Fabrication Of Composite Nanofibers Based Silk Fibroin And Application In Soft Tissue Repair

In living systems, the extracellular matrix (ECMs) plays a pivotal role in controlling cell behavior. It provides resident cells with specific ligands for cell attachment and migration, modulates cell proliferation and function. Therefore, the ideal tissue engineering scaffolds should mimic native ECMs as much as possible. The native ECMs is composed of a cross-linked porous network of multifibril collagens with diameters ranging from 50-500nm and embedded in glycosaminoglycans. Electrospinning could produce polymer fibers with diameters in the range of nanometers to micrometers. Such nanofirous scaffolds could biomimic native ECMs microstructure and have extremely high specific surface area and highly porous with excellent pore interconnection for cell adhesion, proliferation, migration, and differentiated cell function to improve tissue regeneration. Silk fibroin (SF) is an attractive natural fibrous protein for biomedical applications due to its unique properties, including good biocompatibility, biodegradability, lower inflammatory response and commercial availability at relatively low cost. However, since regenerated silk fibroin nanofibers possesses weak mechanical properties, it is difficult to meet their mechanical properties demands as scaffolds for soft tissue repair (skin, blood vessel and nerve guidance conduit), especially tubular scaffolds for blood vessel and nerve guidance conduit. Meanwhile, the optimal design as tisse repair scaffolds shoud biomimic the native ECMs from both the components and the microstructure, then, the mimicking of the function could be made.From the point of view of mimicing native ECMs, electrospinning of biocompatible silk fibroin and hydroxybutyl chitosan blends was studied to biomimic the natural ECMs on both the components and the microstructure. Scanning electronic microscope (SEM) results showed the average nanofibrous diameter increased when the content of HBC was raised from 20% to 100%. Fourier transform infrared spectroscopy (FTIR) and 13C CP/MAS nuclear magnetic resonance (NMR) clarified that SF and HBC molecules existed in H-bond interactions, but HBC did not induce SF conformation to transform from random coil form toβ-sheet structure. X-ray diffraction (XRD) cleared SF/HBC blended nanofibers formed new crystal structure and improved crystallinities. This also further proved the existence of H-bond interactions between SF and HBC. The results of thermogravimetry analysis (TG) demonstrated that the thermal stability of SF/HBC blended nanofiberous scaffolds was improved in comparison with HBC nanofibers. Whereas water contact angle measurements confirmed that SF/HBC nanofibrous scaffolds with different weight ratios were of good hydrophilicity. Both the tensile strength and the elongation at break were improved obviously when the weight ratio of SF to HBC was 20:80.In order to improve stability of SF/HBC nanofibrous scaffolds in vivo and vitro, genipin vapor were used to crosslink electrospun nanofibers, glutaraldehyde(GTA) and ethanol were used for comparison. SEM indicated that crosslinked nanofibers with genipin and GTA vapor for 24h had good water-resistant ability. Characterization of the microstructure (porosity and pore structure) demonstrated crosslinked nanofibrous scaffolds with genipin and GTA vapor had lager porosities and mean diameters than those with ethanol vapor. The average tensile strength of SF/HBC nanofibrous scaffolds with different weight ratios after crosslinking for 24h with genipin were obvious improved, while the average elongation at break decreased slightly. And the mechanical properties of SF/HBC nanofibrous scaffolds with genipin vapor were superior to those crosslinked with GTA and ethanol vapor in same condition. Characterization of fourier transform infrared attenuated total reflectance spectroscopy (FTIR-ATR) and 13C CP/MAS NMR clarified both genipin and GTA acted as crosslinking agents for SF and HBC. Furthermore, genipin could induce SF conformation from random coil to P-sheet. Although GTA could also successfully crosslink SF/HBC nanofibrous scaffolds, in long run, genipin maybe a better method due to its lower cytotoxicity than GTA. Cell viability studies and skin defect repair test in rats