Charles William Archer

The surface of articular cartilage contains a progenitor cell population

It is becoming increasingly apparent that articular cartilage growth is achieved by apposition from the articular surface. For such a mechanism to occur, a population of stem/progenitor cells must reside within the articular cartilage to provide transit amplifying progeny for growth. Here, we report on the isolation of an articular cartilage progenitor cell from the surface zone of articular cartilage using differential adhesion to fibronectin. This population of cells exhibits high affinity for fibronectin, possesses a high colony-forming efficiency and expresses the cell fate selector gene Notch 1. Inhibition of Notch signalling abolishes colony forming ability whilst activated Notch rescues this inhibition. The progenitor population also exhibits phenotypic plasticity in its differentiation pathway in an embryonic chick tracking system, such that chondroprogenitors can engraft into a variety of connective tissue types including bone, tendon and perimysium. The identification of a chondrocyte subpopulation with progenitor-like characteristics will allow for advances in our understanding of both cartilage growth and maintenance as well as provide novel solutions to articular cartilage repair.

Overexpression of BMP-2 and BMP-4 alters the size and shape of developing skeletal elements in the chick limb.

Bone morphogenetic proteins are members of the transforming growth factor beta (TGF beta) superfamily which are involved in a range of developmental processes including modelling of the skeleton. We show here that Bmp-2 is expressed in mesenchyme surrounding early cartilage condensations in the developing chick limb, and that Bmp-4 is expressed in the perichondrium of developing cartilage elements. To investigate their roles during cartilage development, BMP-2 and BMP-4 were expressed ectopically in developing chick limbs using retroviral vectors. Over-expression of BMP-2 or BMP-4 led to a dramatic increase in the volume of cartilage elements, altered their shapes and led to joint fusions. This increase in volume appeared to result from an increase in the amount of matrix and in the number of chondrocytes. The latter did not appear to be due to increased proliferation of chondrocytes, suggesting that it may result from increased recruitment of precursors. BMP-2 and BMP-4 also delayed hypertrophy of chondrocytes and formation of the osteogenic periosteum. These data provide insights into how BMP-2 and BMP-4 may model and control the growth of skeletal elements during normal embryonic development, suggesting roles for both molecules in recruiting non-chondrogenic precursors to chondrogenic fate.

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.

BMP/GDF-signalling interactions during synovial joint development.

Abstract The synovial joint arises from an initial condensation of cells that subsequently develops into distinct skeletal structures, separated by the joint. Bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs) have a fundamental role during skeletogenesis, including joint formation. Development of the joint appears to be dependent on the differential expression/activity of the related BMP and GDF subfamilies. Gdf-5 is expressed in the developing joints and is necessary for the formation of some joints. In contrast, recent data has shown that antagonism of the BMP family is crucial for joint formation. Here, we review mechanisms of how BMP signalling may be antagonised/modified. We also describe the expression of Bmp-2 and Bmp-4 together with two BMP antagonists, chordin and noggin, during chick joint development. Finally, we discuss possible mechanisms of how a joint forms and the evidence that the joint is a ’signalling centre’ that may coordinate the development of adjacent skeletal structures.

The development of articular cartilage: evidence for an appositional growth mechanism.

