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

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

Multiple biochemical biomarkers have been previously investigated for the diagnosis, prognosis and response to treatment of articular cartilage damage, including osteoarthritis (OA). Synovial fluid (SF) biomarker measurement is a potential method to predict treatment response and effectiveness. However, the significance of different biomarkers and their correlation to clinical outcomes remains unclear. This systematic review evaluated current SF biomarkers used in investigation of cartilage degeneration or regeneration in the knee joint and correlated these biomarkers with clinical outcomes following cartilage repair or regeneration interventions. PubMed, Institute of Science Index, Scopus, Cochrane Central Register of Controlled Trials, and Embase databases were searched. Studies evaluating SF biomarkers and clinical outcomes following cartilage repair intervention were included. Two researchers independently performed data extraction and QUADAS-2 analysis. Biomarker inclusion, change following intervention and correlation with clinical outcome was compared. 9 studies were included. Study heterogeneity precluded meta-analysis. There was significant variation in sampling and analysis. 33 biomarkers were evaluated in addition to microRNA and catabolic/anabolic ratios. Five studies reported on correlation of biomarkers with six biomarkers significantly correlated with clinical outcomes following intervention. However, correlation was only demonstrated in isolated studies. This review demonstrates significant difficulties in drawing conclusions regarding the importance of SF biomarkers based on the available literature. Improved standardisation for collection and analysis of SF samples is required. Future publications should also focus on clinical outcome scores and seek to correlate biomarkers with progression to further understand the significance of identified markers in a clinical context

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

Background. Microfracture (MF) and Autologous Chondrocyte Implantation (ACI) are used to repair symptomatic condylar cartilage defects (grade II-IV Outerbridge). Superiority of ACI to MF is still debated. The aim of the study was to conduct a systematic literature review, compare superiority of ACI versus MF in a meta-analysis and investigate the correlation between patient age and outcome of both treatments. Methods. Extended literature search was conducted (papers from January 2001 to present), looking at patient characteristics, pre- and post-operative scores and cartilage repair assessment evaluation. Methodological quality was verified through modified Coleman score and assessment bias. A fixed-effect meta-analysis was conducted, comparing post-operative standardised mean differences between ACI and MF. Pearson correlation coefficient between post-operative score and age was calculated against ACI and MF. Results. of 490 studies systematically analysed, 8 met the inclusion criteria, accounting for 255 patients treated with ACI and 259 with MF. Overall mean postoperative scores were 81.38±8.31 for ACI and 74.9±7.0 for MF, with no significant difference (p=0.13). The average modified Coleman score of the studies was 82.6, with low bias among them. The meta-analysis displayed an overall effect estimate of 0.3 favouring ACI treatment versus MF (95%CI=0.12–0.48, P=0.001). Significant heterogeneity was although observed (I2>70%). Pearson correlation coefficient calculated between mean post-operative score and mean age, surprisingly failed to indicate clear correlation for ACI (r=0.11) and MF (r=0.18) respectively. Conclusions. Minor statistically significant superiority of ACI intervention versus MF in knee cartilage repair was found, together with high levels of heterogeneity, halting the possibility to make full recommendation of ACI versus MF. Level of Evidence. Ia (systematic review and meta-analysis)

