Osteoarthritis (OA) is one of the most prevalent joint diseases involving progressive and degenerative changes to cartilage resulting from a variety of etiologies including post-traumatic incident or aging. OA lesions can be treated at its early stages through cell-based tissue engineering therapies using Mesenchymal Stem Cells (MSCs). In vivo models for evaluating these strategies, have described both chondral (impaction) and osteochondral (biopsy punch) defects. The aim of the investigation was to develop a compact and reproducible defect inducing post-traumatic degenerative changes mimicking early OA. Additionally, a pilot study to evaluate the efficacy of MSC-hydrogel treatment was also assessed. Surgery was performed on New Zealand white rabbits (male, 5–8 months old) with defects created on medial femoral condyle. For developing an appropriate defect, three approaches were used for evaluation: a biopsy punch (n = three at six and twelve weeks), an impaction device1 (n = three at six and twelve weeks) and a dental drill model (n = six at six and twelve weeks). At stated time points, condyles were harvested and decalcified in 10% EDTA, then embedded in Tissue-Tek and sectioned using a cryostat. Upon identification of region of interest, sections were stained with Safranin-O/Fast green and scored using OARSI scoring system by two blinded observers2. For the pilot study, autologous bone marrow was harvested from rabbits and used to isolate and expand MSCs. The Dental drill model was applied to both knee condyles, left untreated for six weeks at which stage, PKH26 fluorescently labelled MSCs were seeded into a hyaluronic acid hydrogel (TETEC). Repair tissue was removed from both condyles and MSC-hydrogel was injected into the left knee, whilst right knee was left empty. Rabbits were sacrificed at one (n = 1), six (n = 3) and twelve (n = 3) weeks post-treatment, processed as previously described and cartilage regeneration evaluated using Sellers score3. Impacted condyles exhibited no observed changes histologically (Mean OARSI score = 1 + 1), whereas biopsy punched and dental drilled defects demonstrated equal signs of cartilage erosion (OARSI score = 3 + 1) at assessed time points. However, biopsy punched condyles formed a diffusive defect, whereas dental drilled condyles showed a more defined, compact and reproducible defect. In the pilot study, PKH-labelled MSCs were observed at one and six weeks post-implantation within the defect space where hydrogel was injected. Tissue regeneration assessment indicated no difference between empty (Mean Sellers score = 14 + 2) and MSC treated defects (Sellers score = 16 + 5) at six weeks post-injection. At twelve weeks, MSC treated defects showed improved tissue regeneration with substantial subchondral bone restoration and good integration of regenerative cartilage with surrounding intact tissue (Sellers score = 10 + 1), whereas untreated defects showed no change in regeneration compared to six weeks (Sellers score = 16 + 2). Dental drill model was found to be the appropriate strategy for investigating early OA progression and treatment. Application of MSCs in defects showed good cartilage regeneration after twelve weeks application, indicating their promise in the treatment of early OA defects

