In an experimental study in rabbits, bone and cartilage regeneration could be achieved with a new class of resorbable bio-implants. These implants consist of an open porous structure made from polylacitdes and an open porous fleece made from polyglactin/polydioxanon. Both layers were not separated from each other, thus allowing mesenchymal cells to penetrate freely from bone into both the bone substitute and the cartilage substitute layer. It could be shown that ostochondral defects of 4mm diameter and 6mm depth in the condyle of the knee of rabbits healed by the process of mesenchymal cell differentiation into osteocytes and chondrocytes triggered by mechanical load induction only. Evaluation of the newly formed cartilage by light microscopy and immunohistology showed hyaline like features. However, in many clinical cases chondral defects occur without substantial accompanying bone loss. In these situations, reconstruction of the cartilage defects only seems to be sufficient. However, fixation of such fleeces onto the bone is difficult. On one hand, adherence of the fleece to the underlying bone is crucial, on the other hand an open connection from the bone to the fleece must be accomplished in order to allow mesenchymal cells to penetrate the fleece. Therefor, any kind of glue fixation is not appropriate. To overcome this problem, a new fixation method was developed which allows a safe connection of the fleece onto the bone while providing an open contact of the fleece to the bone marrow for unhampered migration of mesenchymal cells. The new “Cartilage patches” consist of a fleece (serving as the cartilage substitute layer) made from polyglactin/polydioxanon which had proven its applicability in the above mentioned experiments. Fixation of fleece was achieved by “darts” which were glued onto the fleece. The darts were made from polylacitdes, thus providing sufficient mechanical stability in the bone. During operation, small holes are cut into the bone by a special instrument. The holes are located in such a way that the darts of the cartilage patch fit into them, such resulting in a stable fixation of the fleece onto the underlying bone. Blood containing mesenchymal cells from the bone marrow is able to flow from the holes into the fleece. In a biomechanical analysis the adherence of the cartilage patches were tested with respect to shear resistance and pull-out stabillity. The results of the tests show that the new cartilage patches withstand the mechanical stress exerted onto articular surfaces and can serve as a new class of cartilage substitute layers. In an animal experiment the applicability of the cartilage patches in reconstruction of cartilage defects in the knee joint of sheep will be proven

Glenohumeral joint injuries frequently result in shoulder instability. However, the biomechanical effect of cartilage loss on shoulder stability remains unknown. The aim of the current study was to investigate biomechanically the effect of two severity stages of cartilage loss in different dislocation directions on shoulder stability. Joint dislocation was provoked for 11 human cadaveric glenoids in seven different dislocation directions between 3 o'clock (anterior) to 9 o'clock (posterior) dislocation. Shoulder stability ratio (SSR) and concavity gradient were assessed in intact condition, and after 3 mm and 6 mm simulated cartilage loss. The influence of cartilage loss on SSR and concavity gradient was statistically evaluated. Between intact state and 6 mm cartilage loss, both SSR and concavity gradient decreased significantly in every dislocation direction (p≤0.038), except the concavity gradient in 4 o'clock dislocation direction (p=0.088). Thereby, anterior-inferior dislocation directions were associated with the highest loss of SSR and concavity gradient of up to 59.0% and 49.4%, respectively, being significantly higher for SSR compared to all other dislocation directions (p≤0.04). The correlations between concavity gradient and SSR for pooled dislocation directions were significant for all three conditions of cartilage loss (p<0.001). From a biomechanical perspective, articular cartilage of the glenoid contributes significantly to the concavity gradient, correlating strongly with the associated loss in glenohumeral joint stability. The highest effect of cartilage loss was observed in anterior-inferior dislocation directions, suggesting that surgical intervention should be considered for recurrent shoulder dislocations in the presence of cartilage loss

