Francesco Dell’Accio

Distinct mesenchymal progenitor cell subsets in the adult human synovium

OBJECTIVEnTo analyse the heterogeneity at the single-cell level of human mesenchymal progenitor cells from SM.nnnMETHODSnCell 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnWe 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.

Cellular and molecular mechanisms of cartilage damage and repair.

Cartilage breakdown is the disabling outcome of rheumatic diseases, whether prevalently inflammatory such as rheumatoid arthritis or prevalently mechanical such as osteoarthritis (OA). Despite the differences between immune-mediated arthritides and OA, common mechanisms drive cartilage breakdown. Inflammation, chondrocyte phenotype and homeostatic mechanisms have recently been the focus of research and will be summarised in this review.

Bone morphogenetic proteins and the synovial joints

Bone morphogenetic proteins (BMPs) are involved in a broad array of morpho-genetic processes. These span from the specification of the dorso-ventral body axis to patterning, organogenesis and differentiation of most tissues. Nevertheless, the initial discovery of BMPs as protein preparations that induce ectopically andin vivoa cascade of endochondral bone formation in rats, has strongly stimulated the study of their role in the development of the skeleton and in the patterning of the synovial joints [1, 2, 3]. In addition, with their remarkable cartilage and bone morphogenetic activity, BMPs represent an attractive therapeutic option for skeletal and joint disorders. Indeed, growing scientific evidence supports the concept that tissue repair and regeneration recapitulates to a certain extent the process of tissue formation during embryonic development. Taking advantage of the expanding knowledge in the field of developmental biology to define potential new targeted therapeutic approaches, the role of BMPs in the development of the skeleton and in particular in the patterning and differentiation of joint tissues becomes increasingly clinically relevant.

Articular Chondroprogenitor Cells Maintain Chondrogenic Potential but Fail to Form a Functional Matrix When Implanted Into Muscles of SCID Mice

Objective Articular cartilage is a complex tissue comprising phenotypically distinct zones. Research has identified the presence of a progenitor cell population in the surface zone of immature articular cartilage. The aim of the present study was to determine the in vivo plasticity of articular cartilage progenitor. Design Chondropogenitor cells were isolated from bovine metacarpalphalangeal joints by differential adhesion to fibronectin. Cells were labeled with PKH26 and injected into the thigh muscle of severe-combined immunodeficient (SCID) mice. After 2 weeks, the muscles were dissected and cryosectioned. Sections were stained with safranin O and labeled for sox9 and collagen type II. Polymerase chain reaction analysis was carried out to determine plasticity for a number of tissue-specific markers. Full-depth chondrocytes acted as a control. Results Fluorescent PKH26 labeled cells were detected after 2 weeks in all samples analyzed. A cartilage pellet was present after injection of freshly isolated chondrocytes. After injection with clonal and enriched populations of chondroprogenitors, no distinct pellet was detected, but diffuse cartilage nodules were found with regions of safranin O staining and Sox9. Low levels of collagen type II were also detected. Polymerase chain reaction analysis identified the presence of the endothelial cell marker PECAM-1 in one clonal cell line, demonstrating phenotypic plasticity into the phenotype of the surrounding host tissues. Conclusions The bovine articular cartilage progenitor cells were able to survive in vivo postimplantation, but failed to create a robust cartilage pellet, despite expressing sox9 and type II collagen. This suggests the cells require further signals for chondrogenic differentiation.

Analyses on the mechanisms that underlie the chondroprotective properties of calcitonin

INTRODUCTIONnCalcitonin (CT) has recently been shown to display chondroprotective effects. Here, we investigate the putative mechanisms by which CT delivers these actions.nnnMETHODSnImmortalized C-28/I2 cells or primary adult human articular chondrocytes (AHAC) were cultured in high-density micromasses to investigate: (i) CT anabolic effects using qPCR and immuhistochemistry analysis; (ii) CT anti-apoptotic effects using quantitation of Bax/Bcl gene products ratio, TUNEL assay and caspase-3 expression; (iii) CT effects on CREB, COL2A1 and NFAT transcription factors.nnnRESULTSnCT (10(-10)-10(-8)nM) induced significant up-regulation of cartilage phenotypic markers (SOX9, COL2A1 and ACAN), with down-regulation of catabolic (MMP1 and MMP13 and ADAMTS5) gene products both in resting and inflammatory conditions. This was mirrored by an augmented production of type II collagen and accumulation of glycosaminoglycan- and proteoglycan-rich extracellular matrix in vitro. Mechanistic analyses revealed only partial involvement of cyclic AMP formation in these effects of CT. Congruently, using reporter assays for specific transcription factors, there was no indication for CREB activation, whereas the COL2A1 promoter was genuinely and directly activated by cell exposure to CT. Phenotypically, these mechanisms supported the ability of CT, whilst inactive on its own, to counteract the pro-apoptotic effects of IL-1β, demonstrated by TUNEL-positive staining of chondrocytes and ratio of BAX/BCL genes products.nnnCONCLUSIONnThese data may provide a novel lead for the development of CT-based chondroprotective strategies that rely on the engagement of mechanisms that lead to augmented chondrocyte anabolism and inhibited chondrocyte apoptosis.

