Re-Evaluation of Initial Bone Mineralization from an Engineering Perspective

Bone regeneration was one of the earliest fields to develop in the context of tissue regeneration, and currently, repair of small-sized bone defects has reached a high success rate. Future researches are expected to incorporate more advanced techniques toward achieving rapid bone repair and modulation of the regenerated bone quality. For these purposes, it is important to have a more integrative understanding of the mechanisms of bone formation and maturation from multiple perspectives and to incorporate these new concepts into the development and designing of novel materials and techniques for bone regeneration. This review focuses on the analysis of the earliest stages of bone tissue development from the biology, material science, and engineering perspectives for a more integrative understanding of bone formation and maturation, and for the development of novel biology-based engineering approaches for tissue synthesis in vitro. More specifically, the authors describe the systematic methodology that allowed the understanding of the different nucleation sites in intramembranous and endochondral ossification, the space-making process for mineral formation and growth, as well as the process of apatite crystal cluster growth in vivo in the presence of suppressing biomolecules. A detailed understanding of the developmental process of bone tissue leads to the acquisition of useful information for the bone tissue fabrication. This review summarizes the study of the calcification process of the calvaria and epiphyses from an engineering perspective and provides useful information for the realization of bone tissue biofabrication. Here, we describe the new mechanism of space formation for mineralization such as rupture of chondrocytes and disruption of cell-cell adhesion. We also describe the roles of nucleation site such as cell membrane nanofragments and matrix vesicles.Hara E.S., Okada M., Nagaoka N., et al. Re-Evaluation of Initial Bone Mineralization from an Engineering Perspective. Tissue Engineering - Part B: Reviews, 28, 1, 246. https://doi.org/10.1089/ten.teb.2020.0352

Important roles of odontoblast membrane phospholipids in early dentin mineralization

The objective of this study was to first identify the timing and location of early mineralization of mouse first molar, and subsequently, to characterize the nucleation site for mineral formation in dentin from a materials science viewpoint and evaluate the effect of environmental cues (pH) affecting early dentin formation. Early dentin mineralization in mouse first molars began in the buccal central cusp on post-natal day 0 (P0), and was first hypothesized to involve collagen fibers. However, elemental mapping indicated the co-localization of phospholipids with collagen fibers in the early mineralization area. Co-localization of phosphatidylserine and annexin V, a functional protein that binds to plasma membrane phospholipids, indicated that phospholipids in the pre-dentin matrix were derived from the plasma membrane. A 3-dimensional in vitro biomimetic mineralization assay confirmed that phospholipids from the plasma membrane are critical factors initiating mineralization. Additionally, the direct measurement of the tooth germ pH, indicated it to be alkaline. The alkaline environment markedly enhanced the mineralization of cell membrane phospholipids. These results indicate that cell membrane phospholipids are nucleation sites for mineral formation, and could be important materials for bottom-up approaches aiming for rapid and more complex fabrication of dentin-like structures

Flexural properties, bond ability, and crystallographic phase of highly translucent multi-layered zirconia

This study investigated the mechanical properties, bond ability, and crystallographic forms of different sites in a highly translucent, multi-layered zirconia disk. Flexural properties, bond ability to resin cement, and phase composition were investigated at three sites of a highly translucent, multi-layered zirconia disk: incisal, middle, and cervical. Flexural strength (FS) and flexural modulus (FM) were measured with static three-point flexural test. Shear bond strength (SB) to resin cement was measured after 24 h storage (37°C). Phase composition under mechanical stress was analyzed using X-ray diffraction. Without air abrasion, FS at the incisal site yielded the lowest value and was significantly lower than the middle and cervical sites. Air abrasion lowered the FS of each site. FM at the incisal site without air abrasion showed the significantly lowest value, and air abrasion increased its FM value. At the middle and cervical sites, their FM values were higher than the incisal site but were not significantly affected by air abrasion. SB value did not show significant differences among the sites. After sintering, cubic zirconia was detected at each site. Rhombohedral phase transformation occurred after mirror polishing. In highly translucent, multi-layered zirconia which was mainly composed of cubic zirconia, rhombohedral phase transformation occurred under mechanical stress and resulted in weakened mechanical properties

