Kuihua Zhang

Fabrication of chitosan/silk fibroin composite nanofibers for wound-dressing applications.

Chitosan, a naturally occurring polysaccharide with abundant resources, has been extensively exploited for various biomedical applications, typically as wound dressings owing to its unique biocompatibility, good biodegradability and excellent antibacterial properties. In this work, composite nanofibrous membranes of chitosan (CS) and silk fibroin (SF) were successfully fabricated by electrospinning. The morphology of electrospun blend nanofibers was observed by scanning electron microscopy (SEM) and the fiber diameters decreased with the increasing percentage of chitosan. Further, the mechanical test illustrated that the addition of silk fibroin enhanced the mechanical properties of CS/SF nanofibers. The antibacterial activities against Escherichia coli (Gram negative) and Staphylococcus aureus (Gram positive) were evaluated by the turbidity measurement method; and results suggest that the antibacterial effect of composite nanofibers varied on the type of bacteria. Furthermore, the biocompatibility of murine fibroblast on as-prepared nanofibrous membranes was investigated by hematoxylin and eosin (H&E) staining and MTT assays in vitro, and the membranes were found to promote the cell attachment and proliferation. These results suggest that as-prepared chitosan/silk fibroin (CS/SF) composite nanofibrous membranes could be a promising candidate for wound healing applications.

Electrospun collagen-chitosan-TPU nanofibrous scaffolds for tissue engineered tubular grafts.

The objective of this study is to design a novel kind of scaffolds for blood vessel and nerve repairs. Random and aligned nanofibrous scaffolds based on collagen-chitosan-thermoplastic polyurethane (TPU) blends were electrospun to mimic the componential and structural aspects of the native extracellular matrix, while an optimal proportion was found to keep the balance between biocompatibility and mechanical strength. The scaffolds were crosslinked by glutaraldehyde (GTA) vapor to prevent them from being dissolved in the culture medium. Fiber morphology was characterized using scanning electron microscopy (SEM) and atomic-force microscopy (AFM). Fourier transform infrared spectroscopy (FTIR) showed that the three-material system exhibits no significant differences before and after crosslinking, whereas pore size of crosslinked scaffolds decreased drastically. The mechanical properties of the scaffolds were found to be flexible with a high tensile strength. Cell viability studies with endothelial cells and Schwann cells demonstrated that the blended nanofibrous scaffolds formed by electrospinning process had good biocompatibility and aligned fibers could regulate cell morphology by inducing cell orientation. Vascular grafts and nerve conduits were electrospun or sutured based on the nanofibrous scaffolds and the results indicated that collagen-chitosan-TPU blended nanofibrous scaffolds might be a potential candidate for vascular repair and nerve regeneration.

Fabrication of silk fibroin blended P(LLA‐CL) nanofibrous scaffolds for tissue engineering

Electrospinning using natural proteins and synthetic polymers offers an attractive technique for producing fibrous scaffolds with potential for tissue regeneration and repair. Nanofibrous scaffolds of silk fibroin (SF) and poly(L-lactic acid-co-epsilon-caprolactone) (P(LLA-CL)) blends were fabricated using 1,1,1,3,3,3-hexafluoro-2-propanol as a solvent via electrospinning. The average nanofibrous diameter increased with increasing polymer concentration and decreasing the blend ratio of SF to P(LLA-CL). Characterizations of XPS and (13)C NMR clarified the presence of SF on their surfaces and no obvious chemical bond reaction between SF with P(LLA-CL) and SF in SF/P(LLA-CL) nanofibers was present in a random coil conformation, SF conformation transformed from random coil to beta-sheet when treated with water vapor. Whereas water contact angle measurements conformed greater hydrophilicity than P(LLA-CL). Both the tensile strength and elongation at break increased with the content increasing of P(LLA-CL). Cell viability studies with pig iliac endothelial cells 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. These results suggested that SF/P(LLA-CL) blended nanofibrous scaffolds might be potential candidates for vascular tissue engineering.

Genipin-crosslinked silk fibroin/hydroxybutyl chitosan nanofibrous scaffolds for tissue-engineering application

To improve water-resistant ability and mechanical properties of silk fibroin (SF)/hydroxybutyl chitosan (HBC) nanofibrous scaffolds for tissue-engineering applications, genipin, glutaraldehyde (GTA), and ethanol were used to crosslink electrospun nanofibers, respectively. The mechanical properties of nanofibrous scaffolds were obviously improved after 24 h of crosslinking with genipin and were superior to those crosslinked with GTA and ethanol for 24 h. SEM indicated that crosslinked nanofibers with genipin and GTA vapor 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. Characterization of FTIR-ATR and (13)C NMR clarified both genipin and GTA acted as crosslinking agents for SF and HBC. Furthermore, genipin could induce SF conformation from random coil or α-helix to β-sheet. Although GTA could also successfully crosslink SF/HBC nanofibrous scaffolds, in long run, genipin maybe a better method due to lower cytotoxicity than GTA. Cell viability studies and wound-healing test in rats clarified that the genipin-crosslinked SF/HBC nanofibrous scaffolds had a good biocompatibility both in vitro and in vivo. These results suggested that genipin-crosslinked SF/HBC nanofibrous scaffolds might be potential candidates for wound dressing and tissue-engineering scaffolds.

