BMP9 is a potent inducer of chondrogenesis, volumetric expansion and collagen type II accumulation in bovine auricular cartilage chondroprogenitors.

Reconstruction of the outer ear currently requires harvesting of cartilage from the posterior of the auricle or ribs leading to pain and donor site morbidity. An alternative source for auricular reconstruction is in vitro tissue engineered cartilage using stem/progenitor cells. Several candidate cell-types have been studied with tissue-specific auricular cartilage progenitor cells (AuCPC) of particular interest. Whilst chondrogenic differentiation of competent stem cells using growth factor TGFß1 produces cartilage this tissue is frequently fibrocartilaginous and lacks the morphological features of hyaline cartilage. Recent work has shown that growth factor BMP9 is a potent chondrogenic and morphogenetic factor for articular cartilage progenitor cells, and we hypothesised that this property extends to cartilage-derived progenitors from other tissues. In this study we show monoclonal populations of AuCPCs from immature and mature bovine cartilage cultured with BMP9 produced cartilage pellets have 3-5-fold greater surface area in sections than those grown with TGFß1. Increased volumetric growth using BMP9 was due to greater sGAG deposition in immature pellets and significantly greater collagen accumulation in both immature and mature progenitor pellets. Polarised light microscopy and immunohistochemical analyses revealed that the organisation of collagen fibrils within pellets is an important factor in the growth of pellets. Additionally, chondrocytes in BMP9 stimulated cell pellets had larger lacunae and were more evenly dispersed throughout the extracellular matrix. Interestingly, BMP9 tended to normalise the response of immature AuCPC monoclonal cell lines to differentiation cues whereas cells exhibited more variation under TGFß1. In conclusion, BMP9 appears to be a potent inducer of chondrogenesis and volumetric growth for AuCPCs a property that can be exploited for tissue engineering strategies for reconstructive surgery though with the caveat of negligible elastin production following 21-day treatment with either growth factor.

Articular Cartilage Chondroprogenitors: Isolation and Directed Differentiation.

Experimental data suggests that tissue-specific progenitors are present within hyaline articular cartilage with the potential to contribute to growth, maintenance, and repair. In this chapter, we show how colony-forming progenitor-like cells can be isolated from bovine articular cartilage using differential adhesion to fibronectin. Furthermore, we describe the optimal conditions and factors required to differentiate these progenitor cells to produce hyaline articular cartilage.

In Vitro Preparation of Actively Maturing Bovine Articular Cartilage Explants for X-Ray Phase Contrast Imaging.

Understanding the mechanisms that underpin post-natal maturation of articular cartilage is of crucial importance for designing the next generation of tissue engineering strategies and potentially repairing diseased or damaged cartilage. In general, postnatal maturation of the articular cartilage, which is a wholesale change in collagen structure and function of the tissue to accommodate growth of the organism, occurs over a timescale ranging from months to years. Conversely dissolution of the structural organization of the cartilage that also occurs over long timescales is the hallmark of tissue degeneration. Our ability to study these biological processes in detail have been enhanced by the findings that growth factors can induce precocious in vitro maturation of immature articular cartilage. The developmental and disease related changes that occur in the joint involve bone and cartilage and an ability to co-image these tissues would significantly increase our understanding of their intertwined roles. The simultaneous visualization of soft tissue, cartilage and bone changes is nowadays a challenge to overcome for conventional preclinical imaging modalities used for the joint disease follow-up. Three-dimensional X-ray Phase-Contrast Imaging methods (PCI) have been under perpetual developments for 20 years due to high performance for imaging low density objects and their ability to provide additional information compared to conventional X-ray imaging. In this protocol we detail the procedure used in our experiments from biopsy of the cartilage, generation of in vitro matured cartilage to data analysis of image collected using X-ray phase contrast imaging.

Jellyfish Collagen: A Biocompatible Collagen Source for 3D Scaffold Fabrication and Enhanced Chondrogenicity.

