Study Of The Promoting Effect And Mechanism Of Sema3A Transfected Gingival Mesenchymal Stem Cells On The Repair Bone Defects In Rats

Background and ObjectivesClinically,it is common that some bone defects are difficult to heal.For example,A large area of bone loss around the tooth root leads to loosening and loss of teeth,Jaw defects caused by tumor or trauma or pathological bone resorption leads to insufficient alveolar ridge bone.How to repair a large area of bone defect is still one of the urgent problems in clinical work.The current bone defect repair technology mainly uses tissue or biological materials to reconstruct the defect area,including autologous bone transplantation,allogeneic bone transplantation and bone replacement material transplantation.It is difficult to achieve ideal bone shape repair by using autologous bone such as ribs and iliac bones.It may cause tissue damage and complications such as infection of the donor site.The main problem of allogeneic bone transplantation and bone replacement material transplantation is immune rejection.Its vascularization and bone formation are lower.The main advantage of bone tissue engineering is the low infectivity and low carcinogenicity using self-derived autologous cells.However,due to the special microenvironment and complicated etiology in the oral cavity,there is no satisfactory method to repair alveolar bone defects.With the rapid development of bone tissue engineering based on stem cell technology in recent years,it has provided new ideas for bone regeneration treatment.Seed cells,growth factors and scaffold materials are the main contents of traditional tissue engineering research.With the development of gene therapy,it has overcome the many shortcomings of the introduction of exogenous growth factors,such as complicated implementation process,shorted half-life,easy to be enzymatically decomposed in the body,toxic and side effects,and the diffusion is fast,and it is difficult to maintain an effective therapeutic concentration.Stem cells modified by exogenous genes to express the target protein stably for a long time has become a current research hotspot.This research uses rat gingival-derived mesenchymal stem cells as the seed cell model for in vitro experiments.The aim of the study is to find the effect of semaphorin 3A(Sema3A)transfection on the promotion of osteogenic differentiation of GMSCs and the inhibitory effect on the activation of osteoclast precursor cells,and preliminary study of its mechanism.The Wistar rat skull bone defect model was used as an animal model for in vivo experiments to detect the effect of Sema3A-modified GMSCs on the repair efficiency of rat skull bone defects,in order to provide new ideas for periodontal bone tissue regeneration in clinical treatment.At present,the stem cells used in periodontal tissue regeneration research mainly include odontogenic stem cells and non-odontogenic stem cells.The main odontogenic stem cells are periodontal ligament stem cells(PDLSCs),dental pulp stem cells(DPSCs),gingival mesenchymal stem cells(GMSCs);the main non-dental stem cells are bone marrow stem cells(BMSCs)and adipose stem cells(ADSCs).Gingiva is a special tissue in the oral cavity that covers the alveolar ridge and the posterior area of molars.It is a special component of the oral mucosal immune and biochemical barrier.Under physiological conditions,the gingival tissue shows the ability of scar-free healing and rapid tissue repair and regeneration.In recent years,it has been found that mesenchymal stem cells from gingival tissue have clinical application potential.Compared with other MSCs,GMSCs have many advantages-easy to get,less damage to the body,easy to expand,stable karyotype and so on,which make GMSCs become seed cells with potential practical value.However,there are also contrary research reports that compared with PDLSCs,GMSCs has weak mineralization ability in vitro,which is not conducive to bone regeneration.There are still controversies about this research result.Sema3A is the first semaphorin family protein found in vertebrates,which plays an important role in the physiological or pathological processes such as nervous system,cardiovascular system,tumorigenesis,and immune regulation.In clinical work,we found that patients with craniocerebral trauma combined with fractures formed faster than bone callus with simple fractures,and bone formed faster even for irregular jaw fractures combined with craniocerebral trauma.Later experiments proved that the secretion of multiple cytokines regulates bone repair during traumatic brain injury,among which Sema3A is the most researched regulatory factor.In recent years,it has been found that Sema3A also plays an important role in bone metabolism.We found through literature research that the application of Sema3A recombinant protein in experimental animals can promote local bone formation.The experimental mice with Sema3A gene knockout showed signs of skeletal dysplasia.All of which indicate that Sema3A may have osteoprotective effects.At present,growth factors widely used in tissue engineering include bone morphogenetic protein(BMP),have confirmed that it can effectively enhance the expression of osteogenic related genes in vitro experiments.BMP acts on the defect area to promote bone regeneration in the form of protein combined with scaffold materials or combined with slow-release carriers.Using protein as a bone-promoting factor will face problems