Construction Of Tissue Engineering Cartilage Transfected With BMP7 Gene And Its Role Of Reparation Of Rabbit Articular Cartilage Injury

Cartilage injury is a common disease of orthopaedics, especiallythe articular cartilage injury is usual. Arthrosis disease isdifficult to cure because arthrosis cartilage is short of regenerationability. With the development of a new cross sciencetissue-engineering and gene therapy in the 1970-1980s, a newtherapical method of cartilage injury rises. The merits oftissue-engineering and gene therapy are: ①local gene therapy is ofpertinence due to articular cartilage injury usually occur at thelocal.②The activated protein expressed by objective gene in tissueengineering cartilage can induce cell division and proliferation,infiltrate to the outside of tissue engineering cartilage, inducemesenchyma stem cell differentiate into cartilage cell, acceleratethe integration between the tissue engineering cartilage and adjacenttissues integration. ③The activated protein expressed by objectivegene in tissue engineering cartilage can induce cell synthesizeextracellular matrix, inhibit cell de-differentiation, stabilizecell phenotype, be benefit for the construction of tissue engineeringcartilage which is similar to the normal cartilage. In this study we cloned BMP7 gene from young rabbit articularcartilage chondrocyte, constructed eukarya expression vector plasmid,transfected the plasmid into adult rabbit knee articular cartilagechondrocyte. Using positive clone chondrocyte transfected with BMP7gene, we constructed the tissue engineering cartilage with thechondrocyte-collagen-fibrin gel composite. The result demonstratedBMP7 gene were all expressed in the composite cultured in-vitro ondifferent days cultured. Chondrocyte in tissue engineering cartilagetransfected with BMP7 gene were active, the synthesis of matrix wasvigorous, cell division and proliferation accelerated, which weresuperior to that of non-transfected with BMP7 gene. Chondrocytecultured in-vitro on 14 days were the most active, which weretransplanted into the articular cartilage defect of rabbit. Theresults showed that the repairation of tissue engineering cartilagetransfected with BMP7 gene were better than that of non-transfectedwith BMP7 gene. The study explored the combining of tissue engineeringand gene engineering technique, the results established theoreticaland experimental base for tissue engineering cartilage transfectedwith BMP7 gene applying to clinic.1 Construction of recombinant pcDNA3.1-BMP7 plasmid and itsexpression in rabbit articular cartilage1.1 Construction of recombinant pcDNA3.1-BMP7 plasmid andidentification1.1.1 Clone of BMP7 gene The total RNA was extracted from rabbit knee articular cartilagecell cultured in vitro,A260/A280=0.838,cDNA library was establishedusing RT-PCR method, PCR product was 1.3kb,was purified by gelextraction mini kit.1.1.2 Construction of recombinant pcDNA3.1-BMP7 plasmid Extraction product was ligated into T-vector and ligation wastransformed into JM109 cell,T-JM109 plasmid and pBluescript (M13-)were enzyme digested by HindⅢand Kpn I, ligated. The ligation wastransformed into DH5αcell and achieved pBlue-BMP7. pBlue-BMP7 andpcDNA3.1 were enzyme digested by Not Iand Kpn I,ligated, transformedinto DH5αcell and achieved pcDNA3.1-BMP71.1.3 Sequence analysis Sequence analysis of BMP gene indicated BMP7 DNA was 1.3Kb. BMPgene of T-BMP7,pBlue-BMP7 and pcDNA3.1-BMP7 were consistent withthat of genebank1.1.4 Expression vector pBlue-BMP7 and pcDNA3.1-BMP7 eukarya expression vector wereconstructed.1.2 Recombinant pcDNA3.1-BMP7 plasmid transfected cartilage cell andits expression1.2.1 Transfecting rabbit articular cartilage chondrocyteRecombinant pcDNA3.1-BMP7 transfected adult rabbit cartilage cellsand screened with G418.liposome and pcDNA3.1-BMP7 were 10mland4μg(2.5:1),the efficiency of transfection was high(15～20%),theoptimal concentration of G418400μg/ml. the efficiency of transfectionof primary and second culture cartilage cells were the highest.Positive clones still existed after 28 days screened with G418 andfusion efficiency was 100%.1.2.2 Expressing of BMP7 gene in rabbit articular cartilagechondrocytes BMP7 gene expression of chondrocytes transfected withpcDNA3.1-BMP7 was detected after 7 and 28 days screened by G418.about18KD was achieved by SDS-PAGE and Western blot. immunofluorescencetest and in situ hibrydization were positive.Brief summary: Recombinant pcDNA3.1-BMP7 eurakya expression vectorwas transfected into adult rabbit chondrocytes successfully. Theefficiency of transfection was the highest when the ratio of liposomeand DNA was 2.5:1.the optimal concentration of G418was 400μg/ml. theefficiency of transfection of primary and second chondrocytes werethe highest. Expression protein was stable and was about 18KD.2 Construction of tissue engineering cartilage transfected withBMP7 gene and its role of reparation of rabbit