Osteoarthritis (OA) is a multifactorial disease leading to degeneration of articular cartilage, causing morbidity in approximately 8.5 million of the UK population. As the dense extracellular matrix of articular cartilage is primarily composed of collagen, cartilage repair strategies have exploited the biocompatibility and mechanical strength of bovine and porcine collagen to produce robust scaffolds for procedures such as matrix-induced chondrocyte implantation (MACI). However, mammalian sourced collagens pose safety risks such as bovine spongiform encephalopathy, transmissible spongiform encephalopathy and possible transmission of viral vectors. This study characterised a non-mammalian jellyfish (Rhizostoma pulmo) collagen as an alternative, safer source in scaffold production for clinical use. Jellyfish collagen demonstrated comparable scaffold structural properties and stability when compared to mammalian collagen. Jellyfish collagen also displayed comparable immunogenic responses (platelet and leukocyte activation/cell death) and cytokine release profile in comparison to mammalian collagen in vitro. Further histological analysis of jellyfish collagen revealed bovine chondroprogenitor cell invasion and proliferation in the scaffold structures, where the scaffold supported enhanced chondrogenesis in the presence of TGFß1. This study highlights the potential of jellyfish collagen as a safe and biocompatible biomaterial for both OA repair and further regenerative medicine applications.

Chondrocyte Aging: The Molecular Determinants and Therapeutic Opportunities.

Osteoarthritis (OA) is a joint degenerative disease that is an exceedingly common problem associated with aging. Aging is the principal risk factor for OA, but damage-related physiopathology of articular chondrocytes probably drives the mechanisms of joint degeneration by a progressive decline in the homeostatic and regenerative capacity of cells. Cellular aging is the manifestation of a complex interplay of cellular and molecular pathways underpinned by transcriptional, translational, and epigenetic mechanisms and niche factors, and unraveling this complexity will improve our understanding of underlying molecular changes that affect the ability of the articular cartilage to maintain or regenerate itself. This insight is imperative for developing new cell and drug therapies for OA disease that will target the specific causes of age-related functional decline. This review explores the key age-related changes within articular chondrocytes and discusses the molecular mechanisms that are commonly perturbed as cartilage ages and degenerates. Current efforts and emerging potential therapies in treating OA that are being employed to halt or decelerate the aging processes are also discussed.

Three dimensional printed degradable and conductive polymer scaffolds promote chondrogenic differentiation of chondroprogenitor cells.

Conductive polymers have been used for various biomedical applications including biosensors, tissue engineering and regenerative medicine. However, the poor processability and brittleness of these polymers hinder the fabrication of three-dimensional structures with desirable geometries. Moreover, their application in tissue engineering and regenerative medicine has been so far limited to excitable cells such as neurons and muscle cells. To enable their wider adoption in tissue engineering and regenerative medicine, new materials and formulations that overcome current limitations are required. Herein, a biodegradable conductive block copolymer, tetraaniline-b-polycaprolactone-b-tetraaniline (TPT), is synthesised and 3D printed for the first time into porous scaffolds with defined geometries. Inks are formulated by combining TPT with PCL in solutions which are then directly 3D printed to generate porous scaffolds. TPT and PCL are both biodegradable. The combination of TPT with PCL increases the flexibility of the hybrid material compared to pure TPT, which is critical for applications that need mechanical robustness of the scaffolds. The highest TPT content shows the lowest tensile failure strain. Moreover, the absorption of a cell adhesion-promoting protein (fibronectin) and chondrogenic differentiation of chondroprogenitor cells are found to be dependent on the amount of TPT in the blends. Higher content of TPT in the blends increases both fibronectin adsorption and chondrogenic differentiation, though the highest concentration of TPT in the blends is limited by its solubility in the ink. Despite the contradicting effects of TPT concentration on flexibility and chondrogenic differentiation, a concentration that strikes a balance between the two factors is still available. It is worth noting that the effect on chondrogenic differentiation is found in scaffolds without external electric stimulation. Our work demonstrates the possibility of 3D printing flexible conductive and biodegradable scaffolds and their potential use in cartilage tissue regeneration, and opens up future opportunities in using electric stimulation to control chondrogenesis in these scaffolds.

Bone Morphogenetic Protein-9 Is a Potent Chondrogenic and Morphogenic Factor for Articular Cartilage Chondroprogenitors.