such as protein degradation,effective period of action,high cost and product stability.The process of osteogenesis is a long-term process,and we need a continuous supply of bone-promoting factors.Therefore,we hypothesized that the Sema3A gene was integrated into target cells through viral transfection and continued to play a role in promoting osteogenic differentiation while inhibiting osteoclast activation,and confirmed this hypothesis through in vitro and in vivo experiments.Based on the above understanding,we first isolated and cultured rat GMSCs in vitro,constructed a Sema3A gene lentivirus vector and successfully transfected GMSCs.Then under in vitro mineralization induction conditions,we detected the effect of Sema3A transfection on the osteogenic differentiation of GMSCs.Secondly,we established an indirect co-culture system in vitro to detect the osteoclastic differentiation of GMSCs transfected by Sema3A and explore related mechanisms;At the same time,the bone defect model of rat skull was made.Then,the effect of Sema3A modified GMSCs on the repair of skull defect was further studied by animal experiments in vivo,which provided theoretical basis for the study of its application in periodontal bone regeneration.Methods1.Effects of Sema3A transfection on the osteogenic differentiation of rGMSCs1.1 rGMSCs isolation,culture and identificationFour-week-old SPF grade Wistar rats purchased from the experimental animal center of Shandong University were used to obtain primary cells from buccal and lingual gingival tissues of mandibular molars by enzyme digestion.After digestion and passage,cells with stem cell characteristics were obtained by limited dilution clone method for subculture.The purified rGMSCs were analyzed by colony forming unit(CFU),flow cytometry to detect the expression of cell surface molecules,in vitro lipogenic induction culture and osteogenic induction culture,and the differentiation potential of rGMSCs was identified by oil red O staining and alizarin red staining.1.2 Construction of Sema3 A gene lentivirus vector and transfection of rGMSCsThe specific primers of Sema3A gene were designed and amplified.The target gene and vector(pLenO-GTP)were digested by enzyme and then linked.The linked product was transformed into DH5A.The virus packaging system and transfection reagent were added to 293T cells,and the supernatant of the virus was collected and stored at-80? after super concentration.rGMSCs were seeded in a 96-well plate at a density of 104 cells.The cells were divided into 3 groups:blank group,empty vector group(Lv-NC group),Sema3A gene transfected rGMSCs(Lv-Sema3A group),each group was set to 5 infections Multiplicity of infection(MOI)value(MOI=30,50,80,100,120),to screen the best MOI value of rGMSCs transfected by lentiviral vector;use the best MOI value to transfect rGMSCs in vitro,to observe the transfection efficiency by fluorescence microscopy,flowcytometry,real-time PCR and Western blot analysis of the feasibility of Sema3A gene transfection rGMSCs.1.3 Effect of Sema3A transfection on osteogenic differentiation of rGMSCsUnder the condition of mineralization induction,the mRNA and protein expression levels of ALP,Runx2,OCN were detected after Sema3A transfection.CCK-8 method was used to detect the cell proliferation.The formation of mineralized nodule of rGMSCs were detected,after then the intracellular calcium content was semi-quantitatively measured by enzyme scale after calcium ion was precipitated by cetylpyridinium chloride Analysis.The above experiments were repeated at least three times to analyze the effect of Sema3A transfection on the osteogenic differentiation of rGMSCs in vitro.2.The effect of rGMSCs transfected with Sema3A on the differentiation of osteoclast precursor cells in vitroThe mouse monocyte macrophages-RAW264.7 cells-were selected as osteoclast precursor cells to construct an indirect co culture system in vitro.rGMSCs,Sema3A transfected rGMSCs and empty vector transfected rGMSCs were co-cultured with RAW264.7 cells respectively.siRNA reduced the expression of Nrpl receptor of the Sema3A transfected rGMSCs and then co-cultured with RAW264.7 cells,tartrate resistant acid phosphatase(TRAP)staining1 was used to detect the specific differentiation of osteoclasts;Sema3A in the medium were detected by enzyme-linked immunosorbent assay;RANKL and OPG mRNA and protein of RAW264.7 cells were detected by real-time PCR and Western blot.The above experiments were repeated at least three times.3.Experimental study of Sema3A modified rGMSCs to promote the repair of rat skull defectsFifty-five SPF male Wistar rats of 8 weeks old were selected,and the rats were randomly divided into 5 groups,each with 10 rats,and the remaining 5 were reserved:Sema3A transfected rGMSCs with carrier complex group;empty vector transfection rGMSCs with carrier complex group;rGMSCs with carrier complex group;carrier group;blank group with simple bone defect.An animal model of the bone defect of the rat skull was prepared.Cells were planted in the collagen membrane,and the cell-collagen membrane complex was cultured and transplanted into the rat skull defect.The collagen carrier group was placed with an equal volume of collagen membrane and closed tightly.The group was sutured directly.After 8 weeks of routine feeding,intracardiac perfusion was performed to obtain bone and bone specimens.The effect of bone defect repair in each group was observed by gross observation,X-ray