The zonal organization of articular cartilage is crucial in providing the tissue with mechanical properties to withstand compression and shearing force. Current treatments available for articular cartilage injury are not able to restore the hierarchically organized architecture of the tissue. Implantation of zonal chondrocyte as a multilayer tissue construct could overcome the limitation of current treatments. However, it is impeded by the lack of efficient zonal chondrocyte isolation protocol and dedifferentiation of chondrocytes during expansion on tissue culture plate (TCP). This study aims to develop a protocol to produce an adequate number of high-quality zonal chondrocytes for clinical application via size-based zonal chondrocyte separation using inertial spiral microchannel device and expansion under dynamic microcarrier culture. Full thickness (FT) chondrocytes isolated from porcine femoral condyle cartilage were subjected to two serial of size-based sorting into three subpopulations of different cell sizes, namely small (S1), medium (S2), and large (S3) chondrocytes. Zonal phenotype of the three subpopulations was characterised. To verify the benefit of stratified zonal chondrocyte implantation in the articular cartilage regeneration, a bilayer hydrogel construct composed of S1 chondrocytes overlaying a mixture of S2 and S3 (S2S3) chondrocytes was delivered to the rat osteochondral defect model. For chondrocyte expansion, two dynamic microcarrier cultures, sort-before-expansion and sort-after-expansion, which involved expansion after or before zonal cells sorting, were studied to identify the best sort-expansion strategy. Size-sorted zonal chondrocytes showed zone-specific characteristics in qRT-PCR with a high level of PRG4 expression in S1 and high level of aggrecan, Type II and IX collagen expression in S2 and S3. Cartilage reformation capability of sorted zonal chondrocytes in three-dimensional fibrin hydrogel showed a similar trend in qRT-PCR, histology, extracellular matrix protein quantification and mechanical compression test, indicating the zonal characteristics of S1, S2 and S3 as superficial (SZ), middle (MZ) and deep (DZ) zone chondrocytes, respectively. Implantation of bilayered zonal chondrocytes resulted in better cartilage tissue regeneration in a rat osteochondral defect model than FT control group, with predominantly Type II hyaline cartilage tissue and significantly lower Type I collagen. Dynamic microcarrier expansion of sorted zonal chondrocytes was able to retain the zonal cell size difference that correlate to zonal phenotype, while maintaining the rounded chondrocyte morphology and F-actin distribution similar to that in mature articular cartilage. With the better retention of zonal cell size and zonal phenotype relation on microcarrier, zonal cells separation was achievable in the sort-after-expansion strategy with cells expanded on microcarrier, in comparison to cells expanded on TCP. Inertial spiral microchannel device provides a label-free and high throughput method to separate zonal chondrocytes based on cell size. Stratified implantation of zonal chondrocytes has the potential to improve articular cartilage regeneration. Dynamic microcarrier culture allows for size-based zonal chondrocyte separation to be performed on expanded chondrocytes, thus overcoming the challenge of limited tissue availability from the patients. Our novel zonal chondrocyte isolation and expansion protocol provide a translatable strategy for stratified zonal chondrocyte implantation that could improve articular cartilage regeneration of critical size defects

Introduction and aims. Growth plate cartilage is responsible for bone growth in children. Injury to growth plate can often lead to faulty bony repair and bone growth deformities, which represents a significant clinical problem. This work aims to develop a biological treatment. Methods. Recent studies using rabbit models to investigate the efficacy of bone marrow mesenchymal stem cells (MSC) to promote cartilage regeneration and prevent bone defects following growth plate injury have shown promise. However, translational studies in large animal models (such as lambs), which more closely resemble the human condition, are lacking. Results. Very recently, our labs have shown that ovine bone marrow MSC are multipotential and can form cartilage-like tissue when transplanted into mice. However, using a growth plate injury model in lambs, analogous to those described in the rabbit, autologous marrow MSC seeded into gelatine scaffold containing chondrogenic factor TGF-1, failed to promote growth plate regeneration. T o date, no large animal studies have reported successful regeneration of injured growth plate cartilage using MSC highlighting the possibility that ex vivo expanded MSC may not represent a viable cellular therapy for growth plate injury repair. In addition, using a growth plate injury repair model in young rats, our studies have also focused on understanding mechanisms of the faulty repair and identifying potential targets for enhancing growth plate regeneration using endogenous progenitor cells. We have observed that bony repair of injured growth plate is preceded sequentially by inflammatory, fibrogenic, chondrogenic and osteogenic responses involving both intramembranous and endochondral ossification mechanisms. We have observed infiltration of mesenchymal progenitor cells into the injury site, some of which have the potential to differentiate to osteoblasts or chondrocytes and contribute to the bony repair of the injured growth plate. Conclusion. This presentation will focus on our studies examining the efficacy of ex vivo expanded autologous MSC to enhance growth plate regeneration in the ovine model and work using a rat model aimed at identifying potential targets for enhancing cartilage regeneration by mobilising endogenous stromal progenitor cells

