The Experimental Study Of Repairing Rabbit Meniscus Lesion With Synovial-Derived Mesenchymal Stem Cells And Chitosan Gel

Background:In clinical treatment, the self-healing of meniscus injury is difficult, and there is no better surgical repair way, either; the traditional meniscectomy can easily lead to knee-joint instability, accelerated articular cartilage degeneration and other serious consequences. But the emergence of tissue engineering has provided a viable development for meniscus regeneration.Objective:To explore the feasibility of rabbit meniscus defects repairing with tissue engineering that constructed by chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel composite synovial-derivedmesenchymal stem cellsMethod:Knee joint synovial tissues of healthy New Zealand White Rabbits were obtained under sterile condition, synovial-derivedmesenchymal stem cells was isolated in vitro; after purified by limited dilution method, the synovial-derivedmesenchymal stem cells were cultured and proliferated in vitro, and then multi-directional osteogenic/adipogenic/ chondrogenic differentiation abilities appraisal were conducted under certain conditions; in order for the synovial-derivedmesenchymal stem cells to express key cytokines that can be differentiated to cartilage in vivo for long periods of time, we used TGF-β3/BMP-2 gene transferred by recombinant lentiviruses for the transfection of rabbit synovial-derivedmesenchymal stem cells, and enabled it to stably express TGF-β3/BMP-2; and finally, after in vitro synthesis of chitosan/β-sodium glycerophosphate hydrogel, mixed it with synovial-derivedmesenchymal stem cells that stably expresses TGF-β3/BMP-2, and then injected to rabbit meniscal defect area. The animal shall be executed to death 6 and 12 weeks after surgery with excess anesthesia, at this time, take the repaired meniscus tissue to carry out gross observation, toluidine blue stain, and immunohistochemistry stain of Collagen Ⅰ/Collagen Ⅱ to observe the repairstatus of meniscus.Results:The obtained cultured synovial-derivedmesenchymal stem cells were in uniform morphology, and distributed in flat or swirl shape, besides, under certain induction conditions, it can be differentiated to osteogenesis, adipogenesis and chondrogenesis different directions. After synovial-derivedmesenchymal stem cells were transfected by recombinant lentiviruses, the green fluorescence increased gradually along with the time,96 hours after the transfection, obvious green fluorescence can be seen, and the transfection efficiency was about 100%. Through Real-time PCR, Western blot test, compared it with the control group, it can be found that the transfected synovial-derivedmesenchymal stem cells can stably express TGF-β3 and BMP-2; chitosan and β-sodium glycerophosphate were mixed in vitro to successfully obtain chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel, it was in liquid state at room temperature, and transformed to the solid state at 37℃ for 10 minutes. After mixing the chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel with synovial-derivedmesenchymal stem cells in vitro, injected it into rabbit meniscus defect area, and then executed the animals to death to extract meniscus for observation. Immunohistochemistry result of experimental group showed that the expression of regenerative meniscus tissue Collagen I/Collagen II was positive, and toluidine blue stain also confirmed the component of extracellular matrix of chondrocytes (GAG).Conclusion:The results showed that after the synovial-derivedmesenchymal stem cells were transfected by TGF-β3/BMP-2 gene transferred by recombinant lentiviruses, it can stably express TGF-β3 and BMP-2; and then chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel was used as a scaffold to perform the method of rabbit meniscus defect repairing, which turned out can repair the meniscus defect to a certain extent.