Osteogenic evaluation of glutaraldehyde crosslinked gelatin composite withfetal rat calvarial culture model

The cytotoxicity of the synthetic bone substitute composed of tricalcium ph osphate and glutaraldehyde crosslinked gelatin (GTG) were evaluated by oste oblast 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 solution s (or extracts) were cocultured with osteoblasts to evaluate the cytotoxici ty of GTG composites by alive cell counting. In this study, the extracts we re cocultured with the osteoblasts: thereafter, the concentration of transf orming growth factor-beta (TGF-beta (1)) and prostaglandin E-2 (PGE(2)) in the medium was analyzed to strictly reflect the biological effects of GTG c omposites on the growth of osteoblasts. In order to investigate the osteoco nductive potential of the GTG composites on new bone formation in a relativ e 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 thei r ability for bone regeneration. Deproteinized bovine and porcine cancellou s bone matrixes were used as the controlled materials. All the organ cultur e units were maintained in cultured medium for 5 weeks. Following the cultu re period, the morphology of tissue was observed under an optical microscop e, and the quantitative evaluation of the new generation bone was determine d by using a semiautomatic histomorphometeric method. Except in the initial 4 days, the concentration of TGF-beta (1) of 4% and 8% GTG composites was higher than that of the blank group for all the other experimental time per iods. The PGE(2) concentration for 4% and 8% GTG composites was lower than that of the blank group. It revealed that the 4% and 8% GTG composites woul d not lead to inflammation and would promote osteoblast growth. The morphol ogy 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 vitr o condition was inferior to the blank group and the other 2 GTG composites. Although the concentration of TGF-beta (1) and PGE(2) was gradually back t o normal after 14 days, the morphology of the osteoblasts was abnormal with features such as contracted cytoplast structures. The osteoblast was damag ed perhaps in the initial stage. We suggested that the 4% and 8% GTG compos ites should be soaked in distilled water at least for 4 days before medical applications. The 12% GTG composite and the composites with a concentratio n of glutaraldehyde solution higher than 12% were not recommended as a medi cal prostheses in any condition. The fetal rat calvaria culture also showed the same results with the analysis of TGF-beta (1) and PGE(2). From the st udy, we could predict the results of animal experiments in the future.