Repairing Cartilage Defect With Chitosan Mediated Igf-1 Gene Transfection

Insulin-like growth factor I (IGF-1), a 70 amino acid polypeptide hormone, is largely a mediator of cell growth and differentiation, primarily stimulating DNA synthesis and mitogenic events. In lots of clinical trails IGF-I showed its strong possibility of treating growth delaying, diabetes mellitus, osteoporosis or cartilage injury. However, due to its rapidly in vivo degradation and sometimes severe side effects caused by systemically administration, clinical application of recombinant IGF protein was limited to a large extend. By means of gene transfection especially locally igf-1 gene transfection, which can keep on igf-1 gene expression and hold IGF-1 at a high concentration for a long time, might mostly reduce these adverse reactions. In this article, locally trans-igf-1 gene mediated by chitosan/DNA complex for the therapy of cartilage injury was studied.In vitro experimentation: A DNA fragment encoding IGF-1 mature peptide following with its native signal peptide was subcloned into plasmid pTracer-CMV/Bsd. Thereafter, the recombinant plasmid pTracer-igf-1 was delivered into C2C12 mouse myoblast celline by DNA / chitosan complex. Cell proliferation was detected by ~3H-TdR incorporation. As a result, trans-igf-1 cells showed a markedly increased proliferating rate compared with those no transfected cells. The medium collected from the trans-igf-1 cells could also accelerated the proliferating rate of cultured normal C2C12 cell. A conclusion could be drawn that DNA / CS complex could effectively deliver igf-1 gene into C2C12 cells then IGF-1 protein could be synthesized and secreted out the cells exert its mitogenic function.In vivo Experimentation: We duplicate the mould of articular cartilage damaging by modus operandi. CS,Particles of recombinant plasmids pTracer-igf-1/ CS and normal saline were injected through the patellar tendon into damaging rabbit knee joints every other 72 hours. Cartilage tissue from rabbit joints were harvested 28 days after experimentation. Samples were harvested from the knees for the following detection.(1) Total RNA was isolated for real-time fluorescent quantitative PCR to determine collagen typeâ…¡and aggrecan expression in healing cartilage damage.(2)histologic assessment of tissue healing using H&E staining, toluidine blue histochemical reaction for proteoglycan deposition. (3) immunohistochemistry procedures to demonstrate S-100 protein expression. As a result: (1) The level of gene expression : the compounds of pTracer-igf-1/CS can enhance the expression of collagen type II and aggrecan than ohers groups.(2)a progressive recovery in tissue cellularity and increased tissue organization were observed on H&E,toluidine blue histochemical in trans-igf-1 group, (3)immunohistochemistry stained in trans-igf-1 group appearances immature cartilage cell and contain S-100 less.In general, chitosan can promote genic transfection both in vitro and in vivo. Trans igf-1 gene can significantly stimulate cell proliferation in vitro while accelerate cartilage damage healing in localized in vivo application. The result of this study may give this chitosan mediated igf-1 gene transfection an important theoretical meaning and great clinical value.