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.

Comparison of osteopromoting ability of human tooth powder with the demineralized freeze-dried bone allograft, a bovine xenograft, and a synthetic graft: An in vitro study

“…In a recent in vitro study conducted by Tanwatana et al, 14 several processes, such as thermal and chemical deproteinization of tooth matrix, demonstrated favorable proliferation and attachment of osteoblasts. They concluded that the demineralized and deproteinized tooth matrix is a suitable scaffold for osteoblasts.…”

Tooth as a Bone Graft Material: A Narrative Review

“…The evaluation of annealed toothpowder's direct and indirect cytotoxicity did not show a cytotoxic effect on human alveolar bone marrow stem cells. 41 MC3T3-E1 osteoblasts cell line adheres and proliferates to the surface of chemical (sodium hydroxide) and thermal-treated tooth powder, forming a multilayered and dense cell sheet accumulated after 7 days of culture. 41 Several researchers claimed that deproteinisation of tooth-derived materials decreases immunogenicity and prevents the host's immune following implantation.…”

“…41 MC3T3-E1 osteoblasts cell line adheres and proliferates to the surface of chemical (sodium hydroxide) and thermal-treated tooth powder, forming a multilayered and dense cell sheet accumulated after 7 days of culture. 41 Several researchers claimed that deproteinisation of tooth-derived materials decreases immunogenicity and prevents the host's immune following implantation. However, to the best of our knowledge, any in vitro evaluation of the inflammatory and immune cell response to deproteinized GBM or BBM was found in the literature.…”

Tooth-Derived Matrix Granules for Enhanced Bone Healing: Chemical Composition, Morphological Aspects, and Clinical Outcomes

“…The chemical composition of bone and dentin are quite similar including hydroxyapatite (Hap) and type I collagen showing a high degree of cross-linking with matrix-binding proteins and growth factors such as IGF-II, BMP-2, and TGF [40,41]. Dentin-derived matrix granules are prepared from teeth which have been extracted due to trauma, advanced periodontal bone loss, or other indications such as wisdom teeth or orthodontic treatment.…”

“…In fact, several protocols for the preparation of ATDM granules have been reported in the literature. Therefore, several other terms have been applied for ATDM depending on the processing protocols such as demineralized dentin-matrix (DDM), deproteinized demineralized dentin-matrix (dDDM), tooth-derived dentin-matrix (TDM), mineralized dentin-matrix (MDM), or partially mineralized dentin-matrix (PDM) [37,40,47].…”