Chemical and thermal deproteinization of human demineralized tooth matrix: Physicochemical characterization and osteoblast cell biocompatibility

Abstract

Although human tooth was developed for using as a bone graft material due to their similarity in chemical composition compared to human bone, the clinical application was limited to autologous use. The aims of this study were to develop the deproteinized human demineralized tooth matrix to be used as a bone graft substitution. The physicochemical characteristic and osteoblastic cell biocompatibility of these materials were analyzed and compared. Human demineralized tooth matrix was prepared by using 0.5 M HCl. Deproteinization of demineralized tooth matrix was performed via three protocols; (a) thermal treatment, (b) NaOH/thermal treatment and (c) H₂O₂/thermal treatment. Phase and chemical composition were assessed by X-ray diffraction spectroscopy, X-ray fluorescence spectroscopy and Fourier transform infrared analysis. Structural modification was evaluated by scanning electron microscopy. Degradation behavior was evaluated over 60 days. The adhesion and cell proliferation of osteoblast cells were performed on the most appropriate protocol in which proteins were totally eliminated. The results showed that thermal and chemical-thermal deproteinization yielded no distinct morphological and physical changes in deproteinized human demineralized tooth matrix. The scanning electron microscopy examination demonstrated open dentinal tubules with smooth dentin surface in thermal and H₂O₂/thermal group, while NaOH/thermal group showed rough dentin surface. The X-ray diffraction spectroscopy confirmed that there was only hydroxyapatite phase. The X-ray fluorescence spectroscopy showed that the main components of tooth were calcium (Ca) and phosphorous(P) and the Ca/P molar ratio was about 1.5–1.8. The degradation of the deproteinized human demineralized tooth matrix was slow (Thermal = 9.506 ± 2.568%, NaOH/thermal = 10.244 ± 2.609%, H₂O₂/thermal = 8.976 ± 2.354%, p < 0.05) at day 60. However, only NaOH/thermal deproteinization demonstrated no protein content. The cells attached and grew on the NaOH/thermal deproteinized human demineralized tooth matrix. The deproteinized human demineralized tooth matrix prepared from the NaOH/thermal protocol demonstrated good physicochemical, and degradation properties that were suitable for preparing bone substitution. In addition, the deproteinized human demineralized tooth matrix scaffold is also favorable for cell attachment and proliferation.