Silk Fibroin/Chondroitin Sulfate/Hyaluronic Acid Blend Scaffold For Dermal Tissue Reconstruction

At present, the repair of the dermis tissue deficiency in the clinical medicine mainly depends on the autografting. Though it has a good effect on the defect region, the difficulty in the treatment of extensively and deeply burned patients has been limited by the number of donor sites which are available for autogrfting, and the new wound created on the donor site is painful during healing and may become a cause of long-term morbidity. Thus, the novel dermal substitute that could promote dermal reconstructive activity and had controlled biodegradability is needed urgently. Many studies have initially confirmed that silk fibroin (SF) has good potential for dermal tissue regeneration. It will meet the needs of dermal tissue regeneration better if its ability to regulate cell behavior can be enhanced and the rate of degradation and dermal tissue regeneration can be matched. Accordingly, the Silk fibroin (SF) /Chondroitin sulfate (CS) /Hyaluronic acid (HA) blend scaffold was prepared and its series properties were measured in this dissertation.The SF/CS/HA blend scaffolds were prepared by freeze-drying, and the structures and physic-chemical properties were studied systematically. The results indicated that there were mainly random coil in the SF/CS/HA blend scaffold, with a little Silk II coexistence. Comparing to the SF scaffold, the tensile strength and water binding ability of SF/CS/HA blend scaffold were enhanced obviously. Due to the presence of HA which inhibited the growth of ice crystals, the pore diameter of the blend scaffolds decreased, and because of the network structure of HA, which can hold the stability of morphology of the scaffold during the dry process and decrease the shrinkage ratio, the porosity of the scaffold increased slightly. The average pore diameter and porosity could be adjusted in the range of 95-248μm and 88-93% respectively. The blend scaffolds have a greater resistance to collagenase because of the existence of HA.Then, based on the results of confocal laser microscopy, SEM, histological and MTT, each of the blend scaffolds can promote the L929 fibroblasts adhesion and proliferation better compared to those of the SF scaffold. Especially the blend ratio of SF/CS/HA was 80/5/15, the scaffold was most conducive to cell proliferation, and the cells present a best growth activity with uniform spatial distribution.Further, dermal reconstruction behavior in vivo induced by blend scaffolds was investigated by implanted SF/CS/HA (100/0/0, 80/20/0, and 80/5/15) scaffolds in rat dorsal dermal defects respectively. The results indicated that inflammatory cell infiltration was not observed in all the scaffolds, the immune response and inflammation reactions caused by scaffolds were mild. The repairing cells could migrate to the center of the scaffolds and construct dermal tissue with different speed. The effect of three different scaffolds on inducing dorsal dermal regeneration of the rats was studied, the results showed that SF scaffold incorporated with CS and HA could promote the formation of capillary network and ingrowth of new granulation tissue, and the biodegradation rate of SF/CS/HA (80/5/15) blend scaffold and the tissue regeneration rate could achieved a better match. Masson’s staining showed that collagen fibers formations of SF/CS/HA (80/5/15) blend scaffold were significantly higher than that of SF scaffold.Finally, to study the wound healing mechanism promoted by the scaffold materials, multi-target was tested and analyzed. The results indicated that the CD34, PDGF, VEGF and bFGF in the SF/CS/HA blend scaffold had a higher expression than that in the SF and SF / HA porous scaffolds. This not only illustrated that the SF/CS/HA scaffold had the activity to promote wound angiogenesis and dermal reconstruction, but also pointed out that its molecular mechanism is the stimulation of secretion of PDGF, VEGF and bGFG and accumulation of these growth factors. As well as created a good micro-environment for the repairing cells migration, adhesion and growth.The effect of CS and HA incorporated within SF scaffold on the structures and properties was illuminated in this study. And found that SF/CS/HA (80/5/15) blend scaffold had an obvious activity which could promote angiogenesis and tissue repair and revealed its mechanism. A novel scaffold could be provided for the dermis reconstruction.