There are no efficient treatment options for osteoarthritis (OA) that delay further progression. Besides osteoinduction, there is growing evidence of also anti-inflammatory, angiogenetic and neuroprotective effects of biodegradable magnesium-based biomaterials. Their use for the treatment of cartilage lesions in contrast is not well-evaluated yet. Mg-cylinders were analysed in an in vitro and in vivo OA model. In vitro, SCP-1 stem cell line was analysed under inflammatory conditions and Mg-impact. In vivo, small Mg- and WE43 alloy-cylinders (1mm × 0,5mm) were implanted into the subchondral bone of the knee joint of 24 NZW rabbits after establishment of OA. As control, another 12 rabbits received only drill-holes. µCT-scan were performed and assessed for changes in bone volume and density. After euthanasia, cartilage was evaluated macroscopically and histologically after Safranin-O-staining. Furthermore, staining with CD271 directed antibody was performed to assess neuro-reactivity. In vitro, an increased gene expression of extracellular matrix proteins as collagen II or aggrecan even under inflammatory conditions was observed under Mg-impact. In vivo, µCT evaluation revealed twice-elevated values for bone volume in femoral condyles with Mg-cylinders compared to controls while density remained unchanged. Cartilage showed no significant differences between the groups. Mg- and WE-samples showed significantly lower levels of CD271+ cells in the cartilage and bone of the operated joints than in non-operated joints, which was not the case in the Drilling-group. Furthermore, bone in operated knees of Drilling-group showed a strong trend to an increase in CD271+ cells compared to both Cylinder-groups. Counting of CD271+ vessels revealed that this difference was attributable to a higher amount of these vessels. The in vitro results indicate a potential cartilage regenerative activity of the degradable Mg-based material. While so far there was no positive effect on the cartilage itself in vivo, implantation of Mg-cylinders seemed to reduce pain-mediating vessels. Acknowledgements: This work is funded by the German Research Foundation (DFG, project number 404534760). We thank Björn Wiese for production of the cylinders

Irisin is a hormone-like myokine released from skeletal muscle during exercise. It has also been reported that irisin levels in serum and synovial fluid of knee osteoarthritis (OA) patients were negatively correlated with OA severity. We hypothesized that irisin might play a role in the cartilage homeostasis mediated by physical activity. Therefore, this study aims to explore the cross talk between skeletal muscle and cartilage tissues in human with OA mediated by the myokine irisin. Human articular OA chondrocytes were isolated, expanded and cultured in micro-mass 3-D culture system. Pellets were cultured with or without r-Irisin, and then activated by protein inhibitors of p38-MAPK signalling pathway. After one week the amount of GAG content was evaluated. Quantitative gene expression of Coll-X and Coll-II was performed. WB was utilized to detect expressions of p38-MAPK signalling pathway and Coll-X and Coll-II. In the current study, chondrocytes cultured in r-Irisin showed a significant higher GAG/DNA content compared to control (p<0.05). Moreover, r-Irisin promoted a significant increase of the expression collagen type II and decrease of collagen type X in (p<0.05). This OA chondrocytes recovery was abrogated by the p38 MAPK and ERK signalling pathways. Our observation suggests that Irisin targets chondrocytes promoting GAG content, increasing Collagen Type II and decreasing Collagen type X gene expressions. The observed OA chondrocyte recovery mediated by irisin is obtained through the inactivation of p38/ERK MAP kinase signalling cascades in vitro. This is the first study that demonstrates a cross-talk between muscle and cartilage mediated by irisin

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

Regulation of articular cartilage homeostasis is a complex process in which biologic and mechanical factors are involved. Hyperactivation of Wnt signaling, associated with osteoarthritis (OA), could jeopardize the protective anabolic effect of physiological loading. Here, we investigated the role of excessive Wnt signalling in cartilage molecular responses to loading. Human cartilage explants were harvested from hips of donors without OA. The Wnt agonist CHIR99021 was used to activate Wnt signalling 24 hours before cartilage explants were subjected to a loading protocol consisting of 2 cycles of 1 hour of 10% compression at 1 Hz, followed by 1-hour free swelling. Mechano-responsiveness was evaluated using the expression of type II collagen, aggrecan and MMP-13. Expression of known target genes TCF-1 and c-JUN was evaluated as positive control for Wnt and mechanical stimulation, respectively. In the absence of loading, CHIR99021 decreased the expression of the cartilage anabolic genes type II collagen and aggrecan, and increased the levels of MMP-13, corroborating that Wnt hyperactivation disrupts cartilage homeostasis. In the absence of Wnt hyperactivation, the applied loading protocol, representative for a physiologic stimulation by mechanical loading, led to an increase in type II collagen and aggrecan levels. However, when cartilage explants were subjected to mechanical stimulation in the presence of CHIR99021, the expression of cartilage anabolic genes was decreased, indicating changes to the cells’ mechano-responsiveness. Interestingly, mechanical stimulation was able to reduce the expression levels of MMP-13 compared to the condition of CHIR stimulation without loading. Hyperactivation of Wnt signaling switches the anabolic effect of physiologic compressive loading towards a potential catabolic effect and could contribute to the development and progression of OA

