Co-Culture of Osteoblasts and Endothelial Cells on a Microfiber Scaffold to Construct Bone-Like Tissue with Vascular Networks.

Kouki Inomata,Michiyo Honda

doi:10.3390/ma12182869

Abstract

Bone is based on an elaborate system of mineralization and vascularization. In hard tissue engineering, diverse biomaterials compatible with osteogenesis and angiogenesis have been developed. In the present study, to examine the processes of osteogenesis and angiogenesis, osteoblast-like MG-63 cells were co-cultured with human umbilical vein endothelial cells (HUVECs) on a microfiber scaffold. The percentage of adherent cells on the scaffold was more than 60% compared to the culture plate, regardless of the cell type and culture conditions. Cell viability under both monoculture and co-culture conditions was constantly sustained. During the culture periods, the cells were spread along the fibers and extended pseudopodium-like structures on the microfibers three-dimensionally. Compared to the monoculture results, the alkaline phosphatase activity of the co-culture increased 3–6 fold, whereas the vascular endothelial cell growth factor secretion significantly decreased. Immunofluorescent staining of CD31 showed that HUVECs were well spread along the fibers and formed microcapillary-structures. These results suggest that the activation of HUVECs by co-culture with MG-63 could enhance osteoblastic differentiation in the microfiber scaffold, which mimics the microenvironment of the extracellular matrix. This approach can be effective for the construction of tissue-engineered bone with vascular networks.

Highlights

The reconstruction of structural and functional tissues is highly challenging
Fetal bovine serum (FBS), penicillin, streptomycin, phosphate-buffered saline (PBS, sodium chloride, potassium chloride, disodium hydrogenphosphate, and potassium dihydrogen phosphate), dimethyl sulfoxide (DMSO), 4% paraformaldehyde in PBS, Triton X-100, bovine serum albumin (BSA), 25% glutaraldehyde solution, LabAssayTM alkaline phosphatase (ALP), and protein assay Bradford reagent were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan)
The the concentration of vascular endothelial endothelial growth growth factor factor (VEGF) in the co-culture of the MG-63 cells and human umbilical vein endothelial cells (HUVECs) significantly decreased concentration of VEGF in the co-culture of the MG-63 cells and HUVECs significantly decreased compared with the monoculture. These results suggested that the HUVECs might consume the VEGF, compared with the monoculture

Summary

Introduction

The reconstruction of structural and functional tissues is highly challenging. The ability of excessively damaged organs and tissues to regenerate themselves is low. Since organs and tissues have different structures and functions, treatment should be selected appropriately in consideration of their biochemical properties and anatomical features. An iliac crest, which is used for autologous bone grafting to treat bone fractures, could induce vascularization around the implanted area to promote immediate bone formation [1,2]. This method has many limitations, so new approaches should be developed

Methods

Results

Discussion

Conclusion