Three-dimensional ultrastructural analysis of the interface between an implanted demineralised dentin matrix and the surrounding newly formed bone

Abstract

Previous investigators have reported that transplanted demineralised dentin matrix (DDM) influences bone formation in vivo. However, the specific mechanism of how dentinal tubules contribute to bone formation has not been determined with regard to DDM transplantation therapy. In this study, we ultrastructurally investigated how DDM contacted the surrounding newly formed bone using a scanning electron microscopy (SEM) three-dimensional reconstruction method that is based on focused ion beam slicing and SEM (FIB/SEM). A pulverised and processed DDM derived from human teeth was implanted into rat calvarial bone defects, and a series of X-ray computed tomographic images were obtained over 12 weeks. Implants with surrounding new bone were removed and histologically examined using FIB/SEM. After obtaining objective block-face images, the target boundary face was reconstructed three-dimensionally. The osteocytes of the new bone tissue surrounding the DDM formed a network connected by their cellular processes and formed bone tissue. It is also interesting that the cellular processes of the osteocytes extended into the dentinal tubules, and that bone tissue with canaliculi had formed and filled the DDM surface.

50 years of scanning electron microscopy of bone—a comprehensive overview of the important discoveries made and insights gained into bone material properties in health, disease, and taphonomy

“…From Tanoue et al Reproduced under the terms of the Creative Commons Attribution License (CC BY 4.0). 216 c Sample preparation for transmission electron microscopy using the in situ lift-out technique, starting with deposition of a 30 µm long, 2 µm wide, and 1 µm-thick protective layer, followed by sequential milling, lift-out, and thinning to electron transparency. From Grandfield et al Adapted with permission from John Wiley and Sons.…”

“…215 Likewise, the attachment of osteocytes via cell processes to demineralised dentin matrix particles implanted in the rat calvarium has also been studied using FIB tomography. 216…”

Postulated release profile of recombinant human bone morphogenetic protein-2 (rhBMP-2) from demineralized dentin matrix

“…Urist suggested that, if the dentin matrix undergoes biodegradation or resorption through a remodeling process after grafting, disintegration of covalent bonding occurs and releases osteoinductive proteins, which play a key role in stimulating stem cells in the surrounding tissues and causing phenotypic changes to produce osteoblasts2. This mechanism has been determined by several in vitro and in vivo studies1617183536.…”

“…(Fig. 3; green line) This process is accelerated via both dentinal tubule and interfibrillar space pores and is induced by collagenolytic degradation or osteoclastic resorption, which might start at the beginning of implantation but before the actual release of proteins11736.…”

Large Volume Electron Microscopy and Neural Microcircuit Analysis

“…The FIB can be extremely narrow in diameter (1–1,000 nm) and sputters a very thin layer from the tissue block surface. It can mill even hard tissues, such as tooth or bone (Tanoue et al, 2018 ). These features are valuable and unique to the FIB-SEM; the other microscopies cannot achieve such a thin layer cutting nor hard tissue sectioning (Briggman and Bock, 2012 ; Kubota, 2015 ).…”