The goal of this study was to identify the effect of mismatches in the subchondral bone surface at the native:graft interface on cartilage tissue deformation in human patellar osteochondral allografts (OCA). Hypothesis: large mismatches in the subchondral bone surface will result in higher stresses in the overlying and surrounding cartilage, potentially increasing the risk of graft failure. Nano-CT scans of ten 16mm diameter cadaveric patellar OCA transplants were used to develop simplified and 3D finite element (FE) models to quantify the effect of mismatches in the subchondral bone surface. The simplified model consisted of a cylindrical plug with a 16 mm diameter (graft) and a washer with a 16 mm inner diameter and 36 mm outer diameter (surrounding native cartilage). The thickness of the graft cartilage was varied from 0.33x the thickness of native cartilage (proud graft subchondral bone) to 3x the thickness of native cartilage (sunken graft subchondral bone; Fig. 1). The thickness of the native cartilage was set to 2 mm. The surface of the cartilage in the graft was matched to the surrounding native cartilage. A 1 MPa pressure was applied to the fixed patellar cartilage surface. Scans were segmented using Dragonfly and meshed using HyperMesh. FE simulations were conducted in Abaqus 2019. The simplified model demonstrated that a high stress region occurred in the cartilage at the sharp bony edge between the graft and native subchondral bone, localized to the region with thinner cartilage. A 20% increase in applied pressure occurs up to 50μm away from the graft edge (primarily in the graft cartilage) for grafts with proud subchondral bone but varies little based on the graft cartilage thickness. For grafts with sunken subchondral bone, the size of the high stress region decreases as the difference between graft cartilage and native cartilage thickness decreases (Fig. 2-4), with a 200 μm high stress region occurring when graft cartilage was 3x thicker than native cartilage (i.e., greater graft cartilage thickness produces larger areas of stress in the surrounding native cartilage). The 3D models reproduced the key features demonstrated in the simplified model. Larger differences between native and graft cartilage thickness cause larger high stress regions. Differences between the 3D and simplified models are caused by heterogeneous cartilage surface curvature and thickness. Simplified and 3D FE analysis confirmed our hypothesis that greater cartilage thickness mismatches resulted in higher cartilage stresses for sunken subchondral bone. Unexpectedly, cartilage stresses were independent of the cartilage thickness mismatch for proud subchondral bone. These FE findings did not account for tissue remodeling, patient variability in tissue mechanical properties, or complex tissue loading. In vivo experiments with full-thickness strain measurements should be conducted to confirm these findings. Mismatches in the subchondral bone can therefore produce stress increases large enough to cause local chondrocyte death near the subchondral surface. These stress increases can be reduced by (a) reducing the difference in thickness between graft and native cartilage or (b) using a graft with cartilage that is thinner than the native cartilage. For any figures or tables, please contact the authors directly