There is currently no cure for osteoarthritis (OA), although there are ways to manage it, but most require quite invasive surgeries. There is a resident mesenchymal progenitor cell (MPC) population within the synovial membrane of the joint that have the ability to differentiate into bone, fat, and cartilage. We hypothesise that in vivo and in vitro cell surface marker expression comparisons of the MPCs can determine which population has the highest chondrogenic capacity and is best suited for future clinical trials. Method optimisation protocol: Synovial biopsies (2 or 5mm) were obtained from patients undergoing surgery. The biopsies were digested in either collagenase type I, IA, IV or II at a concentration of 0.5 or 1.0 mg/mL. Digestion was conducted at 37°C for 30, 60, 90 or 120min. To assay for the number of MPCs obtained, the cell suspension was stained with CD90 (a synovial MPC marker) and magnetically purified. The purified cells were then assayed by flow cytometry (Co-stained with a live/dead cell marker, BV510) or bright-field microscopy. Study protocol: Synovial tissues were digested in type IV collagenase for two hours to obtain a single cell suspension. The cells were subsequently stained with mesenchymal stem cell markers, including CD 90, CD 271, CD 44, CD73, and CD105, a macrophage marker, CD68. The macrophages were excluded and the remaining cells were index sorted into 96-well plates. The cells were expanded, and underwent 21-day chondrogenic, adipogenic, and osteogenic differentiation. Differentiation was assayed using RT-qPCR and histological methods. Additionally, the cells were re-analysed for marker expression after culturing. Optimisation: Synovial biopsies of 5mm produced a greater number of live CD90+ cells than 2mm biopsies. It was observed that type IV collagenase at 1mg/ML treatment for 120 min (hip) and 90 min (knee) obtained the greatest number of CD90+ MPCs from the synovium. Results: A single cell was isolated from an OA hip biopsy and was positive for the markers CD90, CD44, CD73, and negative for the markers CD68, CD271, CD105. Following differentiation, PCR analysis suggested that the cell line was able to differentiate into chondrocytes and adipocytes, but not osteoblasts. Histology data agreed with the PCR data with the adipocytes and chondrocytes having positive staining, whereas the osteoblasts were negative. FACS analysis following proliferation showed that the expression in vivo versus in vitro was the same except CD105 that became positive after proliferation in vitro. MPCs express cell surface markers that provide information as to populations have the best cartilage regeneration abilities. By determining the properties of the MPCs in OA hips that allow for better chondrogenic differentiation abilities in vitro, selecting the optimal cells for regenerating cartilage can be done more efficiently for novel cell therapies for OA

The purpose of this study is to assess the improvement in pain and function of the ankle when arthrodiastasis is used for end stage juvenile idiopathic arthritis [JIA] in the paediatric population. All patients treated with ankle arthrodiastasis, 2009–2013 were studied. Clinical, radiological and survivorship data were examined. The Oxford Ankle Foot Questionnaire for Children (OxAFQ-C) and Parents (OxAFQ-P), along with the American Orthopaedic Foot and Ankle Society (AOFAS) Clinical rating system for Ankle-Hindfoot were recorded pre-operatively and at 6 months. Eight patients (9 ankles) with severe tibiotalar JIA, refractory to medical management were treated. Average age at surgery was 14.5 years (range 8–19). Average length of arthrodiastasis was 3.5 months. Length of follow-up averaged 13 months (range 5–28 months). All scores showed an improvement at 6 months. OxAFQ-C scores (out of 60) improved on average from 23 to 43. OxAFQ-P scores also improved from19 to 39. The largest improvement was found for the physical subsection. AOFAS Ankle-Hindfoot score (out of 100) averaged 34 pre-op and 74 at 6 months. Pain scored out of 10 decreased from an average of 7.4 to 4.3 at 6 months. All patients and parents were satisfied with the surgery and would have the procedure performed again. Radiological studies demonstrated cartilage regeneration, joint restoration and deformity correction with arthrodiastasis. Survivorship was good (75%) at 36 months, but 2 patients (3 ankles) had subsequent surgery in the adult sector for progression of disease despite initial improvement following arthrodiastasis. This case series demonstrates the efficacy of ankle arthrodiastasis as a surgical option in severe end-stage ankle inflammatory arthritis in paediatric patients in the short to midterm. It improved functional scores and pain scores which should delay the need for more radical joint fusion or replacement procedures in this challenging surgical condition

