Bio-Absorption of Human Dentin-Derived Biomaterial in Sheep Critical-Size Iliac Defects

Abstract

The aim of this study was to evaluate the bio-absorption and bone regeneration of human tooth-derived dentin scaffold, entitled as perforated root-demineralized dentin matrix (PR-DDM), after in vivo implantation into the critical-size iliac defects. The dentin scaffolds were prepared from human vital, non-functional teeth. Thirty artificial macro-pores (Ø 1 mm) were added after removing the enamel portion. The modified teeth were supersonically demineralized in 0.34 N HNO₃ for 30 min. The microstructure was observed by scanning electron microscope (SEM). The 3D micro-CT and histological analysis were carried out to evaluate the bio-absorption of PR-DDM at 2 and 4 months. A smooth dentin collagen surface with symmetrical macro-pores and tube-type dentinal tubules (Ø 1-2 µm) with micro-cracks were observed on the perforated region. A significant number of custom-made macro-pores disappeared, and the size of the macro-pores became significantly wider at 4 months compared with the 2 months (p < 0.05) evaluated by 3D micro-CT. Histological images revealed the presence of multinucleated giant cells attached to the scalloped border of the PR-DDM. The morphological changes due to bio-absorption by the cellular phagocytes were comparable to the 3D micro-CT and histological images at 2 and 4 months. Altogether, the results demonstrated that the PR-DDM block was gradually absorbed by multinucleated giant cells and regenerated bone. Human PR-DDM might serve as a unique scaffold for extraoral bone regeneration.

Keywords: bio-absorption; bone regeneration; demineralized dentin matrix; tooth-derived scaffold.

Figure 1

Gross images showing the transformation of (a) freshly extracted vital human tooth into (b) perforated demineralized dentin matrix (PR-DDM) (n = 9).

Figure 2

(a) Schematic illustration, and (b) 3D micro-CT images of grafted PR-DDM into critical-size sheep iliac defect.

Figure 3

(a) Scanning electronic microscopy (SEM) image of PR-DDM showing smooth demineralized dentin surface with custom-made artificial macro-pores. (b) Higher magnification of white dotted line in Figure a showing exposed dentinal tubule with micro-cracks (white arrow) in a dense collagen matrix.

Figure 4

Three-dimensional-volume rendering images of PR-DDM. (a) Loss of custom-made perforations (white arrows) at 2 months, (b) Disappearance of perforations along with parts of dentinal wall (white arrows) at 4 months confirmed higher scaffold bio-absorption compared to 2 months.

Figure 5

Three-dimensional micro-CT images of PR-DDM. Size of perforation (arrow) at 2 months (a) and 4 months (b). Loss of structural integrity with demineralized dentin (asterisk) due to bio-absorption at 4 months.

Figure 6

Histological evaluation of sheep iliac defects at 2 months (a–c) and 4 months (d–f) with hematoxylin and eosin (HE) staining. (a) Cellular phagocytosis of PR-DDM (asterisk) by multinucleated giant cells (black arrows), (b) New bone induction (NB) with regular osteoblast layer (arrow) by PR-DDM (asterisk), (c) Well-demarcated boundary (arrow) observed between PR-DDM (asterisk) and new bone (NB); note: osteoblast layer (blue arrow) on new bone, (d) Cellular absorption of PR-DDM (asterisk) by the higher number of multinucleated giant cells (black arrows), (e) New bone (NB) with mosaic-like structure induced by PR-DDM, (f) Simultaneous evidence of new bone formation (NB) with osteoblasts (blue arrow) and PR-DDM bio-absorption (asterisk) by multinucleated giant cells (black arrows).