examination,HE staining,Masson staining and other methods.Results1.Effect of Sema3A transfection on osteogenic differentiation of rGMSCs1.1 Isolation,culture and identification of rGMSCsAfter the primary culture of rGMSCs for three days,the cells were observed to adhere to the wall under the inverted microscope.The cells were scattered and irregular in morphology.They were 80%-90%full of the bottom of the dish about one week,and could be subcultured.After subculture,the cells proliferated as colonies,and the morphology was simple as long fusiform,polygonal and irregular.The monoclonal units were obtained by limited dilution method and the rGMSCs with self-renewal ability were obtained by expanded culture.CFU results showed that the cloning rate of rGMSCs was about 58%.Flow cytometry showed that CD44,CD90 and CD29 were positive,CD45 and CD11b were negative.After 4 weeks of rGMSCs mineralization induction culture,alizarin red staining showed different sizes of mineralized nodules,and after 3 weeks of lipogenesis induction culture,oil red O staining showed the formation of red lipid droplets.1.2 Construction of Sema3A gene lentivirus vector and transfection of rGMSCsThe target gene Sema3A region was amplified by PCR.After purification,the PCR product was digested with BamHI/XbaI and ligated with the same digested vector(pLenO-GTP).The ligated product was transformed into DH5A competent,and the positive clone was identified and the plasmid was purified and extracted.The constructed vector carrying the Sema3A gene and the virus packaging system were co-transfected into 293T cells.After culturing for 48 hours,the cell supernatant was collected and purified and concentrated.The titer of the virus stock was obtained as the experimental group:2.7×109TU/ml;the empty vector group:1.8×109TU/ml,rGMSCs were transfected with MOI:80 and selected by puromycin,the transfection efficiency was over 99%by flow cytometry detection.Real-time PCR and Western blot confirmed that the expression of Sema3A gene is elevated in rGMSCs.1.3 Effect of Sema3A transfection on osteogenic differentiation of rGMSCsCompared with the control group,on the 3rd,7th,and 14th day of osteogenic induction culture,the expression of the bone-related genes ALP,Runx2,OCN mRNA and protein increased by Sema3A transfection of rGMSCs.CCK-8 results showed that compared with the control group,the proliferation activity of the Lv-Sema3A group cell was enhanced.Osteogenesis was induced and cultured for 28 days.Compared with the control group,the formation of mineralized nodules in the rGMSCs transfected with Sema3A was significantly increased compared with the control group.2.The effect of rGMSCs transfected with Sema3A on the differentiation of osteoclast precursor cells in vitroTRAP staining was used to detect the differentiation of RAW264.7 cells in the blank control group(RAW264.7 group)and co-culture group:RAW264.7-Control group,RAW264.7-Lv-NC group,and RAW264.7-Lv-Sema3A group.Compared with the blank control group,the numbers of TRAP+osteoclast-like cells in the co-cultured groups were all reduced;compared with the RAW264.7-Control group,the Sema3A gene transfection with rGMSCs significantly inhibited the TRAP+activity of RAW264.7 cells induced by RANKL.Osteoclast-like cells are formed;after the use of siRNA to reduce the expression of Nrpl receptor,rGMSCs transfected with Sema3A gene significantly weakens the inhibitory effect of RANKL-induced osteoclast differentiation of RAW264.7 cells.ELISA was used to detect the concentration of Sema3A in the co-culture medium after 3d,5d,and 7d induced culture.Compared with the RAW264.7-Control group,the Sema3A concentration in the culture medium of the RAW264.7-Lv-Sema3A group increased significantly and increased with the extension of the culture time.Real-time PCR and Western blot results showed that compared with the RAW264.7 group,the co-culture group had a lower expression level of RANKL mRNA,while the expression level of OPG mRNA increased,and the ratio of OPG/RANKL mRNA increased.Compared with the RAW264.7-Control group,the RANKL mRNA expression level of the RAW264.7-Lv-Sema3A group was significantly reduced,while the OPG mRNA expression level was significantly increased,and the OPG/RANKL mRNA ratio was significantly increased.3.The effect of Sema3A modified rGMSCs on the repair of rat skull defectsAfter gross observation of specimens,X-ray examination,HE staining of paraffin sections,and Masson staining analysis,rats in the experimental group with Sema3A modified rGMSCs as seed cells have a large amount of new bone formation in the skull defect area,new bones are dense and mature,and trabecular bones are arranged neatly,the repair effect is obviously better than the control group.Conclusions1.The lentiviral vector carrying the Sema3A gene could be constructed and transfect into rGMSCs,so that rGMSCs could express Sema3A stably and highly.Sema3A transfection positively regulates the proliferation activity and osteogenic capacity of rGMSCs.2.Sema3A transfection with rGMSCs inhibits the activation of osteoclast precursor cells,specifically in co-culture conditions,increasing the OPG/RANKL ratio and inhibiting the osteoclast differentiation of RAW264.7 cells.After siRNA reduces the expression of Nrpl receptor,Sema3A transfection of rGMSCs significantly reduces the osteoclast differentiation of RAW264.7 cells.3.Sema3A modified rGMSCs can significantly promote the repair of skull defects in rats.