Chondral injuries of the knee are extremely common and present a unique therapeutic challenge due to the poor intrinsic healing of articular cartilage. These injuries can lead to significant functional impairment. There are several treatment modalities for articular osteochondral defects, one of which is autologous chondrocyte implantation. Our study evaluates the mid to long term functional outcomes in a cohort of 828 patients who have undergone an autologous chondrocyte implantation procedure (either ACI or MACI), identifying retrospectively factors that may influence their outcome. The influence of factors including age, sex, presence of osteoarthritis and size and site of lesion have been assessed individually and with multivariate analysis. All patients were assessed using the Bentley Functional Score, Visual Analogue Score and the Cincinnati Functional Score. Assessment were performed pre-operatively and of their status in 2010. The longest follow-up was 12 years (range 24 to 153 months) with a mean age of 34 years at time of procedure. The mean defect size was 409 mm. 2. (range 64 to 2075 mm. 2. ). The distribution of lesions was 51% Medial Femoral Condyle, 12.5% Lateral Femoral Condyle, 18% Patella (single facet), 5% Patella (Multifacet) and 6% Trochlea. 4% had cartilage transplant to multiple sites. High failure rates were noted in those with previous cartilage regenerative procedures or evidence of early osteoarthritis and those with transplantation to multiple sites. Autologous chondrocyte implantation is an effective method of decreasing pain and increasing function, however patient selection plays clear role in the success of such procedure

Purpose. The outcome of idiopathic chondrolysis in South Africa has been reported as a progressive downhill course resulting in a painful, stiff hip (Jones 1971, Sparks&Dall 1982). The cause of the disease remains unknown. Theories suggested are mechanical (decreased movement with loss of synovial nutrition; increased joint pressure) and an auto-immune response in genetically predisposed individuals. Our experience with continuous passive motion (CPM) and anti-inflammatory treatment has been disappointing. Method. In order to improve our understanding of the disease and our results, we prospectively studied 5 consecutive patients. All the patients had a subtotal capsulectomy (Roy&Crawford 1988) to relieve intra-articular pressure and correction of the flexion and abduction deformities. Post-operative treatment was with anti-inflammatories and CPM. Results. The patients were adolescent females between 10 and 12.5 years old. They presented with stiff, painful hips with flexion, abduction and external rotation deformities. They had normal auto-immune markers. Radiographs revealed osteopenia and joint space narrowing. CT confirmed osteopenia and joint space narrowing. Three patients had subchondral erosions (two on either side of the joint and one on the acetabular side only). MRI showed bone oedema and confirmed the erosions. Histology of the synovium showed non-specific chronic inflammation with lymphocyte and plasma cell infiltration suggesting an auto-immune cause. Histology of the cartilage showed a superficial layer of fibrous tissue, then a layer of degenerate chondrocytes, with normal chondrocytes in the deep layer. Post-operatively patients had improved range of motion. At mean follow up of 7.8 months the patients had a repeat MRI to assess cartilage regeneration. NO DISCLOSURES

