Contribution of endothelial cells to human bone-derived cells expansion in coculture.

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.