Preparation And Properties Of Gene Enhanced Tissue-engineered Scaffolds

The tissue and organ damage or functional disorder caused by trauma,tumor and other factors are seriously harmful to the health of human.Tissue engineering(TE),as a new method of tissue repair and regeneration,has the advantages of unlimited source,no immune rejection,no etiology,no risk of infection and so on when compared with the traditional auto/allogenic transplantation,and has made gratifying progress in promoting tissue defect repair and regeneration.However,cytokines are mainly used by directly loading on slow-release scaffolds in traditional TE,they are easy to be inactived rapidly due to the dispersion,transport and degradation in the physiological environment in vivo,so the exogenous cytokines can not produce the expected biological effect,which makes the application of TE seriously limited.In recent years,gene enhanced tissue engineering,which combines tissue engineering and gene engineering,has emerged.It uses gene engineering technology to transfer coding genes of specific cytokines into seed cells or onto scaffolds,so that the cytokines can be expressed in vivo and play the biological role in promoting cell proliferation and differentiation,thus promoting tissue repair and regeneration.However,the research of gene enhanced tissue engineering mostly stays in the stage of using the gene transfected terminal differentiated cells as seed cells or using the coding gene of a known growth factor to transfect the stem cells,which makes it can only be used for the repair and regeneration of a single damaged tissue due to the poor proliferation ability of terminally differentiated cells,or makes its biological function unstable and uncontrollable due to the multiple biological effects of some growth factors,thus seriously limiting its clinical application.In addition,the tumor related researches are mostly focused on tumor inhibition,and there is no report about the repair and regeneration of defect in tumor model.In fact,most tumors are destructive and will cause damage to the surrounding tissues.Therefore,the integrated treatment of tumor inhibition and defect repair agrees with the actual situation of clinical practice,the systematic investigation of tumor inhibition and defect repair can provide a reference for clinical application.In this paper,the infection efficiency of the gene delivery vector to different kinds of bone marrow mesenchymal stem cells(BMSCs)was investigated via the optimization of the target gene and the improved design of the gene delivery vector,and the individualized construction of a variety of gene enhanced tissue-engineered scaffolds was successfully realized.The animal experimental models of osteochondral full-thickness defect of rabbit knee joint,cartilage full-thickness defect of rabbit knee joint,bone defect of proximal tibia of osteosarcoma bearing rat and full-thickness defect of skin of melanoma bearing mouse were established introspectively according to the defect on different types of tissue or caused by the different pathological factors.The potential applications of different types of gene enhanced tissue-engineered scaffolds in the fields of the repair and regeneration of full-thickness defects of osteochondral,the repair and regeneration of full-thickness defects of cartilage,the repair and regeneration of tibia defect and osteosarcoma inhibition,the repair and regeneration of skin full-thickness defect and melanoma inhibition were explored.The main works and results of this paper are as follows:1.The porous HA scaffolds with complete pore structure,uniform pore size and good perforation performance were prepared by the organic foam interface mineralization method,which solved the problems of incomplete pore structure,uneven pore size and frequent plugging in the traditional organic foam impregnation method.The recombinant adenovirus vector carrying BMP-2 and Wnt3? coding genes was constructed by knocking out E1 and E3 genes at the same time,which solved the problems of high cytotoxicity,strong host immune response and low infection efficiency of the traditional recombinant adenovirus vector.The gene enhanced tissue-engineered scaffolds combined with the oxidized sodium alginate/N-succinyl chitosan hydrogel loading with rabbit BMSCs which were infected by the recombinant adenovirus vector carrying Wnt3? coding genes and the porous HA scaffolds loading with rabbit BMSCs which were infected by the recombinant adenovirus vector carrying BMP-2 coding genes were constructed and then applied to the research of repair and regeneration of full-thickness defects of osteochondral.The experimental results showed that the scaffolds promoted the integration between the new osteochondral tissue and the host osteochondral tissue,maintained the stability of cell phenotype,realized the complete repair and regeneration of the full-thickness osteochondral defects,and promoted the recovery of the repaired joint function.2.The polyglutamic acid/poly lysine macroporous hydrogels with intrinsic antibacterial properties were prepared by gas self foaming method.The recombinant lentivirus vector carrying CLECSF1 coding gene was constructed by knocking out tat,rev,nef,vir,vpr and vpu genes at the same time,which fully guaranteed its biological safety and improved its infection efficiency to a certain extent.Rabbit BMSCs infected by recombinant lentiviral vector carrying CLECSF1 encoding gene was combined with polyglutamic acid/poly lysine macroporous hydrogel to construct the gene enhanced tissue engineering scaffolds.Then,it was applied to the research of repair and regeneration of of full-thickness defects of cartilage.The experimental results showed that the gene enhanced cartilage scaffolds promoted the integration of the new cartilage and the host cartilage,promoted the synthesis and secretion of cartilage specific extracellular matrix protein and cartilage specific collagen,achieved the complete repair and regeneration of the full-thickness cartilage defect,and restored the function of absorbing mechanical impact of the repaired joint.3.The porous HA scaffolds with complete pore structure,uniform pore size and uniform pore size and good perforation properties were prepared by using organic foam impregnation interface mineralization method.The recombinant adenovirus vector carrying BMP-2 encoding gene was constructed by knocking out E1 and E3 gene simultaneously.The recombinant lentiviral vector carrying the Bax encoding gene was constructed by knocking out tat,rev,nef,vir,vpr and vpu simultaneously.Then,rats BMSCs infected by recombinant adenovirus vector carrying BMP-2 encoding gene and rats BMSCs infected by recombinant lentivirus vector carrying Bax encoding gene were combined with porous HA scaffolds to construct gene enhanced tissue-engineered bone scaffolds,which were then applied to the research of repair and regeneration of tibia defect and osteosarcoma inhibition.The experimental results showed that the scaffolds could inhibit the development of osteosarcoma and lung metastasis,promote the regeneration of bone defect,and play a positive role in protecting the liver and kidney of osteosarcoma bearing rats.4.The polyglutamic acid/poly lysine macroporous hydrogel with intrinsic antibacterial properties was prepared by gas self foaming method.The recombinant lentiviral vector carrying Bax encoding gene was constructed by simultaneous knockout of tat,rev,nef,vir,vpr and vpu.Mouse BMSCs infected by the recombinant lentiviral vector carrying Bax encoding gene was combined with polyglutamic acid/poly lysine macroporous hydrogel to construct gene enhanced tissue-engineered skin scaffolds,and then applied to the research of the repair and regeneration of skin full-thickness defect and melanoma inhibition.The experimental results showed that the scaffolds could prevent melanoma recurrence and inhibit lung metastasis,promote the complete repair and regeneration of skin defects,and effectively protect the heart,liver and kidney of melanoma bearing mice.The above results indicate that the as-prepared scaffolds can not only promote the regeneration of single or two kinds of tissue defects under physiological conditions,but also achieve the integrated treatment of defect repair and tumor inhibition under tumor conditions.It provides a powerful tool for the study of multiple tissue defect repair and regeneration in clinical practice,and lays a foundation for the study of defect repair and pathogenicity removal and inhibition under other pathological factors.