Effects Of Canine Myoblast Transfected With Human Cartilage-Derived Morphogenetic Protein-2 (hCDMP-2) Gene On The Repair Of Meniscal Fibrocartilage Injury

With the development of modern economy,the type of injury has transformed from battery injury and contusion to traffic accident injury and sports injury among which joint injury is of greatest morbidity. Accordingly,the injury of fibrocartilage in the joint,such as meniscus, glenoid labrun of shoulder and hip,triquetrous cartilage of wrist,became more and more often,which attracted the attention of specialists. Although the above structures are critical to corresponding joint,they cannot heal by themselves if injured and will result in severe consequence of osteoarthritis without prompt treatment.In recent years,tissue engineering combined with stem cell therapy was applied to treat the injury of fibrocartilage;however,there existed the problems of selection, acquisition of seed cells and the quality of repaired tissues.Myoblasts were regarded as ideal donor cells because of their abundant sources, obtainability,potentials of stem cells,strong survival ability and characteristics of rapid proliferation in vitro.Myoblasts were considered better in biopsy,manipulation and clinical application than other stem cells,such as bone mesenchymal stem cells.In addition,myoblasts have been proved to resist ischemia and promote neovascularization of injured tissue,which would be good to the repair of fibrocartilage with insufficient blood supply.Cartilage-derived morphogenic protein-2(CDMP-2)has been proved to promote cartilage repair without ossification and promote the fibro-tissue formation,thus CDMP-2 was considered an ideal cytokine for promoting the repair of fibrocartilage. Our research will transfect the human cartilage-derived morphogenic protein-2(hCDMP-2)gene into the isolated and purified canine myoblasts and transplant the complex of transfected myoblasts and PLA/PGA scaffold to the injured canine meniscus,by which we hope to promote the fibrocartilage-like repair of injured meniscus,improve the quality of healed tissue and look for an effective approach to treat the meniscal injury.Objectives:To explore whether the isolated and purified canine myoblasts would be transformed to fibrochondrocytes in vitro when induced by hCDMP-2 cytokine.If succeeding,we will further explore the effects of canine myoblasts transfected with hCDMP-2 gene on the repair of canine meniscal fibrocartilage by setting up animal experiments with suitable grouped tests.Materials and Methods:The muscle from the hindlimb of adult Beagle canine was obtained,from which myoblasts were isolated and harvested by mechanical decomposition and two-step enzyme digestion. Then the cells were purified by combination of method of differential adherent velocity and flow cytometry.The morphologic properities and the growth state of myoblasts were observed and the growth curves were drawn.In order to precisely characterize the phenotype and purity of these cells,flow cytometry,polymerase reaction with reverse transcription(RT-PCR)and immunocytochemistry analyses were performed.The purified canine myoblasts were induced by hCDMP-2 cytokine in vitro.The morphologic properities and the growth state of cells induced were observed and the growth curves were drawn.In order to precisely characterize the fibrochondrocyte-like phenotype of these cells,RT-PCR and immunocytochemistry analyses were performed. hCDMP-2 CDS sequence was retrieved in PubMed and the sequence was synthesized.The PCR production of hCDMP-2 gene was cloned,and then the positive clone was screened and identified by BLAST.The hCDMP-2 gene was then transfected into the purified canine myoblasts through lentivirus vector.The transfection ratio and expression of hCDMP-2 gene by transfected myoblasts was verified by Real-time Fluorescence Quantitative PCR.The expression of hCDMP-2 protein was also identified by westernblot.The PGA(Sigma,USA)scaffold material was shaped by specific mould and was added with PLA(Sigma, USA)to form the PLA/PGA scaffold.The model of canine meniscal injury was as follows:a defect of 2mm-width,2mm-thickness near the capsule,1.5mm-thickness near the the cavity,whole length of transverse diameter without reservation of basement at the anterior horn of the meniscus 0.5cm distant from the anterior edge was made,which involved the red-red,red-white and white-white areas of meniscus.To perform the animal experiments of canine meniscal repair in vivo,group of pure suture(group A),group of PLA/PGA scaffold containing hCDMP-2 cytokine combined with suture(group B),group of PLA/PGA scaffold containing myoblasts transfected by lentivirus combined with suture (group C)and group of PLA/PGA scaffold containing myoblasts transfected with hCDMP-2 gene combined with suture(group D)were established.The repaired tissues were taken off at 3^rd,8^thand 12^thweek after injury respectively and observed in gross appearance first.Then the morphology(HE staining and Safranin-O staining)was observed, immunocytochemistry analyses for collagenⅡ,S-100 and collagenⅠwere performed,quantative ELISA analyses(n=6)for collagenⅡand collagenⅠwere performed and quantative Alcian Blue analyses(n=6)for GAG were performed.Results:1.Our method had a high possibility of success in obtaining the adult canine myoblasts with high survival rate in vitro and the myoblasts grew very well.Flow cytometry demonstrated that about 98% of P4(purified and passaged by 4 times)adult canine muscle cells were positive for CD56 and negative for fibroblastic marker TE7.RT-PCR analysis showed that P4 cells expressed the myogenic cell-related markers such as MyoD and Myogenin.Immunocytochemistry staining showed that P4 cells were positive for desmin.All these characteristics indicated successful primary culture of adult canine myoblasts with high purification.2.After induced by hCDMP-2 cytokine in vitro,the morphology of the purified P4 canine myoblasts was transformed from