Osteogenic Evaluation of Glutaraldehyde Crosslinked Gelatin Composite with Fetal Rat Calvarial Culture Model

The cytotoxicity of the synthetic bone substitute composed of tricalcium phosphate and glutaraldehyde crosslinked gelatin (GTG) were evaluated by osteoblast cell culture. In a previous study, the GTG composites were soaked in distilled water for 1, 2, 4, 7, 14, 28, and 42 days, and then the solutions (or extracts) were cocultured with osteoblasts to evaluate the cytotoxicity of GTG composites by alive cell counting. In this study, the extracts were cocultured with the osteoblasts; thereafter, the concentration of transforming growth factor-beta (TGF-beta1) and prostaglandin E2 (PGE2) in the medium was analyzed to strictly reflect the biological effects of GTG composites on the growth of osteoblasts. In order to investigate the osteoconductive potential of the GTG composites on new bone formation in a relative short term, a model of neonatal rat calvarial organ culture was designed prior to animal experiments. Three experimental materials of 4, 8, and 12% GTG composites were evaluated by fetal rat calvarial organ culture for their ability for bone regeneration. Deproteinized bovine and porcine cancellous bone matrixes were used as the controlled materials. All the organ culture units were maintained in cultured medium for 5 weeks. Following the culture period, the morphology of tissue was observed under an optical microscope, and the quantitative evaluation of the new generation bone was determined by using a semiautomatic histomorphometeric method. Except in the initial 4 days, the concentration of TGF-beta1 of 4% and 8% GTG composites was higher than that of the blank group for all the other experimental time periods. The PGE2 concentration for 4% and 8% GTG composites was lower than that of the blank group. It revealed that the 4% and 8% GTG composites would not lead to inflammation and would promote osteoblast growth. The morphology and activity of the osteoblasts were not transformed or changed by the 2 GTG composites. For the 12% GTG composite, the performance of the in vitro condition was inferior to the blank group and the other 2 GTG composites. Although the concentration of TGF-beta1 and PGE2 was gradually back to normal after 14 days, the morphology of the osteoblasts was abnormal with features such as contracted cytoplast structures. The osteoblast was damaged perhaps in the initial stage. We suggested that the 4% and 8% GTG composites should be soaked in distilled water at least for 4 days before medical applications. The 12% GTG composite and the composites with a concentration of glutaraldehyde solution higher than 12% were not recommended as a medical prostheses in any condition. The fetal rat calvaria culture also showed the same results with the analysis of TGF-beta1 and PGE2. From the study, we could predict the results of animal experiments in the future