACTIVE study is split into a safety (n=10) and a performance cohort (n=36). The Knee Injury and Osteoarthritis Outcome Score (KOOS), pain (numeric rating scale, NRS), Short-Form Health Survey (SF-36) were compared at baseline and 3, 6, and 12 months after surgery. The primary performance hypothesis is an average change in the KOOS from baseline to 12 months (ΔKOOS) greater than a minimal clinically important change (MIC) of 10. No statistical tests were performed as these are preliminary data on a smaller portion of the total study. Result. All patients of the safety cohort (n=10, mean age±SD, 30±9 years) were treated with the hydrogel for a symptomatic (NRS≥4) cartilage defect on the femoral condyle or trochlear groove (mean size±SD, 1.2±0.4cm2). No signs of an adverse foreign tissue reaction or serious adverse events were recorded within the safety cohort. At final follow-up mean KOOS±SD was 66.9±23.5, mean NRS resting±SD was 1.3±1.9, NRS activity±SD was 3.8±2.9 and mean SF-36±SD was 72.0±10.9. ΔKOOS was 21. One patient sustained new knee trauma prior to final follow-up, affecting final scores considerably. When excluded, ΔKOOS was 24(n=9). Conclusion. These promising initial findings provide a solid basis for continuation and expansion of this unique cartilage treatment. The MIC of 10 was surpassed. Though, results should be interpreted cautiously as they are based solely on preliminary data of the first 10 patients. Acknowledgements. Study is sponsored by Hy2Care, producer of the CartRevive®(dextran-tyramine) Hydrogel implant

Aims. To explore the efficacy of extracorporeal shockwave therapy (ESWT) in the treatment of osteochondral defect (OCD), and its effects on the levels of transforming growth factor (TGF)-β, bone morphogenetic protein (BMP)-2, -3, -4, -5, and -7 in terms of cartilage and bone regeneration. Methods. The OCD lesion was created on the trochlear groove of left articular cartilage of femur per rat (40 rats in total). The experimental groups were Sham, OCD, and ESWT (0.25 mJ/mm. 2. , 800 impulses, 4 Hz). The animals were euthanized at 2, 4, 8, and 12 weeks post-treatment, and histopathological analysis, micro-CT scanning, and immunohistochemical staining were performed for the specimens. Results. In the histopathological analysis, the macro-morphological grading scale showed a significant increase, while the histological score and cartilage repair scale of ESWT exhibited a significant decrease compared to OCD at the 8- and 12-week timepoints. At the 12-week follow-up, ESWT exhibited a significant improvement in the volume of damaged bone compared to OCD. Furthermore, immunohistochemistry analysis revealed a significant decrease in type I collagen and a significant increase in type II collagen within the newly formed hyaline cartilage following ESWT, compared to OCD. Finally, SRY-box transcription factor 9 (SOX9), aggrecan, and TGF-β, BMP-2, -3, -4, -5, and -7 were significantly higher in ESWT than in OCD at 12 weeks. Conclusion. ESWT promoted the effect of TGF-β/BMPs, thereby modulating the production of extracellular matrix proteins and transcription factor involved in the regeneration of articular cartilage and subchondral bone in an OCD rat model. Cite this article: Bone Joint Res 2024;13(7):342–352

Aims

This study aims to identify the top unanswered research priorities in the field of knee surgery using consensus-based methodology.

Aims

Adenosine, lidocaine, and Mg²⁺ (ALM) therapy exerts differential immuno-inflammatory responses in males and females early after anterior cruciate ligament (ACL) reconstruction (ACLR). Our aim was to investigate sex-specific effects of ALM therapy on joint tissue repair and recovery 28 days after surgery.

Aims

This study aimed to determine the expression and clinical significance of a cartilage protein, cartilage oligomeric matrix protein (COMP), in knee osteoarthritis (OA) patients.

