Study On Fibrication Of Micro RNA-loaded Tissue Engineering Scaffolds For Osteoinduction

Bone defects caused by trauma,bone disease or bone tumors are serious clinical problems that affect human life and health.Although the traditional bone defect repair materials can partially meet the needs to replace the defective parts,there still exists some drowbacks such as lack of function and angiogenesis,which make it almost impossible to achieve "complete repair" in structure and function,in other words,"regeneration".The induction on stem cells to grow into scaffolds and differentiate into osteoblasts to generate new bone is the key to "regeneration".Therefore,the development of adjustable stem cell behavior and bone tissue regeneration process of bioactive materials with the ability to regulate the behaviour of stem cells and new bone formation is crucial to bone regeneration.microRNA plays an important role in the regulation of stem cell differentiation,bone formation and homeostasis.Compared with growth factors,mi RNA has the advantages such as simple structure,easy to synthesize and operate,uneasy to inactivate,and it can specifically genetically regulate the behavior of cells.Thus microRNA becomes the most popular research field of medicine in the past decade.The main purpose of this research is to construct a bioactive scaffold with precise structure and osteoinductive properties by integrating the bioacitve signal,miRNA,with the three-dimensional porous scaffold.Then futher study its effect on osteoblastic differentiation of stem cells and ectopic osteogenesis in vivo.(1)Construction of mi RNA nano-delivery vectorThe efficiency of miRNA delivery is highly dependent on the design of nano-vectors.Therefore,polyethyleneimine-capped gold nanoparticles(AuNPs),an effective vector for small nucleic acid delivery,was applied in this study to intracellularly deliver miRNA into stem cells and regulate osteoblastic differentiation.The results show that AuNPs can protect and efficiently transport miRNAs into human bone marrow mesenchymal stem cells(hMSCs)and mouse preimplantation osteoblasts(MC3T3-E1)with negligible cytotoxicity,compared to commercially available lipofectamine,which has significant cytotoxicity.It was found that AuNPs / miRNA composite particles could synergetically enhance osteoblastic differentiation and promote the mineralization process.It was further observed that AuNPs were mainly distributed on the endoplasmic reticulum,suggesting that the possible mechanism of induction effect of AuNPs on osteogenic differentiation might be that Au NPs may exert appropriate stress stimuli on the endoplasmic reticulum,activating endoplasmic reticulum stress,and thus affect the expression of osteo-related proteins.(2)Construction of non-AuNPs / miRNA loaded three-dimensional gel scaffoldsIn order to further realize the application of miRNA in bone regeneration and repair,the introduction of three-dimensional porous scaffold is essential,which can work as the carrier for drug release and temporary template for the ingrowth of cells and tissues.Considering the excellent drug loading and release ability of gelatin and sodium alginate,the mixure of these two kind of gels was used as the scaffold material to load miRNAs.The three-dimensional fiber deposition technology was used to generate the 3D scaffold,and the effect of gel crosslinking degree on the physical and chemical properties of the scaffolds was also systematically studied.The results show that the crosslinking degree of gel has no significant effect on the morphology,pore sturcture,porosity and pore size distribution of the scaffolds,but can significantly affect the swelling rate,degradation rate and mechanical properties.On the other hand,scaffolds with different pore structures were obtained by controlling the oritations of fiber deposition.It was found that the pore structure had no significant effect on the porosity,water swelling and degradation properties of scaffolds,but had a significant effect on the pore size distribution and mechanical properties.The establishment of the above system provides a material basis for the construction of AuNPs / mi RNA loaded three-dimensional scaffolds.(3)Construction and Osteogenic induction on stem cells of AuNPs/miRNAs-loaded gel scaffoldsUsing the swelling properties of hydrogel,AuNPs/miR-29 b nanocomposite were successfully loaded onto scaffolds with different crosslinking properties by physical adsorption.It was confirmed that the nanocomposites were uniformly distributed on the surface as well as inner side of scaffolds.The scaffolds could store and release the nanocomposites.The surface of gel scaffolds can be mineralized by forming calcium and phosphorus nanostructures,which is conducive to osteogenic differentiation and bone matrix formation.And we further explored the synergistic effect of miRNA and gel crosslinking on the osteogenic differentiation of stem cells.In vitro studies showed that the crosslinking degree of scaffolds had a significant effect on the adhesion and spread of hMSCs.On the stiffer scaffolds,there were more adhered cells and the cell spreading area increased.In the early stage of osteogenesis differentiation,scaffolds with low crosslinking degree could release more miRNAs,and thus the effect on osteoblastic differentiation of hMSCs was more obvious.In the middle and late stages,under the synergistic effect of miRNA release and crosslinking properties of scaffolds,the effect of scaffolds with high crosslinking degree on the osteogenesis of hMSCs was more significant.The scaffold system was further applied to the in vivo heterotopic osteogenesis model of subcutaneous implantation in nude mice to study its effect on stem cell differentiation and new bone formation.After two and four weeks of implantation in nude mice,both blank scaffolds and miRNA-loaded scaffolds were able to induce fast ingrowth of cells and tissues,and the degradation rates of scaffolds controlled by the crosslinking degree had an obvious effect on the ingrowth of cells and tissues.Tissue staining and immunohistochemistry showed a positive correlation between osteogenesis and angiogenesis,consistently demonstrating that miRNAloaded scaffolds with high crosslinking degree were more favorable for osteogenesis and angiogenesis.The physicochemical properties(permeability,degradation rate,stiffness,etc.)of the scaffolds regulated by the crosslinking degree and the release behavior of miRNAs provide the mechanical and biological signals for the osteogenic differentiation of hMSCs,and synergistically regulate the differentiation behavior of hMSCs and new bone formation.The miRNA-loaded three-dimensional porous gel scaffolds are a new exploration of the combination of mi RNA technology and regenerative medicine,and give the the new functions to regenerative scaffolds by cross-domain.This study can also provide theoretical basis and new ideas on the application of mi RNA to bone regeneration and the new generation of bone repair materials.