The Influence of Demineralization Process on Physicochemical Characteristics,BMP-2 Quantification and In vitro Biocompatibility of Human Demineralized Tooth Matrix

Abstract

Objectives: To examine the influence of different hydrochloric acid (HCl) demineralization processes on the physicochemical characteristic of the demineralized tooth matrix (DTM) in the aspect of physicochemical properties, degradation kinetic, the amount of bone morphogenetic protein-2 (BMP-2) and cell biocompatibility on the demineralized tooth matrix scaffold using osteoblastic cells. Materials and methods: Caries-free permanent teeth were divided and pulp tissue were removed. The teeth were pulverized into small particles range from 500-1,000 um. The particles were pooled and assigned to 5 groups with different protocols of HCI concentration and reaction time [0M/0min(TM), 0.5M/10min (0.5M/10DTM), 0.5M/20min (0.5M/20DTM), 1M/10min (1M/10DTM) and 1M/20min (1M/20DTM)]. The study was divided into 2 parts, physicochemical properties evaluation, and in vitro osteoblast cell biocompatibility. In physicochemical characterization, the chemical compositions were analyzed by X-ray diffraction and X-ray fluorescence spectroscopy. Structure topography and surface area were evaluated by scanning electron microscopy and BET analysis respectively. Protein extraction was performed for 24 hrs. Bradford protein assay was used to quantify total protein and ELISA was used for BMP-2 quantification. For degradation rate assessment, TM and DTM scaffolds were fabricated using 5% wt/v of polyvinyl alcohol (PVA) as a biding reagent. All groups were assessed at 60 days using 50 mM Tris-HCl solution (pH 7.4) at 37°c. Base on the physicochemical properties, DTM group from proper physicochemical properties and degradation rate was chosen for further study. For cell biocompatibility, MC3T3-E1 cells were seeded on the scaffolds. The cell-scaffold constructs of each group were divided for culturing in proliferation medium for evaluating cell viability and osteogenic induction medium for assessing cell differentiation on day 1, 3, 7, 14, and day 21. The morphology of the empty scaffolds and cell adherence were examined using SEM at 1, 7 and 14 days after cell seeding. The data was presented as mean±SD. The compared means analysis (p<0.05) was used for comparing total protein and BMP-2 quantification, degradation rate, cell proliferation and cell differentiation. Results: All tooth particles were white to yellow in color with hard in consistency. DTM processed with the longer demineralization reaction times revealed less opacity. From SEM images, 0.5M/20DTM and 1M/20DTM groups exhibited a smooth surface with larger dentinal tubules and abundant of exposed collagen fibrils. Calcium and Phosphorous content in all DTM were lower compared to TM group. The lowest calcium and phosphate content were found in 1M/20DTM group (Ca=3.43%, P=1.07%) compared to TM (Ca=15.60%, P=8.08%). The Ca/P ratio for TM, 0.5M/10DTM, 0.5M/20DTM, 1M/10DTM and 1M/20DTM were 1.49, 1.97, 2.39, 2.35 and 2.48 respectively. The XRD confirmed that there was only hydroxyapatite phase. The crystallinity in TM, 0.5M/10DTM, 0.5M/20DTM, 1M/10DTM and 1M/20DTM were 63.09%, 62.12%, 56.84%, 61.915% and 54.33% respectively. From BET analysis demonstrated the average pore distributions from 42.37-68.17A° with the highest in 1M/20DTM group. In contrast to the surface areas range from 1.77-5.43 m2/g with the lowest in 1M/20m group. The highest total protein (3.53±0.24 μg/ml) and BMP-2 concentration (1,181.53±193.77 pg/ml) were found in 1M/20DTM group compared to TM (0.98±0.081 μg/ml, 2.67±5.34 pg/ml, p=0.000). The degradation at 60 days of OM/0DTM, 0.5M/10DTM, 0.5M/20DTM, 1M/10DTM, and 1M/20DTM were 6.41+0.30%, 9.19±1.92%, 8.46±0.28%, 19.27+2.74%, 22.90±10.10% respectively (p<0.05). From physicochemical characteristics, 0.5M/10DTM and 0.5M/20DTM were selected for further in vitro study compared to TM as control. SEM analysis of cell-scaffold construct in TM group demonstrated irregular shape of cells and small size on day 1 and day 7. Contrary to 0.5M/10DTM and 0.5M/20DTM groups that the cells showed larger size and propagation. At day 14, the cells proliferated to multilayers on the surface of the scaffolds in all groups. Cell proliferation of all groups increased from day 1 to day 14 but slightly decreased at day 21. There was a statistically significant difference between day 1, 14 and day 21 (p<0.05) in TM group. Whereas, in 0.5M/10DTM and 0.5M/20DTM group showed significant difference at day 14 compare to day 1,3 and day 7 (p<0.05). The cell number in 0.5M/10DTM group was significantly higher than TM group at day 1 and day 3 (p<0.05). The ALP activity of 0.5M/10DTM group showed significantly different increasing at day 14 and 21. However, there was no significantly different of osteocalcin (OCN) concentration between 0.5M/10DTM, 0.5M/20DTM and TM group in all time point. Conclusions: The reaction time of HCl has effect on tooth matrix in aspect of surface morphology, crystallinity, element components, Ca/P ratio and the quantity BMP-2. The HCl acid concentration affect degradation rate of the material. The tooth matrix which demineralized using 0.5M/10min and 0.5M/20min protocol demonstrated superior physicochemical characteristic and cell biocompatibility compared with human tooth matrix.