Coculture of Osteoblasts and Endothelial Cells: Optimization of Culture Medium and Cell Ratio

Abstract

Vascularization strategies in cell-based bone tissue engineering depend on optimal culture conditions. The present study aimed to determine optimal cell culture medium and cell ratio for cocultures of human marrow stromal cells (HMSCs) and human umbilical vein endothelial cells (HUVECs) in view of both osteogenic and angiogenic outcome parameters upon two-dimensional and three-dimensional culture conditions. Cultures were performed in four different media: osteoblastic cell proliferation medium, osteogenic medium (OM), endothelial medium, and a 1:1 mixture of the latter two media. Mineralization within the cocultures was observed only in OM. Subsequent experiments in OM showed that alkaline phosphatase activity, mineralization, and CD31(+) staining were highest for cocultures at a 50:50 HMSC/HUVEC ratio. Therefore, the results from the present study show that a HMSC/HUVEC coculture ratio of 50:50 in OM is the best combination to obtain both osteogenic and angiogenic differentiation.

Fluorapatite Enhances Mineralization of Mesenchymal/Endothelial Cocultures

“…Their results showed that a HMSC/HUVEC coculture ratio of 1:1 appeared to be optimal. 26 However, most recently, a study did show that under dynamic culture conditions, coculture of human osteoblast-like cells with a low ratio of HUVECs on a copolymer scaffold significantly influenced the expression of osteogenic markers. 27 It would be interesting to explore, in the future, the effects of HMVEC seeding density, that is, the HMVEC:ASC ratio on the growth and differentiation of ASCs on the OR-FA and SSE surfaces.…”

Enhanced Osteogenesis in Cocultures with Human Mesenchymal Stem Cells and Endothelial Cells on Polymeric Microfiber Scaffolds

“…HUVECs were selected for this study because they are an established model of ECs and have been shown to stimulate osteogenesis in vitro. [20][21][22][23]27 A general hMSC expansion medium with osteogenic supplements was used because of its proven ability to support osteogensis in cocultures of HUVECs and hMSCs, 20 as opposed to other media formulations that have demonstrated greater utility in maintaining and promoting angiogenesis of ECs in coculture. 17,20,28 While the absence of angiogenic growth factors in osteogenic media limits the EC metabolic activity and proliferation, 20 it is still sufficient for manifestation of the angiogenic potential of HUVECs in cocultures with hMSCs.…”

“…[20][21][22][23]27 A general hMSC expansion medium with osteogenic supplements was used because of its proven ability to support osteogensis in cocultures of HUVECs and hMSCs, 20 as opposed to other media formulations that have demonstrated greater utility in maintaining and promoting angiogenesis of ECs in coculture. 17,20,28 While the absence of angiogenic growth factors in osteogenic media limits the EC metabolic activity and proliferation, 20 it is still sufficient for manifestation of the angiogenic potential of HUVECs in cocultures with hMSCs. 15,20,22 From previous studies, it is known that coculturing ECs with bone-forming cells promotes cellular proliferation in monolayer 21,23 as well as in 3D.…”

“…[15][16][17][18][19] Furthermore, previous studies have demonstrated that ECs are capable of enhancing the proliferation and osteogenic differentiation of MSCs. [20][21][22][23][24] Thus, cocultures of MSCs and ECs are currently being investigated for their ability to enhance bone formation and have shown that trophic regulation from ECs can provide necessary components for MSC osteogenic differentiation. 5,6 The addition of ECs to MSC or osteoblast cultures has been shown to enhance both proangiogenic and pro-osteogenic gene expression, stimulate the alkaline phosphatase (ALP) activity, and increase mineralization.…”

“…5,6 The addition of ECs to MSC or osteoblast cultures has been shown to enhance both proangiogenic and pro-osteogenic gene expression, stimulate the alkaline phosphatase (ALP) activity, and increase mineralization. [15][16][17][20][21][22][23][25][26][27] While numerous studies have evaluated such cocultures in two dimensions, data obtained from three-dimensional (3D) conditions are still very limited, as studies emphasizing the osteogenic outcome have primarily been performed in monolayer 19,21,23,24 or pellet cultures, 20,22,26 but not on porous scaffolds. Studies performed with cocultures of ECs and bone-forming cells on porous scaffolds have investigated primary osteoblasts 16,17,28 or an osteoblast cancer cell line, 27,28 and have focused on the survival of the ECs, 28 angiogenic gene expression, the development of scaffold vascularization with ECs 16,17,28 or the properties of the scaffold material, 27 but not on the osteogenic differentiation of boneforming cells (e.g., by quantifying bone-like matrix production and maturation).…”