In vitro and in vivo effects of rat kidney vascular endothelial cells on osteogenesis of rat bone marrow mesenchymal stem cells growing on polylactide-glycoli acid (PLGA) scaffolds

Abstract

It is well established that vascularization is critical for osteogenesis. However, adequate vascularization also remains one of the major challenges in tissue engineering of bone. This problem is further accentuated in regeneration of large volume of tissue. Although a complex process, vascularization involves reciprocal regulation and functional interaction between endothelial and osteoblast-like cells during osteogenesis. This prompted us to investigate the possibility of producing bone tissue both in vitro and ectopically in vivo using vascular endothelial cells because we hypothesized that the direct contact or interaction between vascular endothelial cells and bone marrow mesenchymal stem cells are of benefit to osteogenesis in vitro and in vivo. For that purpose we co-cultured rat bone marrow mesenchymal stem cells (MSC) and kidney vascular endothelial cells (VEC) with polylactide-glycolic acid scaffolds. In vitro experiments using alkaline phosphatase and osteocalcin assays demonstrated the proliferation and differentiation of MSC into osteoblast-like cells, especially the direct contact between VEC and MSC. In addition, histochemical analysis with CD31 and von-Willebrand factor staining showed that VEC retained their endothelial characteristics. In vivo implantation of MSC and VEC co-cultures into rat's muscle resulted in prevascular network-like structure established by the VEC in the PLGA. These structures developed into vascularized tissue, and increased the amount and size of the new bone compared to the control group (p < 0.05). These results suggest that the vascular endothelial cells could efficiently stimulate the in vitro proliferation and differentiation of osteoblast-like cells and promote osteogenesis in vivo by the direct contact or interaction with the MSC. This technique for optimal regeneration of bone should be further investigated.

Osteogenic Differentiation of Mesenchymal Stem Cells by Mimicking the Cellular Niche of the Endochondral Template

“…However, unlike previous studies, there was also an increase in late osteogenic markers (calcium) compared with the control group (osteogenic). Importantly, and in contrast to previous studies, 37,[40][41][42][43][44][46][47][48][49]69 this increase in ALP and calcium content was achieved in a 3D scaffoldless construct, without the use of any osteogenic supplements. When compared with the calcium content results, 49 the increase in calcium content in the coculture group with added MSCs/ HUVECs, but no osteogenic supplements, was significantly higher than in the same group cultured in osteogenic supplements as reported by Freeman et al 49 There was significantly higher calcium content in the group cultured with osteogenic supplements than in the group without Quantitative analysis of cross-sectional area of the rudimentary vessels present in the aggregates.…”

“…Moreover, similar to previous studies, 37,[40][41][42][43][44][46][47][48][49]69 the coculture group with both HUVECs and MSCs added had significantly higher ALP production than the control groups (osteogenic and noncoculture group) after 3 weeks of coculture. However, unlike previous studies, there was also an increase in late osteogenic markers (calcium) compared with the control group (osteogenic).…”

“…[4][5][6][7][8]53,77,78 Previous studies have investigated whether prevascularizing 3D constructs in vitro would eradicate this limitation, through the coculture of endothelial stem cells with MSCs [40][41][42][43][79][80][81][82][83] or osteoblasts, 45,84,85 and found that prevascular networks are formed both in vitro and in vivo. Our previous study previously found that rudimentary vessels were formed in vitro when both MSCs and HUVECs were added to the already formed cartilage template.…”

“…Previous in vitro studies have shown that direct coculture of MSCs or osteoblasts with endothelial cells can upregulate production of the early osteogenic marker ALP, [36][37][38][39] without the presence of osteogenic supplements. Other studies have investigated whether coculture of MSCs and endothelial cells can increase ALP production in three-dimensional (3D) polymer scaffolds 40,41 or 3D cellular aggregates, [42][43][44][45] but the majority of these studies were conducted in the presence of osteogenic growth supplements. [42][43][44][45] The osteogenic potential of MSC/chondrocyte or osteoblast/ chondrocyte cocultures has been variable and inconclusive in both two-dimensional (2D) and 3D cultures.…”

Indirect co-culture with tenocytes promotes proliferation and mRNA expression of tendon/ligament related genes in rat bone marrow mesenchymal stem cells

“…It has been illustrated that growth factors such as FGF-2, FGF-4, BMP-2, EGF, PDGF, promoted the proliferation of in vitro cultured MSCs (Martin et al 1997;Liu et al 2009;Choi et al 2008;Tamama et al 2006). Meanwhile, when co-cultured with ligament cells (Mizuno et al 2008) or vascular endothelial cells (Sun et al 2007), the proliferation of MSCs was elevated which was believed due to the humoral factors released from the co-culture cells. Our previous researches also elucidated cyclic mechanical stretch enhanced the proliferation of rat and human bone marrow derived MSCs (Song et al 2007a, b).…”