It is well-established that cartilage grows by a combination of matrix secretion, cell hypertrophy and cell proliferation. The extent to which this growth is by appositional, as opposed to interstitial mechanisms, however, remains unclear. Using the knee joints of the marsupial Monodelphis domestica to study cartilage growth, we have combined an immunohistochemical study of the TGF-β family of cartilage growth and differentiation factors between 30 days postpartum to 8 months, together with a stereological analysis of cartilage morphology during growth. Furthermore, to gain an insight into the generation of the characteristic zones within cartilage, we have examined the effects of intra-articular administration of bromodeoxyuridine, an agent that is incorporated into DNA during cell division and blocks further cell cycling. During early growth, TGF-β2 and -β3 were widely expressed but TGF-β1 was less so. After the formation of the secondary centre of ossification, all isoforms became more restricted to the upper half of the tissue depth and their distribution was similar to that previously described for IGFs, and PCNA-positive cells. Stereological analysis of tissue sections from the femoral condylar cartilage at 3 and 6 months showed that there was a 17% increase in total cartilage volume but a 31% decrease in cell density on a unit volume basis. Finally, cell-cycle perturbation with BrDU, which was injected into the knee joints of 3-month-old animals and analysed 1 and 4 months post-injection, revealed that the chondrocytes occupying the transitional zone were depleted 1 month post-injection, resulting in thinning of the articular cartilage. This effect was reversed 4 months post-injection. Immunohistochemical analysis revealed that BrDU-treatment altered the expression patterns of all TGF-β isoforms, with a marked reduction in labelling of TGF-β1 and -β3 isoforms in the upper half of the cartilage depth. Overall, the data lends further support to the notion of articular cartilage growing by apposition from the articular surface rather than by interstitial mechanisms.

Cell shape and cartilage differentiation of early chick limb bud cells in culture.

The onset of chondrogenesis in te embryonic chick is preceded by a prechondrogenic condensation of the prospective cartilage cells. Similarly, in culture, the chondrogenic phenotype is only expressed by limb mesoderm cells plated at densities above confluence. This has led to the proposal that chondrogenic differentiation requires immediate histogenic interactions prior to overt chondrogenesis (Solursh et al., 1978, In Vitro 14, 51-61; and, 1980, Dev. Biol. 78, 141-150). It is noticeable that in both the in vitro situations cells secreting a cartilaginous matrix are rounded in shape. Recently, Glowacki, Trepman and Folkman (personal communication) have shown a dependence of phenotypic expression by mature chondrocytes on cell shape. Cells maintained in a rounded configuration by culturing on a semi-adhesive substratum (poly(HEMA)) synthesised more sulphur-containing extracellular matrix than cells allowed to flatten on normal tissue culture plastic. We have investigated whether there is a similar shape-dependent relationship in the differentiation of chick embryonic mesoblasts into actively secreting chondroblasts. Our results show that a rounded cell shape is conductive to the synthesis of a sulphated matrix.

A biomarker-based mathematical model to predict bone-forming potency of human synovial and periosteal mesenchymal stem cells.

OBJECTIVE To develop a biomarker-based model to predict osteogenic potency of human mesenchymal stem cells (MSCs) from synovial membrane and periosteum. METHODS MSC populations were derived from adult synovium and periosteum. Phenotype analysis was performed by fluorescence-activated cell sorting and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Telomere lengths were determined by Southern blot analysis. In vitro osteogenesis was assessed quantitatively by measurements of alkaline phosphatase activity and calcium deposits. To investigate bone formation in vivo, MSCs were seeded onto osteoinductive scaffolds and implanted subcutaneously in nude mice. Bone was assessed by histology, and the human origin investigated by in situ hybridization for human Alu genomic repeats. Quantitation was achieved by histomorphometry and real-time RT-PCR for human osteocalcin. Analysis at the single-cell level was performed with clonal populations obtained by limiting dilution. Multiple regressions were used to explore the incremental predictive value of the markers. RESULTS Periosteal MSCs had significantly greater osteogenic potency than did synovial MSCs inherent to the single cell. Bone was largely of human origin in vivo. Within the same tissue type, there was variability between different donors. To identify predictors of osteogenic potency, we measured the expression levels of osteoblast lineage genes in synovial and periosteal clonal MSCs prior to osteogenic treatment. We identified biomarkers that correlated with osteogenic outcome and developed a mathematical model based on type I collagen and osteoprotegerin expression that predicts the bone-forming potency of MSC preparations, independent of donor-related variables and tissue source. CONCLUSION Our findings indicate that our quality-control mathematical model estimates the bone-forming potency of MSC preparations for bone repair.

