Construction And In Vitro Study On Proliferation,Mineralization Of Alginate Injectable Scaffold Based Bone Tissue Engineering Mixed With PRP

Objective:To study the effects of Platelet-Rich Plasma in Alginate based Bone Tissue Engineering on osteoblasts viabilities and mineralization in vitro and confirm whether PRP can be used as the growth factor resource of injectable Alginate scaffold based Bone Tissue Engineering.Material and methods:①The isolation,culture and identification of osteoblasts in vitro:New born rat calvarid osteoblasts were isolated by the enzyme digesting for several times and cultured in vitro.The cells were observed by inverted phase contrast microscope.Osteoblasts were identified by ALP dyeing and mineralization nodules dyeing.②The study of osteoblastic proliferation by MTT assay:Osteoblastic suspension was prepared by combining the third passage of OBs with 1.5% Alginate.The cells were divided into three groups:1.the experimental group (1.5%Alginate/OBs/20%PRP)2.the control group(1.5%Alginate/OBs)3.the monolayer culture group,and cultured in vitro for 14 days.On the days 2,3,4,5,6,7,10,12,14d,the proliferation of OBs was measured by MTT assay respectively,then the growth curves were drawn according to OD values.③The study of osteoblastic mineralization by tetracycline hydrochloride staining: Osteoblastic suspension was prepared by combining the third passage of OBs with 1.5%Alginate.The cells were divided into three groups:1.the experimental group (1.5%Alginate/OBs/20%PRP)2.the control group(1.5%Alginate/OBs)3.the monolayer culture group,and cultured in vitro for 31 days.On the days 14,24,31d, mineralization nodules of osteoblasts were observed by tetracycline hydrochloride staining under fluorescence inverted phase contrast microscope.Mineralization nodules numbers and mineralizing surface percentage were measured respectively per field of vision in every group at different time.Results:①The isolation,culture and identification of osteoblasts in vitro:The cells possessed the characteristics of osteoblastic typical morphology.ALP dyeing and mineralization nodules dyeing were positive.The cells can form bone nodules in vitro.②The study of osteoblastic proliferation by MTT assay:Osteoblastic proliferation inclination in Alginate three-dimensionally cultured group remained climbing on,in contrast to the monolayer culture one,and Alginate three-dimension culture system's biocompatibility was no worse than that of the monolayer culture one.Moreover MTT assay results(OD values)indicated cytoactive of the experimental group was much higher than that of the controlled,which difference had statistical significance (P＜0.05).③The study of osteoblastic mineralization by tetracycline hydrochloride staining:The intensity,numbers and volume of fluorescence in Alginate three-dimensionally cultured group remained going on,in contrast to the monolayer culture group.And this inclination became obvious with culture lasting.Moreover, between the experimental group and the control one,there was significant difference in mineralization nodules number and mineralizing surface percentage on the culture days of 24th and 31st(P＜0.05).Conclusions:Construction of an injectable tissue engineered bone with Alginate, osteoblasts and PRP does not require sophisticated techniques and ensures good biological property in vitro of the artificial bone that can be easily shaped and allows better growth,better function of the OBs,suggesting great potential of this technique for use.Platelet-Rich Plasma can be used as growth factor resource of alginate bone tissue engineering.