Cell Sheets And Extracellular Matrix Sheets Derived From Pre-osteoblasts For Bone Defect Repair

Bone regeneration has always been a challenge for orthopedics and craniofacial surgery.Currently,bone substitutes are increasingly used in surgical procedures.In this study,the temperature-sensitive polydimethylsiloxane(PDMS)grafted with nano-temperature-sensitive polymer molecular brushes was prepared.The pre-osteoblast(MC3T3-E1)cell sheet could be obtained by culturing MC3T3-E1 cells on the temperature-sensitive PDMS and detaching cell sheet through lowering temperature.The MC3T3-E1-derived extracellular matrix sheet could also be obtained through culturing MC3T3-E1 cells on the temperature-sensitive PDMS and acellular processing.Further,the application of cell sheet and extracellular matrix sheet in the repair of bone defects was explored in this study.This study is mainly divided into the following three parts:In the first part of this study,we prepared a temperature-sensitive PDMS by grafting N-isopropylacrylamide(NIPAAm)onto the surface of PDMS by UV induced graft polymerization.After culturing the cells on the temperature-sensitive PDMS,a complete MC3T3-E1 cell sheet was obtained by reducing the temperature to room temperature,and the morphology of the obtained cell sheet was evaluated.In addition,MC3T3-E1-derived extracellular matrix sheet were obtained by decellularizing the cell sheets.The growth,chemotaxis effect and in vitro osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)on the MC3T3-E1-derived extracellular matrix sheet were evaluated.The results showed that complete matrix sheet can be obtained by culturing MC3T3-E1 cells on PDMS and then conducting decellularized.The harvested matrix sheets showed a chemotactic effect on BMSCs in a concentration-dependent manner,compared with the control group and the gelatin methacryloyl(GelMA)group.MC3T3-E1-derived extracellular matrix sheet could also promote osteogenic differentiation of BMSCs in vitro,and had higher osteogenesis-related gene expression and enhanced mineralization.In the second part of this study,we explored the effect of appropriate mechanical stimulation on cell sheet,the results showed that there was no significant difference in the surface morphology of the cell sheet undergoing appropriate mechanical stimulation and without appropriate mechanical stimulation,but the mechanically stimulated cell sheets had higher modulus.In addition,a double-layer cell sheet was constructed using gelatin transfer method in vitro,and a double-layer cell sheet after appropriate mechanical stimulation and static culture were constructed respectively.We used a mouse skull defect model to evaluate the in vivo bone repair effect of a double-layered cell sheet composed of MC3T3-E1 cell sheets that were cultured normally and stimulated with appropriate mechanics.The results of micro-CT and H&E staining showed that the double-layered cell sheet after proper mechanical stimulation had better bone repair effect.In the third part of this study,we used the MC3T3-E1-derived extracellular matrix sheet as a biomimetic periosteum and further wrapped it on the surface of GelMA hydrogel to construct a biomimetic periosteum-bone substitute.The rabbit large radius bone defect model was used to evaluate the repair effect of the substitute.The results of micro-CT and H&E staining showed that repair of large segmental bone defects could be greatly improved by wrapping extracellular matrix sheets on the surface of GelMA.Moreover,combining extracellular matrix sheet with GelMA hydrogel may provide a promising periosteum-bone biomimetic bone substitute for bone tissue engineering.