Contribution of Endothelial Cells to Human Bone-Derived Cells Expansion in Coculture

Abstract

Creating a functional vascularized bone tissue remains one of the main goals of bone tissue engineering. Recently, a growing interest in the crosstalk between endothelial cells (EC) and osteoblasts (OB), the two main players in a new bone formation, has been observed. However, only a few reports have addressed a mutual influence of OB and EC on cell proliferation. Our study focuses on this issue by investigating cocultures of human bone-derived cells (HBDC) and human umbilical vein endothelial cells (HUVEC). Three various proportions of cells have been used that is, HBDC:HUVEC 1:1, 1:4, and 4:1 and the cocultures were investigated on day 1, 4, and 7, while HUVEC and HBDC monocultures served as reference. We have detected enhanced alkaline phosphatase (ALP) activity in a direct HBDC-HUVEC coculture. This effect was not observed when cells were separated by an insert, which is consistent with other reports on various OB-EC lineages. The appearance of gap-junctions in coculture was confirmed by a positive staining for connexin 43. The number of cells of both phenotypes has been determined by flow cytometry: CD-31-positive cells have been considered EC, while CD-31-negative have been counted as OB. We have observed an over 14-fold increase in OB number after a week in the 1:4 HBDC:HUVEC coculture as compared with less than fourfold in monoculture. The increase in HBDC number in 1:1 coculture has been less pronounced and has reached the value of about sevenfold. These results correspond well with the cell proliferation rate, which has been measured by 5-bromo-2¢-deoxyuridine incorporation. Moreover, at day 7 EC have been still present in the coculture, which is inconsistent with some other reports. Real-time polymerase chain reaction analysis has revealed the upregulation of ALP and collagen type I genes, but not osteocalcin gene, in all the cocultures grown without pro-osteogenic additives. Our study indicates that HUVEC significantly promote HBDC expansion and upregulate collagen I gene expression in these cells. We believe that these findings have application potency in bone tissue engineering.

Dual Role of Mesenchymal Stem Cells Allows for Microvascularized Bone Tissue‐Like Environments in PEG Hydrogels

“…While monocultures of hBM-MSCs without growth factors did not show ALP activity (Figure 3 a), we observed a basal activity of ALP in cocultures without growth factors (Figure 4 a). It has been shown that HUVECs can produce BMP-2 [ 49,50 ] and induce ALP activity in BMMSCs, [ 49,51 ] adipose-derived stem cells, [ 50 ] and osteoblasts [ 52 ] when cocultivated. However, in two of these studies it was also shown that HUVECs themselves have weak but detectable amounts of ALP.…”

Overcoming physical constraints in bone engineering: ‘the importance of being vascularized’

“…Two-dimensional co-culture approaches (by the use of two chambers-based systems or conditioned media) are providing increasing mechanistic details on the paracrine cross-talk between ECs and osteoblast cell lines Leszczynska et al, 2013;Pedersen et al, 2013a;Santos et al, 2009;Shi et al, 2014;Tao et al, 2009). Outgrowth of endothelial cells and primary osteoblasts in vitro enhances both angiogenesis and osteogenesis (Herzog, Dohle, Bischoff, & Kirkpatrick, 2014).…”

“…The feasibility of co-culture or co-implantation strategies has been so far tested in several studies, focusing on the quality of the engineered bone substitute (Dariima, Jin, Lee, Wall, & Kim, 2013;Fu et al, 2014;Kokemüller et al, 2014;Leszczynska et al, 2013;R P Pirraco et al, 2013;Rogério P Pirraco et al, 2014). Interestingly, mineralization can be achieved without osteogenic media or additional growth factors through co-cultures of normal human primary osteoblats and Human umbilical vein ECs (HUVECs) mainly by recurring to the manipulation of the coculture ratio (Shah, Wenke, & Agrawal, 2014).…”

Cell interactions between human progenitor-derived endothelial cells and human mesenchymal stem cells in a three-dimensional macroporous polysaccharide-based scaffold promote osteogenesis

“…In 3-D matrices, a number of studies using co-cultures of two lineages have been published, with cell lines from different sources (human and murine), including differentiated cells mixed with undifferentiated cells (MSCs or progenitors). Among the cell culture models used to develop these hybrid constructs, human umbilical vein endothelial cells (HUVEC) [22], human outgrowth endothelial cells (OEC) [23], human microcapillary endothelial cells [24] and endothelial progenitor cells from cord blood have been used for co-cultures with human bone-derived cells [25], osteoblast-like MG63 cells [26], primary human osteoblast cells [27], human osteoprogenitor cells from bone marrow [28], MSCs from adipose tissue [29] or bone marrow [30]. However, combining differentiated cells with undifferentiated cells arising from different sources (mouse, human) in co-culture assay does not mimic the physiological situation that occurs in a clinical situation of bone repair.…”