In Vitro Cell Viability Tests on a Composite Graft Containing Alpha Tricalcium Phosphate, Chondroitin Sulfate and Disodium Succinate

Abstract

A granular bone substitute composed of alpha tricalcium phosphate, chondroitin sulfate and disodium succinate is biocompatible in vivo. The aim of this in vitro study was to analyze cellular aspects related to bone regeneration. The response of MG63 and L929 cells to liquid extracts of the respective material was reported. Viability and integrity of the cell membrane were determined by MTT and trypan blue exclusion assay. Cytoskeletal changes were followed by phalloidin staining. Expression of apoptotic, mitogenic and osteogenic differentiation genes was detected by RT-PCR. Supernatants obtained at 24 hours of extraction, but not later, reduced formazan formation in L929 and MG63 cells (p<0.05). Also sodium chondroitin sulfate at 10 % and disodium succinate at 2.5% significantly decreased cell viability (p<0.05). Moreover, we observed impaired integrity of the cell membrane and cytoskeletal changes. Expression of mitogenic genes was partially reduced while apoptotic and differentiation genes were unchanged. Liquid extracts of TCP and TCP failed to cause any substantial effects. Thus, the composite bone substitute can cause an early, but temporary cell necrosis. Since bone regeneration is initiated by a transient catabolic phase, the observed effects are in line with this biological principle.