While skeletal development can occur by either intramembranous or endochondral bone formation, all current tissue engineering approaches for bone repair and regeneration try to mimic intramembranous ossification. In this study, we propose to create an in vitro cartilage template as the transient model for in vivo endochondral bone formation. The goals of this study are to (1) establish a method of growing chondrocytes in a well-characterized macroporous biphasic calcium phosphate (MBCP) scaffold and (2) induce maturation of chondrocytes grown in the MBCP scaffold. Chondrocytes isolated from chick embryonic tibia were grown on MBCP particles and treated with retinoic acid to induce chondrocyte maturation and extracellular matrix deposition. Chondrocytes were observed to attach and proliferate on the MBCP scaffold. The thickness of the chondrocyte and extracellular matrix layer increased in the presence of the retinoid. Alkaline phosphatase activity and expression, proteoglycans synthesis, cbfa1 and type I collagen mRNA levels also increased in the presence of retinoic acid. These results demonstrated for the first time the proliferation, maturation of chondrocytes, and matrix deposition on MBCP, suggesting the potential for such scaffold in tissue engineering via the endochondral bone formation mechanism.

The major aim of the current investigation was to evaluate the role of thiols during chondrocyte maturation and apoptosis. Using a thiol-sensitive fluorescent probe, we found that in chick growth plate chondrocytes, hypertrophy is accompanied by a decrease in the glutathione content. In this study, we show that the maturation-dependent loss of thiol, although not causing death of maturing chondrocytes, drastically increases susceptibility to apoptosis by oxidative and nitrosoactive stress. To investigate how the loss of thiol content in cultured chondrocytes affects the expression of the hypertrophic phenotype, we chemically manipulated intracellular thiol levels and analyzed the expression of important maturation markers. We found that thiol depletion causes a decrease in the expression of osteopontin, type X and type II collagen and a significant loss of alkaline phosphatase activity, suggesting that the expression of the hypertrophic phenotype is tightly regulated by redox levels in chondrocytes.

Objective: To determine the role of mitochondria in chondrocyte apoptosis induced by inorganic phosphate (Pi). Materials and Methods: Chondrocytes isolated from the growth plates of chick embryo tibia were treated with Pi in serum-free media; chondrocyte viability, mitochondrial membrane potential, cytochrome c release from mitochondria, caspase 3 activity, endonuclease activity, and DNA fragmentation were investigated. Results: Exposure to Pi for 24 hours induced apoptosis in growth plate chondrocytes through a pathway that involved loss of mitochondrial function, release of cytochrome c into the cytoplasm, increases in caspase 3 and endonuclease activities, and fragmentation of DNA. Conclusions: This study suggests that mitochondria are important players in Pi-induced apoptosis.

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