Abstract: BACKGROUND: The development of tissue engineering has provided a new method for the clinical treatment of bone defects, but the problems of slow formation and slow vascularization of tissue engineered bone have always existed. Previous studies have shown that the combined culture system of vascular endothelial cells and adipose stem cells has better ability to repair bone defects than the single type of cells. Fibroblasts have excellent proliferation characteristics, the ability to secrete and synthesize collagen, contain a variety of regulatory factors, can differentiate into osteogenesis, and have the potential to become excellent seed cells to participate in the construction of tissue engineered bone.
OBJECTIVE: To investigate the effects of combined culture of fibroblasts, vascular endothelial cells, and adipose stem cells on the proliferation and osteogenic differentiation of adipose stem cells.
METHODS:  (1) The cells were divided into four groups: adipose stem cell group, adipose stem cell + vascular endothelial cell co-culture group, adipose stem cell + fibroblast co-culture group, adipose stem cell + vascular endothelial cell + fibroblast co-culture group. The morphological changes of the cells were observed under an inverted microscope. (2) At 1, 3, 5, 7, and 9 days of co-culture, the proliferation of adipose stem cells in each group was detected by CCK-8 assay and the growth curve was plotted. (3) Alizarin red staining and alkaline phosphatase staining were performed on adipose stem cells in each group at 7, 14, 21, and 28 days of co-culture. (4) At 3 weeks of co-culture, the expression level of bone morphogenetic protein 2 in adipose stem cells in each group was detected by western blot assay.  
RESULTS AND CONCLUSION: (1) After 14 days of culture, in the adipose stem cell + vascular endothelial cell + fibroblast co-culture group, some cells fused into clumps and distributed in nests, while in the adipose stem cell group, the cell morphology was single and no cell clusters were found. (2) The morphology of the cell growth curve was basically the same in each group, and the absorbance value increased gradually. The absorbance value of the adipose stem cell + vascular endothelial cell + fibroblast co-culture group was highest, followed by the adipose stem cell + vascular endothelial cell co-culture group, followed by the adipose stem cell + fibroblast co-culture group. (3) At 28 days of co-culture, all the cells in each group showed red positive cells, and the most cells in the adipose stem cell + vascular endothelial cell + fibroblast co-culture group showed red focus. The adipose stem cell group was the least. At 28 days of co-culture, each cell had red positive particles. The number of particles was most in the adipose stem cell + vascular endothelial cell + fibroblast group and adipose stem cell + fibroblast co-culture group, and that in the adipose stem cell group was least. (4) Bone morphogenetic protein 2 was expressed in all cells of each group, and it was more obvious in the adipose stem cell + fibroblast co-culture group and the adipose stem cell + vascular endothelial cell + fibroblast co-culture group. (5) Results confirmed that under the condition of co-culture in vitro, fibroblasts promoted osteogenic differentiation of adipose stem cells, which is more strongly than vascular endothelial cells, but the promotion of proliferation was not as good as that of vascular endothelial cells. The co-culture system of fibroblasts combined with vascular endothelial cells and adipose stem cells has the strongest ability to promote the proliferation of adipose stem cells and the rapid and efficient differentiation into osteoblasts.

Key words: stem cells, co-culture, adipose stem cells, vascular endothelial cells, fibroblasts, bone tissue engineering