Fabrication of a Fish-Bone-Inspired Inorganic-Organic Composite Membrane

Biological materials have properties like great strength and flexibility that are not present in synthetic materials. Using the ribs of crucian carp as a reference, we investigated the mechanisms behind the high mechanical properties of this rib bone, and found highly oriented layers of calcium phosphate (CaP) and collagen fibers. To fabricate a fish-rib-bone-mimicking membrane with similar structure and mechanical properties, this study involves (1) the rapid synthesis of plate-like CaP crystals, (2) the layering of CaP-gelatin hydrogels by gradual drying, and (3) controlling the shape of composite membranes using porous gypsum molds. Finally, as a result of optimizing the compositional ratio of CaP filler and gelatin hydrogel, a CaP filler content of 40% provided the optimal mechanical properties of toughness and stiffness similar to fish bone. Due to the rigidity, flexibility, and ease of shape control of the composite membrane materials, this membrane could be applied as a guided bone regeneration (GBR) membrane

Three-dimensional observation and analysis of remineralization in dentinal caries lesions

The remineralization mechanism in dental caries lesions is not completely understood. This study reports on ultrastructural and chemical changes observed within arrested caries lesions. Carious human teeth were observed using scanning electron microscopy (SEM) and focused-ion-beam (FIB)-SEM. The crystals detected in the caries lesions were characterized by transmission electron microscopy (TEM), along with chemical element mapping using energy-dispersive spectroscopy (EDS)-STEM. FIB-SEM 3D reconstructions revealed a severely damaged dentin surface abundantly covered by bacteria. Although the dentin tubules were clogged up to a depth of 100 mu m, bacterial invasion into dentin tubules was not observed. TEM crystal analysis and EDS-STEM revealed the presence of Ca and P, as well as of Mg within the HAp crystals deposited inside the dentin tubules. It was concluded that extensive remineralization with deposition of Mg-HAp crystals had occurred in dentin tubules of caries-arrested dentin. Understanding the natural remineralization process is thought to be helpful for developing clinical biomimetic remineralization protocols

Micro-Architectural Investigation of Teleost Fish Rib Inducing Pliant Mechanical Property

Despite the fact that various reports have been discussing bone tissue regeneration, precise bone tissue manipulation, such as controlling the physical properties of the regenerated bone tissue, still remains a big challenge. Here, we focused on the teleost fish ribs showing flexible and tough mechanical properties to obtain a deeper insight into the structural and functional features of bone tissue from different species, which would be valuable for the superior design of bone-mimicking materials. Herein, we examined their compositions, microstructure, histology, and mechanical properties. The first rib of Carassius langsdorfii showed a higher Young's modulus with a small region of chondrocyte clusters compared with other smaller ribs. In addition, highly oriented collagen fibers and osteocytes were observed in the first rib, indicating that the longest first rib would be more mature. Moreover, the layer-by-layer structure of the oriented bone collagen was observed in each rib. These microarchitectural and compositional findings of fish rib bone would give one the useful idea to reproduce such a highly flexible rib bone-like material

Eco-Benign Orange-Hued Pigment Derived from Aluminum-Enriched Biogenous Iron Oxide Sheaths

Inorganic pigments have been widely used due to their low cost of production, strong hiding power, and chemical resistance; nevertheless, they have limited hue width and chromaticity. To eliminate these disadvantages, we herein propose the use of an ingenious biotemplate technique to produce Al-enriched biogenic iron oxide (BIOX) materials. Spectrophotometric color analysis showed that high levels of Al inclusion on heat-treated BIOX samples produced heightened yellowish hues and lightness. The Al-enriched BIOX sheaths exhibited a stable tubular structure and excellent thermal stability of color tones after heating at high temperatures and repetitive heat treatments. Ultrastructural analysis and mechanical destruction experiments revealed that the highly chromatic orange-hue of these pigments are ascribed probably to an ingenious cylindrical nanocomposite architecture composed of putative Fe-included low crystalline Al oxide regions and hematite particles embedded therein. The present work therefore demonstrates that the bioengineered material can serve as an epochal orange-hued inorganic pigment with low toxicity and marked thermostability that should meet large industrial demand

