| Engineering complex tissues is important but difficult to achieve in tissue regeneration. Osteochondral tissue engineering for the repair of osteochondral defect, involving simultaneous regeneration of bone and cartilage, has attracted considerable attention and also serve as an optimal model system for developing effective strategies aimed at regenerating complex tissues. In the present study, we formulated a bilayered gene-activated osteochondral scaffold consisting of plasmid TGF-β1-activated chitosan-gelatin scaffold for chondrogenic layer and plasmid BMP-2-activated hydroxyapatite/chitosan-gelatin scaffold for osteogenic layer. Mesenchymal stem cells seeded in each layer of the bilayered gene-activated osteochondral scaffold showed significant cell proliferation, high expression of TGF-β1 protein and BMP-2 protein respectively. The results showed that spatially controlled and localized gene delivery system in the bilayered integrated scaffolds could induce the mesenchymal stem cells in different layers to differentiate into chondrocytes and osteoblasts in vitro, respectively, and simultaneously support the articular cartilage and subchondral bone regeneration in the rabbit knee ostochondral defect model. This study give the evidence that multi-tissue regeneration through the combination of biomimetic and multiphasic scaffold design, spatially controlled and localized gene delivery system and multilineage differentiation of a single stem cell population represents a promising strategy for facilitating the development of complex tissue or organ systems.Cationic polymers with nucleic acid drug delivery ability are widely used in experimental and clinical studies. However, their interactions with the immune systems are rarely studied. In the present study, cationic polymers including PEI, polylysine, cationic dextran and cationic gelatin exhibited strong stimulation on Th1 response which was characterized by the induction of the proliferation of CD4+ T cells and the secretion of Thl related cytokines. Experiments performed on macrophages demonstrated that cationic polymers specifically stimulated the macrophage to secret IL-12 which is one of the main Thl-inducing cytokines. The result that MyD88 inhibitor remarkably reduced the IL-12 expression induced by cationic polymers suggested that this stimulation was mainly mediated by toll-like receptor (TLR) pathway. Additionally, cationic polymers could strongly inhibit LPS induced TNF-a secretion in macrophages. This result implied that cationic polymers may interact with macrophages through TLR-4 which is the receptor of LPS. The following test of inhibiting IL-12 expression stimulated by cationic polymers using TLR-4 antibody proved that the stimulation was mainly mediated by TLR-4. Data in the present study demonstrated that the stimulation ability of cationic polymer was related with its cationic degree and neutralizing cationic polymer with anionic polymer completely abrogated the stimulation effect. The molecular weight of the polymers also influenced their stimulation ability, larger molecular means stronger stimulation ability. In conclusion, the present study revealed that cationic polymers could promote Thl responses in vivo via TLR-4 mediated IL-12 secretion and the molecular weight and cationic degree of the polymers determined the stimulation ability.Basing on the specific stimulation on IL-12 and IFN-γ, cationic materials have remarkable anti-inflammation activity in experimental arthritis models. The concentrations of IFN-γand IL-12 in animal serum samples were enhanced accordingly following C-dextran injection, which was the same as those in the cellular experiments. Using IFN-γR-/- mice and macrophage-depletion rats, we demonstrated that IFN-y and macrophage played the key roles in the materials activity of anti-inflammation. We also noticed that neutrophil infiltration at the inflammatory sites was reduced when AIA animals were treated with C-dextran. In conclusion, cationic polymers act through toll-like receptor-4 on macrophage to induce IL-12 secretion, and IL-12 promotes the expression of IFN-y by NK cells (or T cell). The resulting elevated systemic levels of IFN-y inhibit arthritis development by preventing neutrophil recruitment to the inflammatory sites.
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