Articular cartilage is a multi-zonal tissue that coats the epiphysis of long bones and avoids its wear during motion. An unusual friction could micro-fracture this connective membrane and progress into an osteochondral defect (OD), where the affected cartilage suffers inflammation, fibrillation, and forfeiture of its anisotropic structure. Clinical treatment for ODs has been focused on micro-fracture techniques, where the defect area is removed and small incisions are performed in the subchondral bone, which allows the exudation of mesenchymal stem cells (hMSCs) to the abraded zone. However, hMSCs represent less than 0.01% of the total cell population and are not able to self-organise coherently, so the treatments fail in the long term. To select, support and steer hMSCs from the bone marrow into a specific differentiation stage, and recreate the cartilage anisotropic microenvironment, multilayer dual-porosity 3D-printed scaffolds were developed. Dual-porosity scaffolds were printed using prepared inks, containing specific ratios of poly-(d,l)lactide-co-caprolactone copolymer and gelatine microspheres of different diameters, which acted as sacrificial micro-pore templates and were leached after printing. The cell adhesion capability was investigated showing an increased cell number in dual-porosity scaffolds as compared to non-porous ones. To mimic the stiffness of the three cartilage zones, several patterns were designed, printed, and checked by dynamic-mechanical analysis under compression at 37 ºC. Three patterns with specific formulations were chosen as candidates to recreate the mechanical properties of the cartilage layers. Differentiation studies in the selected scaffolds showed the formation of mature cartilage by gene expression, protein deposition and biomolecular analysis. Given the obtained results, designed scaffolds were able to guide hMSC behaviour. In conclusion, biocompatible, multilayer and dual-porosity scaffolds with cell entrapment capability were manufactured. These anisotropic scaffolds were able to recreate the physical microenvironment of the natural cartilage, which in turn stimulated cell differentiation and the formation of mature cartilage. Acknowledgments: This work was supported by the EMAKIKER grant

Introduction. Successful joint preservation surgery requires the ability to accurately assess the health of the articular cartilage pre-operatively. Traditional radiological methods allow morphological assessment of the cartilage and therefore only identify those with established degeneration. Biophysical properties of cartilage are now being used to identify these changes occurring earlier in the disease processes. Prior literature states that healthy cartilage has a transverse relaxation time of between 15–60 ms (16). Our study aims to establish the correlation and accuracy of MRI with T2 cartilage mapping with observed intra-operative chondral defects. Methods. We routinely request MRI with T2 mapping on all patients with suspected or confirmed femoroacetabular impingement (FAI). A review was performed on all patients who underwent both pre-operative imaging and subsequent hip arthroscopy for FAI over a 24-month period. Using linear regression we correlated intra-operatively observed chondral defects of the femoral head and acetabulum (Outerbridge classification scores) with the pre-operative transverse relaxation times. Statistical analysis of 66 chondral points was undertaken. Results. Results show that there is a significant association between an increase in transverse relaxation time and higher acetabular Outerbridge classification (p = 0.0141). Discussion. This study has identified that MRI with T2 cartilage mapping is an accurate predictor of acetabular cartilage health. Our findings suggest that 3T MRI with T2 cartilage mapping is a useful tool in joint preservation surgery and provides accurate information allowing hip arthroscopists to identify patients who may benefit most from conservative operative intervention

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

The lifetime prevalence of symptomatic osteoarthritis at the knee is 50% osteoarthritis of the ankle occurs in only 1% of the population. This variation in prevalence has been hypothesised to result from the differential responsiveness of the joint cartilages to catabolic stimuli. Human cartilage explants were taken from the talar domes (n=12) and the femoral condyles (n=7) following surgical amputation. Explants were cultured in the presence of either a combination of high concentration cytokines (TNFα, OSM, IL-1α) to resemble a post traumatic environment or low concentration cytokines to resemble a chronic osteoarthritic joint. Cartilage breakdown was measured by the percentage loss of Sulphated glycosaminoglycan (sGAG) from the explant to the media during culture. Expression levels of the pro-inflammatory molecules nitric oxide and prostaglandin E. 2. were also measured. Significantly more sGAG was lost from knee cartilage exposed to TNFα (22.2% vs 13.2%, P=0.01) and TNFα in combination with IL-1α (27.5% vs 16.0%, P=0.02) compared to the ankle; low cytokine concentrations did not affect sGAG release. Significantly more PGE. 2. was produced by knee cartilage compared to ankle cartilage however no significant difference in nitrite production was noted. Cartilage from the knee and ankle has a divergent response to stimulation by pro-inflammatory cytokines, with high concentrations of TNFα alone, or in combination with IL-1α amplifying cartilage degeneration. This differential response may account for the high prevalence of knee arthritis compared to ankle OA and provide a future pharmacological target to treat post traumatic arthritis of the knee

Nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is key in maintaining redox homeostasis and the pathogenesis of osteoarthritis (OA) involves oxidative distress. We thus investigated whether Nrf2/ARE signaling may control expression of key chondrogenic differentiation and hyaline cartilage maintenance factor SOX9. In human C-28/I2 chondrocytes SOX9 expression was measured by RT–qPCR after shRNA-mediated knockdown of Nrf2 or its antagonist the Kelch-like erythroid cell-derived protein with cap “n” collar homology-associated protein 1 (Keap1). Putative ARE-binding sites in the proximal SOX9 promoter region were inactivated, cloned into pGL3, and co-transfected with phRL–TK for dual-luciferase assays to verify whether Nrf2 transcriptionally regulates SOX9. SOX9 promoter activity without and with Nrf2-inducer methysticin were analyzed. Sox9 expression in articular chondrocytes was correlated to cartilage thickness and degeneration in wild-type (WT) and Nrf2-knockout mice. Data were analyzed by one-way ANOVA, a Student's t-test, or Wilcoxon rank-sum test, according to the normal distribution. Statistical significance was set to p < 0.05. While Keap1-specific RNAi increased SOX9 expression, Nrf2-specific RNAi significantly decreased it. Putative ARE sites (ARE. 1. , ARE. 2. ) were identified in the SOX9 promoter region. ARE. 2. mutagenesis significantly reduced SOX9 promoter activity, while truncation of ARE. 1. did not. A functional ARE. 2. site was thus essential for methysticin-mediated induction of SOX9 promoter activity. Knee cartilage of young Nrf2-knockout mice further revealed significantly fewer Sox9-positive chondrocytes as compared to old Nrf2-knockout animals, which further showed thinner cartilage and more severe cartilage erosion. Our data suggest that SOX9 expression in articular cartilage is directly Nrf2-dependent and that pharmacological Nrf2 activation may hold potential to diminish age-dependent osteoarthritic changes in knee cartilage through improving protective SOX9 expression

In this study, we developed biocompatible adhesive which enables implanted chondrogenic-enhanced hASCs being strongly fixed to the lesion site of defected cartilage. The bioengineered mussel adhesive protein (MAP) was produced and purified using a bacterial expression system as previously reported. The cell encapsulated coacervate was formulated with two polyelectrolyte, the MAP and 723kDa hyaluronic acid (HA). MAP formed liquid microdroplets with HA and subsequently gelated into microparticles, which is highly viscous and strongly adhesive. The MAP with chondro-induced hASCs were implanted on the osteochondral defect created in the patellar groove/condyle of OA-induced rabbits. Rabbits were allocated to three different groups as follows: Group1 – Fibrin only; Group2 – Fibrin with hASCs (1.5×10. 6. chondro-induced hASCs); Group3; MAP with hASCs. The implanted cells were labeled with a fluorescent dye for in vivo visualization. After 35 days, fluorescent signals were more potently detected for MAP with hASCs group than Fibrin with hASCs group in osteochondral defect model. Moreover, histological assessment showed that MAP with hASCs group had the best healing and covered with hyaline cartilage-like tissue. The staining image shows that MAP with hASCs group were filled with perfectly differentiated chondrocytes. Although Fibrin with hASCs group had better healing than fibrin only group, it was filled with fibrous cartilage which owes its flexibility and toughness. As MAP with hASCs group has higher possibility of differentiating to complete cartilage, Fibrin only group and Fibrin with hASCs group have failed to treat OA by rehabilitating cartilage. In order to clarify the evidence of remaining human cell proving efficacy of newly developed bioadhesive, human nuclear staining was proceeded with sectioned rabbit cartilage tissue. The results explicitly showed MAP with hASCs group have retained more human cells than Fibrin only and Fibrin with hASCs groups. We investigated the waterproof bioadhesive supporting transplanted cells to attach to defect lengthily in harsh environment, which prevents cells from leaked to other region of cartilage. Collectively, the newly developed bio-adhesive, MAP, could be successfully applied in OA treatment as a waterproof bioadhesive with the capability of the strong adhesion to target defect sites

