Objective To construct recombinant lentiviral vectors of porcine bone morphogenetic protein 2 (BMP-2) gene and to detect BMP-2 gene activity and bone marrow mesenchymal stem cells (BMSCs) osteogenetic differentiation so as to lay a foundation of the further study of osteochondral tissue engineering. Methods BMSCs were isolated from bone marrow of 2-month-old Bama miniature porcines (weighing, 15 kg), and the 2nd generation of BMSCs were harvested for experiments. The porcine BMP-2 gene lentiviral vector was constructed by recombinant DNA technology and was used to transfect BMSCs at multiplicity of infection (MOI) of 10, 25, 50, 100, and 200, then the optimal value of MOI was determined by fluorescent microscope and inverted phase contrast microscope. BMSCs transfected by BMP-2 recombinant lentiviral vectors served as experimental group (BMP-2 vector group); BMSCs transfected by empty vector (empty vector group), and non-transfected BMSCs (non-transfection group) were used as control groups. RT-PCR, immunohistochemistry staining, and Western blot were performed to detect the expressions of BMP-2 mRNA and protein. Then the BMSCs osteogenesis was detected by alkaline phosphatase (ALP) staining, ALP activities, and Alizarin red staining. Results The recombinant lentiviral vectors of porcine BMP-2 gene was successfully constructed and identified by RT-PCR and gene sequencing, and BMSCs were successfully transfected by BMP-2 recombinant lentiviral vectors. Green fluorescent protein could be seen in the transfected BMSCs, especially at MOI of 100 with best expression. The immunohistochemistry staining and Western blot showed that BMSCs transfected by BMP-2 recombinant lentiviral vectors could express BMP-2 protein continuously and stably at a high level. After cultivation of 2 weeks, the expression of ALP and the form of calcium nodules were observed. Conclusion The porcine BMP- 2 gene lentiviral vector is successfully constructed and transfected into the BMSCs, which can express BMP-2 gene and protein continuously and stably at a high level and induce BMSCs differentiation into osteoblasts.
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.