Articular cartilage contains a subpopulation of tissue-specific progenitors that are an ideal cell type for cell therapies and generating neocartilage for tissue engineering applications. However, it is unclear whether the standard chondrogenic medium using transforming growth factor beta (TGFß) isoforms is optimal to differentiate these cells. We therefore used pellet culture to screen progenitors from immature bovine articular cartilage with a number of chondrogenic factors and discovered that bone morphogenetic protein-9 (BMP9) precociously induces their differentiation. This difference was apparent with toluidine blue staining and confirmed by biochemical and transcriptional analyses with BMP9-treated progenitors exhibiting 11-fold and 5-fold greater aggrecan and collagen type II (COL2A1) gene expression than TGFß1-treated progenitors. Quantitative gene expression analysis over 14 days highlighted the rapid and phased nature of BMP9-induced chondrogenesis with sequential activation of aggrecan then collagen type II, and negligible collagen type X gene expression. The extracellular matrix of TGFß1-treated progenitors analyzed using atomic force microscopy was fibrillar and stiff whist BMP9-induced matrix of cells more compliant and correspondingly less fibrillar. Polarized light microscopy revealed an annular pattern of collagen fibril deposition typified by TGFß1-treated pellets, whereas BMP9-treated pellets displayed a birefringence pattern that was more anisotropic. Remarkably, differentiated immature chondrocytes incubated as high-density cultures in vitro with BMP9 generated a pronounced anisotropic organization of collagen fibrils indistinguishable from mature adult articular cartilage, with cells in deeper zones arranged in columnar manner. This contrasted with cells grown with TGFß1, where a concentric pattern of collagen fibrils was visualized within tissue pellets. In summary, BMP9 is a potent chondrogenic factor for articular cartilage progenitors and is also capable of inducing morphogenesis of adult-like cartilage, a highly desirable attribute for in vitro tissue-engineered cartilage.

In vitro tissue culture model validation-the influence of tissue culture components on IPL energy output.

Intense pulsed light (IPL) has been used therapeutically in a number of clinical settings and has been shown to have a photobiomodulatory effect on connective tissue cells, such as those derived from skin and tendon. In vitro cell culture models are essential tools preclinically in investigating such treatment modalities, as they help in optimising parameters for successful treatment. However, as culture system components have been reported to absorb part of the irradiated energy, which in turn has a bearing on the amount of light reaching the cells, it is important to establish specific parameters for the particular in vitro model used. This study, therefore, investigates the effect of our tissue culture system components on the IPL energy delivered. Individual wells of multi-well plates were irradiated with IPL at different device settings and under variable culture conditions (e.g. in the absence or presence of cell culture media with or without the pH indicator dye, phenol red), and the energy lost through the culture system determined. Our data demonstrated that the IPL device delivered significantly lower outputs than those published, and energy absorption by the culture equipment would further reduce fluencies delivered to the cell monolayer. Furthermore, energy absorption by media containing phenol red was marginally greater than clear media and resulted in only a small increase in temperature, which would not be harmful to cells. The use of phenol red-containing media therefore is valid and physiologically relevant when examining light-culture system interactions.

Survival and immunomodulation of stem cells from human extracted deciduous teeth expanded in pooled human and foetal bovine sera.

The immunomodulatory properties of mesenchymal stem cells (MSCs) from autologous and allogeneic sources are useful in stimulating tissue regeneration and repair. To obtain a high number of MSCs for transplantation requires extensive in vitro expansion with culture media supplements that can cause xeno-contamination of cells potentially compromising function and clinical outcomes. In this study stem cells from human extracted deciduous teeth (SHED) were cultured in Knockout™ DMEM supplemented with either pooled human serum (pHS) or foetal bovine serum (FBS) to compare their suitability in maintaining immunomodulatory properties of cells during in vitro expansion. No significant difference in cell survival of SHED grown in pHS (pHS-SHED) or FBS (FBS-SHED) was observed when co-cultured with complement, monocytes or lymphocytes. However, significant changes in the expression of sixteen paracrine factors involved in immunomodulation were observed in the supernatants of FBS-SHED co-cultures with monocytes or lymphocytes compared to that in pHS-SHEDs after both 24 and 120 h of incubation. Further analysis of changing protein levels of paracrine factors in co-cultures using biological pathway analysis software predicted upregulation of functions associated with immunogenicity in FBS-SHED and lymphocyte co-cultures compared to pHS-SHED co-cultures. Pathway analysis also predicted significant stimulation of HMGB1 and TREM1 signalling pathways in FBS-SHED co-cultures indicating activation of immune cells and inflammation. Though FBS supplementation does not impact survival of SHED, our combinatorial biological pathway analysis supports the idea that in vitro expansion of SHEDs in pHS provides optimal conditions to minimise xeno-contamination and inflammation and maintain their immunomodulatory properties.