Cartilage diseases have a significant impact on the patient's quality of life and are a heavy burden for the healthcare system. Better understanding, early detection and proper follow-up could improve quality of life and reduce healthcare related costs. Therefore, the aim of this study was to evaluate if difference between osteoarthritic (OA) and non-osteoarthritic (non-OA) knees can be detected quantitatively on cartilage and subchondral bone levels with advanced but clinical available imaging techniques. Two OA (mean age = 88.3 years) and three non-OA (mean age = 51.0 years) human cadaveric knees were scanned two times. A high-resolution peripheral quantitative computed tomography (HR-pQCT) scan (XtremeCT, Scanco Medical AG, Switzerland) was performed to quantify the bone microstructure. A contrast-enhanced clinical CT scan (GE Revolution Evo, GE Medical Systems AG, Switzerland) was acquired with the contrast agent Visipaque 320 (60 ml) to measure cartilage. Subregions dividing the condyle in four parts were identified semi-automatically and the images were segmented using adaptive thresholding. Microstructural parameters of subchondral bone and cartilage thickness were quantified. The overall cartilage thickness was reduced by 0.27 mm between the OA and non-OA knees and the subchondral bone quality decreased accordingly (reduction of 33.52 % in BV/TV in the layer from 3 to 8 mm below the cartilage) for the femoral medial condyle. The largest differences were observed at the medial part of the femoral medial condyle both for cartilage and for bone parameters, corresponding to clinical observations. Subchondral bone microstructural parameters and cartilage thickness were quantified using in vivo available imaging and apparent differences between the OA and non-OA knees were detected. Those results may improve OA follow-up and diagnosis and could lead to a better understanding of OA. However, further in vivo studies are needed to validate these methods in clinical practice

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

Lesions in the joint surface are commonly treated with osteoarticular autograft transfer system (OATS), autologous cell implantation (ACI/MACI), or microfracture. Tissue formed buy the latter commonly results in mechanically inferior fibrocartilage that fails to integrate with the surrounding native cartilage, rather than durable hyaline cartilage. Fractional laser treatment to make sub-millimeter (<500 µm) channels has been employed for tissue regeneration in the skin to facilitate rejuvenation without typical scarring. Additionally, we have pioneered a means to generate articular cartilage matrix from chondrocytes—dynamic Self-Regenerating Cartilage (dSRC). Combining these two approaches by performing fractional laser treatment of the joint cartilage and treating with dSRC is a new paradigm for joint surface restoration. This approach was refined in a series of in vitro experiments and tested in swine knee defects during a 6-month study in 12 swine. dSRC are generated by placing 10. 7. swine knee chondrocytes into sealed 15-mL polypropylene tubes and cultured on a rocker at 40 cycles per minute for 14 days at 37°C. The chondrocytes aggregate and generate new extracellular matrix to form a pellet of dSRC. Channels of approximately 300-500 µm diameter were created by infrared laser ablation in swine cartilage (in vitro) and swine knees (in vivo). The diameter and depth of the ablated channel in the cartilage was controlled by the light delivery parameters (power, spot size, pulse duration) from a fractional 2.94 µm Erbium laser. The specimens were evaluated with histology (H&E, safranin O, toluidine blue) and polarized-sensitive optical coherence tomography for collagen orientation. We can consistently create laser-ablated channels in the swine knee and successfully implant new cartilage from dSRC to generate typical hyaline cartilage in terms of morphology and biochemical properties. The neocartilage integrates with host cartilage in vivo. These findings demonstrate our novel combinatorial approach for articular cartilage rejuvenation

