The Application Of Biodegradable Fiber Scaffold And MiR-495 In The Repair Of Bone And Cartilage Injury

With the acceleration of human production and life rhythm andaging of population,the patients with bone and cartilage injuries show an increasing trend year by year.There are many causes of bone and cartilage damage,including accidents,bone tumors,congenital diseases,aging and other factors.In these diseases of bone injury,how to treat the large size bone defect is a difficult problem in clinical practice,such bone injury diseases are difficult to heal,and it is easy to cause bone nonunion.At present,there are mainly autologous and allogeneic bone transplantation.Although there is no immune reaction and osteogenic ability,the source of autologous bone is limited,and the patient still has to experience the pain caused by the second surgery for bone extraction.Allogeneic bone has sufficient sources,which can meet the large demand of clinical treatment for bone defects.However,autogenous bone is not the best solution for bone defects due to the risk of immune rejection and viral infection.In order to solve the above problems,using bone tissue engineering technology to prepare bone defect substitutes has become an important issue in this field.As bone substitutes should possess the following characteristics:(1)the porous structure of osteoblast adhesion and proliferation,(2)the suitable mechanical properties and mechanical strength,(3)materials should have good biocompatibility,and absorbability in vivo,(4)has a good ability of osteoblast differentiation.Therefore,a good substitute for large bone defect should possess these characteristics.In the aspect of cartilage injury,this study mainly focused on OA caused by aging,apoptosis,differentiation and other factors.As a kind of endogenous non-coding RNA,miRNAs can regulate the expression of multiple genes in the body,thus affecting the function of these cell.miRNA can specifically interact with mRNA,promote the degradation of mRNA and inhibit the translation of mRNA.Many studies have found that many miRNAs are up-regulated and down-regulated in chondrocytes of patients with OA,indicating that miRNAs can be involved in the formation of OA and the progression of OA.This study intends to study the mechanism and relationship between miR-495 and the formation of OA,so as to find a new target for the treatment of OA.Based on the above ideas,the content of this study is as follows:(1)Melt-spinning method was used to prepare the bone defect repair scaffolds with micro-nano diameter.Compared with the traditional electrospinning technology,the fiber scaffolds prepared by this technology with higher porosity and better three-dimensional structure.PLGA and HA were used to prepare the fibers.PLGA has good biological activity and in vivo degradability,and HA has good osteogenic ability and mineralization.From the aspect of materials and preparation technology,PLGA and HA used in this study can fully meet the requirements of being used as bone repair substitutes.In order to further improve the hydrophilicity,cell adhesion and proliferation ability of the material surface,a layer of PDA was formed on the surface of the fiber scaffold by polymerization of DOPA under alkaline conditions,and BMP-2 growth factor was adhered on the PDA with the adhesion ability of PDA.The results showedthat the surface hydrophilic ability was significantly improved,and the absorption ability of BMP-2 was significantly increased.After 28 days of release detection,the Bmp-2 presented a slow release characteristic.These results indicate that we have successfully prepared a bone repair scaffold with slow release of BMP-2,good hydrophilicity and high porosity.(2)MC-3T3-E1 cells were used to evaluate the cell adhesion,proliferation and osteogenic induction ability of the scaffold.The PLGA group showed the lowest cell proliferation rate,while the PLGA/HA group showed significant improvement.This is mainly because HA can improve the hydrophilicity of the material surface and increase the surface roughness,all of which are conducive to cell adhesion and proliferation.When PDA was adhered to the material surface,the cell proliferation rate increased compared with the control group.The group with the highest cell proliferation rate occurred in PDA-PLGA/HA/BMP-2.In the effect of osteogenic gene expression,the expression levels of RUNX2 genes in PLGA/HA and PDA-PLGA/HA were 26% and 61% higher than those in the PLGA group.The RUNX2 gene expression of PDA-PLGA/HA/BMP-2 was 1.25 times higher than that of PDA-PLGA/HA.These results suggest that the introduction of HA and BMP-2 can effectively promote the expression of RUNX2.The expression of OPN gene was the lowest in PLGA,the expression of OPN in PDA-PLGA/HA was 4 times higher than that in PLGA group,and the expression of OPN in PDA-PLGA/HA/BMP-2 was the highest.(3)The effects of overexpression or downregulation of miR-495 on apoptosis,growth and wound repair of chondrocytes were evaluated in vitro.The results showed that the high expression of miR-495 significantly promoted the apoptosis of chondrocytes and inhibited their proliferation.The transfection of miR-495 significantly inhibited the regeneration ofscratch wounds,resulting in reduced regeneration ability of chondrocytes.In addition,we also observed that the transfection of miR-495 can promote the activation of caspase-3 and further promote the apoptosis of chondrocytes.miR-495 directly down-regulated the expression of AKT1 in chondrocytes,while miR-495 inhibitor effectively enhanced the expression of AKT1.Therefore,miR-495 may promote chondrocyte apoptosis by targeting AKT1,and AKT1 overexpression inhibited the effects of miR-495 transfection on chondrocyte apoptosis,growth.This study provides a new drug target for the treatment of OA caused by the apoptosis of chondrocytes.In this study,a kind of bone defect repair scaffold was prepared by using melt-spinning method and BMP-2 binding via PDA.This scaffold can be used for large size bone defect repair.Meanwhile,the role of miR-495 in the apoptosis of chondrocytes was further studied,and the molecular mechanism of miR-495 inducing the apoptosis of chondrocytes was discovered.