Collagen hydrogel scaffold promotes mesenchymal stem cell and endothelial cell coculture for bone tissue engineering

Abstract

The generation of functional, vascularized tissues is a key challenge for the field of tissue engineering. Before clinical implantations of such tissue engineered bone constructs can succeed, tactics to promote neovascularization need to be strengthened. We have previously demonstrated that the tubular perfusion system (TPS) bioreactor is an effective culturing method to augment osteogenic differentiation and maintain viability of human mesenchymal stem cells (hMSC). Here, we devised a strategy to address the need for a functional microvasculature by designing an in vitro coculture system that simultaneously cultures osteogenic differentiating hMSCs with endothelial cells (ECs). We utilized the TPS bioreactor as a dynamic coculture environment, which we hypothesize will encourage prevascularization of endothelial cells and early formation of bone tissue and could aid in anastomosis of the graft with the host vasculature after patient implantation. To evaluate the effect of different natural scaffolds for this coculture system, the cells were encapsulated in alginate and/or collagen hydrogel scaffolds. We discovered the necessity of cell-to-cell proximity between the two cell types as well as preference for the natural cell binding capabilities of hydrogels like collagen. We discovered increased osteogenic and angiogenic potential as seen by amplified gene and protein expression of ALP, BMP-2, VEGF, and PECAM. The TPS bioreactor further augmented these expressions, indicating a synergistic effect between coculture and applied shear stress. The development of this dynamic coculture platform for the prevascularization of engineered bone, emphasizing the importance of the construct microenvironments and will advance the clinical use of tissue engineered constructs. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1123-1131, 2017.

Keywords: TPS bioreactor; bone regeneration; coculture; endothelial cells; hydrogel scaffold; mesenchymal stem cells.

Figure 1

Coculture in Alginate and Collagen Scaffolds. A) Experimental setup depicts a 7 day static preculture of hMSCs encapsulated in alginate scaffolds (labeled Day -7), followed by a 14 day dynamic or static coculture with HUVECs encapsulated in collagen scaffolds (Day 0 - Day 14). B) BMP-2 immunostaining demonstrates increase in BMP-2 production (brown) in hMSCs (dark blue nucleus). C) BMP-2 mRNA expressions in hMSCs significantly increases over 14 days in dynamic culture but stays constant static culture. D) VEGF mRNA expressions in HUVECs significantly increases over 14 days in dynamic culture and shows an increase on day 7 in static before decreasing back close to basal levels. The symbol ‘*’ indicates statistical significance within groups at a timepoint (p<0.05).

Figure 2

Cell Adhesion and Osteogenic Differentiation in Collagen Scaffolds. A) Experimental setup for cell adhesion study on alginate and collagen substrates, and TCPS as positive control. B) Experimental setup to investigate the effect of collagen encapsulation on osteogenic differentiation compared to TCPS as a control. C) Fluorescence images of hMSCs or HUVECs seeded on alginate, collagen, or TCPS substrates, taken at 2.5× magnification. D) Quantification of fluorescence signal read via a spectrophotometer at excitation of 494nm and emission of 517nm for both hMSCs and HUVECs. Units are listed as RFU (relative fluorescence units). E) Fluorescence images of hMSCs labeled with live (green) and dead (red) stain on TCPS or encapsulated in 3D collagen scaffolds. F) Gene expression of osteocalcin (OCN) mRNA in hMSCs over 14 days. Production was statistically greater in collagen scaffolds compared to the TCPS control. G) Gene expression of BMP-2 mRNA in hMSCs shows significantly increased expression in 3D collagen compared to TCPS. The symbol ‘*’ indicates statistical significance within groups at a timepoint (p<0.05).

Figure 3

Effect of Dynamic Culture on hMSCs and HUVEC coculture. A) Experimental setup of cell encapsulation groups: hMSCs in collagen, HUVECs in collagen, or hMSCs and HUVECs in collagen. Cell-seeded scaffolds were cultured in static well plates or in the TPS bioreactor under dynamic flow conditions. B) E) Fluorescence viability images of hMSCs labeled with live (green) and dead (red) stain on 3D collagen scaffolds after 1, 3, or 7 days of static or dynamic culture. Scale bar represents 100 µm. C) Gene expression of ALP and BMP-2 in hMSCs monocultured in static (solid bars) or dynamic (striped bars). Overall, dynamic coculture resulted in the highest expression of ALP and BMP-2 expression by day 7. D) Gene expression of PECAM and VEGF in HUVEC monocultured in static (solid bars) or dynamic (striped bars). Overall, dynamic coculture resulted in the highest expression of PECAM and VEGF expression by day 7. E) Gene expression of ALP, BMP-2, PECAM, and VEGF in hMSC and HUVEC cocultured in static (solid bars) or dynamic (striped bars). Overall, the synergistic effect of coculture and dynamic coculture resulted in the highest expression of all four markers by day 7. The symbol ‘*’ indicates statistical significance within groups at a timepoint (p<0.05). F) Immunofluorescence staining of hMSCs, HUVECs, and coculture in static and dynamic. hMSCs were stained for BMP-2 (pink/red), counterstained with DAPI (blue). HUVECs were stained for CD31 (green), VEGF (red), and counterstained with DAPI (blue). Cocultured samples were stained for BMP-2 (pink/red), CD31 (green), and counterstained with DAPI (blue). Images were taken at 40× with an additional 2× zoom (right panel).