Objective: To investigate the effect of phosphorylatable short peptide ((P)SP) conjugated chitosan (CS) ((P)SP-CS)mediated insulin-like growth factor 1 (IGF-1) gene and human interleukin 1 receptor antagonist (IL-1Ra) gene local transfection on the repair of articular cartilage defect.
Methods: Co-expression plasmid pBudCE4.1-IL-1Ra + IGF-1, single gene expression plasmid pBudCE4.1-IL-1Ra and pBudCE4.1-IGF-1 were constructed and combined with (P)SP-CS to form (P)SP-CS/pDNA complexes. Thirty 3-month-old healthy male New Zealand white rabbits, weighing 2.0-2.5 kg, double legs were randomly divided into 5 groups (n = 12). Lateral femoral condyle articular surface was only exposed in sham-operated group (group A); full-thickness cartilage defects were created in the articular surface of the lateral femoral condyle of the knee in 4 intervention groups: (P)SP-CS/pBudCE4.1 (group B), (P)SP-CS/pBudCE4.1-IL-1Ra (group C), (P)SP-CS/pBudCE4.1-IGF-1 (group D), and (P)SP-CS/pBudCE4.1-IL-1Ra + IGF-1 (group E). At 1 week after operation, intra-articular injection of (P)SP-CS/pDNA complexes was administrated 2 times a week for 7 weeks in each intervention group, the same volume normal saline in group A. The general condition of animal was observed after operation, and rabbits were sacrificed at 8 weeks. Knee joint synovial fluid was collected to measure the concentrations of the IL-1Ra and IGF-1 by ELISA; mRNA expressions of Aggrecan, matrix metalloproteinase 3 (MMP-3), and MMP inhibitor 1 (TIMP-1) were detected by real-time fluorescent quantitative PCR; the chondrogenic phenotype of nascent cells in the damage zone was identified by alcian blue-periodic acid/schiff (AB-PAS) histochemistry and Aggrecan immunohistochemistry staining.
Results: Thirty experimental rabbits all survived to the end of experiment, without infection and death. Large amounts of exogenous proteins of IGF-1 and IL-1Ra were detected in the synovial fluid of 4 intervention groups. There were significant differences between groups D, E and group A in IGF-1 protein expression, and between goups C, E and group A in IL-1Ra protein expression (P < 0.05). Aggrecan and TIMP-1 mRNA expressions were significantly up-regulated in group E, simultaneously MMP-3 mRNA expression was significantly down-regulated when compared with groups C and D (P < 0.05). Varying degrees of cartilage repair appeared in groups C, D, and E, showing positive staining of AB-PAS and Aggrecan, and group E had better results than groups C and D (P < 0.05); inflammatory cell infiltration and fibrous tissue proliferation were seen in the defect region of group B, without significant cartilage repairing.
Conclusion: (P)SP-CS is an ideal gene delivery system for cartilage defect gene therapy; IL-1Ra and IGF-1 double gene transfection has better biologic effect on cartilage defect repair.