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

Abstract. Objectives. Osteoarthritis (OA) is a complex joint disorder characterised by the loss of extracellular matrix (ECM) leading to cartilage degeneration. Changes to cartilage cell (chondrocyte) behaviour occur including cell swelling, the development of fine cytoplasmic processes and cell clustering leading to changes in cell phenotype and development of focal areas of mechanically-weak fibrocartilaginous matrix. [1]. To study the sequence of events in more detail, we have investigated the changes to in situ chondrocytes within human cartilage which has been lightly scraped and then cultured with serum. Methods. Human femoral heads were obtained with Ethical permission and consent from four female patients (mean age 74 yrs) undergoing hip arthroplasty following femoral neck fracture. Osteochondral explants of macroscopically-normal cartilage were cultured as a non-scraped control, or scraped gently six times with a scalpel blade and both maintained in culture for up to 2wks in Dulbecco's Modified Eagle's Medium (DMEM) with 25% human serum (HS). Explants were then labelled with CMFDA (5-chloromethylfluorescein-diacetate) and PI (propidium iodide) (10μM each) to identify the morphology of living or dead chondrocytes respectively. Explants were imaged using confocal microscopy and in situ chondrocyte morphology, volume and clustering assessed quantitatively within standardised regions of interest (ROI) using Imaris. ®. imaging software. Results. Within 2wks of culture with HS, chondrocyte volume increased significantly from 412±9.3µm. 3. (unscraped) at day 0 to 724±16.6 µm. 3. (scraped) [N(n) = 4(380)] (P=0.0002). Chondrocyte clustering was a prominent feature of HS culture as the percentage of clusters in the cell population increased with scraping from 4.8±1.4% to 14.9±3.9% [N(n) = 4(999)] at week 2 (P=0.0116). In addition, the % of the chondrocyte population within clusters increased from approximately 38% to 60%, and the number of cells per cluster increased significantly from 3.2±0.08 to 4±0.22 (P=0.031). The development of abnormal ‘fibroblastic-like’ chondrocyte morphology demonstrating long (>5µm) cytoplasmic processes also occurred, however the time course of this was more variable. For some samples, clustering occurred before abnormal morphology, but for others the opposite occurred. Typically, by the second week, 17±2.64% of the cell population had processes and this increased to 22±4.02% [N(n) = 4(759)] with scraping. Conclusions. Scraping the cartilage will remove surface constituents including lubricants (e.g. lubricin, hyaluronic acid, phospholipids), extracellular matrix constituents (collagen, proteoglycans – potentially the ‘lamina splendens’) and cells (chondrocytes and mesenchymal stromal cells (MSCs)). Although we do not know which of these component(s) is important, the effect is to dramatically increase the permeation of serum factors into the cartilage matrix and signal the development of cytoplasmic processes, cell clustering and swelling. It is notable that these cellular changes are similar to those occurring in early OA. [1]. This raises the interesting possibility that scraped cartilage cultured with human serum recapitulates some of the changes to in situ chondrocytes during early stages of cartilage degeneration and as such, could be a useful model for following the deleterious changes to matrix metabolism. 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

Large cartilage lesions in younger patients can be treated by fresh osteochondral allograft transplantation, a surgical technique that relies on stable initial fixation and a minimum chondrocyte viability of 70% in the donor tissue to be successful. The Missouri Osteochondral Allograft Preservation System (MOPS) may extend the time when stored osteochondral tissues remain viable. This study aimed to provide an independent evaluation of MOPS storage by evaluating chondrocyte viability, chondrocyte metabolism, and the cartilage extracellular matrix using an ovine model. Femoral condyles from twelve female Arcott sheep (6 years, 70 ± 15 kg) were assigned to storage times of 0 (control), 14, 28, or 56 days. Sheep were assigned to standard of care [SOC, Lactated Ringer's solution, cefazolin (1 g/L), bacitracin (50,000 U/L), 4°C storage] or MOPS [proprietary media, 22-25°C storage]. Samples underwent weekly media changes. Chondrocyte viability was assessed using Calcein AM/Ethidium Homodimer and reported as percent live cells and viable cell density (VCD). Metabolism was evaluated with the Alamar blue assay and reported as Relative Fluorescent Units (RFU)/mg. Electromechanical properties were measured with the Arthro-BST, a device used to non-destructively compress cartilage and calculate a quantitative parameter (QP) that is inversely proportional to stiffness. Proteoglycan content was quantified using the dimethylmethylene blue assay of digested cartilage and distribution visualized by Safranin-O/Fast Green staining of histological sections. A two-way ANOVA and Tukey's post hoc were performed. Compared to controls, MOPS samples had fewer live cells (p=0.0002) and lower VCD (p=0.0004) after 56 days of storage, while SOC samples had fewer live cells (p=0.0004, 28 days; p=0.0002, 56 days) and lower VCD (p=0.0002, 28 days; p=0.0001, 56 days) after both 28 and 56 days (Table 1). At 14 days, the percentage of viable cells in SOC samples were statistically the same as controls but VCD was lower (p=0.0197). Cell metabolism in MOPS samples remained the same over the study duration but SOC had lower RFU/mg after 28 (p=0.0005) and 56 (p=0.0001) days in storage compared to controls. These data show that MOPS maintained viability up to 28 days yet metabolism was sustained for 56 days, suggesting that the conditions provided by MOPS storage allowed fewer cells to achieve the same metabolic levels as fresh cartilage. Electromechanical QP measurements revealed no differences between storage methods at any individual time point. QP data could not be used to interpret changes over time because a mix of medial and lateral condyles were used and they have intrinsically different properties. Proteoglycan content in MOPS samples remained the same over time but SOC was significantly lower after 56 days (p=0.0086) compared to controls. Safranin-O/Fast Green showed proteoglycan diminished gradually beginning at the articular surface and progressing towards bone in SOC samples, while MOPS maintained proteoglycan over the study duration (Figure 1). MOPS exhibited superior viability, metabolic activity and proteoglycan retention compared to SOC, but did not maintain viability for 56 days. Elucidating the effects of prolonged MOPS storage on cartilage properties supports efforts to increase the supply of fresh osteochondral allografts for clinical use. For any figures or tables, please contact the authors directly