clarified that the genipin-crosslinked SF/HBC nanofibrous scaffolds had a good biocompatibility both in vitro and in vivo and could promote wound healing.The nanofibrous scaffolds of SF and HBC blends still remain poor mechanical properties to meet the requirements of high mechanical properties of blood vessels and nerve guidance conduit. Therefore, nanofibrous scaffolds of SF and poly(L-lactic acid-co-s-caprolactone) (P(LLA-CL) (50:50)) blends were fabricated via electrospinning. The average nanofibrous diameter increased with increasing polymer concentration and decreasing the blend ratio of SF to P(LLA-CL). Characterizations of X-ray photoelectron spectroscopy (XPS), FTIR and 13C CP/MAS NMR clarified the presence of SF on their surfaces and no obvious covalent bond reaction between SF with P(LLA-CL) and SF in SF/P(LLA-CL) nanofibers was present in a random coil conformation. Whereas, water contact angle measurements conformed greater hydrophilicity than P(LLA-CL). Compared to pure SF and P(LLA-CL) nanofibrous scaffolds, the mechanical properties were obviously improved and readily were tailored to meet the requirement of specific application through changing the blend ratio of SF to P(LLA-CL).In vitro degradation of SF/P(LLA-CL) nanofibrous scaffolds were studied in phosphate buffered saline (pH 7.4±0.1) at 37℃for 6 months. Through a series of analysis and characterizations (including loss weight, pH changes of PBS solutions, TG, DSC, XRD and FTIR-ATR) to fiberous scaffolds after degradation for different time, the results showed that the pure SF nanofibrous scaffolds were not degradable in PBS, the degradation rate of the pure P(LLA-CL) fibrous scaffolds were faster than SF/P(LLA-CL) blended nanofibrous scaffolds. The adding of SF reduced the degradation rate of P(LLA-CL). This is probably caused by the intermolecular interactions between SF and P(LLA-CL) to hinder the P(LLA-CL) molecular chains movement.Cell viability studies with pig iliac endothelial cells (PIECs) demonstrated that SF/P(LLA-CL) blended nanofibrous scaffolds significantly promoted cell growth in comparison with P(LLA-CL), especially when the weight ratio of SF to P(LLA-CL) was 25:75. SEM showed that PEICs more easily spread to develop an endothelial cell layer on the surface of SF/P(LLA-CL) (25:75) nanofibrous scaffolds than on pure (PLLA-CL) scaffolds. SF/P(LLA-CL) blended nanofibrous scaffolds were implanted in the back of New Zealand rabbits and evaluated their tissue compatibility in vivo. The results demonstrated that SF/P(LLA-CL) blended nanofibrous scaffolds had good tissue compatibilityAligned nanofibrous scaffolds of SF/P(LLA-CL) (25:75) were fabricated by electrospinning technique under optimum condition. The degree of orientation increased with the increase in the rotating drum rates from 500 to 4000rpm. The diameter of nanofibers had no obvious difference with increasing rotating rates. Mechanical test showed aligned nanofibrous scaffolds possessed good mechanical properties. The tensile strength was much higher at parallel than at perpendicular directions. MTT results showed Schwann cells (SCs) had greater proliferation on random/aligned SF/P(LLA-CL) nanofibrous scaffolds than that on random/aligned P(LLA-CL) fibrous scaffolds. And SCs had greater proliferation on aligned SF/P(LLA-CL) nanofibrous scaffolds than random SF/P(LLA-CL) nanofibrous scaffolds. The alignment of the SF/P(LLA-CL) nanofibers could control cell orientation and strengthen the interaction between the cell body and the fibers in the longitudinal direction of the fibers.Adult male Sprague-Dawley (SD) rats were used for animal models. The aligned SF/P(LLA-CL) (25:75) nanofibrous nerve guidance conduit (NGC) was used for bridge implantation across a 10-mm long sciatic nerve defect in rats, P(LLA-CL) nanofibrous nerve guidance conduit (NGC) and nerve autografts were used for control. The outcome of regenerated nerve at 4 and 8 weeks was evaluated by a combination of electrophysiological assessment, histological and immunohistological analysis, as well as electron microscopy study. The results of the functional and morphological parameters demonstrated that the aligned SF/P(LLA-CL) NGC could promote greater peripheral nerve regeneration in comparison with the aligned P(LLA-CL) NGC, while, was slightly inferior to nerve autografts.