In vitro reproduction of endochondral ossification using a 3D mesenchymal stem cell construct

Abstract

Endochondral ossification is one of the essential bone development processes in vertebrates. Although researchers from a variety of fields, including cellular/molecular biology, chemistry, and materials science, have worked to gain a better understanding of the tissue development, integration of findings from these different fields remains a major challenge. An in vitro model system that reproduces endochondral ossification would be a valuable tool for overcoming this problem, because an in vitro standardized model system can be easily accessed by researchers from different fields. Here, we fabricated a large 3D mesenchymal stem cell (MSC) construct with a ball-like morphology, which is termed a cell ball, and cultured it under a hypoxia condition, since hypoxia causes chondrogenic differentiation of MSCs in primordial cartilage, which is crucial for endochondral ossification. Region-specific chondrogenic differentiation of MSCs and mineralization within the cartilage tissue were observed in the cell ball. The precipitated minerals were detected as hydroxyapatite. Consequently, a 3D construct consisting of mineralized tissue surrounded by cartilage tissue was obtained. Moreover, the angiogenic activity of this synthesized tissue changed depending on the chondrogenic phenotype remains in the tissue, which is similar to what happens in the ossification process. Thus, this MSC cell ball system clearly reproduced the initial stage of endochondral ossification in vitro. This system is a promising tool for use as an in vitro model for investigating bone tissue development.

Effects of substrate stiffness and cell-cell contact on mesenchymal stem cell differentiation

“…Cells at an earlier time point stained negative for Alcian Blue, but were positive at a later time point, indicating that cells with chondrogenic characteristics may have developed as well (Supp Fig. 3), as has been observed in some MSC cultures [31]. Under media conditioned with osteogenic supplements, single cells, doublets, and triplets showed no significant differences in ALP expression when differentiated on 0.51 kPa and on 3.7 kPa substrates, and all exhibited low levels of ALP.…”

Comparative Analysis of Mouse-Induced Pluripotent Stem Cells and Mesenchymal Stem Cells During Osteogenic Differentiation In Vitro

“…This iPSC clone (clone mGF-iPS-3F-3) presented germline transmission, which is proof that the generated iPSCs were of high quality [18]. The clonal MSCs (mBMSC-4), which we established from mouse femur bone marrow, were multipotent, as demonstrated by their ability to differentiate specifically into osteoblast, adipocyte, chondrocyte, and myoblast lineages [19,20].…”

Identifying the Cellular Mechanisms Leading to Heterotopic Ossification

“…MSCs have frequently been shown to form endochondral bone when cultured under appropriate conditions (e.g. under hypoxia and/or in the presence of TGF-β) [28,29]. Furthermore, in some instances the application of MSCs in vivo following an initial period of in vitro chondrogenic differentiation has led to the formation of endochondral bone that contained a central bone marrow cavity [30].…”