Osteoarthritis (OA) of the spine and diarthrodial joints is by far the most common cause of chronic disability in people over 50 years of age. The disease has a striking impact on quality of life and represents an enormous societal and economic cost, a burden that will increase greatly as populations age. OA is a complex condition with broad pathology. Damage to the articular cartilage is a consistent feature, accompanied by changes to the subchondral bone and synovium. Progression of the disease involves further degeneration of the articular cartilage, damage to the underlying bone and morphological changes that include subchondral bone thickening, development of cysts, osteophytes and inflammation of the synovium. Enhanced production of proinflammatory cytokines and matrix metalloproteinases accelerates degradation of the articular cartilage. It is striking that no approved pharmacological intervention, biological therapy or procedure prevents the progressive destruction of the OA joint. All current treatments, without exception, produce symptomatic rather than regenerative results. While there have been some exciting developments in the search for OA treatments in the last decade, including matrix metalloproteinase inhibitors, anti-TNF and anti-IL1 drugs for example, none of these has to date emerged as an effective medicinal product. There is thus an urgent and compelling need to identify, validate and test new biological therapeutics. Stromal cell therapy represents one such compelling approach. The results from several early clinical studies have indicated that this approach holds a great deal of promise for the treatment of OA. Most studies have involved direct intraarticular injection of a suspension of mesenchymal stromal cells (MSCs) for treatment of knee OA. Results from a number of controlled patient studies have suggested that this treatment results in an effective repair response. Although data regarding mechanism of action are limited, it appears that the cells have an anti-inflammatory effect, possibly targeting cells within the synovium, rather than a direct cartilage repair effect. Several recent reports have highlighted a dramatic and sustained response in patients receiving MSC treatment. For example, allogeneic expanded adipose-derived MSCs have been shown to be safe and effective in the treatment of complex perianal fistulas in Crohn's disease. Also, allogeneic bone marrow-derived MSCs has a been shown to have a positive effect in pediatric acute graft versus host disease. These observations point to a mechanism of action that involves host immunomodulation, but this needs further examination. Within the field of musculoskeletal disease effective translation of MSC technology has been hindered by a lack of randomized controlled patient studies, severe inconsistencies regarding the preparation and characterization of the cell product, and an incomplete understanding of the therapeutic mechanism. Direct to consumer clinics have flourished in some countries, providing cell treatments to OA patients. Most or all of these utilize unexpanded cell fractions from marrow or fat without even rudimentary product characterization and may report an exaggerated clinical outcome. Data from these clinics is not likely to yield information that will be useful. In fact, a recent systemic review of clinical trials involving MSC treatment in OA indicated that only a limited number of studies provided high quality evidence and long term follow up. Many suffered from a lack of consistency, including a diversity of methods for MSC preparation, and thus did not contribute to a supporting evidence base. There is a compelling need to provide clear and unambiguous clinical proof of concept relating to MSC treatment for OA. The ADIPOA2 study, currently active in Europe, will go some way towards achieving this. This is a 150 patient, phase 2b study designed to to assess the efficacy of a single injection of autologous adipose-derived MSCs in the treatment of mild to moderate OA of the knee, active and unresponsive to conservative therapy for at least 12 months

Articular cartilage repair is assumed to improve by covering the cartilage lesion with a biomaterial scaffold tailored to the specific requirements of the weight-bearing joint surface. We have tested the feasibility of a novel composite collagen-polylactide scaffold rhCo-PLA in cartilage repair. To confirm these results and further challenge the scaffold, we tested it in a large porcine cartilage defect. A critical-sized full-thickness chondral defect was made in the medial femoral condyle of 18 domestic pigs. This technically widest possible defect size of 11×17 mm was determined in a pilot test. Five weeks later, the defect was either treated with the novel rhCo-PLA scaffold or left untreated to heal spontaneously. After four months, the medial condyles were evaluated macroscopically using Goebel's score, in which the worst possible result receives a total of 20 points and imaged with µCT to evaluate subchondral bone. Macroscopic score and subchondral bone microstructure were similar in both study groups. The total Goebel score was higher in spontaneous group (9.75±3.9 for spontaneous and 9.1±3.7 for rhCo-PLA, respectively) but differences between individual animals were large. Subchondral bone volume fraction was 48.2±3.6% for rhCo-PLA and 44.2±3.4% for spontaneous. Trabecular thickness was greater in operated joints (207.9±18.8 µm for spontaneous and 242.9±32.9 µm for rhCo-PLA) than in contralateral non-operated joints (193.3±15.1 µm and 213.4±33.2 µm, respectively). These preliminary data demonstrate that individual differences in the macroscopic appearance were large but there were no significant differences between the two study groups in the score or subchondral bone structure

