Study On Differentiation Of BMSCs Into Osteochondral Composites Induced By Gradient Mineralized Hydrogel

Objective:Gradient mineralized hydrogels?GM?were prepared and used as the carriers of bone marrow mesenchymal stem cells?BMSCs?,aiming at inducing them to differentiate into osteoblasts and chondrocytes,finally promoted the formation of osteochondral composites.This study would provide a better idea for the treatment of the articular cartilage defects.Methods:1)Preparation and characterization of GM hydrogels:Dual network hydrogels were prepared via covalent cross-linking and ionic cross-linking,and then they were immersed in the5%,10%,and 15%CaCl₂&NaH₂PO₄ mineralized aqueous solution to conduct biomimetic mineralization,respectively.The synthesized mineralized hydrogels were characterized by stress-strain,Fourier transform infrared spectroscopy?FTIR?,scanning electron microscope?SEM?and X-ray diffraction?XRD?.2)Cytocompatibility of hydrogels and characterization of differentiation of BMSCs into osteochondral composites induced by GM hydrogel.BMSCs were cocultured with various mineralized hydrogels for different periods,and then they were assayed via CCK-8,AO/EB,Alizarin Red staining,Alcian Blue staining and immunofluorescence staining?IF?,aiming at confirming the cytocompatibility of hydrogels and the differentiation potential of BMSCs into chondrocytes and osteoblasts upon the induction of GM hydrogels.Results:The results of FTIR and XRD confirmed that the characteristic absorption peaks of calcium alginate and polyacrylamide appeared in the spectra of FTIR and XRD.Meanwhile the intensity of the characteristic absorption peaks of hydroxyapatite increased with the enhancement of mineralized degree.SEM results verified that calcium nodules became more obvious with the enhancement of mineralized degree.Stress-strain test results showed that the compression strength of hydrogels with different mineralized degree varied from 30 KPa to 380 KPa,which fully met the needs of increased mechanical strength of articular cartilage from the surface to the deep layer.2)The results of CCK-8 and AO/EB staining showed that the cellular survival rate of the hydrogel group was more than 90%,indicating the synthesized hydrogels were non-toxic and had a good cytocompatibility.The results of Alizarin Red staining,Alcian Blue staining and Immunofluorescence staining?IF?showed that the hydrogels with higher mineralized degree will induce more BMSCs to differentiate into osteoblasts,conversely,more BMSCs would differentiate into chondrocytes.Conclusion:BMSCs can be induced to differentiate into osteochondral composites,a gradient transition layer changed from chondrocytes to osteoblasts by gradient mineralized hydrogel under the action of scaffold materials with different elastic modulus and different mineralized degree.This study will provide a new idea for the regeneration of articular cartilage tissue and its application in the clinic.