Subchondral drillings for articular cartilage defects usually result in fibrocartilage repair, which is inferior biomechanically compared to hyaline cartilage. We postulate that intra-articular injections with autologous marrow-derived stem cells (MSC) and hyaluronic acid (HA) can improve the quality of repair cartilage. We tested this hypothesis in a goat model by creating an articular cartilage defect in the stifle joint and conducted subchondral drillings. The animals were divided into three groups: Group A (control) no injections, Group B (HA) weekly injection of 1 ml sodium hyaluronate for three weeks, Group C (HA+MSC) similar to Group B but with 2 mls autologous MSC in addition to HA. MSC were obtained by bone marrow aspiration, centrifuged, and divided into aliquots, which were cryopreserved. Fifteen animals were equally divided between the groups and sacrificed at 24 weeks after surgery where the joint was harvested and examined macroscopically and histologically. Of the 15 animals, two had died in Group A and one was excluded from Group C due to an infection. In Group A, repair constituted mainly of scar tissue, while in Group B, there was less scar tissue, with small amounts of proteoglycan and collagen II at the osteochondral junction. In contrast, repair cartilage from Group C animals demonstrated almost complete coverage of the defect with evidence of hyaline cartilage regeneration. Histology as assessed by Gill scoring was significantly better in Group C with one-way ANOVA giving an F-statistic of 10.611 with a p-value of 0.004, which was highly significant. Post-operative intra-articular injections of autologous MSC in combination with HA following subchondral drillings into chondral defects resulted in better cartilage repair

Subchondral drillings for articular cartilage repair give functional improvement that peaks at 24 months after surgery. We postulate that intra-articular injections with autologous peripheral blood stem cells (PBSC) and hyaluronic acid (HA) following subchondral drillings can improve the repair process. Thirty-four patients with full thickness chondral defects of the knee joint underwent subchondral drillings. The operated knees were then placed on continuous passive motion for a period of two hours per day for four weeks, with partial weight-bearing for the first six weeks. PBSC were harvested by apheresis and divided into aliquots which were cryopreserved. One week after surgery, weekly intra-articular injections of 2.5 mLs PBSC mixed with 2 mLs of sodium hyaluronate were given for five weeks after surgery. Patients were followed up for an average of 11 months (range 6–20) and assessed using serial MRI scans. Second look arthroscopy and chondral biopsies were obtained in five patients. International Knee Documentation Committee (IKDC) scores were compared with previous microfractures results from the Mithoefer cohort study using linear interpolation to generate time-based predicted values. The difference was compared using a two-tailed, one-sample T-test against a value of zero. Serial MRI scans showed healing of subchondral bone and evidence of cartilage regeneration that was confirmed on arthroscopy with good integration into surrounding cartilage with no delamination. Biopsy specimens showed attributes typical of hyaline cartilage with good cellular morphology, abundant proteoglycans and Type II collagen. No oedema or degenerative changes were seen. The IKDC data was on average 12.8 points (95% CI 6.5-19.1) higher than the Mithoefer group with p=0.0002. Intra-articular injections of PBSC and HA following subchondral drillings resulted in good repair tissue based on MRI, arthroscopic, and histological criteria, with IKDC scores superior to standard microfracture surgery

INTRODUCTION. Osteochondral defects are still a challenge for the orthopaedic surgeon, since most of the current surgical techniques lead to fibrocartilage formation and poor subchondral regeneration, often associated to joint stiffness and/or pain. Thinking of the ideal osteochondral graft from both the surgical an commercial point of view, it should be an off-the-shelf product; this is the research direction and the explanation for the new biomaterials recently proposed to repair osteochondral defect inducing an “in situ” cartilage regeneration starting from the time of the implantation into the defect site. For the clinical pilot study we performed, a newly developed nanostructured biomimetic scaffold was used to treat chondral and osteochondral lesions of the knee; its safety and manageability, as much as the surgical procedure reproducibility and the clinical outcome, were evaluated in order to test its intrinsic potential without any cells colture aid. MATERIALS AND METHODS. A new osteochondral scaffold was obtained by enucleating equine collagen type 1 fibrils with hydroxyapatite nanoparticles in 3 different layers with 3 different gradient ratios at physiological conditions. 30 patients (9F, 21M, mean age 29,3yy) affected by either chondral or osteochondral lesions of the knee (8 medial femoral condyles, 5 lateral femoral condyles, 12 patellae, 8 femoral throcleas) underwent the scaffold implantation from January to July 2007. The sizes of the lesions were in between 2 and 6 squared cm. All patients and their clinical outcome were analyzed prospectively at 6, 12, 24 and 36 months using the Cartilage standard Evaluation Form as proposed by ICRS and an high resolution MRI. RESULTS. We observed a statistically significant scores improvement and function recovery comparing the pre-operative to the follow-up parameters evaluated. Moreover, we noticed a better improvement from 12 to 24mm follow up while the good results gained at 2yy were confirmed at 3yy follow up evaluation. The MOCART scoring scale was used to analyze the MRIs. In 80% of cases we obtained a complete filling of the cartilage defect and in some patients we even appreciated articular surface congruency. In this series we report 1 failure followed by a re-operation with different technique. CONCLUSIONS. This new minimally invasive one-step surgical approach to osteochondral defects seems to be an easy and effective procedure. The results obtained are very encouraging and this procedure show satisfactory outcomes even in big osteochondral defects