Summary Statement. Hypoxia enhances chondrocyte phenotype of cells migrating from cartilage fragments, thus supporting the use of chondral fragment as a potential cell source for one-stage cartilage repair. Introduction. Minced cartilage fragments are a viable cell source for one stage cartilage repair, as shown in both in preclinical and clinical studies. However, the joint microenvironment, in which the repair process takes place, is hypoxic and no evidences are present in literature regarding the behaviour of cartilage fragments in a hypoxic environment. Aim of the study is to verify if hypoxia could influence chondrocyte outgrowth from cartilage fragments into a Hyaluronic-Acid/fibrin scaffold and evaluate its effects on migrating chondrocyte behaviour, compared to normoxic condition. This could be significant in the perspective of a wide clinical application of human chondral fragments for single stage repair. Materials and methods. Constructs were prepared with minced cartilage fragments harvested from the trochlear region of 20 human young donors during ACL reconstruction, loaded onto a non-woven esterified Hyaluronic-Acid-derivative felt (Hyaff-11) and retained with a coating of fibrin glue (Tisseel). Constructs were cultured either in normoxic or in hypoxic (10% O. 2. ) condition. The growth medium contained DMEM-high-glucose (4500mg/l)with 10% fetal-bovine-serum. After 1 month, construct sections were stained with haematoxylin/eosin and Safranin-O and examined for cell counting. Expression of chondrocyte markers (SOX9, collagen-II, collagen-I), hypoxic markers(HIFs) and proliferative markers (beta-catenin, PCNA) was assessed using immunofluorescence. Results. Migrating cells predominantly showed a spindle-like shape when close to the fragments and a more roundish shape when embedded into the scaffold. A slight decrease in chondrocyte migration and proliferation was observed in hypoxic cultures, albeit not statistically significant. Conversely, an increase in the expression of SOX9, beta-catenin, HIFs, collagen-II (p<0.05) in migrating chondrocytes from hypoxic cultures was shown by immunofluorescence. Discussion/Conclusion. Hypoxia seems to improve the chondrocyte phenotype/behaviour of cell outgrowing from cartilage fragments onto a HA/fibrin scaffold. Moreover, hypoxic condition did not hamper the ability and the mechanisms by which chondrocytes migrate from cartilage fragments and proliferate into the surrounding environment. This is clinically relevant in order to validate one-step repair techniques by means of human cartilage fragments loaded into composite scaffolds

Abstract

Osteoarthritis is the most common chronic condition of the joints. It is characterized by the degeneration of articular cartilage, formation of osteophytes and alterations in the synovium. This process has a severe impact on the quality of life of the patients and the currently available treatments are unsatisfactory and often merely focused on pain relief. In our group we are working on the development of in situ cross-linkable hydrogel platforms that could be used for resurfacing the damaged articular cartilage using a minimally invasive arthroscopic procedure. Stable fixation of the gel at the joint surface, facilitating the ingrowth of local stem and progenitor cell populations and supporting intrinsic repair mechanisms are considered minimal design parameters. To achieve this, we are exploring the use of enzymatically cross-linkable natural polymer-tyramine conjugates.

Dextran-tyramine conjugates were prepared by activation of dextran-OH and subsequent reaction with tyramine. Hyaluronic acid-tyramine and protein-tyramine conjugates were prepared using DMTMM coupling. In situ crosslinking is achieved by mixing the polymer conjugates with the enzyme HRP and minute, non-toxic amounts of H2O2 as oxidizing agent. Support of cartilage formation was studied after mixing of the polymer conjugates with mesenchymal stem cells, chondrocytes or combinations of both prior to crosslinking. Cell ingrowth was studied by implanting the hydrogels in an ex-vivo cartilage defect while mechanically loading the explant in a bioreactor and cell migration in the hydrogels was evaluated by tracking the sprouting of fluorescently labelled cell-spheroids.

We prepared dextran-tyramine conjugates with a degree of substitution of 10 tyramine residues per 100 monosaccharide units. The conjugated hyaluronic acid-tyramine had a degree of substitution of 10% of the carboxylic acid groups, while for the proteins the substitution was dependent on the protein type.

Enzymatically crosslinked hydrogels, based on dextran and hyaluronic acid, with the addition of co-cross linkable proteins show excellent properties for application in the regeneration of damaged cartilage.

