Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche

Abstract

Mesenchymal stem cells (MSCs) within their native environment of the stem cell niche in bone receive biochemical stimuli from surrounding cells. These stimuli likely infl uence how MSCs differentiate to become bone precursors. The ability of MSCs to undergo osteogenic differentiation is well established in vitro; however, the role of the natural cues from bone's regulatory cells, osteocytes and osteoblasts in regulating the osteogenic differentiation of MSCs in vivo are unclear. In this study we delineate the role of biochemical signalling from osteocytes and osteoblasts, using conditioned media and co-culture experiments, to understand how they direct osteogenic differentiation of MSCs. Furthermore, the synergistic relationship between osteocytes and osteoblasts is examined by transwell co-culturing of MSCs with both simultaneously. Osteogenic differentiation of MSCs was quantified by monitoring alkaline phosphatase (ALP) activity, calcium deposition and cell number. Intracellular ALP was found to peak earlier and there was greater calcium deposition when MSCs were co-cultured with osteocytes rather than osteoblasts, suggesting that osteocytes are more infl uential than osteoblasts in stimulating osteogenesis in MSCs. Osteoblasts initially stimulated an increase in the number of MSCs, but ultimately regulated MSC differentiation down the same pathway. Our novel coculture system confi rmed a synergistic relationship between osteocytes and osteoblasts in producing biochemical signals to stimulate the osteogenic differentiation of MSCs. This study provides important insights into the mechanisms at work within the native stem cell niche to stimulate osteogenic differentiation and outlines a possible role for the use of co-culture or conditioned media methodologies for tissue engineering applications.

Bone tissue engineering by using a combination of polymer/Bioglass composites with human adipose-derived stem cells

“…There were no significant difference for the cell attachment and spreading on the different scaffold groups, which indicated that the additional Bioglass ® didn't effect the cell attachment. However, the 40 wt% Bioglass ® constructs appeared to significantly enhance human ADSCs alkaline phosphatase activity compared with the 5 wt% Bioglass ® and PDLLA only constructs after 4 weeks, suggesting stimulated cell osteogenic differentiation towards the osteogenic lineage as ALP activity has been extensively used as one of the indicators for stem cell osteogenic differentiation (Birmingham et al 2012;Mauney et al 2004). After 6 weeks in vitro culture, Alcian blue/Sirius red staining revealed that extensive collagenous matrix was produced along with the increase of Bioglass ® concentration.…”

Characterization of the growth modulatory activities of osteoblast conditioned media on cord blood progenitor cells

“…The OCM HSPC expansion protocol is based on the differentiation of MSC into immature M-OST after a 6 day osteogenic differentiation period induced in standard osteogenic medium (Birmingham et al 2012). Non-filtered centrifuged supernatant is then frozen without cryoprotectant until use (Dumont et al 2014).…”

Coculture of Spheroids/2D Cell Layers Using a Miniaturized Patterned Platform as a Versatile Method to Produce Scaffold‐Free Tissue Engineering Building Blocks

“…Results reported by Birmingham et al may shed light into the nonstatistically relevant difference observed in the ALP expression of hASCs, while cultured with Saos-2: the results showed that, in 2D cultures, the transwell-mediated coculture of osteoblasts with mesenchymal stem cells leads to an initial peak in cell proliferation, and a delayed induction of osteogenic differentiation. [30] Another plausible explanation for the results relies in the recently reported ability of tumorogenic cells, namely Saos-2, to prolong hASCs stemness while cultured in direct 2D coculture, as compared to ASCs monocultures. [36] For HUVEC coculture, ALP activity was reduced for half of the value detected on the control condition (M199), as shown in Figure 1c.…”

“…Both are known to drive an increased expression of this marker in early time points. [30][31][32][33][34][35] Double stranded DNA (dsDNA) quantification showed that the stem cell spheroids produced in different coculture procedures presented approximately three times more dsDNA than the control conditions (cultured as monocultures in either α-MEM or M199) ( Figure 1c). Similar results were previously reported for other indirect coculture methods, where stem cell proliferation was increased via coculture with osteoblast cells [30,31] and HUVEC cells.…”

“…[30][31][32][33][34][35] Double stranded DNA (dsDNA) quantification showed that the stem cell spheroids produced in different coculture procedures presented approximately three times more dsDNA than the control conditions (cultured as monocultures in either α-MEM or M199) ( Figure 1c). Similar results were previously reported for other indirect coculture methods, where stem cell proliferation was increased via coculture with osteoblast cells [30,31] and HUVEC cells. [33,34] However, these differences detected on dsDNA amount did not originate significant differences on spheroid diameters, suggesting that coculture conditions promoted the formation of more compact spheroids.…”