Purpose. The prevalence of focal chondral lesions reported inthe literature during knee arhroscopy can be as high as 63%. Of these, more than half are either grade III or grade IV lesions (Outerbridge). Full thickness cartilage lesions ranging from 2cm2 to 10cm2 are the most challenging to treat. To goal of this study was to evaluate clinical outcomes of pain, function and quality of life, along with radiological outcomes of cartilage repair using microfracture, autologous minced cartilage and polymeric scaffold. Method. A cohort of thirty-eight patients with Outerbridge grade III or IV cartilage injuries larger than 2cm2 in the knee's femoral condyle, trochlea or patella were prospectively folowed since 2008. They were all treated with microfracture, fresh minced autologous cartilage grafting and a polymeric scaffold technique through mini-arthrotomy of the knee. Autografts and scaffolds were secured to subchondral bone using fibrin glue and tran-sosseous resorbable sutures. Patients were evaluated pre and postoperatively using VAS scores for pain, WOMAC and IKDC scores for knee function and SF-36 questionnaire for quality of life. Clinical evaluations were done by physical examination, and imaging was done using X-Rays, MRI and arthro-CT. Results. Mean follow-up time was14.64.6 months. Mean age was 48.39.3 years old. Pre-op lesions averaged 3.51.5 cm2. VAS pain scores were significantly reduced after surgery (7,62 to 2,52.3, p<. 0001). Improvement in knee function using IKDC score improved from 26,717.5 to 55,415.3, p<. 001). In addition, WOMAC total scores showed significant reduction from 55,520.3 to 27,517.6. SF-36 quality of life Physical Component Summary improved from 26,411.4 to 45,812.3, p<. 01; Mental Component Summary improved from 41,916.8 to 49,411.2, p<. 048). Imaging results indicate sustained cartilage thickness from 6 to 18 months. One patient was an early failure due to scaffold loosening, and two patients had no clinical improvement and no significant cartilage regeneration on MRI and Arthro-CT imaging at 6 months post-op. Conclusion. The combination of microfractures, fresh minced autologous cartilage grafting and polymeric scaffold fixation seems to be an effective treatment option for post-traumatic and focal cartilage lesions of the knee in the short term. A longer-term follow-up to evaluate the sustainability of these results is ongoing. Shortcomings of this study are its short term, the lack of second look arthroscopies and cartilage biopsies to evaluate cartilage microstructure, and the absence of a gold standard treatment for full-thickness cartilage lesions larger than 2cm2 that could be used as a control

Hyaline articular cartilage has been known to be a troublesome tissue to repair once damaged. Since the introduction of autologous chondrocyte implantation (ACI) in 1994, a renewed interest in the field of cartilage repair with new repair techniques and the hope for products that are regenerative have blossomed. This article reviews the basic science structure and function of articular cartilage, and techniques that are presently available to effect repair and their expected outcomes.