Unique progenitor cells have been identified recently and successfully cultured in vitro from human articular cartilage. These cells are able to maintain chondrogenic potential upon extensive expansion. In this study, we have developed a sheep, ex-vivo model of cartilage damage and repair, using these progenitor cells. This study addresses the question can such a model be used to determine factors required for progenitor cell proliferation, differentiation and integration of matrix onto bone. The hypothesis was that sheep allogenic cartilage derived progenitor cells could regenerate artificially damaged sheep articular cartilage in an osteochondral culture model. Progenitor cells were derived from ovine articular cartilage using a differential adhesion assay to fibronectin and expanded clonally. These clonal cells were marked with lentiviral vectors derived from the Human Immunodeficiency Virus-1. When a self-inactivating lentiviral vector encoding a ubiquitous phosphoglycerate kinase promoter, driving a Green Fluorescent Protein (GFP) reporter gene, was used to transduce these cells, up to 80% of these progenitor cells expressed GFP. Normal sheep medial femoral condyles containing about 2mm thick sub-condral bone were obtained and 4mm circular defects created on the cartilage surface using a biopsy punch. Condyles were cultured for two weeks in vitro with GFP labelled progenitor cells within a fibrin glue scaffold (Tisseel Lyo) and matrix production (collagen) as determined by spatially offset Raman spectroscopy and immunohistochemistry was demonstrated. Progenitor cells were able to proliferate and differentiate into collagen producing cells. Such an ex-vivo model system is an effective tool for the analysis of cartilage repair from various sources of stem cells. These ex-vivo experiments and variations on defect type, size, titration of scaffold and progenitor cell numbers requirements can further be used as a basis for screening prior to in vivo experiments

Recent advances in tissue engineering have made progress towards the development of biomaterials with the capability for delivery of growth factors to promote enhanced tissue repair. However, controlling the release of these growth factors is a major challenge and the associated high costs and side effects of uncontrolled delivery of has proved increasingly problematic in clinical orthopaedics. Gene therapy might be a valuable tool to avoid these limitations. While non-viral vectors are typically inefficient at transfecting cells, our group have had significant success in this area using a scaffold-mediated gene therapy approach for regenerative applications. These gene activated scaffold platforms not only act as a template for cell infiltration and tissue formation, but also as a ‘factory’ to provoke autologous host cells to take up specific genes and then engineer therapeutic proteins in a sustained but eventually transient fashion. Alternatively, scaffold-mediated delivery of siRNAs and miRNAs can be used to silence specific genes associated with pathological states in orthopaedics. This presentation will provide an overview of some of this research with a particular focus on gene-activated biomaterials for promoting stable cartilage formation in joint repair and on scaffold-based delivery of therapeutics for enhancing vascularization & bone repair.

Damage to articular cartilage is difficult to treat, as it has a low capacity to regenerate. Biomimetic natural polymer scaffolds can potentially be used to regenerate cartilage. Collagen hyaluronic acid (CHyA) scaffolds have been developed in our laboratory to promote cell infiltration and repair of articular cartilage. However, the low mechanical properties of such scaffolds potentially limit their use to the treatment of small cartilage defects. 3D-printed polymers can provide a reinforcing framework in these scaffolds, thus allowing their application in the treatment of larger defects. The aim of this study was to create mechanically functional biomaterial scaffolds by incorporating a CHyA matrix into 3D-printed polymer meshes resulting in an integrated porous material composite with improved mechanical properties for repair of large cartilage defects. 3D-printed meshes were developed to facilitate an architecture suitable for nutrient flow, cell infiltration, and even CHyA incorporation. And the meshes were freeze dried in custom made moulds to create a pore structure suitable for chondrogenesis. Uniaxial compressive testing of the scaffolds revealed improved mechanical properties following reinforcement with printed meshes, with the compressive modulus increasing from 0.8kPa (alone) to 0.5MPa (reinforced structure). The reinforced scaffolds maintained interconnected pores with the mean pore diameter increasing from 130 to 175µm. The reinforcement had no negative impact on MSC viability, with 90.1% viability in reinforced scaffolds at day 7. The compressive modulus of the reinforced CHyA scaffold is close to native articular cartilage, suggesting that this approach can be used for treatment of large cartilage defects.

