Osteogenic Differentiation Of ADSCs Encapsulated In The Calcium Alginate/bone-powder Microbeads In Spinner Flask

The widespread use of traditional autogenous bone graft and allogeneic bone graft techniques is limited by their disadvantages, such as, limited donor source, immune rejection, patients suffering and so on. Recently, bone tissue engineering receives considerable attention. The location and shape of bone defects vary greatly from different causes. However, calcium alginate microbeads can better adapt to the requirements of different shapes. To explore the feasibility of constructing bone microbeads in vitro, ADSCs encapsulated in the calcium alginate/bone-powder microbeads were cultured in the spinner flask.In this study, calcium alginate/bone-powder microbeads with better mechanical strength and mass transfer performance were prepared by optimizing the concentration of sodium alginate solution, calcium chloride solution and the density of bone-powder. The results of orthogonal experiments revealed that the concentration of sodium alginate solution determined the mechanical strength and mass transfer performance of microbeads, followed by the concentration of calcium chloride solution, and the impact of bone power was minimal. The optimal preparation conditions were that the concentration of the sodium alginate solution was2.5%, the concentration of calcium chloride was4%and the the bone power density was5.0mg/mL. Calcein-AM/PI Staining, growth curve and osteogenic detection showed ADSCs used in the experiments owned good growth state, cells activity and osteogenic differentiation potential. The cells proliferation experiments showed that calcium alginate/bone-powder microbeads were biocompatible, and ADSCs can maintain good growth state and cell viability in microbeads and the optimal embedding density was5×106cells/mL To construct bone microbeads in vitro, one experimental group and two control groups were designed in this study. The experimental group was the osteogenesis differentiation of ADSCs encapsulated in the calcium alginate/bone-powder microbeads in spinner flask, control group I was the osteogenesis differentiation of ADSCs encapsulated in the calcium alginate/bone-powder microbeads in T-flask and control group Ⅱ was the osteogenesis differentiation of ADSCs encapsulated in the calcium alginate microbeads in T-flask. After induced14days, ALP kit and ALP staining were respectively used to quantitatively and qualitatively detect the ALP in the culture medium. After induced21days, alizarin red staining and von-Kossa staining were used to detect the mineralized nodules in cells. There was no ALP in the culture medium after inducing2days, and ALP was detected on the third day in three groups. The content of ALP reached the maximum on the14th day, when mineralized nodules began to generate. ALP quantitative detection, ALP staining, alizarin red staining and von-kossa staining showed in both quantitative and qualitative aspects that ADSCs of the experimental group obviously obtained the higher ostcogenic capacity than the other two control groups. In conclusion, the three-dimensional dynamic environment and bone power can work together to promote osteogenic differentiation of ADSCs encapsulated in microbeads, constructing bone microbeads successfully.