Paracrine Effects Influenced by Cell Culture Medium and Consequences on Microvessel-Like Structures in Cocultures of Mesenchymal Stem Cells and Outgrowth Endothelial Cells

Abstract

Mesenchymal stem cells (MSC) from bone marrow and outgrowth endothelial cells (OEC) from peripheral blood are considered as attractive cell types for applications in regenerative medicine aiming to build up complex vascularized tissue-engineered constructs. MSC provide several advantages such as the potential to differentiate to osteoblasts and to support the neovascularization process by release of proangiogenic factors. On the other hand, the neovascularization process can be actively supported by OEC forming perfused vascular structures after co-implantation with other cell types. In this study the formation of angiogenic structures in vitro was investigated in cocultures of MSC and OEC, cultured either in the medium for osteogenic differentiation of MSC (ODM) or in the medium for OEC cultivation endothelial cell growth medium-2 (EGM2 Bullet Kit). After 2 weeks, cocultures in EGM2 formed more microvessel-like structures compared to cocultures in ODM as demonstrated by immunofluorescence staining for the endothelial marker CD31. Increased expression of CD31 and CD146 in quantitative real-time polymerase chain reaction as well as a higher percentage of CD31- and CD146-positive cells in flow cytometry indicated a beneficial influence of EGM2 on endothelial cell growth and function. In addition, the improved formation of vascular structures in EGM2 correlates with higher levels of the proangiogenic factor vascular endothelial growth factor and platelet-derived growth factor in the supernatant of cocultures as well as in monocultures of MSC when cultivated in EGM-2. Nevertheless, ODM was more suitable for the differentiation of MSC to osteoblastic lineages in the cocultures, whereas EGM2 favored factors involved in vessel stabilization by pericytes. In conclusion, this study highlights the importance of medium components for cell interaction triggering the formation of angiogenic structures.

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

“…23,25 However, no upregulation in collagen expression was found in the coculture of outgrowth ECs and MSCs. 15 To our knowledge, no other studies have reported increased GAG production in cocultures of MSCs and ECs in either 2D or 3D. GAGs have a variety of biological functions necessary for osteogenesis and cell-to-cell communications.…”

“…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. 27 However, since most of these findings were obtained using endothelial growth media, we evaluated the effect of 3D culture in osteogenic media.…”

“…Studies evaluating the use of ECs in MSC or osteoblast cultures have observed the formation of microvessels in the engineered construct. [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. [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).…”

Endothelialization of Acellular Porcine ECM with Chemical Modification

“…Results generated from both the AlamarBlue™ assay and the H&E staining were found to be consistent with each other, implying that an incubation time of 45 to 90 minutes, after seeding, should be applied. Healthy and functional blood vessel should be covered by a complete confluent mono-layer of endothelial cells [14], [15]. The cell layer after 45 minutes incubation time (Fig.…”