spindle to polygon;the Sox9 gene which is specific for chondrocytes began to be expressed at the 20^thday after induction(the 11^thday of PI2)and the expression increased with time lapsing by RT-PCR;collagenⅡ,aggrecan which are also specific for chondrocytes and collagenⅠwhich is specific for fibrochondrocytes began to be expressed at the 25^thday after induction(the 5^thday of PI3)and the positive ratio increased with time lapsing by immunocytochemistry.The results above indicated that the purified canine myoblasts would have the phenotype of fibrochondrocytes when induced by hCDMP-2 cytokine in vitro,which ensure the feasibility of our further experiments.3.The hCDMP-2 plasmid were successfully constructed with artificially synthesized CDS sequence and then the hCDMP-2 gene was transfected to the purified canine myoblasts through lentivirus vector in vitro with the transfection ratio of about 71%by Real-time Fluorescence Quantitative PCR.Myoblasts transfected with hCDMP-2 gene expressed hCDMP-2 gene by Real-time Fluorescence Quantitative PCR and hCDMP-2 protein by westernblot.4.The animal experiments of canine meniscal repair in vivo showed: (1)There were no newborn tissue at the defective area of canine meniscus of group A at the 3^rd,8^thand 12^thweek after injury and the size of the area was nearly unchanged.At the 3^rdweek after injury,the scaffold material still existed at the defective area with smaller size in group A,B and C. At the 8^thweek,the scaffold material of group A,B and C disappeared completely and an identical defect was left.The above results showed that there was no newborn tissue in the canine meniscal defect by the treatment of group A,B and C and the PLA/PGA scaffold material could degrade with the degradation time of 8 weeks.(2)At the 3^rdweek,the scaffold material still could be seen with smaller size at the defective area in group D.A little cartilage lacuna could be seen at the red-red area of repaired tissue by HE staining while the white-white area was not stained. The result of red-red area of repaired tissue stained by Safranin-O was positive while the white-white area was negtive.The collageⅡ,S-100 protein and collagenⅠwere weakly expressed at the red-red area of repaired tissue by immunohistochemistry.At the 8^thweek,the repaired tissue in group D whose color and luster was dark and textile was soft still existed full of the defective area without scaffold material.More cartilage lacuna and a little fiber could be seen at the red-red area of repaired tissue by HE staining and a little cartilage lacuna without fiber could be seen at the white-white area.A larger scale of red-red area of repaired tissue with deeper color than at the 3^rdweek was stained by Safranin-O staining and the white-white area was also stained with red corlor.The collageⅡ,S-100 protein and collagenⅠwere all expressed at both the red-red area and white-white area of repaired tissue by immunohistochemistry.At the 12^thweek,the repaired tissue in group D whose color and luster approximated to the normal meniscus,texitile was harder and density was higher still existed with size unchanged.Obvious cartilage lacuna and fiber could be seen at the red-red area of repaired tissue by HE staining and more cartilage lacuna still without fiber could be seen at the white-white area.Both the red-red area and the white-white area of repaired tissue were stained by Safranin-O staining with a larger scale and deeper color than at the 3^rdweek.The collageⅡ,S-100 protein and collagenⅠwere all expressed at both the red-red area and white-white area of repaired tissue by immunohistochemistry too.These results demonstrated that there were newborn tissues at the defective area by treatment of group D and the repaired tissue had the characteristics of fibrocartilage.(3)At the 3^rdweek after injury,some collageⅡ,collagenⅠand GAG were detected at the red-red area of repaired tissue in group D while they were not detected at the white-white area.At the 8^thweek,the content of collageⅡ,collagenⅠand GAG at red-red area of repaired tissue in group D were higher than at the 3^rdweek(P＜0.01)and these proteins were also detected at the white-white area.At the 12^thweek, collageⅡ,collagenⅠand GAG with higher contents than at 8^thweek (P＜0.01)were detected both at the red-red area and at the white-white area of repaired tissue in group D.At the same time during the whole process of repair in group D,the content of these proteins at the red-red area of repaired tissue was significantly higher than at the white-white area(P＜0.01),which indicated that the repair speed at the red-red area of repaired tissue was higher than that at the white-white area.However,at the same time during the whole process of repair in group D,the content of these proteins at the red-red and the white-white area was significantly lower than that of at respective area of normal meniscus(P＜0.01).Conclusions:1.Our method for isolating and purifying myoblasts in vitro proved to be convenient and practical with high possibility of success and low price.Studies of cell therapy using primary myoblasts can now be broadly applied to canine models of human muscle and non-muscle diseases.2.The purified canine myoblasts could have the phenotype of fibrochondrocytes after induced by hCDMP-2 cytokine.3. The canine meniscal defect in our research could not be repaired by pure suture,applying hCDMP-2 cytokine only or using purified myoblast therapy only.4.The purified myoblasts transfected with hCDMP-2 gene could promote the repair of canine meniscal fibrocartilage of each area and the repair speed of the red-red area was higher than that of the white-white area.5.The method used in our research may provide a new approach to treat large meniscal defects.6.The therapy of purified myoblasts combined with hCDMP-2 cytokine could promote the the repair of canine meniscal fibrocartilage,however related therapeutic mechanism and approach of future clinical application remains unknown.