articular cartilagedefect.2.1 Construction of tissue engineering cartilage transfected withBMP7 gene. Rabbit chondrocytes were isolated and cultured with IMDM for 3-4days. recombinant pcDNA3.1-BMP7 was transfected and screened withG418 for 14 days. Positive clones fusion efficiency was 100%.cellsupernatant and 0.5% collagen from the tails of rabbits mixed equally,added 8%NaHCO3 , thrombin 20mg/ml(4:1), chondrocytes with BMP7 gene-collagen-fibrin gel composite are cultured, negative was done. Theresult showed that the culture was about 16.0mm×16.0mm×3.0mm,thecells were round and well distributed, the cultures were ivory,transparency degraded, the support began to shrink after cultured 3-4days in vitro, and shrinked distinctly for 5～7 days, after that itstopped shrink, the culture with non-transfected cells was about 8.0mm×8.0mm×1.5mm while the culture with transfected cells was about9.0mm×9.0mm×2.0mm, tissue engineering cartilages transfected withBMP7 gene were bouncy, were superior to that of non-transfectedwith BMP7 gene.2.2 Structural and functional analysis of tissue engineeringcartilage transfected with BMP7 gene The constructed tissue engineering cartilage was identified bystaining of HE ,II-type collagen mRNA in situ hybridization andtolonium chloride. Chondrocytes in tissue engineering cartilagetransfected with BMP7 gene were active, organelle were abundant, thesynthesis of matrix was vigorous, which were superior to that ofnon-transfected with BMP7 gene. Chondrocytes cultured in-vitro on 14days were the most active. It showed that the BMP7 protein was ofbiological activity, which induced cell division and proliferationand the synthesis of extracellular matrix, elapsed biochemistricalanalysis indicated that the contents of DNA were the highest afterthe cartilage were cultured for 21 days, having no significance ofdifference compared with 14 days; the contents of GAG were thehighest on 14 days, similar to that of 21 days. furthermore, comparedwith the control, tissue engineering cartilage transfected with BMP7gene had abundant DNA and GAG. in situ hybridization andimmunohistochemical demonstrated that tissue engineering cartilagetransfected with BMP7 gene expressed BMP mRNA and protein, the controlexpressed weakly. All the above proved that the division and proliferation ofchondrocytes in tissue engineering cartilage transfected with BMP7gene accelerated, organelle were abundant, the synthesis of matrixwas vigorous and expressed BMP mRNA and protein, At the same time,the BMP7 protein was of biological activity. It showed that weconstructed tissue engineering cartilage successfully, whichprovided bases for transgene tissue engineering cartilagetransplantation and transplantation time.2.3 Reparation of rabbit articular cartilage injury by tissueengineering cartilage transfected with BMP7 gene5.0mm×5.0mm×3.0mm full thickness defect was made on thesurface of patella of distal end of femur of adult rabbit articularcartilage. Transplantation by tissue engineering cartilagetransfected with BMP7 gene were better than that of non-transfectedwith BMP7 gene. The results showed that the defect region of reparationgroup of tissue engineering cartilage transfected with BMP7 gene andthat of non-transfected with BMP7 gene were all filled up by ivorygrafts after 4 weeks, but the former was lighter , the height ofrepaired tissue were equal to the surface of the surrounding articularcartilage, having bound between the tissue and peripheral normaltissue, but no gap and fissure, having smoothy surface; the repairedtissue of the group of non-transfected with BMP7 gene were lower thanthe surface of the surrounding articular cartilage, although thelatter could repair rabbit articular cartilage injury. The studyexplored the combining of tissue engineering and gene engineeringtechnique, peripheral tissue of impairment region of the twogroupshad no apparent immunologic rejection, impairment region ofnon-therapy group sunk, filled by fibrous tissue. morphology showedthat the filler was identified by staining of HE and toloniumchloride to be cartilage tissue.brief summary: the reparation of articular cartilage defecttransfected with BMP7 gene were superior to that of non-transfectedwith BMP7 gene. in situ hybridization and immunohistochemistry provedthat the repaired tissue after transplantation for 4 weeks expressedBMP mRNA and protein in the region of defect. Further reparation workwould still be studied on.Summary 1.We cloned BMP7 gene from young rabbit knee articular cartilagechondrocyte, sequence analysis of BMP gene indicated BMP7 DNA was1.3Kb, BMP7 gene cloned were consistent with that in genebank; 2.Recombinant pcDNA3.1-BMP7 eurakya expression vector wasconstruted and transfected into adult rabbit articular cartilagecells successfully. we identified mRNA and protein of BMP7 wereexpressed in the cells; 3.We constructed tissue engineering cartilage transfected with...