In vitro studies to evaluate the effect of varying culture conditions and IPL fluencies on tenocyte activities.

Tendons are dense, fibrous connective tissues which carry out the essential physiological role of transmitting mechanical forces from skeletal muscle to bone. From a clinical perspective, tendinopathy is very common, both within the sporting arena and amongst the sedentary population. Studies have shown that light therapy may stimulate tendon healing, and more recently, intense pulsed light (IPL) has attracted attention as a potential treatment modality for tendinopathy; however, its mechanism of action and effect on the tendon cells (tenocytes) is poorly understood. The present study therefore investigates the influence of IPL on an in vitro bovine tendon model. Tenocytes were irradiated with IPL at different devise settings and under variable culture conditions (e.g. utilising cell culture media with or without the pH indicator dye phenol red), and changes in tenocyte viability and migration were subsequently investigated using Alamar blue and scratch assays, respectively. Our data demonstrated that IPL fluencies of up to 15.9 J/cm2 proved harmless to the tenocyte cultures (this was the case using culture media with or without phenol red) and resulted in a significant increase in cell viability under certain culture conditions. Furthermore, IPL treatment of tenocytes did not affect the rate of cell migration. This study demonstrates that irradiation with IPL is not detrimental to the tenocytes and may increase their viability under certain conditions, thus validating our in vitro model. Further studies are required to elucidate the effects of IPL application in the clinical situation.

The bio in the ink: cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells.

Cell-laden hydrogels are the primary building blocks for bioprinting, and, also termed bioinks, are the foundations for creating structures that can potentially recapitulate the architecture of articular cartilage. To be functional, hydrogel constructs need to unlock the regenerative capacity of encapsulated cells. The recent identification of multipotent articular cartilage-resident chondroprogenitor cells (ACPCs), which share important traits with adult stem cells, represents a new opportunity for cartilage regeneration. However, little is known about the suitability of ACPCs for tissue engineering, especially in combination with biomaterials. This study aimed to investigate the potential of ACPCs in hydrogels for cartilage regeneration and biofabrication, and to evaluate their ability for zone-specific matrix production. Gelatin methacryloyl (gelMA)-based hydrogels were used to culture ACPCs, bone marrow mesenchymal stromal cells (MSCs) and chondrocytes, and as bioinks for printing. Our data shows ACPCs outperformed chondrocytes in terms of neo-cartilage production and unlike MSCs, ACPCs had the lowest gene expression levels of hypertrophy marker collagen type X, and the highest expression of PRG4, a key factor in joint lubrication. Co-cultures of the cell types in multi-compartment hydrogels allowed generating constructs with a layered distribution of collagens and glycosaminoglycans. By combining ACPC- and MSC-laden bioinks, a bioprinted model of articular cartilage was generated, consisting of defined superficial and deep regions, each with distinct cellular and extracellular matrix composition. Taken together, these results provide important information for the use of ACPC-laden hydrogels in regenerative medicine, and pave the way to the biofabrication of 3D constructs with multiple cell types for cartilage regeneration or in vitro tissue models. STATEMENT OF SIGNIFICANCE: Despite its limited ability to repair, articular cartilage harbors an endogenous population of progenitor cells (ACPCs), that to date, received limited attention in biomaterials and tissue engineering applications. Harnessing the potential of these cells in 3D hydrogels can open new avenues for biomaterial-based regenerative therapies, especially with advanced biofabrication technologies (e.g. bioprinting). This study highlights the potential of ACPCs to generate neo-cartilage in a gelatin-based hydrogel and bioink. The ACPC-laden hydrogel is a suitable substrate for chondrogenesis and data shows it has a bias in directing cells towards a superficial zone phenotype. For the first time, ACPC-hydrogels are evaluated both as alternative for and in combination with chondrocytes and MSCs, using co-cultures and bioprinting for cartilage regeneration in vitro. This study provides important cues on ACPCs, indicating they represent a promising cell source for the next generation of cartilage constructs with increased biomimicry.

