Osteopontin Induces Differentiation Of Mouse Bone Mesenchymal Stem Cells Into Epithelial Cells And Promotes The Wound Healing

BackgroundA variety of cells and cytokines participate in the wound healing of skin which involves the complex physiological process of inflammation, granulation, angiogenesis and tissue remodeling. The skin damage repair required epithelial cells to maintain skin integrity. Bone marrow mesenchymal stem cells have high abilities of self-renewal and multi-directional differentiation potential after isolated and cultured in vitro, and the transform to epidermal cells has been used in wound tissue repair.Osteopontin is an extracellular matrix protein, studies have shown that osteopontin plays an important role in promoting the healing caused by inflammatory cells, endothelial cells and some other cells. Previous studies found that bone marrow mesenchymal stem cells are closely related to the high expression of osteopontin in the wound tissues. To understand if osteopontin plays an important role in the differentiation of bone mesenchymal stem cells into epithelial cells and boosting the wound healing, which has a vital significance in complete repairing of the skin function and structure.ObjectiveIn the experiment wild type and osteopontin knockout mice were used to study the role of osteopontin played in the differentiation of bone mesenchymal stem cells into epithelial cells in vitro and vivo.MethodsIn vitro experiment: experiment was divided into two groups, wild type group(WT) and osteopontin knockout group(KO). First, the wild type and osteopontin knockout newborn mice(age one day old) BMSCs were isolated and cultured and the purity was measured by flow cytometry.Second, the cultured cells of the third-generation were mixed with epithelial transformation culture medium respectively and then incubated in the incubator. To compare the expression level of keratinocyte protein 14(CK14) between KO and WT groups after induction of differentiation,the microscopic observation,flow cytometry method,cells immunofluorescence staining,and western blot method were used by us.Animal experiment: there were four groups in the experiment, such as the group of wild type mice injected GFP-MSCs around the wound(WT/MSCs), the group of wild type mice injected PBS around the wound(WT/PBS), the group of osteopontin knockout mice injected GFP-MSCs around the wound(KO/MSCs), and the group of osteopontin knockout mice injected PBS around the wound(KO/PBS).Every group is consist of 8 male mice, who’s weight is about 20 to 25 g and the age is about 6 to 8 weeks old. The isolated and cultured GFP-MSCs and PBS were injected to the wound around according to the grouping. After observed for seven days, the rates of wound closure in each group were calculated and the migration of GFP-MSCs were observed from the wound by small animal imaging system. Then the third, 5th, and 7th days after surgery, tissues of each group were cut for HE staining to observe the growth of the wound and the GFP-MSCs to differentiate into epithelial cells were detected by immunofluorescence staining.Results1. The purity of isolated and cultured BMSCs is over 90%,and the cells were seen as a type of fusiform or fibroblast-like.2. Immunofluorescence staining of the induced cells showed that CK14 was expressed in both WT and KO groups,and the WT group is obviously higher than the KO group(P <0.01).3. The expression of CK14 measured by western blot from the induced cells showed that KO group is significantly lower than WT group(P<0.01).4. The migrated speed of the injected GFP-MSCs, WT/MSCs group is faster than KO/MSCs group and the amount of migrated cells is the same(P<0.01).5. Animal experiments: The WT/PBS group was set as a control, the wound healing of KO/PBS group is significantly slower than the control(P<0.05)and the wound healing of WT/MSCs group is faster than the control(P<0.05).6. The injected GFP-MSCs around the wound have the ability to differentiate into epidermal cells and the differentiated ability of WT/MSCs group is stronger than KO/MSCs(P< 0.05).Conclusions1. Migrated ability of bone mesenchymal stem cells can be enhanced by osteopontin.2. In vitro and vivo,osteopontin plays a vital role in the differentiation of bone mesenchymal stem cells into epithelial cells.3. The wound healing can be promoted by osteopontin induces BMSCs differentiate into epidermal cells.