Feasibility Of Non-viral Transfection Of RBMMSC Sheets And Enhanced Osteogenesis Of AntimiR-138Transfected RBMMSC Sheets-implant Complex

Dental implant has become an important strategy to restore missing tooth. However，it still takes a long time to obtain implant osseointegration (usually3-6months), especiallyfor some special patients with metabolic bone diseases (such as osteoporosis, diabetes, andpostoperative radiotherapy etc.), who often suffer from the planting contraindicationsbecause of high risks of implant failure. Our research is to verify the feasibility oftransfecting the cell sheets using the non-viral way and to confirm the osteogenesisenhancing effect of antimiR-138on cell sheets composed of bone marrow mesenchymalstem cells (BMSCs). Then, we try to construct the antimiR-138transfected rBMMSCsheets-implant complex, and to comform the osteoinduction effect of the antimiR-138transfected rBMMSC sheets-implant complex. We hope this technique would betterpromote bone formation around the implant (i.e.,osseointegration) by providing macro-and micro-molecular environment including stem cells, extracellular matrix, and thebioactive molecules at gene level. PartⅠCultivation and characterization of rBMMSCsObjective:Isolation and identification the biological characters of rBMMSCs.Methods:rBMMSCs were isolated from rat bone marrow tissues and were observed under aninverted microscope. The CD markers of rBMMSCs were analyzed by flow cytometryanalysis. MTT and clone formation assay was used to detected proliferation capacity ofrBMMSCs. ALP, alizarin red and oil red O staining tests was used to investigate theosteoblastic/adipogenic differentiation potential of rBMMSCs.Results:rBMMSCs were successfully obtained in the present study, which positivelyexpressed mesenchymal stem cell (MSC) markers such as CD29, CD90and CD105, whilenegative for hematopoietic cells markers such as CD34, CD31and CD45. The cloneformation ability was11.6%. Following osteogenic/adipogenic induction, the calcifiednodule and fat drops could be detected after Alizarin red staining and Oil red O staining.Conclusions:(1)The rBMMSCs in our experiment showed self-renewal and muti-potentialdifferentiation characters.(2)The obtained rBMMSCs could be used as gene therapy vector cells to buildmiRNA genetically modified BMMSCs-sheets.PartⅡ Feasibility of non-viral transfection of cell sheets andenhanced osteogenesis of mesenchymal stem cell sheets byantimiR-138transfectionObjective:To verify the feasibility of transfecting the cell sheets with the non-viral way; Toconfirm the osteogenesis enhancing effect of antimiR-138on cell sheets composed of bone marrow mesenchymal stem cells (BMSCs).Methods:1)We cultured the rBMMSC sheets using a continuous Vc inducing method, whichwas later transfected with different Lipofectamine2000-miRNA lipoplexes. Thetransfection efficiencies of different formulations were assessed by the flow cytometry andmiRNA RT-PCR. H&E staining and SEM observation were performed to evaluate theharvested sheets.2)To assess the in vitro osteogenesis of the antimiR-138transfected rBMMSC sheets,the control group using antimiR negative control (antimiR-control) and that withoutperforming transfection (no agent control) were set. Alizard red, sirius red staining, ALPstaining, Real-time PCR and Western blot were used to compare the difference ofosteoblastic differentiation capacity of these sheets.3)The ectopic bone regeneration ability of the rBMMSC sheets was assessed inimmunocompromised mice: Micro-CT scanning, H&E or Masson’s Trichrome stainingwere used to assess the osteogenesis of the rBMMSC sheet/FDB complexes in SCID mice8weeks after implantation.Results:The rBMMSC sheets are fabricated by a vitamin C inducing method. They can besuccessfully transfected with antimiR-138to achieve a transfection efficiency of nearly100%by a Lipofactamine2000based transfection formulation after proper adaption andoptimization. The antimiR-138transfection significantly enhances the osteogenesis of therBMMSC sheets. In the in vitro study, the animiR-138transfection significantly enhancesthe osteogenic differentiation of the rBMMSC sheets, indicated by the higher alkalinephosphatase (ALP) production, denser extracellular matrix mineralization andup-regulated osteogenesis related genes including runt-related transcription factor-2(RUNX2), osterix, ALP, osteocalcin and bone morphogenetic protein-2at both mRNA andprotein levels, compared to controls. Regarding the underlying mechanism, theantimiR-138transfection down-regulates the endogenous miR-138levels in rBMMSCsheets, activate the extracellular signal regulated kinases1/2(ERK1/2) pathway and enhance the expression of RUNX2. The in vivo ectopic bone regeneration results confirmthe robust enhancing effect of antimiR-138transfection on the bone regeneration ability ofrBMMSC sheets combined with freeze dried allograft bone, showing great clinicalsignificance for bone repair and regeneration.Conclusions:1) Satisfactory transfection efficiency of nearly100%, good cytocompatibility andunimpaired cell sheet structure can be obtained by properly optimizing theLipofactamine2000based transfection formulation.2) The antimiR-138transfection significantly enhances the osteogenesis of therBMMSC sheets both in vitro and in vivo, with the ERK1/2pathway involved.PartⅢ Construction and enhanced osteogenesis of antimiR-138transfected rBMMSC sheets-implant complexObjective:To construct the antimiR-138transfected rBMMSC sheets-implant complex;Tocomform the osteoinduction effect of the antimiR-138transfected rBMMSCsheets-implant complex.Methods:1)The rBMMSC sheets were harvested and wrapped around the MAO-treatedimplants to construct the complexes. The SEM observation was performed to evaluate thecomplexes.2)The effects of the antimiR-138transfected rBMMSC sheets-implant complexosteogenetic differentiation were assessed: The alkaline phosphatase (ALP) production,collagen secretion, extracellular matrix (ECM) mineralization, osteogenesis-related geneexpression and protein product were systemically assessed.3)The ectopic bone regeneration ability of the rBMMSC sheet-implant complexs wasassessed: Micro-CT scanning, H&E or Masson’s Trichrome staining andimmunofluorescence staining were used to assess the osteogenesis of the rBMMSC sheet-implant complexs in SCID mice8weeks after implantation.Results:The antimiR-138transfected rBMMSC sheets integrated well with MAO-treatedimplants with abundant cells and extracellular matrix covered on the porous structure ofthe implants. In the in vitro study, the osteogenic differentiation of the antimiR-138transfected rBMMSC sheets-implant complex was significantly enhanced, indicated bythe higher alkaline phosphatase (ALP) production, denser extracellular matrixmineralization and up-regulated osteogenesis related genes including runt-relatedtranscription factor-2(RUNX2), osterix, ALP, osteocalcin and bone morphogeneticprotein-2at both mRNA and protein levels, compared to controls. The in vivo ectopic boneregeneration results showed the robust osteoinduction effect of the antimiR-138transfected rBMMSC sheets-implant complex, showing great clinical significance for oralimplantology.Conclusions:1) It is feasible to construct the antimiR-138transfected rBMMSC sheets-implantcomplex.2) The antimiR-138transfected rBMMSC sheets-implant complex showd excellentosteogenic as well as osteoinductive ability both in vitro and in vivo.3) Most importantly, the tecnique would become the most promising and effectivemethod to improve the success rate of oral implantology.