Identification and characterization of human cell populations capable of forming stable hyaline cartilage in vivo

Articular cartilage of adult individuals has a limited capacity for repair. Although not well studied, it appears likely that in many cases joint surface defects (JSD) progress over the years, ultimately evolving into frank osteoarthritic joints. Developing parameters that will identify the individuals at risk, i.e., those individuals who will develop osteoarthritis after joint injury, is a challenge in modern medicine, and using new technologies such as genomics, proteomics and bioinformatics may lead in the coming decade to the identification of prognostic predictors. In addition, to prevent this joint deterioration, several therapeutic strategies have been developed, microfracture, mosaicplasty, and autologous chondrocyte transplantation (ACT) presently being the most popular. The microfracture technique is mostly reserved to small size lesions, and consists in perforating the bottom of the JSD to recruit skeletal precursors present in the underlying bone marrow. The mosaicplasty procedure consists of collecting several osteochondral plugs obtained from low weight bearing areas of the joint, and subsequently fitting them into the JSD, like in a mosaic. This transforms a relatively large defect into several small defects that can be repaired spontaneously by the surrounding tissue and by the invading bone marrow derived skeletal precursors/mesenchymal stem cells [1].

A8.13 Syndecan-4 Function is Essential for Matrix Remodelling Under Inflammatory Conditions, But Dispensable During Embryogenesis

Objective The heparan sulphate proteoglycan syndecan-4 (Sdc4) has been associated strongly with osteoarthritis, a disease that mimics key aspects of early cartilage remodelling during endochondral ossification, but its role in embryonic and adult bone formation remains unclear. Therefore, we used Sdc4 -/- mice to analyse the distribution and functional role of Scd4 in endochondral ossification of mouse embryos and in adult fracture repair, which recapitulates endochondral ossification, but like osteoarthritis, involves an inflammatory component. Methods Sdc4 promoter activity was analysed in Sdc4 -/- /LacZ knock-in animals using β-galactosidase stainings. E16.5 embyros were used for histological (alcian blue/alizarin red) and immunohistological (PCNA, Col10a1, ADAMTS-4, BC-3, Sdc2) staining and the calcified bone area was quantified using whole mount staining of these embryos. Histological (Masson-Goldner, alcian blue) and immunohistological (Col10a1, Sdc2, PCNA) staining at day 7, 14 and 28 fracture calli were performed. These experiments were repeated with anti-TNF treatment during fracture healing. Callus size and cartilage area were quantified using image J Chondrocytes were isolated from neonatal knee joints and embyronal cartilage. Proliferation was investigated using MTT assay. Gene expression analysis for Sdc-2, Sdc-4 with and without stimulation using TNFα and WNT3a was performed using quantitative RT-PCR. Results In Sdc4 -/- /LacZ knock-in animals, Sdc4 promoter activity was detectable in all stages of chondrocyte differentiation during embryogenesis. Sdc4 deficiency inhibited chondrocyte proliferation both in vivo and in vitro, but this did not lead to a growth phenotype at birth. In contrast to embryogenesis, fracture healing in adult mice was markedly delayed in Sdc4 -/- animals and accompanied by increased callus formation. Analysing the discrepancy between the mild embryonic and the severe adult phenotype, we found a compensatory up-regulation of Sdc2 in the developing cartilage of Sdc4 -/- mice that was absent in adult tissue. Stimulation of chondrocytes with Wnt3a in vitro, led to an increased expression of Sdc2, while stimulation with TNFα resulted in an up-regulation of Sdc4 but a decreased expression of Sdc2. In consequence treatment with a blocking anti-TNF antibody during fracture healing abolished the difference in callus size between wildtype and sdc4 -/- mice. Conclusions We conclude that Sdc4 is functionally involved in endochondral ossification and that the loss of Sdc4 impairs adult fracture healing due to the inhibition of compensatory mechanisms under inflammatory conditions.