Human endothelial and foetal femur-derived stem cell co-cultures modulate osteogenesis and angiogenesis

Abstract

BackgroundA dynamic vasculature is a prerequisite for bone formation where the interaction of bone cells and endothelial cells is essential for both the development and the healing process of bone. Enhanced understanding of the specific mediators involved in bone cell and endothelial cell interactions offers new avenues for skeletal regenerative applications. This study has investigated the osteogenic and angiogenic potential of co-cultures of human foetal diaphyseal or epiphyseal cells with human umbilical vein endothelial cells (HUVEC) in the presence and absence of vascular endothelial growth factor (VEGF) supplementation.MethodsEarly osteogenic activities of the co-cultures (±VEGF) were assessed by alkaline phosphatase (ALP) activity. Osteogenic and angiogenic gene expression was measured using quantitative polymerase chain reaction. An ex vivo organotypic embryonic chick (E11) femur culture model was used to determine the osteogenic effects of VEGF as determined using micro-computed tomography (μCT) and Alcian blue/Sirius red histochemistry and immunocytochemistry for expression of CD31.ResultsALP activity and gene expression of ALP and Type-1 collagen was enhanced in foetal skeletal/HUVECs co-cultures. In foetal diaphyseal/HUVECs co-cultures, VEGF reduced the levels of ALP activity and displayed a negligible effect on von Willebrand factor (vWF) and VEGF gene expression. In contrast, VEGF supplementation was observed to significantly increase FLT-1 and KDR gene expression in co-cultures with modulation of expression enhanced, compared to VEGF skeletal monocultures. In the organotypic chick model, addition of VEGF significantly enhanced bone formation, which coincided with elevated levels of CD31-positive cells in the mid-diaphyseal region of the femurs.ConclusionThese studies demonstrate a differential skeletal response of early foetal skeletal cells, when co-cultured with endothelial cells and the potential of co-culture models for bone repair. The differential effect of VEGF supplementation on markers of angiogenesis and osteogenesis in co-cultures and organ cultures, demonstrate the importance of the intricate temporal coordination of osteogenic and angiogenic processes during bone formation and implications therein for effective approaches to bone regenerative therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0270-3) contains supplementary material, which is available to authorized users.

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

“…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.…”

“…[35] In contrast, direct coculture of HUVEC cells with stem cells has been established as enhancing its osteoblast phenotypic markers, resulting, for example, in an increase of ALP expression. [34,35] Villars and co-workers highlighted these differences by comparing direct and indirect cocultures of HUVEC and BMSC cells. [33] Using an equivalent clean platform to the one employed on spheroid production, by aligning and touching with the spheroid production platform on the empty platform, the spheroids could be transferred -see the Experimental Section; and Figure S1, Supporting Information.…”

“…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.…”

3D human bone marrow stromal and endothelial cell spheres promote bone healing in an osteogenic niche

“…The most significant increase in bone-forming parameters was observed in defects implanted with HUVEC and HBMSC monoculture pellets ( P < 0.001) compared with sham controls. The current studies used previously acquired knowledge from 2D coculture studies (18) of human skeletal and endothelial cells and enhanced stimulation of osteogenic markers ALP and type I collagen while recognizing the typical limitation of a 2D coculture and the absence of spatial dynamics and cell motility.…”

The effect of modified electrospun PCL‐nHA‐nZnO scaffolds on osteogenesis and angiogenesis

“…in vitro approaches for prevascularization in tissue‐constructs consist of cocultured cell seeding on the scaffolds, spheroids generation, and cell sheet technology (Laschke & Menger, ). In recent years, several studies have focused on coculturing of osteogenic and angiogenic cells, such as coculturing of fetal femur‐derived stem cells and human umbilical vein endothelial cells (HUVECs) (Inglis, Christensen, Wilson, Kanczler, & Oreffo, ), rat bone marrow stem cells and rat aortic endothelial cells (Gurel Pekozer et al, ), HUVECs and osteoblast‐like cells (MG‐63 cells; Shi, Andrukhov, Berner, Schedle, & Rausch‐Fan, ), human bone marrow stem cells (hBMSCs) and HUVECs (Kang, Kim, Fahrenholtz, Khademhosseini, & Yang, ), and also hBMSCs and human progenitor‐derived endothelial cells (PDECs; Guerrero et al, ). Almost all of them showed upregulation of osteogenesis and angiogenesis in coculturing conditions.…”