Biomimetic mineralization using matrix vesicle nanofragments

In vitro synthesis of bone tissue has been paid attention in recent years; however, current methods to fabricate bone tissue are still ineffective due to some remaining gaps in the understanding of real in vivo bone formation process, and application of the knowledge in bone synthesis. Therefore, the objectives of this study were first, to perform a systematic and ultrastructural investigation of the initial mineral formation during intramembranous ossification of mouse calvaria from a material scientists' viewpoint, and to develop novel mineralization methods based on the in vivo findings. First, the very initial mineral deposition was found to occur at embryonic day E14.0 in mouse calvaria. Analysis of the initial bone formation process showed that it involved the following distinct steps: collagen secretion, matrix vesicle (MV) release, MV mineralization, MV rupture, and collagen fiber mineralization. Next, we performed in vitro mineralization experiments using MVs and hydrogel scaffolds. Intact MVs embedded in collagen gel did not mineralize, whereas, interestingly, MV nanofragments obtained by ultrasonication could promote rapid mineralization. These results indicate that mechanically ruptured MV membrane can be a promising material for in vitro bone tissue synthesis. © 2019 The Authors. journal Of Biomedical Materials Research Part A Published By Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1021–1030, 2019.Kunitomi Y., Hara E.S., Okada M., et al. Biomimetic mineralization using matrix vesicle nanofragments. Journal of Biomedical Materials Research - Part A, 107, 5, 1021. https://doi.org/10.1002/jbm.a.36618

Bundling of collagen fibrils influences osteocyte network formation during bone modeling

Osteocytes form a cellular network by gap junctions between their cell processes. This network is important since intercellular communication via the network is essential for bone metabolism. However, the factors that influence the formation of this osteocyte network remain unknown. As the early stage of osteocyte network formation occurs on the bone surface, we observed a newly formed trabecular bone surface by orthogonal focused ion beam-scanning electron microscopy. The embedding late osteoblast processes tended to avoid bundled collagen fibrils and elongate into sparse collagen fibrils. Then, we examined whether the inhibition of bundling of collagen fibrils using a potent lysyl oxidase inhibitor, beta-aminopropionitrile (BAPN) changed the cellular network of the chick calvaria. The osteocyte shape of the control group was spindle-shape, while that of the BAPN group was sphere-shaped. In addition, the osteocyte processes of the control group were elongated vertically to the long axis of the cell body, whereas the osteocyte processes of the BAPN group were elongated radially. Therefore, it was suggested that the bundling of collagen fibrils influences normal osteocyte network formation during bone modeling

Sandblasting may damage the surface of composite CAD-CAM blocks

　OBJECTIVE:　 CAD-CAM blocks to fabricate semi-direct and indirect restorations are available in different sorts of ceramics as well as composite. In order to bond restorations prepared out of composite blocks into tooth cavities, it is recommended to gently sandblast the surface prior to the application of a primer/adhesive. Today, the effect of sandblasting composite block surfaces has not thoroughly been investigated. In this study, the ultra-structure of composite CAD-CAM blocks was investigated with special attention to the effect of sandblasting on the surface topography and of silanization on the bonding performance. 　METHODS:　 Five different composite CAD-CAM blocks were involved. We correlatively investigated their structural and chemical composition using X-ray diffraction (XRD), energy dispersion spectroscopy (EDS), scanning electron microscopy (SEM) and (scanning) transmission electron microscopy ((S)TEM). The effect of sandblasting was also imaged in cross-section and at the interface with composite cement. Finally, we measured the shear bond strength to the sandblasted block surface with and without silanization. 　RESULTS:　 All composite blocks revealed a different ultra-structure. Sandblasting increased surface roughness and resulted in an irregular surface with some filler exposure. Sandblasting also damaged the surface. When the sandblasted composite blocks were silanized, superior bonding receptiveness in terms of higher bond strength was achieved except for Shofu Block HC. 　SIGNIFICANCE:　 Sandblasting followed by silanization improved the bond strength to composite CAD-CAM blocks. However, sandblasting may also damage the composite CAD-CAM block surface. For the composite CAD-CAM block Shofu Block HC, the damage was so severe that silanization did not improve bond strength