Introduction. Autologous Matrix Induced Chondrogenesis (AMIC) for surgical treatment of osteochondral lesions of the talus (OCLT) has shown excellent clinical and radiological results at short term follow up two years after surgery. However, no mid-term follow up data is available. Aim. 1. To evaluate the clinical outcome after AMIC-aided reconstruction of osteochondral lesions of the talus at a minimum follow up time of five years. 2. To evaluate the morphology and quality of the regenerated cartilage by magnetic resonance imaging (MRI) at on at a minimum follow up time of five years. Methods. Seventeen patients prospectively underwent surgery receiving a AMIC-aided repair of OCLT consisting of debridement, autologous grafting, and sealing of the defect with a collagen scaffold (Chondro-Gide, Geistlich Surgery, Wolhusen, Switzerland). Clinical and radiological assessment was performed before and after a minimum of 60 months after surgery (average 78 months, range, 60–120). Clinical examination included the American Orthopaedic Foot & Ankle Society (AOFAS) ankle score and the Visual Analogue Scale (VAS). Radiological imaging consisted of MRI. The Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score was applied. Results. The AOFAS ankle score improved significantly from a mean of 60 points preoperatively (range, 17–79) to 91 points (range, 70–100) postoperatively (p< 0.01). The preoperative pain score averaged a VAS of 5 (range, 2–8), improving to an average of 1.1 (range 0–8) (p< 0.01). The MOCART score for cartilage repair tissue on postoperative MRI averaged 71 points (range, 50–90). Conclusion. The AMIC-procedure is safe for the treatment of OCLT with overall good clinical and magnetic resonance imaging results at five years follow up

Aims. The purpose of this study was to evaluate the mid-term outcomes of autologous matrix-induced chondrogenesis (AMIC) for the treatment of larger cartilage lesions and deformity correction in hips suffering from symptomatic femoroacetabular impingement (FAI). Methods. This single-centre study focused on a cohort of 24 patients with cam- or pincer-type FAI, full-thickness femoral or acetabular chondral lesions, or osteochondral lesions ≥ 2 cm. 2. , who underwent surgical hip dislocation for FAI correction in combination with AMIC between March 2009 and February 2016. Baseline data were retrospectively obtained from patient files. Mid-term outcomes were prospectively collected at a follow-up in 2020: cartilage repair tissue quality was evaluated by MRI using the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score. Patient-reported outcome measures (PROMs) included the Oxford Hip Score (OHS) and Core Outcome Measure Index (COMI). Clinical examination included range of motion, impingement tests, and pain. Results. A total of 12 hips from 11 patients were included (ten males, one female, mean age 26.8 years (SD 5.0), mean follow-up 6.2 years (SD 5.2 months)). The mean postoperative MOCART score was 66.3 (SD 16.3). None of the patients required conversion to total hip arthroplasty. Two patients had anterior impingement. External hip rotation was moderately limited in four patients. There was a correlation between MOCART and follow-up time (r. s. = -0.61; p = 0.035), but not with initial cartilage damage, age, BMI, or imaging time delay before surgery. PROMs improved significantly: OHS from 37.4 to 42.7 (p = 0.014) and COMI from 4.1 to 1.6 (p = 0.025). There was no correlation between MOCART and PROMs. Conclusion. Based on the reported mid-term results, we consider AMIC as an encouraging treatment option for large cartilage lesions of the hip. Nonetheless, the clinical evidence of AMIC in FAI patients remains to be determined, ideally in the context of randomized controlled trials. Cite this article: Bone Joint J 2024;106-B(5 Supple B):32–39

The April 2024 Foot & Ankle Roundup³⁶⁰ looks at: Safety of arthroscopy combined with radial extracorporeal shockwave therapy for osteochondritis of the talus; Bipolar allograft transplantation of the ankle; Identifying risk factors for osteonecrosis after talar fracture; Balancing act: immediate versus delayed weightbearing in ankle fracture recovery; Levelling the field: proximal supination osteotomy’s efficacy in severe and super-severe hallux valgus; Restoring balance: how adjusting the tibiotalar joint line influences movement after ankle surgery.