Summary. The findings demonstrate that culture expanded human mesenchymal stem cells (MSCs) incorporated and proliferated in clinically relevant cell scaffolds better than freshly isolated bone marrow mononucleated cells (MNCs); in fact, only in MSC cultures were cells present for longer term chondrogenic inductions. Introduction. The treatment of chondral defects poses a significant clinical problem and a variety of cell sources and techniques have been studied and practiced to regenerate cartilage. Preclinical and clinical evidence suggests that MSCs can help regenerate cartilage when transplanted into cartilage lesions. However, the uptake of MSCs for cell therapies is limited due to the need for their culture expansion to generate subsequent numbers for transplantation. An alternative is to use minimally manipulated MNCs, which avoids the costs and regulatory implications of culture expansion and would enable the treatment of cartilage defects in a one-step procedure. Therefore, this study has focused on comparing these two cell types within three different scaffolds that can currently be used as cell delivery systems. Methods. Culture expanded human MSCs and MNCs freshly isolated from bone marrow were seeded at a density of 50,000 cells in 3mm. 2. scaffolds of Chondro-Gide® (type I/III collagen), Alpha Chondro Shield® (polyglycolic acid) and Hyalofast™ (hyaluronic acid). The cell-seeded scaffolds were incubated for 2 hours to permit initial cell adhesion and then treated with or without chondrogenic inducers (100nM dexamethasone, 10ng/ml TGF-β1, 37.5µg/ml ascorbic acid and ITS-X in DMEM/10% serum) for 28 days at 37°C. The Cell incorporation, growth and viability was assessed using Live/Dead staining and confocal microscopy, along with histological stains of the sectioned scaffolds. Proteoglycan synthesis was measured using DMMB assay of glycosaminoglycan (GAG) into the harvested culture medium. Results. MSCs adhered to the scaffolds to a much greater extent than the MNCs. In fact, the low number of MNCs initially incorporated into the scaffolds diminished over time such that no viable MNCs were seen during long term cultures and in all cases. MSCs incorporated into the Chondro-Gide® scaffold better than into the Alpha Chondro Shield® or Hyalofast™, and during long term cultures the MSCs in Chondro-Gide® proliferated to become significantly greater in number than those in the other two scaffolds. There was no clear matrix deposition. However, the MSCs in Hyalofast™ were rounded in shape, which is consistent with the morphology of chondrocytes, in the presence of chondrogenic inducers only. Furthermore, a significantly greater level of GAG was detected in the medium harvested from Chondro-Gide® and Hyalofast™ cultures under chondrogenic conditions compared with non chondrogenic conditions. Discussion/Conclusion. This study has shown that human MSCs incorporated, adhered and proliferated better in clinically utilised cell scaffolds compared to MNCs, enabling the induction of chondrogenesis in the longer term. Freshly isolated MNCs from bone marrow contain only 0.01–0.001% of MSCs in addition to non-adherent cell types, e.g. hematopoietic cells, which may account for their low cellular incorporation and decreased cell proliferation in the scaffolds. This outcome for MNCs may be improved using prospective MSC isolation techniques, where in vivo studies are also required to properly examine the chondrogenic potential. Nonetheless, our initial work suggests that culture expanded MSCs are a better option than minimally manipulated cells for cartilage repair

Mesenchymal stem cells (MSCs) have potential for therapeutic repair of cartilage and bone but still require optimization in terms of their capacity to deposit an appropriate extracellular matrix (ECM). Adult human cartilage has a limited capacity for repair and is unusual in that it is one of the few tissues where injury is not followed by an influx of monocytes. We are studying the effects of co-culturing primary monocytes with MSCs differentiating along chondrogenic lineage but in addition we needed to investigate the effects of the monocytes on the mature chondrocytes that will result from the MSCs and will also be present in the host tissue.

Human articular cartilage chondrocytes were isolated from human donors undergoing knee replacement surgery for osteoarthritis (OA) with full ethical consent. Cultures were expanded and cells used below passage five for co-culture experiments. Monocytes were prepared from fresh heparinized human blood samples by Ficoll gradient. Co-cultures consisted of either chondrocyte micromasses overlaid with monocytes, or chondrocytes and monocytes seeded together within a collagen/glycosaminoglycan scaffold (Chondromimetic, Tigenix UK). Media, cell pellets and scaffolds were analysed for extracellular matrix (ECM) proteins and proteases by dot blot, western blot, zymography and immunohistochemistry.

