Bone Regeneration Using Dentin Matrix Depends on the Degree of Demineralization and Particle Size

Abstract

Objectives: This study aimed to examine the influence of particle size and extent of demineralization of dentin matrix on bone regeneration.

Materials and methods: Extracted human teeth were pulverized and divided into 3 groups according to particle size; 200, 500, and 1000 μm. Each group was divided into 3 groups depending on the extent of demineralization; undemineralized dentin (UDD), partially demineralized dentin matrix (PDDM), and completely demineralized dentin matrix (CDDM). The dentin sample was implanted into rat calvarial bone defects. After 4 and 8 weeks, the bone regeneration was evaluated with micro-CT images, histomorphometric and immunohistochemical analyses. Osteoblasts were cultured on UDD and DDM to evaluate the cell attachment using electron microscope.

Results: Micro-CT images and histological observation revealed that CDDM had largely resorbed but UDD had not, and both of them induced little bone formation, whereas all particle sizes of PDDM induced more new bone, especially the 1000 μm. Electron microscopic observation showed osteoblasts attached to DDM but not to UDD.

Conclusions: PDDM with larger particle size induced prominent bone regeneration, probably because PDDM possessed a suitable surface for cell attachment. There might be an exquisite balance between its resorption and bone formation on it. PDDM could be considered as a potential bone substitute.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. The percentage of elution Ca in solution and residual Ca in dentin in the process of demineralization by 2% HNO3.

The time setting for the duration of demineralization was based on the results of the percentage of elution Ca in solution and residual Ca in the dentin which were measured over time using the Calcium E test kit in order to estimate the degree of demineralization.

Fig 2. Radiographic analysis of specimens using μCT.

New bone formation around PDDM is enhanced in all particle sizes compared with CDDM in a time-dependent manner. The results of 1000 μm PDDM are especially noteworthy. On the contrary, almost all samples of UDD, with the exception of 1000 μm at 8 weeks, resulted in the defect being mostly occupied with dentin particles.

Fig 3

A) Histological findings of HE stained sections at low-power magnification. The black arrowhead represents the edge of the cortical bone defect. In the UDD group, the area of newly formed bone was limited around the UDD in almost all sections (A-c, d, e, f, g). In the PDDM group, new bone formation surrounding the PDDM was evident at 4 weeks (A-i, j, k), especially in the 1000 μm samples (A-k). At 8 weeks, the newly formed bone is present in variable extents around PDDM, and complete closure in the surgical defect by consecutive new bone formation with PDDM is confirmed from end to end of the defect (A-l, m, n). In the CDDM group, the new bone formation is confirmed to be not only surrounding the CDDM, but also in the gaps between CDDMs (A-t) at 8 weeks in the 1000 μm samples. B) Quantitative micrograph analysis. Histomorphometric findings regarding n-Bone% and r-Imp% are shown. At 4 weeks, significant difference of n-Bone% is observed between the PDDM and other groups in each particle size. Within the PDDM and CDDM groups, the n-Bone% of 1000 μm particles showed a significant difference compared with those of the 200 μm and 500 μm groups. A significant difference in r-Imp% was observed between the CDDM and the other groups in the 200 μm and 500 μm samples. At 8 weeks, a significant difference in the n-Bone% was observed between the PDDM and the other groups in the 200 and 500 μm samples. In the 1000 μm group, the n-Bone% of PDDM and CDDM shows a significant increase in comparison with that of UDD. C) Histological findings of 1000 μm UDD, PDDM and CDDM at 4 weeks. This is the high-power magnification view of 1000 μm UDD, PDDM and CDDM sections stained with HE (C-a, b, c), Osteocalcin (C-d, e, f) and TRAP (C-g, h, i) at 4 weeks. New bone formation (* in C-b, c) surrounding PDDM and CDDM (D in C-b, c) was evident, and they were amalgamated. The maturation of new bone with many lacunae containing osteocytes (C-b) and some lining cells on the surface of the newly formed bone was evident. The presence of Osteocalcin–positive cells (arrowheads in C-e, f) and some TRAP-positive multinucleated giant cells (arrowheads in C-h, i) with Howship’s lacunae in that area was confirmed. Bars = 100 μm.

Fig 4. Osteoblast culture on the surface of UDD and DDM.

SEM micrographs show that cell morphology is sensitive to surface nanostructure. Dentinal tubes are clearly observed on the DDM surface, but not on the UDD surface. Osteoblasts were cultured on the surfaces of UDD and DDM; however, osteoblast attachment was observed only on the surface of DDM.