The Development of Synovial Joints

Synovial joints arise through two main processes. In long bone elements, cartilaginous differentiation occurs across the locations of the prospective joints that then segment secondarily. This process occurs through the development of a noncartilaginous region known as the interzone. The interzone becomes an important signaling center to the opposing elements, which can regulate growth through such factors as GDF-5. The interzone also expresses bone morphogenetic proteins (BMPs) and their antagonists, such as noggin. Overexpression of BMPs, or the loss of noggin leads to joint fusions. The interzone also expresses Wnt-14, which appears to be specific for this region in the developing anlagen, and regulates its nonchondrogenic nature. Cavitation of the joint follows, driven by selective high-level synthesis of hyaluronan by interzone cells and presumptive synovial cells. In addition, as the interzone disperses during cavity enlargement, data are now accruing that suggest that both the synovium and articular cartilage develop from this population. Finally, the development of articular cartilage progresses through appositional growth driven by a progenitor/stem cell subpopulation that resides in the articular surface. The individual elements of the skeleton are connected together at regions termed joints or articulations. Classically, there are three broad categories of joints: immovable joints (syntharthroses); mixed articulations, in which the range of movement is limited (amphiarthroses); and the movable, or synovial, joints (diarthroses). This review concentrates on the development of the synovial joints.

The distribution of Notch receptors and their ligands during articular cartilage development

We examined the distribution of Notch family members and their ligands during the development of articular cartilage and the growth plate. Notch 1 was expressed by the chondrocytes of the developing articular surface but became increasingly restricted to the deeper layers after birth whilst expression of this family member was restricted to hypertrophic chondrocytes in the growth plate. Notch 2 and 4, Delta and Jagged 2 showed a broadly similar distribution, being present throughout the articular cartilage during development and becoming increasingly restricted to deeper layers with age. Hypertrophic chondrocytes within the growth plate also expressed Notch 2 and 4, Delta and Jagged 2 (which was also expressed in prehypertrophs). Notch 3 and Jagged 1 were absent from developing articular cartilage but were present in deeper layers at later time points (> 1 month) and both receptor and ligand were expressed in hypertrophic chondrocytes at all ages examined. These results highlight the complex Notch signalling interactions that result in the formation of the heterogeneous articular cartilage and allow for the co‐ordinated ossification and elongation of the growth plate. Mechanisms by which these processes are controlled are discussed in light of recent advances in the understanding of Notch signalling pathways.

The comparison of equine articular cartilage progenitor cells and bone marrow-derived stromal cells as potential cell sources for cartilage repair in the horse.

A chondrocyte progenitor population isolated from the surface zone of articular cartilage presents a promising cell source for cell-based cartilage repair. In this study, equine articular cartilage progenitor cells (ACPCs) and equine bone marrow-derived stromal cells (BMSCs) were compared as potential cell sources for repair. Clonally derived BMSCs and ACPCs demonstrated expression of the cell fate selector gene, Notch-1, and the putative stem cell markers STRO-1, CD90 and CD166. Chondrogenic induction revealed positive labelling for collagen type II and aggrecan. Collagen type X was not detected in ACPC pellets but was observed in all BMSC pellets. In addition, it was observed that BMSCs labelled for Runx2 and matrilin-1 antibodies, whereas ACPC labelling was significantly less or absent. For both cell types, osteogenic induction revealed positive von Kossa staining in addition to positive labelling for osteocalcin. Adipogenic induction revealed a positive result via oil red O staining in both cell types. ACPCs and BMSCs have demonstrated functional equivalence in their multipotent differentiation capacity. Chondrogenic induction of BMSCs resulted in a hypertrophic cartilage (endochondral) phenotype, which can limit cartilage repair as the tissue can undergo mineralisation. ACPCs may therefore be considered superior to BMSCs in producing cartilage capable of functional repair.