Platelet-rich plasma induces post-natal maturation of immature articular cartilage and correlates with LOXL1 activation.

Platelet-rich plasma (PRP) is used to stimulate the repair of acute and chronic cartilage damage even though there is no definitive evidence of how this is achieved. Chondrocytes in injured and diseased situations frequently re-express phenotypic biomarkers of immature cartilage so tissue maturation is a potential pathway for restoration of normal structure and function. We used an in vitro model of growth factor-induced maturation to perform a comparative study in order to determine whether PRP can also induce this specific form of remodeling that is characterised by increased cellular proliferation and tissue stiffness. Gene expression patterns specific for maturation were mimicked in PRP treated cartilage, with chondromodulin, collagen types II/X downregulated, deiodinase II and netrin-1 upregulated. PRP increased cartilage surface cell density 1.5-fold (P < 0.05), confirmed by bromodeoxyuridine incorporation and proportionate increases in proliferating cell nuclear antigen gene expression. Atomic force microscopy analysis of PRP and growth factor treated cartilage gave a 5-fold increase in stiffness correlating with a 10-fold upregulation of lysyl oxidase like-1 gene expression (P < 0.001). These data show PRP induces key aspects of post-natal maturation in immature cartilage and provides the basis to evaluate a new biological rationale for its activity when used clinically to initiate joint repair.

Characterisation of a divergent progenitor cell sub-populations in human osteoarthritic cartilage: the role of telomere erosion and replicative senescence.

In recent years it has become increasingly clear that articular cartilage harbours a viable pool of progenitor cells and interest has focussed on their role during development and disease. Analysis of progenitor numbers using fluorescence-activated sorting techniques has resulted in wide-ranging estimates, which may be the result of context-dependent expression of cell surface markers. We have used a colony-forming assay to reliably determine chondroprogenitor numbers in normal and osteoarthritic cartilage where we observed a 2-fold increase in diseased tissue (P < 0.0001). Intriguingly, cell kinetic analysis of clonal isolates derived from single and multiple donors of osteoarthritic cartilage revealed the presence of a divergent progenitor subpopulation characterised by an early senescent phenotype. Divergent sub-populations displayed increased senescence-associated ß-galactosidase activity, lower average telomere lengths but retained the capacity to undergo multi-lineage differentiation. Osteoarthritis is an age-related disease and cellular senescence is predicted to be a significant component of the pathological process. This study shows that although early senescence is an inherent property of a subset of activated progenitors, there is also a pool of progenitors with extended viability and regenerative potential residing within osteoarthritic cartilage.

Combining regenerative medicine strategies to provide durable reconstructive options: auricular cartilage tissue engineering.

Recent advances in regenerative medicine place us in a unique position to improve the quality of engineered tissue. We use auricular cartilage as an exemplar to illustrate how the use of tissue-specific adult stem cells, assembly through additive manufacturing and improved understanding of postnatal tissue maturation will allow us to more accurately replicate native tissue anisotropy. This review highlights the limitations of autologous auricular reconstruction, including donor site morbidity, technical considerations and long-term complications. Current tissue-engineered auricular constructs implanted into immune-competent animal models have been observed to undergo inflammation, fibrosis, foreign body reaction, calcification and degradation. Combining biomimetic regenerative medicine strategies will allow us to improve tissue-engineered auricular cartilage with respect to biochemical composition and functionality, as well as microstructural organization and overall shape. Creating functional and durable tissue has the potential to shift the paradigm in reconstructive surgery by obviating the need for donor sites.

Adipose, Bone Marrow and Synovial Joint-Derived Mesenchymal Stem Cells for Cartilage Repair.

