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Injuries to articular cartilage are one of the most challenging issues of musculoskeletal medicine due to the poor intrinsic ability of this tissue for repair. Despite progress in orthopaedic surgery, the lack of efficient modalities of treatment for large chondral defects has prompted research on tissue engineering combining chondrogenic cells, scaffold materials and environmental factors. The aim of this review is to focus on the recent advances made in exploiting the potentials of cell therapy for cartilage engineering. These include: 1) defining the best cell candidates between chondrocytes or multipotent progenitor cells, such as multipotent mesenchymal stromal cells (MSC), in terms of readily available sources for isolation, expansion and repair potential; 2) engineering biocompatible and biodegradable natural or artificial matrix scaffolds as cell carriers, chondrogenic factors releasing factories and supports for defect filling, 3) identifying more specific growth factors and the appropriate scheme of application that will promote both chondrogenic differentiation and then maintain the differentiated phenotype overtime and 4) evaluating the optimal combinations that will answer to the functional demand placed upon cartilage tissue replacement in animal models and in clinics. Finally, some of the major obstacles generally encountered in cartilage engineering are discussed as well as future trends to overcome these limiting issues for clinical applications.

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Involvement of Pi and Ca in chondrocyte maturation was studied because their levels increase in cartilage growth plate. In vitro results showed that Pi increases type X collagen expression, and together with Ca, induces apoptosis-associated mineralization, which is similar to that analyzed in vivo, thus suggesting a role for both ions and apoptosis during endochondral ossification.Introduction: During endochondral ossification, regulation of chondrocyte maturation governs the growth of the cartilage plate. The role of inorganic phosphate (Pi), whose levels strongly increase in the hypertrophic zone of the growth plate both in intra-and extracellular compartments, on chondrocyte maturation and mineralization of the extracellular matrix has not yet been deciphered. Materials and Methods:The murine chondrogenic cell line ATDC5 was used. Various Pi and calcium concentrations were obtained by adding NaH 2 PO 4 /Na 2 HPO 4 and CaCl 2 , respectively. Mineralization was investigated by measuring calcium content in cell layer by atomic absorption spectroscopy and by analyzing crystals with transmission electron microscopy and Fourier transform infrared microspectroscopy. Cell differentiation was investigated at the mRNA level (reverse transcriptase-polymerase chain reaction [RT-PCR] analysis). Cell viability was assessed by methyl tetrazolium salt (MTS) assay and staining with cell tracker green (CTG) and ethidium homodimer-1 (EthD-1). Apoptosis was evidenced by DNA fragmentation and caspase activation observed in confocal microscopy, as well as Bcl-2/Bax mRNA ratio (RT-PCR analysis). Results: We showed that Pi increases expression of the hypertrophic marker, type X collagen. When calcium concentration is slightly increased (like in cartilage growth plate), Pi also induces matrix mineralization that seems identical to that observed in murine growth plate cartilage and stimulates apoptosis of differentiated ATDC5 cells, with a decrease in Bcl-2/Bax mRNA ratio, DNA fragmentation, characteristic morphological features, and caspase-3 activation. In addition, the use of a competitive inhibitor of phosphate transport showed that these effects are likely dependent on Pi entry into cells through phosphate transporters. Finally, inhibition of apoptosis with ZVAD-fmk reduces -induced mineralization. Conclusions: These findings suggest that Pi regulates chondrocyte maturation and apoptosis-associated mineralization, highlighting a possible role for Pi in the control of skeletal development.

Guicheux J. Differential effects of hypoxia on osteochondrogenic potential of human adipose-derived stem cells. Am J Physiol Cell Physiol 298: C355-C364, 2010. First published November 25, 2009 doi:10.1152/ajpcell.00398.2009.-Human adipose tissue-derived stem cells (hATSC) have been contemplated as reparative cells for cartilage engineering. Chondrogenic differentiation of hATSC can be induced by an enriched culture medium and a three-dimensional environment. Given that bone is vascularized and cartilage is not, oxygen tension has been suggested as a regulatory factor for osteochondrogenic differentiation. Our work aimed at determining whether hypoxia affects the osteochondrogenic potential of hATSC. hATSC were cultured in chondrogenic or osteogenic medium for 28 days, in pellets or monolayers, and under 5% or 20% oxygen tension. Cell differentiation was monitored by real-time PCR (COL2A1, aggrecan, Runx2, and osteocalcin). The chondrogenic differentiation was further evaluated by Alcian blue and immunohistological staining for glycosaminoglycans (GAGs) and type II collagen, respectively. Osteogenic differentiation was also assessed by the staining of mineralized matrix (Alizarin Red) and measurement of alkaline phosphatase (ALP) activity. The expression of chondrogenic markers was upregulated when hATSC were exposed to hypoxia in chondrogenic medium. Conversely, osteocalcin expression, mineralization, and ALP activity were severely reduced under hypoxic conditions even in the presence of osteogenic medium. Our data strongly suggest that hypoxia favors the chondrogenic differentiation of hATSC as evidenced by the expression of the chondrogenic markers, whereas it could alter their osteogenic potential. Our results highlight the differential regulatory role of hypoxia on the chondrogenic and osteogenic differentiation processes of hATSC. These data could help us exploit the potential of tissue engineering and stem cells to replace or restore the function of osteoarticular tissues.

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