Human chondrocytes maintained stable micromasses and laid down an ECM for at least 40 days. Human monocytes eventually formed a proliferating cell population with a rounded morphology on top of the chondrocyte micromasses. These cells established an adherent population with a fibroblastic morphology when replated on plastic. Analysis of chondrocyte ECM proteins indicated that monocytes affected deposition of types I and II collagen, decorin and fibronectin and the overall amounts of gelatinases released. RTPCR demonstrated a decrease in type I collagen expression and a concomitant increase in MMP13 expression.

The precise interaction between monocytes and and chondrocytes has yet to be established but is thought to involve a mixture of contact and paracrine factors. In this study co-culture of monocytes with chondrocytes resulted in phenotypic changes to the chondrocytes which may warrant the inclusion of monocytes in cartilage/bone repair and also provide information as to the responses of OA chondrocytes to external stimuli.

Introduction

Articular hyaline cartilage has a unique structural composition that allows it to endure high load, distribute load to bone and enables low friction movement in joints. A novel acellular xenogenic graft is proposed as a biological cartilage replacement, for repair of osteochondral defects. Acellular porcine cartilage has been produced using repeated freeze thaw cycles and washing using hypotonic buffers and sodium dodecyl sulphate solution (SDS; Keir, 2008). DNA content of the acellular matrix was reduced by 93.3% compared to native cartilage as measured by nanodrop spectrophotometry of extracted DNA, with a corresponding reduction in glycosaminoglycan (GAG) content.

One reason why NICE (National Institute for Clinical Excellence) does not support operations by the NHS to heal hyaline cartilage lesions using a patients own cells is because there is no clear evidence to show that these operations are beneficial and cost-effective in the long term. Specifically, NICE identified a deficiency of high quality cartilage being produced in repaired joints. The presence of high quality cartilage is linked to long-lasting and functional repair of cartilage. The benchmark for quality, NICE stipulate, is repair cartilage that is stiff and strong and looks similar to the normal tissue surrounding it, i.e. mature hyaline articular cartilage.

Biopsy material from autologous cartilage implantation surgical procedures has the appearance of immature articular cartilage and is frequently a mixture of hyaline and fibrocartilage. Osteoarthritic cartilage, in its early stages, also exhibits characteristics of immature articular cartilage in that it expresses proteins found in embryonic and foetal developmental stages, and is highly cellular as evidenced through the presence of chondrocyte clusters. Therefore, an ability to modulate the phenotype and the structure of the extracellular matrix of articular cartilage could positively affect the course of repair and regeneration of articular cartilage lesions. In order to do this, the biochemical stimuli that induce the transition of an essentially unstructured amorphous cartilage mass (immature articular cartilage) to one that is highly structured and ordered, and biomechanically adapted to its particular function (mature articular cartilage) has to be identified.

We show for the first time, that fibroblast growth factor-2 and transforming growth factor beta-1 induce precocious maturation of immature articular cartilage. Our data demonstrates that it is possible to significantly enhance maturation of cartilage tissue using growth factor stimulation; consequently this may have applications in transplantation therapy, or through phenotypic modulation of osteoarthritic chondrocytes in diseased cartilage in order to stimulate growth and maturation of repair tissue.

We have developed a novel, two-layered, collagen matrix seeded with chondrocytes for repair of articular cartilage. It consists of a dense collagen layer which is in contact with bone and a porous matrix to support the seeded chondrocytes. The matrices were implanted in rabbit femoral trochleas for up to 24 weeks. The control groups received either a matrix without cells or no implant.

The best histological repair was seen with cell-seeded implants. The permeability and glycosaminoglycan content of both implant groups were nearly normal, but were significantly less in tissue from empty defects. The type-II collagen content of the seeded implants was normal. For unseeded implants it was 74.3% of the normal and for empty defects only 20%. The current treatments for articular injury often result in a fibrous repair which deteriorates with time. This bilayer implant allowed sustained hyaline-like repair of articular defects during the entire six-month period of observation.