Aims

Pigment epithelium-derived factor (PEDF) is known to induce several types of tissue regeneration by activating tissue-specific stem cells. Here, we investigated the therapeutic potential of PEDF 29-mer peptide in the damaged articular cartilage (AC) in rat osteoarthritis (OA).

More and more evidences showed that cartilage harbored local progenitor cells that could differentiate toward osteoblast, chondrocyte, and adipocyte. However, our previous results showed that osteoarthritis derived chondroprogenitor cells (OA-CPC) exhibited strong osteogenic potential even in chondrogenic condition. How to promote their chondrogenic potential is the key for cartilage repair and regeneration in osteoarthritis. Recently, lipid availability was proved to determine skeletal progenitor fate. Therefore, we aim to determine whether lipid inhibition under 3D culture condition could enhance OA-CPC chondrogenesis. Moreover, glucose concentration was also evaluated for chondrogenic capacity. Although there are many researches showed that lower glucose promotes chondrogenesis, in our results, we found that OA-CPC in high concentration of glucose (4.5g/L) with lipid inhibitor (GW1100) showed strongest chondrogenic potential, which could form largest cell pellet with strong proteoglycan staining, COL II expression and no COL I expression. Besides, COL2A1 was increased and COL10A1 was decreased significantly by GW1100 under high glucose condition in 2D culture. Interestingly, although the expression level of MMP13 was not changed by GW1100 at RNA and protein level, less MMP13 protein secreted out of cell nuclear. In summary, we estimated that higher glucose and lower lipid supplies benefit OA-CPC chondrogenesis and cartilage repair

In relation to regenerative therapies in osteoarthritis and cartilage repair, mesenchymal stromal cells (MSCs) have immunomodulatory functions and influence macrophage behaviour. Macrophages exist as a spectrum of pro-(M1) and anti-(M2) inflammatory phenotypic subsets. In the context of cartilage repair, we investigated MSC-macrophage crosstalk, including specifically the priming of cartilage cells by macrophages to achieve a regenerative rather than fibrotic outcome. Human monocytes were isolated from blood cones and differentiated towards M1 and M2 macrophages. Monocytes (Mo), M1 and M2 macrophages were cultured directly and indirectly (trans-well system) with human bone marrow derived MSCs. MSCs were added during M1 polarisation and separately to already induced M1 cells. Outcomes (M1/M2 markers and ligands/receptors) were evaluated using RT-qPCR and flow cytometry. Influence on chondrogenesis was assessed by applying M1 and M2 macrophage conditioned media (CM) sequentially to cartilage derived cells (recapitulating an acute injury environment). RT-qPCR was used to evaluate chondrogenic/fibrogenic gene transcription. The ratio of M2 markers (CD206 or CD163) to M1 markers (CD38) increased when MSCs were added to Mo/M1 macrophages, regardless of culture system used (direct or indirect). Pro-inflammatory markers (including TNFβ) decreased. CXCR2 expression by both M1 macrophages and MSCs decreased when MSCs were added to differentiated M1 macrophages in transwell. When adding initially M1 CM (for 12 hours) followed by M2 CM (for 12 hours) sequentially to chondrocytes, there was a significant increase of Aggrecan and Collagen type 2 gene expression and decrease in fibroblastic cell surface markers (PDPN/CD90). Mo/M1 macrophages cultured with MSCs, directly or indirectly, are shifted towards a more M2 phenotype. Indirect culture suggests this effect can occur via soluble signaling mediators. Sequential exposure of M1CM followed by M2CM to chondrocytes resulted in increased chondrogenic and reduced fibrotic gene expression, suggesting that an acute pro-inflammatory stimulus may prime chondrocytes before repair

