Biomimetic mineralization using matrix vesicle nanofragments

Abstract

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.

Re-Evaluation of Initial Bone Mineralization from an Engineering Perspective

“…For a minute analysis of the mineralization site, the trimmed samples were embedded in resin and observed by a scanning electron microscope (SEM) using a back-scattered electron detector and transmission electron microscope (TEM). 28 As a result, we found that the earliest mineralization proceeded through three stages: The initially highly compacted cells, demonstrated by the expression of intercellular Connexin 43 gap junctions, lose cell-cell contact. Concomitant with the decrease in the expression of Connexin 43, the expression of integrins and the secretion of collagen fibers are observed at the initial mineralization site.…”

“…The initial size of the minerals was the same as those of MVs, but after growth, the minerals showed a diameter of less than 1 mm in diameter, which has been previously named as ''calcospherites.'' A large amount of these minerals are formed in the vicinity of the collagen fibrils, grow and become densely packed, and eventually, go through a maturation stage 28 (Fig. 4).…”

Matrix Vesicles: Role in Bone Mineralization and Potential Use as Therapeutics

“…Therefore, to isolate MtVs from tissue, a digestion step with collagenase should be performed prior to MtV isolation [ 107 , 132 , 133 ]. MtVs can also be isolated from cell culture samples [ 17 , 102 , 144 ]. Some of the literature reported that a collagenase digestion step was added to release MtVs or mineralizing-EVs from the cell culture matrix [ 102 , 134 , 145 , 146 , 147 ].…”

“…It also has been demonstrated that AFM operated in peak force quantitative nanomechanical property mapping (AFM-PFQNM) was able to measure the nanomechanical and morphological properties of individual MtVs under both mineralizing (with addition of calcium ions) and non-mineralizing fluid conditions [ 152 ]. TEM is a common method to characterize the physical properties of MtVs, including the size and the crystals present inside MtVs [ 17 , 23 , 134 , 144 , 149 ]. When the characterization of certain surface proteins on MtVs is needed, immunogold labeling of MtVs prior to TEM can also be performed.…”

Cellular Fragments as Biomaterial for Rapid In Vitro Bone-Like Tissue Synthesis

“…Cell-free approaches for rapid bone-like mineral formation have also been attempted, such as those based on the isolation of osteoblast-secreted MVs [21,22]. Nevertheless, the application of the isolated MVs in bone tissue engineering still faced hindrances, as previous studies were unable to induce mineral formation in vitro using MVs [21,22], or it still required long term culture for mineralization [20].…”