Current cell-based repair strategies have proven unsuccessful for treating cartilage defects and osteoarthritic lesions, consequently advances in innovative therapeutics are required and mesenchymal stem cell-based (MSC) therapies are an expanding area of investigation. MSCs are capable of differentiating into multiple cell lineages and exerting paracrine effects. Due to their easy isolation, expansion, and low immunogenicity, MSCs are an attractive option for regenerative medicine for joint repair. Recent studies have identified several MSC tissue reservoirs including in adipose tissue, bone marrow, cartilage, periosteum, and muscle. MSCs isolated from these discrete tissue niches exhibit distinct biological activities, and have enhanced regenerative potentials for different tissue types. Each MSC type has advantages and disadvantages for cartilage repair and their use in a clinical setting is a balance between expediency and effectiveness. In this review we explore the challenges associated with cartilage repair and regeneration using MSC-based cell therapies and provide an overview of phenotype, biological activities, and functional properties for each MSC population. This paper also specifically explores the therapeutic potential of each type of MSC, particularly focusing on which cells are capable of producing stratified hyaline-like articular cartilage regeneration. Finally we highlight areas for future investigation. Given that patients present with a variety of problems it is unlikely that cartilage regeneration will be a simple "one size fits all," but more likely an array of solutions that need to be applied systematically to achieve regeneration of a biomechanically competent repair tissue.

In vitro growth factor-induced bio engineering of mature articular cartilage.

Articular cartilage maturation is the postnatal development process that adapts joint surfaces to their site-specific biomechanical demands. Maturation involves gross morphological changes that occur through a process of synchronised growth and resorption of cartilage and generally ends at sexual maturity. The inability to induce maturation in biomaterial constructs designed for cartilage repair has been cited as a major cause for their failure in producing persistent cell-based repair of joint lesions. The combination of growth factors FGF2 and TGFß1 induces accelerated articular cartilage maturation in vitro such that many molecular and morphological characteristics of tissue maturation are observable. We hypothesised that experimental growth factor-induced maturation of immature cartilage would result in a biophysical and biochemical composition consistent with a mature phenotype. Using native immature and mature cartilage as reference, we observed that growth factor-treated immature cartilages displayed increased nano-compressive stiffness, decreased surface adhesion, decreased water content, increased collagen content and smoother surfaces, correlating with a convergence to the mature cartilage phenotype. Furthermore, increased gene expression of surface structural protein collagen type I in growth factor-treated explants compared to reference cartilages demonstrates that they are still in the dynamic phase of the postnatal developmental transition. These data provide a basis for understanding the regulation of postnatal maturation of articular cartilage and the application of growth factor-induced maturation in vitro and in vivo in order to repair and regenerate cartilage defects.

Tribological characteristics of healthy tendon.

Tendons transfer muscular forces efficiently and painlessly, facilitating joint motion. Whilst the tribology of articular cartilage is constantly explored, a poorer understanding remains of tendon lubrication and friction. This study reports experimental data describing the tribological characteristics of tendon and its surrounding tissue, before presenting an arithmetic solution to facilitate numerical modelling. The experimental characteristics of the tensile (i.e. mid-substance) and compressive (i.e. fibrocartilaginous) regions of bovine flexor tendon were investigated using a pin-on-plate tribometer, with immunofluroscence analysis describing the relative intensity and distribution of surface-bound lubricin. Arithmetic analysis considering the digital extensor tendon determined that, in physiological conditions, the tensile tendon region was able to generate elastohydrodynamic lubrication (EHL). The equivalent region of compressive tendon exhibited a higher intensity of surface-bound lubricin which, it is hypothesised, serves to minimise the increased frictional resistance due to generating only mixed or boundary lubrication regimes. Arithmetic analysis indicates that, given a more favourable biomechanical environment, this region can also generate EHL. Whilst acknowledging the limitations of transferring data from an animal model to a clinical environment, by providing the first data and equations detailing the film thicknesses and lubrication regime for these two tendon regions it is hoped that clinicians, engineers and scientists can consider improved clinical strategies to tackle both tendinopathy and tendon rupture.

Fibroblast growth factor 2 and transforming growth factor ß1 induce precocious maturation of articular cartilage.

