Biological effects and cytotoxicity of tricalcium phosphate and formaldehyde cross-linked gelatin composite

Abstract The purpose of this study was to prepare and evaluate a composite, GTF, combining tricalcium phosphate (TCP) ceramic particles and formaldehyde cross-linked gelatin, as bone substitute. The content of formaldehyde in the developed material was used to control the physical and mechanical properties of the gelatin structure, in terms of cross-linked gelatin molecules, solubility, and biodegradation of the reconstituted matrices. Formaldehyde, however, is known to be a potentially toxic substance that would cause inflammation and severe tissue response. Thus, it is necessary to know whether the substances released from the GTF composite will cause severe tissue response. In this study, the biocompatibility and cytotoxicity of the GTF composite were examined by the in vitro method of human myoblast cell culture. Extracts were obtained by soaking the GTF composite in normal saline for 1, 2, 4, 6, and 8 days. The extract mixed with complete medium in a ratio of 1:1 was added to cell culture wells each containing 1 × 10 5 myoblasts. After being cultured for 2 days, the cells were trypsinized and counted in a Neubauer countingchamber under an optical microscope. The culture dishes were washed with 0.185 M sodium cacodylate buffer (pH 7.2) and fixed with 4% glutaraldehyde solution for 30 min. They were then dehydrated through a graded ethanol series and stained with hematoxylin/eosin for optical microscopic examination. The substances released from the GTF composite were analyzed by gas chromatography (GC), capillary electrophoresis (CE), and inductive coupled plasma-atomic emission spectrometry (ICP-AES). The results showed that the GTF composite was well tolerated by the myoblast. The unchained or uncross-linked formaldehyde was completely released from the GTF composites after being soaked in the normal saline for 4 days. The extract was thought to inhibit the cell growth for the initial 4 days of soaking time. After soaking for 4–6 days, the GTF composite gradually began to release some nutritious elements which were beneficial to the myoblast growth and caused cell numbers to increase substantially. The released nutritious constituents were analyzed by the above-mentioned instruments. The results were in agreement with the above observations.