Cell culture on tissue culture plastic (TCP) is widely used across biomedical research to understand the in vivo environment of a targeted biological system. However, growing evidence indicates that the characteristics of cells investigated in this way differ substantially from their characteristics in the human body. The limitations of TCP monolayer cell cultures are especially relevant for chondrocytes, the cell population responsible for producing cartilage matrix, because their zonal organization in hyaline cartilage is not preserved in a flattened monolayer assay. Here, we contrast the response of primary human chondrocytes to inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma, via transcriptional, translational, and histological profiling, when grown either on TCP or within a 3D cell pellet (scaffold-less). We focus on anti-apoptotic (Bcl2), pro-apoptotic (Bax, Mff, Fis1), and senescent (MMP13, MMP1, PCNA, p16, p21) markers. We find that the 3D environment of the chondrocyte has a profound effect on the behavior and fate of the cell; in TCP monolayer cultures, chondrocytes become anti-apoptotic and undergo senescence in response to inflammatory cytokines, whereas in 3D cell pellet cultures, they exhibit a pro-apoptotic response. Our findings demonstrate that chondrocyte culture environment plays a pivotal role in cell behavior, which has important implications for the clinical applicability of in vitro research of cartilage repair. Although there are practical advantages to 2D cell cultures, our data suggest researchers should be cautious when drawing conclusions if they intend to extrapolate findings to in vivo phenomena. Our data demonstrates opposing chondrocyte responses in relation to apoptosis and senescence, which appear to be solely reliant on the environment of the culture system. This biological observation highlights that proper experimental design is crucial to increase the clinical utility of cartilage repair experiments and streamline their translation to therapy development

Osteoarthritis (OA) is an inflammatory disease affecting the complete synovial joint including the cartilage layer and the subchondral bone plate. Due to the multifactorial causes and the not yet completely resolved molecular mechanisms, it lacks a gold standard treatment to mitigate OA. Hence, biomaterials capable of delaying or preventing OA are a promising alternative or supplement to antiphlogistic and surgical interventions. Magnesium (Mg) and its alloys are among the promising biomaterials with osteoinductive effects. This work investigated the impact of Mg micro cylinders (length ≈of 1.0 mm and width of 0.5 mm) in vitro, in favoring joint regeneration together with preventing OA progression. Therefore, a mesenchymal stem cell line (SCP-1) was applied in order to assess the compatibility of the degradable material. Furthermore, an in vitro OA model utilizing SCP-1 cells based on the supplementation of the cytokines; IL-1β, TNF-α was established and disclosed the capability of Mg microparticles in differentiating SCP-1 cells into chondrogenic and osteogenic lineages proven through extracellular matrix staining and gene marker analysis. A concentration above 10 mM revealed a reduction in the cell viability by 50 %. An increase in the expression of collagens especially and proteoglycans (COL2A1, Aggrecan) as extracellular matrix proteins as well as an increase in osteogenic marker (ALP, BMP2) favoring the mineralization process were observed. The inflammatory condition reduced the viability and productivity of the applied stem cell line. However, the application of Mg microparticles induced a cell recovery and reduction of inflammation marker such as MMP1 and IL6. The cytocompatible and the ability of Mg microparticles in supporting bone and cartilage repair mechanisms in vitro even under inflammatory conditions make biodegradable Mg microparticles a suitable implant material to treat OA therapy. Acknowledgements: This project OAMag was funded by the German Research Foundation (project number 404534760). The author thank Dr. Björn Wiese (hereon) for the production of Mg based material and Prof. Böcker (MUM Musculoskeletal University Center Munich) for the provision of SCP-1 cell line

