For the repair of critical-sized bone defect, the difficulties are mainly focused on the formation of vascularized bone, especially the center of the defect. To construct bone tissue accompanying vascular system, our team co-cultured ASCs and EPCs to establish a dual stem cell system. In this present study, it was found that the ASCs/EPCs co-culture system can enhance the expression of osteogenic and angiogenic genes in vitro, and furthermore, by supporting vascularized bone regeneration, it can significantly accelerate bone healing of critical-sized bone defects in vivo.
EPCs are precursor cells of vascular endothelial cells, which have the ability to proliferate, migrate and differentiate into cells arranged along the lumen of blood vessels and can be isolated from peripheral blood and spleen. ASCs also have high proliferative growth characteristics and multi-differential potential, can be extracted from autologous subcutaneous fat, and have a wide range of tissue sources. But to date, specific markers for each cell type are still lacking. A number of surface proteins have been used to identify rat adipose stem cells, including CD73, CD90, CD105, CD44, etc[17–20]. Here, CD73 and CD90 are used as positive markers to identify ASCs. By using hematopoietic stem cell (HSCs) marker CD45 and EPCs marker CD34 to identify ASCs free of HSCs and EPCs, our results demonstrated that ASCs expressed a cell-surface protein profile positive for CD73 and CD90 and negative for CD45 and CD34. For EPCs, we detected the cell markers CD34 and CD133 (AC133) , which are highly expressed in EPCs, but not expressed after the EPCs differentiated into mature vascular endothelial cells. Meanwhile, we also detected CD31, which is not expressed in EPCs, but highly expressed in mature endothelial cells, as well as, CD11b, which is expressed in monocytes, but not in EPCs[22–25]. Our results showed that EPCs expressed a surface protein profile positive for CD133 and CD34 and negative for CD11b and CD31. These results confirmed the ASCs and EPC phenotypes, suggesting that the above markers can be used for the identification of ASCs and EPCs.
To determine the effect of these two kinds of cells in osteogenic and angiogenic differentiation, ASCs and EPCs were co-cultured in vitro, and then the osteogenesis-related genes (OCN, Col I and BMP2) and angiogenesis-related genes (VEGF, cdh5 and vWF) were analyzed by real-time RT-PCR. The results showed that the ASCs/EPCs co-culture system can increase gene expression of osteogenesis and angiogenesis. Von kossa staining was used to detect the formation of mineralized nodules in vitro, which also confirmed that ASCs/EPCs co-culture can improve the osteogenesis of this cell system.
Based on the in vitro results, we further performed an in vivo experiment on the repair of critical-sized bone defects in rats. The Micro-CT scans of the rat's cranial bones were performed after 5 weeks after surgery. The results clearly showed that the amount of new bone in the defect area was much greater in the HA/Col + ASCs + EPCs group than in the other groups, including HA/Col + ASCs group. In addition, BMD also showed that there was more bone tissue formation in the HA/Col + ASCs + EPCs group. Moreover, as confirmed by immunohistochemical analysis, the blood vessel density in the defect area was higher in the HA/Col + ASCs + EPCs group than in other three groups. The possible reason for this phenomenon is that the lack of EPCs in the ASCs group resulted in a relative decrease in vascularization and bone formation, which relied solely on the inward growth of the host blood vessels. However, the distance between the blood vessels of the host tissue and the center of the bone defect is far from sufficient to achieve bone regeneration, especially for critical-sized defect. As a result, nutrients, metabolites and other molecules cannot be delivered to the center area of defect, which severely impedes bone regeneration. In the ASCs/EPCs group, EPCs directly increased the invasion of blood vessels and promoted the differentiation of ASCs to osteogenesis and, meanwhile, pre-osteogenic ASCs could promote the recruitment of EPCs and enhance the ability of EPCs to form blood vessels. The interaction accelerates vascularization and bone formation in the meantime, making nutrients, cytokines, and other molecular factors involved in the bone healing process more accessible. Taken together, these results indicated that the ASCs/EPCs co-culture system can promote the formation of vascularized bone during bone regeneration and achieve the repair of the critical-sized bone defect.