Objective To construct recombinant lentiviral expression vectors of porcine transforming growth factor β1 (TGF-β1) gene and transfect bone marrow mesenchymal stem cells (BMSCs) so as to provide TGF-β1 gene-modified BMSCs for bone and cartilage tissue engineering. Methods The TGF-β1 cDNA was extracted and packed into lentiviral vector, and positive clones were identified by PCR and gene sequencing, then the virus titer was determined. BMSCs were isolated frombone marrow of the 2-month-old Bama miniature pigs (weighing 15 kg), and the 2nd and 3rd generations of BMSCs wereharvested for experiments. BMSCs were then transfected by TGF-β1 recombinant lentiviral vectors (TGF-β1 vector group)respectively at multi pl icity of infection (MOI) of 10, 50, 70, 100, and 150; then the effects of transfection were detected bylaser confocal microscope and Western blot was used to determine the optimal value of MOI. BMSCs transfected by empty vector (empty vector group) and non-transfected BMSCs (non-transfection group) were used as control group. RT-PCR, immunocytochemistry, and ELISA were performed to detect the expressions of TGF-β1 mRNA, TGF-β1 protein, and collagen type II. Results Successful construction of recombinant lentiviral vectors of porcine TGF-β1 gene was identified by PCR and gene sequencing, and BMSCs were successfully transfected by TGF-β1 recombinant lentiviral vectors. Green fluorescence was observed by laser confocal microscope. Western blot showed the optimal value of MOI was 70. The expression of TGF-β1 mRNA was significantly higher in TGF-β1 vector group than in empty vector group and non-transfection group (P lt; 0.05). Immunocytochemistry results revealed positive expression of TGF-β1 protein and collagen type II in BMSCs of TGF-β1 vector group, but negative expression in empty vector group and non-transfection group. At 21 days after transfection, high expression of TGF-β1 protein still could be detected by ELISA in TGF-β1 vector group. Conclusion TGF-β1 gene can be successfully transfected into BMSCs via lentiviral vectors, and long-term stable expression of TGF-β1 protein can be observed, prompting BMSCs differentiation into chondrocytes.
Objective To prepare collagen-chitosan /nano-hydroxyapatite-collagen-polylactic acid (Col-CS/ nHAC-PLA) biomimetic scaffold and to examine its biocompatibility so as to lay the foundation for its application on the treatment of osteochondral defect. Methods PLA was dissolved in dioxane for getting final concentration of 8%, and the nHAC power was added at a weight ratio of nHAC to PLA, 1 ∶ 1. The solution was poured into a mold and frozen. CS and Col were dissolved in 2% acetum for getting the final concentrations of 2% and 1% respectively, then compounded at a weight ratio of CS to Col, 20 ∶ 1. The solution was poured into the frozen mold containing nHAC-PLA, and then biomimetic osteochondral scaffold of Col-CS/nHAC-PLA was prepared by freeze-drying. Acute systemic toxicity test, intracutaneous stimulation test, pyrogen test, hemolysis test, cytotoxicity test, and bone implant test were performed to evaluate its biocompatibility. Results Col-CS/nHAC-PLA had no acute systemic toxicity. Primary irritation index was 0, indicating that Col-CS/nHAC-PLA had very slight skin irritation. In pyrogen test, the increasing temperature of each rabbit was less than 0.6℃, and the increasing temperature sum of 3 rabbits was less than 1.3℃, which was consistent with the evaluation criteria. Hemolytic rate of Col-CS/nHAC-PLA was 1.38% (far less than 5%). The toxicity grade of Col-CS/nHAC-PLA was classified as grade I. Bone implant test showed that Col-CS/nHAC-PLA had good biocompatibility with the surrounding tissue. Conclusion Col-CS/ nHAC-PLA scaffold has good biocompatibility, which can be used as an alternative osteochondral scaffold.