Objective To explore the osteogenic potential of cervical intervertebral disc fibroblasts in vitro, to investigate the regulatory factors of recombinant human bone morphogenetic protein 2(rhBMP-2) and tumor necrosis factor α(TNF-α) on osteogenic phenotype of fibroblasts and to discuss the condition that facilitates osteogenesis of fibroblasts. Methods Theannulus fibroblasts cell lines of experiment goats were established in vitro and the biologicspecificity was found. According to different medias, 4 groups were included in this experiment: control group, TNF-α group ( 50 U/ml TNF-α), rhBMP-2 group (0.1 μg/ml rhBMP-2) and TNF-α+rhBMP-2 group (50 U/ml TNF-α+0.1 μg/ml rhBMP-2). Thefibroblasts were incubated in the media for about 3 weeks,and then the markers for osteogenic features were investigated by biochemistry, histochemistry observations. Results rhBMP-2 and TNF-α had no effect on the proliferation of fibroblasts from the experiment goats. rhBMP-2 or TNF-α could stimulate fibroblasts to secrete alkaline phosphatase and collagen type Ⅰ. The combined use of rhBMP-2 and TNF-α or the single use of rhBMP-2 could make fibroblasts to secrete osteocalin and the morphological changes of the fibroblasts were very obvious. Histochemical study of the nodules with specific new bone labeler(Alizarin red S) revealed positive reaction, denoting that the nodules produced by the fibroblasts werebone tissues. There was statistically significant difference(Plt;0.05) inALP activity between 3 experimental groups and control group and in secretion of osteocalcin between rhBMP-2 group, TNF-α+rhBMP-2 group and control group. Conclusion The results point out clearly that rhBMP-2 can induce theosteogenic potential of annulus fibroblasts in vitro.
OBJECTIVE: To prevent the senescence of ’seed cells’ for tissue engineering, the life span of human fibroblasts is extended by reconstitution of telomerase activity, and the osteogenic potential of these fibroblasts are tested. METHODS: The pGRN145 plasmids encoding human telomerase reverse transcriptase (hTERT) were introduced into the normal human primary fibroblasts by electroporation. Telomerase activity was analyzed by TRAP-PCR assay. The beta-galactosidase stain was used to indicate the signs of cell senescence. The hTERT positive fibroblasts were then induced to form bone nodules. The bone nodules were stained by tetracycline and Alizarin Red S. RESULTS: Stable telomerase activity could be detected in the transfected fibroblasts and no signs of cell senescence were found in the fibroblasts cultured for more than 50 doublings. The hTERT positive fibroblasts could form bone nodules when they were cultured in vitro induced by bone morphogentic protein 2 and tumor necrosis factor-alpha. CONCLUSION: The fibroblasts with reconstituted telomerase activity reserve their osteogenic potential.
The osteogenc potential of bone marrow has been proved by experiment. To investigate more in details, bone marrow was obtained from the trochanteric region of femur of NewZealand rabbit in 4 to 8 weeks old. After being cultured in vitro for one week, the hematopoietic component of the bone marrow had disappeared, thus the stromal cells were obtained. Then the stromal cells were subcultured in cultural fluid containing dexamethasone (10-8 mol/L) and natrium glycerophosphate (10mmol/L). Under the phasecontrast microscope, it was found that being cultured for 15 days. The stromal cells were lined up in one layer and late the secretion activity was increased and gradually transformed into multilayer structure and was congregated into diffused opaque clusters in twenty days. During culture, the cells were examined by tetracycline fluorescence label, histochemistry stains, transmission electron microscopy, scanning electron microscopy and energy dispersive X-ray microanalysis. The results showed that the morphological and biological characteristics of the cultured stromal cells derived from the bone marrow were similiar to those of osteoblasts and could synthesized mineralized new bone tissue in vitro.