Meniscal cartilage provides joint stabilisation, load distribution, impact absorption and decreased friction in joints that have a complex movement such as the knee. If the meniscal cartilage degrades or is surgically removed, there is a strong probability, over time, of damage to the articular surface. The ability to regenerate damaged meniscal cartilage with an implanted device that replaces the biological equivalent would allow for joint stabilisation, robust movement and reduce the risk of damage to the articular cartilage. An implant with many of the characteristics of meniscus and with the ability to integrate correctly and firmly with the surrounding tissue, would be advantageous.

Inclusion of Platelet Rich Plasma (PRP) into the scaffolds to provide a concentrated source of matrix proteins and autologous growth factors may further enhance the regenerative repair process. To investigate the suitability of the collagen scaffolds, addition of meniscal chondrocytes and or PRP was examined in vitro.

Human meniscal chondrocyte cells were isolated, via collagenase digestion, from meniscal cartilage recovered from total knee replacement surgery. Meniscal chondrocytes were cultured in vitro to expand cell numbers. PRP was produced from volunteer's blood using a centrifuge and density based platelet recovery system. Release of Platelet Derived Growth Factor type AB (PDGF-AB) was measured by ELISA as an indicator of the behaviour of the peptide growth factor component. Combinations of scaffold, meniscal chondrocytes and PRP were tested for interaction, suitability and viability.

Experiments so far have shown good biocompatibility, in vitro, as meniscal chondrocytes were able to grow within the range of scaffolds produced. Cell retention could be enhanced by addition of PRP to the scaffolds. PDGF-AB was released over 5 days from the scaffold and PRP combination.

Further studies are in progress to derive relevant scaffold modifications and combinations for practical, robust, treatment strategies.

Arthroscopic management of femoroacetabular impingement (FAI) has become the mainstay of treatment. However, chondral lesions are frequently encountered and have become a determinant of less favourable outcomes following arthroscopic intervention. The aim of this systematic review and meta-analysis was to assess the outcomes of hip arthroscopy (HA) in patients with FAI and concomitant chondral lesions classified as per Outerbridge. A systematic search was performed using the PRISMA guidelines on four databases including MEDLINE, EMBASE, Cochrane Library and Web of Science. Studies which included HA as the primary intervention for management of FAI and classified chondral lesions according to the Outerbridge classification were included. Patients treated with open procedures, for osteonecrosis, Legg-Calve-Perthes disease, and previous ipsilateral hip fractures were excluded. From a total of 863 articles, twenty-four were included for final analysis. Demographic data, PROMs, and radiological outcomes and rates of conversion to total hip arthroplasty (THA) were collected. Risk of bias was assessed using ROBINS-I. Improved post-operative PROMs included mHHS (mean difference:-2.42; 95%CI:-2.99 to −1.85; p<0.001), NAHS (mean difference:-1.73; 95%CI: −2.23 to −1.23; p<0.001), VAS (mean difference: 2.03; 95%CI: 0.93-3.13; p<0.001). Pooled rate of revision surgery was 10% (95%CI: 7%-14%). Most of this included conversion to THA, with a 7% pooled rate (95%CI: 4%-11%). Patients had worse PROMs if they underwent HA with labral debridement (p=0.015), had Outerbridge 3 and 4 lesions (p=0.012), concomitant lesions of the femoral head and acetabulum lesions (p=0.029). Reconstructive cartilage techniques were superior to microfracture (p=0.042). Even in concomitant lesions of the femoral head and acetabulum, employing either microfracture or cartilage repair/reconstruction provided a benefit in PROMs (p=0.027). Acceptable post-operative outcomes following HA with labral repair/reconstruction and cartilage repair in patients with FAI and concomitant moderate-to-severe chondral lesions, can be achieved. Patients suffering from Outerbridge 3 and 4 lesions, concomitant acetabular rim and femoral head chondral lesions that underwent HA with labral debridement, had worse PROMs. Reconstructive cartilage techniques were superior to microfracture. Even in concomitant acetabular and femoral head chondral lesions, employing either microfracture or cartilage repair/reconstruction was deemed to provide a benefit in PROMs