Faculty Bibliography

In recent years artificial bone replacement materials have been developed that can be coated with growth factors and loaded with osteoblasts to induce osteogenesis. All these attempts from the bone tissue engineering field disregard the fact that skeletal development can proceed by two pathways: intramembranous or endochondral bone formation. Here we propose to mimic nature by creating an in vitro cartilage template that could after implantation in vivo, remodel into bone, as is normally carried out during the process of endochondral bone formation. The specific aims of this study are: (1) to establish a method of growing chondrocytes in a well characterized biphasic calcium phosphate (BCP) scaffold, and (2) to induce chondrocyte hypertrophy and cartilage matrix deposition in cells growing in the BCP scaffold. Materials and Methods: Chondrocytes isolated from 18-day chick embryo tibial growth plates were grown on particles of macroporous biphasic calcium phosphate (MBCP(R), Biomatlante, France). Chondrocytes were grown in culture continuously for 3 weeks, and fed every day with Dulbecco's modified Eagle medium (GIBCO) containing 10% Nu serum (Fisher), 2mM L-glutamine, 100 U/ml penicillin/streptomycin, and 50 mug/ml ascorbic acid. After one week, cultures were treated daily with 100 nM all trans-retinoic acid (RA), to induce chondrocyte maturation, and extracellular matrix synthesis. Chondrocyte proliferation was observed using scanning electron microscopy (SEM) on appropriately prepared specimens. Alkaline phosphatase (AP) activity was measured spectrophotometrically. Levels of proteoglycans and levels of type X Collagen were determined using Alcian blue staining and Western blot analysis, respectively. Results: Chondrocytes attached, and proliferated on the BCP scaffold. Levels of AP, proteoglycans, and type X Collagen increased in the presence of RA. An unexpected observation was made regarding changes in chondrocyte morphology in the presence of RA. Chondrocytes grown on BCP scaffold assumed an elongated morphology in an intricate net of collagenous proteins, while cells grown on tissue culture plate surfaces maintained a polygonal and flat morphology. Conclusion: Results from this study demonstrated for the first time the proliferation, maturation of chondrocytes, and cartilage matrix deposition on a macroporous calcium phosphate scaffold, and the potential of such a scaffold in tissue engineering through 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. Furthermore, severe thiol depletion profoundly affected cell survival under oxidative and nitrosoactive stress. It was concluded that the loss of thiol reserve is not only linked to the expression of the hypertrophic phenotype but also influenced chondrocyte survival, linking chondrocyte maturation and the activation of the apoptotic pathway

The homozygous form of beta-thalassemia, the most common single gene disorder, is treated by red cell transfusion therapy. Following transfusion, the chelator, deferoximine, is administered to patients to remove excess iron. However, when this drug is given to young children, metaphyseal dysplasia and abnormalities of linear growth are frequently observed. To explore the notion that deferoximine interferes with endochondral growth by chelating zinc, we examined the effect of the drug on chondrocytes maintained in long-term culture. We found that deferoximine caused a dose-dependent inhibition of a wide range of functions including cell proliferation, protein synthesis (and possibly under-hydroxylation of type X collagen), and mineral deposition. Directly relevant to the mineralization process was the observation that the drug dramatically lowered the activity of alkaline phosphatase, a zinc-requiring enzyme. To test the hypothesis that enzyme inhibition was due to chelation of zinc by deferoximine, the cell culture medium was supplemented with excess zinc. However, this treatment did not overcome the deferoximine-dependent change in enzyme activity. We next examined the possibility that deferoximine, in the presence of ascorbate, could form a free radical system that would serve to inactivate the enzyme. Using alkaline phosphatase extracted from chick cartilage, we noted that the activity of the phosphatase was markedly reduced in the presence of deferoximine and ascorbate. These effects were consistant with the notion that deferoximine and ascorbate can act as a prooxidant couple. This conclusion was confirmed when we measured the oxidative activities of the system using nitrobule tetrazolium and cytochrome c. Indeed, we noted that deferoximine markedly activates the autocatalytic oxidation of ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)