In Vitro Study Of Fibrocartilage Tissue Engineering Using TGF-β1 Modified Synovial Mesenchymal Stem Cells Of Rabbit Temporomandibular Joint

PartⅠConstruction of Recombinant Adeno-associated Virus with TGF-β1Objective To construct and confirm the recombinant adeno-associated virus with transforming growth factorβ1 (TGF-β1).Methods Both plasmids of pcDNA3-TGF-β1 and pAAV-MCS were linked after being digested by enzyme of EcoR I+Xba I, and the recombinant plasmids of pAVV-MCS-TGF-β1 were collected from the colon bacillus DH5a transformed by the linked plasmids. The recombinant plasmids were confirmed by enzyme digestion and DNA sequence examination. The recombinant virus rAAV-TGF-β1 were collected from the transfected AAV-293 cells which were co-transfected by pAAV-MCS-TGF-β1, pAAV-RC and pHelper through the process of calcium-phosphate coprecipitation. The titers of recombinant virus were detected.Results The recombinant plasmid pAAV-MCS-TGF-β1 with TGF-β1 were reconstructed, and the recombinant adeno-associated virus with TGF-β1 were also achieved. After being treated by enzyme digestion and sequence analysis, the recombinant pAAV-MCS-TGF-β1 was confirmed that TGF-β1 was inserted correctly into the vector and the sequence of this gene was also corrected. The titers of the recombinant virus (rAAV-TGF-β1) were detected as 3×108pfu/ml and TGF-β1 of rAAV-TGF-β1 was detected by PCR. Conclusion The recombinant adeno-associated virus vector with TGF-β1 (rAAV-TGF-β1) were reconstructed successfully and might be employed to transduce the synovial mesenchymal stem cells in the tissue engineering of fibrocartilage in the following experiments.PartⅡIsolation and culture of synovial mesenchymal stem cells of rabbit temporomandibular jointObjective To explore the isolation and culture of synovial mesenchymal stem cells(SMSCs) for the tissue engineering of temporomandibular joint.Methods Synovium membrane was harvested from New-Zealand white rabbits (3-month old) under the condition of asepsis. SMSCs were harvested through the method of enzyme digestion and isolated by limited dilution. The proliferation and viability of the isolated SMSCs were detected. For evaluation of the multipotential differentiation of SMSCs, the passage 3 SMSCs were cultured in the defined medium to be differentiated into adipose cells, osteoblasts and chondrocytes. After being cultured in defined medium for 14d, the differentiation of SMSCs into adipose cells and osteoblasts were tested by Oil red O staining or Alizarin red staining. Being induced by TGF-β1 for 21d in vitro, the differentiation of SMSCs into chondrocyte were evaluated by Safranin O staining and collagen typeⅡimmunohistochemical staining.Results Rabbit SMSCs maintained the fibroblast-like morphology after being cultured 10 passages. Primary SMSCs began attaching on the floor of the flash after 24h, and the attachment ability of SMSCs reached to 80% after 48h. It was found that calcified nodules and lipid drops in the SMSCs after SMSCs being induced and differentiated into osteogenic cells and adipogenic cells. And special extracellular matrix of cartilage could be found in the SMSCs stained by Safranin O and collagen typeⅡstained positive immunohistochemically after the chondrogenic differentiation of SMSCs.Conclusion It was found that the SMSCs could maintain the fibroblast-like morphology after being cultured several passages, and had multipotential differentiation cultured in the defined medium. Therefore, it suggested that SMSCs might be a choice of seeding cells for temporomandibular joint tissue engineering.PartⅢStudy on transduction of SMSCs in vitro using recombinant adeno-associated virus with TGF-β1Objective To detect the expression of TGF-β1 mRNA and protein of the SMSCs transduced by recombinant adeno-associated virus with TGF-β1 (rAAV-TGF-β1) in vitro and investigate the ability of the transduced SMSCs differentiation into fibrocartilage.Methods Three groups were divided as experimental group of SMSCs transduced by rAAV-TGF-β1, control group of SMSCs transduced by empty vector of adeno-associated virus, and blank control group of SMSCs non-transduced. MTT assay was employed to detect the proliferation of SMSCs after being transduced 1～7d. RT-PCR was applied to explore the expression of TGF-β1, and Western blot was used to evaluate the protein of TGF-β1 of the three groups. Pellet culture of tansduced SMSCs was adopted to detect the expression of mRNA of collagen typeⅠ,ⅡandⅩ, sox9 and aggrecan using RT-PCR, and the collagen typeⅠproteins and collagen typeⅡprotein were investigated by the method of immunocytochemistry staining.Results Both TGF-β1 mRNA and protein could express in the experimental group of SMSCs transduced by rAAV-TGF-β1; the mRNA expression of collagen typeⅠ,Ⅱ, Sox9 and aggrecan were detected, and the expression of collagen typeⅠprotein is stronger than collagen typeⅡby the method of immunocytochemistry staining. No collagen typeⅩexpressed in transduced SMSCs at any time point during the experiment. In the conrol group, the secretion of cartilage matrixes were not detected. These date suggest that the SMSCs transfected by rAAV-TGF-β1 could differentiate into fibrocartilage.Conclusion It could be concluded from this study that TGF-β1 could be transduced by adeno-associated virus vector into SMSCs; the transduced cells could express more TGF-β1 mRNA and protein than that of non-transduced cells; and transduced SMSCs could differentiate into fibrocartilage.PartⅣIn vitro study on fibrocartilage tissue engineering using gene TGF-β1 modified SMSCs and chitosan/collagen typeⅠscaffoldObjective To investigate the potential of the SMSCs transduced by recombinant adeno-associated virus rAAV-TGF-β1 cultured in the fabricated chitosan/collagen typeⅠ(CS/COL-I) scaffold for fibrocartilage tissue engineering.Methods The SMSCs were transduced by rAAV-TGF-β1 and cultured onto the fabricated chitosan/collagen typeⅠscaffold. After being cultured in defined medium, histological HE staining was applied to examine the cell morphology of SMSCs-CS/COL-I constructs. Safranin O/Fast green staining and immunocyto-chemistry staining were used to detect the the cartilage matrix secreted by transduced SMSCs. The cell morphology and attachment of the SMSCs-CS/COL-I constructs were examined through scanning electronic microscope (SEM). And the proliferation of SMSCs on scaffold was detected by MTT.Results The SMSCs transduced by rAAV-TGF-β1 attached onto the scaffolds and secreted much extracellular matrix around the cells. The cartilage-like matrix filled in the pores of the scaffolds and was stained red by Safranin O/Fast green. It was found that collagen typeⅡwas immunohistochemically positive in the transduced SMSCs or around the cells. The cells in the constructs communicated each other in the pores of the scaffolds, and matrix surrounded the cells examined through SEM. And the transduced SMSCs cultured in the scaffolds presented good viability and proliferation indicated by MTT assay.Conclusion The fabricated CS/COL-I coploymer could be a proper three-dimensional scaffold for the SMSCs transduced by rAAV-TGF-β1 to proliferate and differentiate.