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

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

Aims. Osteochondral lesions of the talus (OLT) are a common cause of disability and chronic ankle pain. Many operative treatment strategies have been introduced; however, they have their own disadvantages. Recently lesion repair using autologous cartilage chip has emerged therefore we investigated the efficacy of particulated autologous cartilage transplantation (PACT) in OLT. Methods. We retrospectively analyzed 32 consecutive symptomatic patients with OLT who underwent PACT with minimum one-year follow-up. Standard preoperative radiography and MRI were performed for all patients. Follow-up second-look arthroscopy or MRI was performed with patient consent approximately one-year postoperatively. Magnetic resonance Observation of Cartilage Repair Tissue (MOCART) score and International Cartilage Repair Society (ICRS) grades were used to evaluate the quality of the regenerated cartilage. Clinical outcomes were assessed using the pain visual analogue scale (VAS), Foot Function Index (FFI), and Foot Ankle Outcome Scale (FAOS). Results. All patients had ICRS grade IV cartilage lesions, except for one (ICRS grade III). The paired MOCART scores significantly improved from 42.5 (SD 1.53) to 63.5 (SD 22.60) (p = 0.025) in ten patients. Seven patients agreed to undergo second-look arthroscopy; 5 patients had grade I (normal) ICRS scores and two patients had grade II (nearly normal) ICRS scores. VAS, FFI, and all subscales of FAOS were significantly improved postoperatively (p ≤ 0.003). Conclusion. PACT significantly improved the clinical, radiological, and morphological outcomes of OLT. We consider this to be a safe and effective surgical method based on the short-term clinical results of this study. Cite this article: Bone Jt Open 2023;4(12):942–947

