Osteogenic Differentiation of Mesenchymal Stem Cells by Mimicking the Cellular Niche of the Endochondral Template

Abstract

In vitro bone regeneration strategies that prime mesenchymal stem cells (MSCs) with chondrogenic factors, to mimic aspects of the endochondral ossification process, have been shown to promote mineralization and vascularization by MSCs both in vitro and when implanted in vivo. However, these approaches required the use of osteogenic supplements, namely dexamethasone, ascorbic acid, and β-glycerophosphate, none of which are endogenous mediators of bone formation in vivo. Rather MSCs, endothelial progenitor cells, and chondrocytes all reside in proximity within the cartilage template and might paracrineally regulate osteogenic differentiation. Thus, this study tests the hypothesis that an in vitro bone regeneration approach that mimics the cellular niche existing during endochondral ossification, through coculture of MSCs, endothelial cells, and chondrocytes, will obviate the need for extraneous osteogenic supplements and provide an alternative strategy to elicit osteogenic differentiation of MSCs and mineral production. The specific objectives of this study were to (1) mimic the cellular niche existing during endochondral ossification and (2) investigate whether osteogenic differentiation could be induced without the use of any external growth factors. To test the hypothesis, we evaluated the mineralization and vessel formation potential of (a) a novel methodology involving both chondrogenic priming and the coculture of human umbilical vein endothelial cells (HUVECs) and MSCs compared with (b) chondrogenic priming of MSCs alone, (c) addition of HUVECs to chondrogenically primed MSC aggregates, (d-f) the same experimental groups cultured in the presence of osteogenic supplements and (g) a noncoculture group cultured in the presence of osteogenic growth factors alone. Biochemical (DNA, alkaline phosphatase [ALP], calcium, CD31⁺, vascular endothelial growth factor [VEGF]), histological (alcian blue, alizarin red), and immunohistological (CD31⁺) analyses were conducted to investigate osteogenic differentiation and vascularization at various time points (1, 2, and 3 weeks). The coculture methodology enhanced both osteogenesis and vasculogenesis compared with osteogenic differentiation alone, whereas osteogenic supplements inhibited the osteogenesis and vascularization (ALP, calcium, and VEGF) induced through coculture alone. Taken together, these results suggest that chondrogenic and vascular priming can obviate the need for osteogenic supplements to induce osteogenesis of human MSCs in vitro, while allowing for the formation of rudimentary vessels.

FIG. 1.

DNA content/sample of all three experimental conditions cultured without osteogenic supplements (-OM) compared with the results from our previous study of the same groups cultured in the presence of osteogenic supplements (+OM). ^p < 0.05 versus CP21+HUVECs group, ^ap < 0.05 versus CP21-HUVECs.*p < 0.05, and ****p < 0.0001. (n = 6 samples per group per time point.) Error bars denote standard deviation. HUVECs, human umbilical vein endothelial cells. Color images available online at www.liebertpub.com/tea

FIG. 2.

Alcian blue staining of all three groups cultured with (+OM) and without osteogenic supplements (-OM) over the course of the experiment. Each of the images was imaged at a magnification of 20 × . Color images available online at www.liebertpub.com/tea

FIG. 3.

(A) ALP activity of all of the experimental groups cultured without osteogenic supplements compared with osteogenic group alone at 1, 2, and 3 weeks after the addition of HUVECs/MSCs. ^p < 0.05 versus CP21+HUVECs group, ^ap < 0.05 versus CP21-HUVECs, and ^bp < 0.05 versus Osteo alone. (B) ALP activity of all three experimental groups cultured without osteogenic supplements compared with the same groups cultured in the presence of osteogenic supplements and the osteogenic group alone. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (n = 6 samples per group per time point.) Error bars denote standard deviation. ALP, alkaline phosphatase; MSCs, mesenchymal stem cells. Color images available online at www.liebertpub.com/tea

FIG. 4.

(A) Calcium content of all of the experimental groups cultured without osteogenic supplements (-OM) compared with osteogenic group alone at 1, 2, and 3 weeks after the addition of HUVECs/MSCs. ^bp < 0.05 versus Osteo alone. (B) Calcium content of all three experimental groups cultured without osteogenic supplements compared with the same groups cultured in the presence of osteogenic supplements (+OM) and the osteogenic group alone. **p < 0.01, n = 6 samples per group per time point. Error bars denote standard deviation. Color images available online at www.liebertpub.com/tea

FIG. 5.

Alizarin red staining of the aggregates cultured without osteogenic supplements before addition of cells and all three groups cultured with (+OM) and without osteogenic supplements (-OM) at 1, 2, and 3 weeks after the addition of cells. Each of the images was imaged at a magnification of 20 × . Arrows denote mineralization nodules present within the cellular aggregates. Color images available online at www.liebertpub.com/tea

FIG. 6.

An ELISA of VEGF expression of all three experimental groups cultured without osteogenic supplements (-OM) compared with the same groups cultured in the presence of osteogenic supplements (+OM). ^p < 0.05 versus CP21+HUVECs group, ^ap < 0.05 versus CP21-HUVECs, **p < 0.01 and ****p < 0.0001, n = 6 samples per group per time point. Error bars denote standard deviation. ELISA, enzyme-linked immunosorbent assay; VEGF, vascular endothelial growth factor. Color images available online at www.liebertpub.com/tea

FIG. 7.

An ELISA of CD31 content of all of the experimental groups cultured without osteogenic supplements at 1, 2, and 3 weeks after the addition of HUVECs/MSCs. ^ap < 0.05 versus CP21-HUVECs, and *p < CP21+HUVECs:MSCs. (n = 6 samples per group per time point.) Error bars denote standard deviation. Color images available online at www.liebertpub.com/tea

FIG. 8.

(A) CD31 staining (green) of all three experimental groups cultured with (+OM) and without (-OM) osteogenic supplements over the course of the experiment. Each of the images was imaged at a magnification of 40× with either a nuclei counterstain DAPI (blue) or propidium iodide (red). Boxes denote examples of the rudimentary vessels present within the aggregates. (B) Quantitative analysis of cross-sectional area of the rudimentary vessels present in the aggregates. ***p < 0.001 and error bars denote standard deviation (n = 9). DAPI, 4′,6-diamidino-2-phenylindole. Color images available online at www.liebertpub.com/tea