Introduction and Objective. Local cartilage defects in the knee are painful and mostly followed by arthritis. In order to avoid impaired mobility, the osteochondral defect might be bridged by a synthetic compound material: An osteoconductive titanium foam as an anchoring material in the subchondral bone and an infiltrated polymer as gliding material in contact with the surrounding natural cartilage. Materials and Methods. Titanium foam cylinders (Ø38 mm) with porosities ranging from 57% to 77% were produced by powder metallurgy with two different grain sizes of the space holder (fine: 340 ± 110 μm, coarse: 530 ± 160 μm). The sintered titanium foam cylinders were infiltrated with UHMWPE powder on one end and UHMWPE bulk at the other end, at two different temperatures (160 °C, 200 °C), using a pressure of 20 MPa for 15 minutes. Smaller cylinders (Ø16 mm) were retrieved from the compound material by water jet cutting. The infiltration depths were determined by optical microscopy. The anchoring of the UHMWPE was measured by a shear test and the mechanical properties of the titanium foam were verified by a subsequent compression test. The tribological behaviour was investigated in protein containing liquid using fresh cartilage pins (Ø5 mm) sliding against a UHMWPE disc with or without a notch to simulate the gap between the implant and the surrounding cartilage. Friction coefficients were determined in a rotation tribometer and the cartilage wear in a multidirectional six-station tribometer from AMTI (load 10 – 50 N, sliding speed 20 mm/s, 37 °C). Results. UHMWPE could be infiltrated into titanium foam by 1.1 – 1.3 mm with fine pores and by 1.5 – 1.8 mm with coarse pores. The infiltration was neither dependent on the type of UHMWPE (powder or bulk) nor on the temperature. The polymer was so well anchored inside the titanium foam pores that the shear forces for the compounds exceeded the shear strength obtained for a UHMWPE-cylinder. This effect was due to the increased stiffness of the compound plug. Uniaxial compression of the titanium foams after the shear-off of the polymer revealed yield strengths ranging from 50 – 88 MPa for porosities of 62 – 73%. The Ø16 mm samples yielded beyond physiological loads in the knee (≥ 10x body weight) and behaved in a strain hardening and fully ductile manner, reaching deformations of at least 50 % of their initial height without the appearance of macroscopically visible cracks. For smaller plug diameters down to Ø8 mm, however, the lower porosity / higher strength foam should be used to limit elastic deformation of the compound to < 0.1 mm. Pore size did not significantly influence the strength and stiffness values. The elevated coefficient of friction between cartilage and UHMWPE of about 1 was not negatively affected by the presence of the gap. The height loss of the cartilage pin after 1 hour (respectively after 3600 reciproque wear cycles) was 0.2 ± 0.1 mm using a flat disc. For discs with a 1 mm wide V-notch, the wear increased to 0.9 ± 0.3 mm. Conclusions. The tested titanium foams are well suited to act as an anchoring material in the subchondral bone as mechanical properties can be tailored by choosing the adequate porosity and as bone ingrowth has previously been demonstrated for the used pore sizes. UHMWPE is not an ideal gliding partner against cartilage because the friction coefficients of frictions were high. The presence of a V-notched gap was detrimental for cartilage wear. More hydrophilic polymers like PCU should be tested as potential gliding materials