Study Induced Osteogenic And Chondrogenic Differentiation Of Human Mesenchymal Stem Cells On Polylactic Glycolic Acid/tricalcium Phosphate(PLGA/TCP) Scaffolds In The Feeder-cultivar Bioreactors

Bone tissue engineering is a research field involving multiple subjects which shows well development prospect in reparing and restablishing bone defect and bone ununion.The research of bone tissue engineering focuses on three fields as below:(1)seeded cells;(2)scaffold materials;(3)in vitro engineering bone tissue construction.In vitro construction of cell-scaffold complex is one of the most important parts of bone tissue engineering.The research issue “Personalized bone tissue cultured in bioreactors and applied in animal” of our laboratory also focuses on in vitro construction of cell-scaffold complex.Human bone mesenchymal stem cells(hBMSCs)were used as seeded cells in this study.It was testified for the proliferation,osteogenic differentiation and chondrogenic differentiation capacity of hBMSCs cultured on well plate.The results of VON KASSA and alcain bule staining,ALP activity and GAG content test,and western blotting of marker protein showed that differentiation group had great difference with control group on cell morphology,biochemical index and marker protein expression.It could be concluded that cells of differentiation group had taken osteogenic differentiation and chondrogenic differentiation which testified the viability of using hBMSCs as seeded cells.Four cytokines EPO,NGF,IGF-1 and IFN-β were investigated for their ability to induce chondrogenic differentiation of hBMSCs in this study.It was found that cell morphology of IFN-β group had taken great change.The results of alcain blue staining and GAG content test testified that IFN-β had the ability of inducing chondrogenic differentiation of hBMSCs.After different combinations of 100ng/m L IFN-β and 10ng/m L TGF-β3 were added in classical chondrogenic differentiation medium to culture hBMSCs,GAG content,marker gene expression and marker protein expression were tested at different time points.The data showed that IFN-β had stronger ability of promoting chondrogenic differentiation of hBMSCs than TGF-β3 and co-work of both would be better than sole-work.We also investigated the working concentration of IFN-β and found that 100ng/mL wad the most suitable.Polylactic acid-glycolic acid/tricalcium phosphate(PLGA/TCP)was choosed as scaffold material for proliferation and differentiation of hBMSCs in this study.At first,the biocompatibility of PLGA/TCP was testified.The result of SEM showed that hBMSCs could use the porosity characteristic of PLGA/TCP and proliferate better than 2D control.The results of MTT and ALP activity test showed that PLGA/TCP had favorable biocompatibility not only in supporting adherence and growth of hBMSCs,but also in promoting proliferation and differentiation of h BMSCs.Then the biosecurity of PLGA/TCP in animal was testified.The results of pyrogenic reaction test,hemolytic test,acute systemic toxicity test,intracutaneous stimulation test and subcutaneous implantation test showed that PLGA/TCP had good biosecurity,suitable degradation rate and harmless degradation product in animal and could be planted as scaffold for h BMSCs.The self-design feeder-cultivar perfusion bioreactors system was used to culture multiple hBMSCs and PLGA/TCP scaffold complex in this study.The scaffolds were fixed in cultivar bioreactors and the medium containing sufficient nutrition and oxygen with suitable pH and temperature was provided by feeder bioreactor.The osteogenic differentiation and chondrogenic differentiation of hBMSCs and PLGA/TCP scaffold complex cultured in feeder-cultivar perfusion bioreactors system was investigated.The results of SEM,ALP activity and GAG content test,qRT-PCR of marker gene and western blotting of marker protein showed that h BMSCs in 3D bioreactor culture group had a higher level of differentiation than 3D static culture group.The feasibility of applying feeder-cultivar perfusion bioreactors system in bone tissue engineering was testified.The feasibility of using hBMSCs as seeded cells,using PLGA/TCP material as scaffold,and using feeder-cultivar perfusion bioreactors system to dynamically culture hBMSCs and PLGA/TCP scaffold complex and in vitro large-scale constructing cell-scaffold complex for bone tissue engineering is testified in this study.The discovery of ability of IFN-β promoting chondrogenic differentiation of MSCs open a new window for bone tissue engineering.