Staphylococcal Enterotoxin C Injection In Combination With Ascorbic Acid Promotes The Differentiation Of Bone Marrow-Derived Mesenchymal Stem Cells Into Osteoblasts In Vitro

BackgroundMesenchymal stem cells(MSCs)are multipotent adult stem cells that mainly exist in the bone marrow.When isolated and cultured in vitro,MSCs possess the potential to differentiate into osteoblasts,chondrocytes,neurocytes, adipocytes and myocardial cells,and the differentiated cells can proliferate potently.This multipotentiality for differentiation makes these cells ideal candidates as target cells for application to tissue engineering and gene therapy.Bone fractures are common clinical conditions.Regarding their therapy, staphylococcal enterotoxin C(SEC)injection has been found to be beneficial for fracture healing when used in broken ends of fractured bone or raw surface.The effects of SEC injection have been attributed to its dominant component,namely enterotoxin C.Zhu et al.reported that,although SEC injection has no significant effects on the proliferation and differentiation of osteoblasts,it has marked effects on the mineralization of osteoblasts.Both animal experiments and clinical studies have demonstrated that SEC injection can accelerate the absorption and organization of hematomas on the ends of bone fractures and promote porosis,thereby benefiting the fracture healing process.On the other hand,it is well known that ascorbic acid can enhance the activity of alkaline phosphatase(AKP)and improve the vitality of osteoblastic cells.However,no report to date has investigated whether staphylococcal enterotoxin C injection has any effects on MSCs and whether combination of staphylococcal enterotoxin C injection with ascorbic acid can promote the differentiation of MSCs into osteoblasts,thereby resulting in improved fracture healing.ObjectiveThe present study aimed to investigate the effects of staphylococcal enterotoxin C injection in combination with ascorbic acid(SEC-AA)on the differentiation of MSCs and the cytopoiesis and mineralization of osteoblasts, thus providing data and a basis for clinical therapy of bone fractures with SEC-AA.MethodsThe cells identified as MSCs in the same batch were induced as the following grouping:(1)Groups with SEC injection alone(the concentration of enterotoxin C was 10 ng/L,30 ng/L and 100 ng/L respectively);(2)Groups with SEC injection(10 ng/L,30 ng/L,100 ng/L)plus ascorbic acid(final concentration:50μg/ml).In addition,the cells cultured in normal media without any inducer acted as control.The alkaline phosphatase(AKP)activities,the alizarin red-stained calcified nodules and the changes in cellular morphology and ultrastructure observed by scanning and transmission electron microscopy were used to investigated the abilities of the differentiation of MSCs.ResultsAfter induced by SEC injection,the detected activity of AKP increased along with the increase of the concentration of SEC injection,but there was no statistically significant difference among the different groups.However,when combined with the induction of ascorbic acid,the activity of AKP among various groups increased significantly.After induced for 28 days and stained with 0.1%alizarin red-Tris-HCl,it was found that in SEC injection alone group,there were a few scattered red calcium nodi in the cells,but in the SEC injection plus ascorbic acid group,it was found that the calcium nodi distributed in cluster,with more nodi and longer diameter of nodi.Under scanning electron microscope,the group of SEC-AA exhibited gross cytoplasmic process and flat and the microvilli on the surface became more plentiful compared to the SEC injection alone group.Under transmission electron microscope,in the SEC injection alone group, MSCs showed more mitochondria and endoplasmic reticulum,with plentiful ribosome.In the SEC-AA group,MSCs showed more mitochondria and increased density of stroma,with more plentiful ribosome and rough endoplasmic reticulum.ConclusionsAfter induced for 28 days,it was found that SEC-AA induced increased level of AKP activities in MSCs and the number of alizarin red-stained calcium nodi,and the increases were significant statistically compare with that in the SEC alone group.The changes of cellular morphology and ultrastructure under scanning and transmission electron microscopes indicated an enhanced differentiation of MSCs to osteoblasts.The findings demonstrated that SEC-AA can promote the differentiation of MSCs to osteoblasts and accelerate the cytopoiesis of osteoblasts.