OBJECTIVE: We have discovered that a combination of fibroblast growth factor 2 and transforming growth factor ß1 induce profound morphologic changes in immature articular cartilage. The purpose of this study was to test the hypothesis that these changes represent accelerated postnatal maturation. METHODS: Histochemical and biochemical assays were used to confirm the nature of the morphologic changes that accompany growth factor stimulation of immature bovine articular cartilage explants in serum-free culture medium. Growth factor-induced apoptosis, cellular proliferation, and changes in the collagen network were also quantitatively analyzed. RESULTS: Growth factor stimulation resulted in rapid resorption from the basal aspect of immature cartilage explants that was simultaneously opposed by cellular proliferation from the apical aspect driven from a pool of chondroprogenitor cells we have previously described. Maturation-dependent changes in tissue stiffness, collagen crosslinking, and collagen fibril architecture as well as differentiation of the extracellular matrix into distinct pericellular, territorial, and interterritorial domains were all present in growth factor-stimulated cartilage samples and absent in control samples. CONCLUSION: Our data demonstrate that it is possible to significantly enhance the maturation of cartilage tissue using specific growth factor stimulation. This may have applications in transplantation therapy or in the treatment of diseased cartilage, through phenotype modulation of osteoarthritic chondrocytes in order to stimulate growth and maturation of cartilage repair tissue.

Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage.

BACKGROUND: Articular cartilage displays a poor repair capacity. The aim of cell-based therapies for cartilage defects is to repair damaged joint surfaces with a functional replacement tissue. Currently, chondrocytes removed from a healthy region of the cartilage are used but they are unable to retain their phenotype in expanded culture. The resulting repair tissue is fibrocartilaginous rather than hyaline, potentially compromising long-term repair. Mesenchymal stem cells, particularly bone marrow stromal cells (BMSC), are of interest for cartilage repair due to their inherent replicative potential. However, chondrocyte differentiated BMSCs display an endochondral phenotype, that is, can terminally differentiate and form a calcified matrix, leading to failure in long-term defect repair. Here, we investigate the isolation and characterisation of a human cartilage progenitor population that is resident within permanent adult articular cartilage. METHODS AND FINDINGS: Human articular cartilage samples were digested and clonal populations isolated using a differential adhesion assay to fibronectin. Clonal cell lines were expanded in growth media to high population doublings and karyotype analysis performed. We present data to show that this cell population demonstrates a restricted differential potential during chondrogenic induction in a 3D pellet culture system. Furthermore, evidence of high telomerase activity and maintenance of telomere length, characteristic of a mesenchymal stem cell population, were observed in this clonal cell population. Lastly, as proof of principle, we carried out a pilot repair study in a goat in vivo model demonstrating the ability of goat cartilage progenitors to form a cartilage-like repair tissue in a chondral defect. CONCLUSIONS: In conclusion, we propose that we have identified and characterised a novel cartilage progenitor population resident in human articular cartilage which will greatly benefit future cell-based cartilage repair therapies due to its ability to maintain chondrogenicity upon extensive expansion unlike full-depth chondrocytes that lose this ability at only seven population doublings.

Distinct mesenchymal progenitor cell subsets in the adult human synovium.

OBJECTIVE: To analyse the heterogeneity at the single-cell level of human mesenchymal progenitor cells from SM. METHODS: Cell populations were enzymatically released from the knee joint synovium of adult human individuals. Single cell-derived clonal populations were obtained by limiting dilution and serially passaged to determine growth rates. Phenotypic analysis was carried out by flow cytometry. Replicative senescence was assessed by the senescence-associated beta-galactosidase staining. Telomere lengths were determined semiquantitatively by Southern blotting. Telomerase activity was measured using a real-time quantitative telomerase repeat amplification procedure. Culture-expanded clonal populations were subjected to in vitro differentiation assays to investigate their mesenchymal multipotency. RESULTS: The 50 clones analysed displayed wide variations in the proliferation rates, even within the same donor sample. The time taken to reach 20 population doublings ranged from 44 to 130 days. The phenotype of the clones tested was compatible with that of mesenchymal stem cells. Mean telomere lengths ranged from 5.2 to 10.9 kb with positive linear trend with telomerase activity, but no correlation with proliferative rates or cell senescence. All clones tested were capable of chondrogenic and osteogenic differentiation, though with large variability in potency. In contrast, only 30% of the clones were adipogenic. CONCLUSIONS: We report for the first time the co-existence, within the synovium, of progenitor cell subsets with distinct mesenchymal differentiation potency. Our findings further emphasize the need for strategies to purify cell populations with the clinically desired tissue formation potentials.