| Bone damages caused by accident,infection and cancer seriously affect human life.Although bone tissue engineering offers great promise for bone defect repair,there are still many bone defects that cannot be repaired completely clinically.Scaffold-based gene therapy promotes bone regeneration by regulating the expression of target proteins in cells at the genetic level,which can overcome the limitations caused by protein therapy such as cytokines and growth factors.Message RNA(mRNA)encoding bone-associated protein has the unique advantage of being able to perform related functions directly in cytoplasm,providing a prospective method for bone regeneration.Previous studies have confirmed the high expression of osteogenesis-related mRNAs in the cytoplasm of mesenchymal stem cells(MSCs)after pre-osteogenic differentiation,whose expression levels changes with the time of differentiation induction of MSCs.Therefore,this study intended to extract osteoinductive mRNA(Oi-mRNA)drived from osteogenically pre-differentiated MSCs at different days and detect the effect of Oi-mRNA on the osteogenic differentiation of MSCs.Subsequently,Oi-mRNA was loaded into the demineralized bone matrix(DBM)scaffold with a matrix stiffness of0.67 ± 0.14 MPa,and the effect of Oi-mRNA modified DBM(Oi-mRNA/DBM)scaffold on the bone regeneration was studied.The main contents and results of this paper are as follows:(1)The Oi-mRNA was extracted from MSCs that were induced osteogenic differentiation for 1 d,3 d,7 d,14 d,and 21 d(marked as Oi1-mRNA,Oi3-mRNA,Oi7-mRNA,Oi14-mRNA,and Oi21-mRNA,respectively).The Oi-mRNAs were transfected into MSCs to explore their effects on the osteogenic differentiation of MSCs.The results showed that the Oi-mRNAs had no obvious effect on the activity of MSCs after transfection into MSCs.The Oi1-mRNA,Oi3-mRNA,and Oi21-mRNA had no significant effect on the osteogenic differentiation of MSCs.Besides,the Oi7-mRNA could significantly promote the alkaline phosphatase(ALP)expression of MSCs in the early stage of osteogenic differentiation(7 d),while the Oi14-mRNA could promote the calcium deposition and secretion of extracellular matrix of MSCs in the middle and later stages of osteogenic differentiation(14 d and 21 d).(2)The Oi7-mRNA and Oi14-mRNA were selected to be combined with the DBM scaffolds to form the Oi7-mRNA/DBM and Oi14-mRNA/DBM scaffolds.MSCs were cultured on the scaffolds of DBM,Oi7-mRNA/DBM and Oi14-mRNA/DBM for 7 d,14 d and 21 d.ALP staining results showed that the Oi7-mRNA/DBM scaffold could promote the expression of ALP in MSCs(7 d).The results of histological and immunohistochemical staining showed that the Oi14-mRNA/DBM scaffold could significantly promote the secretion of extracellular matrix,the formation of collagen and the expression of osteogenic markers osteocalcin(OC)and osteopontin(OPN)(14d and 21 d).(3)A rat cranial defect model was established to examine the bone regeneration capacity of the DBM and Oi14-mRNA/DBM scaffolds via micro computed tomography(μ-CT)scanning and histological staining after implanted for 1 month,2 months and 3months,respectively.The results showed that the Oi14-mRNA/DBM scaffold could promote the infiltration of cells and repair of bone defect in vivo when comparied with the DBM scaffold.In summary,this study explored the effects of mRNAs derived from osteogenically pre-differentiated MSCs at different stages on the osteogenic differentiation of MSCs.The Oi14-mRNA that derived from MSCs in the middle stage of osteogenic differentiation possessed pro-osteogenic effect on MSCs.The Oi14-mRNA/DBM scaffold could significantly promote osteogenic differentiation of MSCs in vitro and the repair of bone defect in vivo.The DBM scaffold loaded with mRNAs that encoding osteoinductive proteins in this study is expected to provide a powerful tool for bone regeneration. |
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