Abstract. Objectives. In relation to regenerative therapies in osteoarthritis and cartilage repair, mesenchymal stromal cells (MSCs) have immunomodulatory functions and influence macrophage behaviour. Macrophages exist as a spectrum of pro-(M1) and anti-(M2) inflammatory phenotypic subsets. In the context of cartilage repair, we investigated MSC-macrophage crosstalk, including specifically the priming of cartilage cells by macrophages to achieve a regenerative rather than fibrotic outcome. Methods. Human monocytes were isolated from blood cones and differentiated towards M1 and M2 macrophages. Monocytes (Mo), M1 and M2 macrophages were cultured directly and indirectly (trans-well system) with human bone marrow derived MSCs. MSCs were added during M1 polarisation and separately to already induced M1 cells. Outcomes (M1/M2 markers and ligands/receptors) were evaluated using RT-qPCR and flow cytometry. Influence on chondrogenesis was assessed by applying M1 and M2 macrophage conditioned media (CM) sequentially to cartilage derived cells (recapitulating an acute injury environment). RT-qPCR was used to evaluate chondrogenic/fibrogenic gene transcription. Results. The ratio of M2 markers (CD206 or CD163) to M1 markers (CD38) increased when MSCs were added to Mo/M1 macrophages, regardless of culture system used (direct or indirect). Pro-inflammatory markers (including TNFa) decreased. CXCR2 expression by both M1 macrophages and MSCs decreased when MSCs were added to differentiated M1 macrophages in transwell. When adding initially M1 CM (for 12 hours) followed by M2 CM (for 12 hours) sequentially to chondrocytes, there was a significant increase of Aggrecan and Collagen type 2 gene expression and decrease in fibroblastic cell surface markers (PDPN/CD90). Conclusions. Mo/M1 macrophages cultured with MSCs, directly or indirectly, are shifted towards a more M2 phenotype. Indirect culture suggests this effect can occur via soluble signaling mediators. Sequential exposure of M1CM followed by M2CM to chondrocytes resulted in increased chondrogenic and reduced fibrotic gene expression, suggesting that an acute pro-inflammatory stimulus may prime chondrocytes before repair. 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. BACKGROUND. Cell culture on tissue culture plastic (TCP) is widely used across biomedical research to understand the in vivo environment of a targeted biological system. However, growing evidence indicates that the characteristics of cells investigated in this way differ substantially from their characteristics in the human body. The limitations of TCP monolayer cell cultures are especially relevant for chondrocytes, the cell population responsible for producing cartilage matrix, because their zonal organization in hyaline cartilage is not preserved in a flattened monolayer assay. OBJECTIVE. Here, we contrast the response of primary human chondrocytes to inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma, via transcriptional, translational, and histological profiling, when grown either on TCP or within a 3D cell pellet (scaffold-less). We focus on anti-apoptotic (Bcl2), pro-apoptotic (Bax, Mff, Fis1), and senescent (MMP13, MMP1, PCNA, p16, p21) markers. RESULTS. We find that the 3D environment of the chondrocyte has a profound effect on the behavior and fate of the cell; in TCP monolayer cultures, chondrocytes become anti-apoptotic and undergo senescence in response to inflammatory cytokines, whereas in 3D cell pellet cultures, they exhibit a pro-apoptotic response. CONCLUSION. Our findings demonstrate that chondrocyte culture environment plays a pivotal role in cell behavior, which has important implications for the clinical applicability of in vitro research of cartilage repair. Although there are practical advantages to 2D cell cultures, our data suggest researchers should be cautious when drawing conclusions if they intend to extrapolate findings to in vivo phenomena. Our data demonstrates opposing chondrocyte responses in relation to apoptosis and senescence, which appear to be solely reliant on the environment of the culture system. This biological observation highlights that proper experimental design is crucial to increase the clinical utility of cartilage repair experiments and streamline their translation to therapy development. 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. Objective. The preparation of host degenerate cartilage for repair typically requires cutting and/or scraping to remove the damaged tissue. This can lead to mechanical injury and cartilage cell (chondrocytes) death, potentially limiting the integration of repair material. This study evaluated cell death at the site of cutting injury and determined whether raising the osmotic pressure (hyper-osmolarity) prior to injury could be chondroprotective. Methods. Ex vivo human femoral head cartilage was obtained from 13 patients (5 males and 8 females: 71.8 years old) with Ethical Permission and Patient consent. Cartilage wells were created using 3 or 5mm biopsy punches. Cell death at the wounded edge of the host cartilage and the edge of the extracted explants were assessed by quantifying the percentage of cell death (PCD) and measuring the width of the cell death zone at identified regions of interest (ROI) using the confocal laser scanning microscopy and image analysis software. To assess the chondroprotective effect of hyper-osmolarity, cartilage specimens were incubated in 340 or 600mOsm media, five minutes prior to injury to allow the chondrocytes to respond to the altered osmolarity. Wounded cartilage explants and cartilage wells were then cultured for a further 150 minutes following injury. Results. In 340mOsm media, the PCD around the 3mm cartilage wells was significantly less compared to the corresponding explants (20.05±10.24% vs 35.25±4.86%; P=0.0003). When using the 5mm biopsy punch, the PCD at the wound edges was significantly lower when compared to the 3mm cartilage wells (13.33±7.80% vs 20.05±10.24%; P=0.0121) at the same osmolarity. The width of the cell death zone for the well edges for both 3 and 5mm punches was significantly narrower when compared to their corresponding harvested cartilage explants in 340mOsm media (P<0.0001; P=0.0218, respectively). Exposing cartilage to raised osmolarity (600mOsm) prior to injury significantly reduced the PCD for cartilage wells produced by the 3mm biopsy punches (from 20.05±10.24% to 12.24±6.00%; P=0.0025). In addition, the zone of cell death was marginally reduced at the edges of the 5mm cartilage wells (19.25±15.78mm to 12.72±9.09mm; P=0.0499). Conclusions. The choice of biopsy punch and the osmolarity of the incubation medium prior to cartilage injury markedly affected the extent of chondrocyte death both at the edges of the cartilage wells and the explants. The smaller biopsy punch caused more chondrocyte death in the native cartilage wells compared to the larger punch, but this could be compensated for by the chondroprotective effect of raising the osmotic pressure. In general, there was less cell death at the wounded edges of the cartilage wells, compared to the explants. These results suggest that there is scope for further optimising the cutting implements used to create the cartilage wells and protecting chondrocytes by hyper-osmolarity in order to minimize cell death at cut edges and potentially enhance integration between cartilage repair material and host cartilage. 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