Objective: To observe the heterotopic osteogenesis of the autogenous marrow stromal cells (MSCs) on the ceramic bovine bone (CBB)/hydrogel scaffold (HG) and the effects of the recombinant human bone morphogenetic protein-2 (rhBMP-2) and the transforming growth factor beta (TGF-beta) on osteogenesis.
Methods: The autogenous marrow stromal cells were cultured by the mineralized condition medium (10% FBS, dexamethasone 10 nmol, L-vitamin C 50 mg/L, beta-sodium glycerophosphate DMEM culture medium 10 mmol). At 5 days, the MSCs differentiation was observed by Type I collagen, the Mend calcium-cobalt staining, and the Von-Kossa staining. The cell suspension of 5 x 10(6)/ml was obtained. There were three groups: Group A: added in rhBMP-2 (10 microg)-TGF-beta (0.05 microg); Group B: added in TGF-beta (0.05 microg); and Group C (the control group): without the growth factor. Then, the MSCs loading on CBB/HG were embedded in the autogenous subcutaneous area at 4 and 8 weeks, and the osteogenesis was observed by the HE staining and the modified Mallory's trichrome staining, with an image analysis. Type I collagen and the bone morphogenetic synthesis were examined by the immunohistochemistry stains.
Results: Most MSCs induced by the mineralized condition medium at 5 days became smaller and polygon-shaped, and the cytodendrite became shorter. The MSCs were observed by the Mend calcium-cobalt staining. Some brown and black grains were found in the cytochylema. The MSCs were positive for the Type I collagen immunohistochemistry stains. At 20 days, the mineralized nubs were found by the Von Kossas stains. At 4 weeks, some strips of the new bone were observed by the HE staining and the modified Mallory's trichrome staining in all the groups. The bone matrix area was significantly larger in Group A than in Group B(P < 0.01). The average gray degrees of Type I collagen were lower in Groups A and B than in Group C. However, there was no significant difference in the bone morphogenesis among the three groups. At 8 weeks, there were significantly more snatch strips and macula mature bone formation in Groups A and B than in Group C. The Type I collagen and the bone morphogenesis were not significantly different among the three groups.
Conclusion: The autogenous marrow stromal cells on the ceramic bovine bone /hydrogel scaffold can promote the heterotopic osteogenesis, and the combined use of rhBMP-2 and TGF-beta is better than the only use of rhBMP-2 or TGF-beta in promoting osteogenesis.