Electrospinning collagen/chitosan/poly(L-lactic acid-co-ϵ-caprolactone) to form a vascular graft: Mechanical and biological characterization†

For blood vessel tissue engineering, an ideal vascular graft should possess excellent biocompatibility and mechanical properties. For this study, a elastic material of poly (L-lactic acid-co-ε-caprolactone) (P(LLA-CL)), collagen and chitosan blended scaffold at different ratios were fabricated by electrospinning. Upon fabrication, the scaffolds were evaluated to determine the tensile strength, burst pressure, and dynamic compliance. In addition, the contact angle and endothelial cell proliferation on the scaffolds were evaluated to demonstrate the structures potential to serve as a vascular prosthetic capable of in situ regeneration. The collagen/chitosan/P(LLA-CL) scaffold with the ratio of 20:5:75 reached the highest tensile strength with the value of 16.9 MPa, and it was elastic with strain at break values of ~112%, elastic modulus of 10.3 MPa. The burst pressure strength of the scaffold was greater than 3365 mmHg and compliance value was 0.7%/100 mmHg. Endothelial cells proliferation was significantly increased on the blended scaffolds versus the P(LLA-CL). Meanwhile, the endothelial cells were more adherent based on the increase in the degree of cell spreading on the surface of collagen/chitosan/P(LLA-CL) scaffolds. Such blended scaffold especially with the ratio of 20:5:75 thus has the potential for vascular graft applications.

The aligned core–sheath nanofibers with electrical conductivity for neural tissue engineering

Currently, electroactive biomaterials have often been fabricated as tissue engineering scaffolds to provide electrical stimulation for neural tissue engineering. The goal of this work was to study the synergistic effect of electrical stimulation and nerve growth factor (NGF) on neuron growth. The composite meshes of polyaniline (PANi) and well-blended poly(l-lactic acid-co-ε-caprolactone)/silk fibroin (PS) incorporated with nerve growth factor (NGF) were prepared by coaxial electrospinning. The results showed that the increased concentration of PANi had a large effect on the fiber diameter, which was significantly reduced from 683 ± 138 nm to 411 ± 98 nm and then increased to 498 ± 100 nm. The contact angles and Youngs modulus decreased to 28.3°± 5.4° and 7.2 ± 1.2 MPa, respectively, and the conductance increased to 30.5 ± 3.1 mS cm-1. The results of the viability and morphology of mouse Schwann cells on the nanofibrous meshes showed that PS-PANi-1 loaded with NGF exhibited the highest cell number after 5 days culture and the aligned nanofibers could guide cell orientation. The synergistic effects of electrical stimulation and NGF were also investigated via the growth and differentiation of rat pheochromocytoma 12 (PC12) cells. The scaffolds loaded with NGF under electrical stimulation could effectively support PC12 neurite outgrowth and increase the percentage of neurite-bearing cells as well as the median neurite length. More importantly, the NGF release from the conductive core-shell structure nanofiber could be increased by electrical stimulation. These promising results demonstrated that there was a potential use of this functional scaffold for nerve tissue regeneration.

Vitamin E-loaded silk fibroin nanofibrous mats fabricated by green process for skin care application

In the present study, we reported fabrication and skin benefit of a novel vitamin E (VE)-loaded silk fibroin (SF) nanofibrous mats. RRR-α-Tocopherol polyethylene glycol 1000 succinate (VE TPGS), a water-soluble derivative of VE, was incorporated into SF nanofiber successfully by aqua solution electrospinning for the first time. Morphology of the composite nanofibers changed with the different amount of VE TPGS: a ribbon-like shape for lower loading dose of VE TPGS, while a round shape for higher loading dose (more than 4% (wt/wt) based on the weight of SF). After treated with 75% (v/v) ethanol vapor, the composite nanofibrous mats showed an excellent water-resistant ability. In vitro study disclosed a sustained release behavior of VE TPGS disassociated from the nanofibrous mats. The mouse skin fibroblasts (L929 cells) cultured on the VE-loaded SF nanofibrous mats spread and proliferated much better than on cover slips. Moreover, the incorporation of VE TPGS was found strengthening the ability of SF nanofibrous mats on protecting the cells against oxidation stress induced by tert-butyl hydroperoxide. Our data presented impressive skin benefits of this VE-loaded SF nanofibrous mats, suggesting a promising applicative potential of this novel product on personal skin care, tissue regeneration and other related area.

Electrospun scaffolds from silk fibroin and their cellular compatibility.

Electrospinning offers an attractive opportunity for producing silk fibroin (SF) nano/micro fibrous scaffolds with potential for tissue regeneration and repair. Electrospun scaffolds of silk fibroin were fabricated as a biomimetic scaffold for tissue engineering. The morphology of the electrospun scaffolds was investigated with SEM and AFM. The SEM images indicated that electrospun SF fibers were ribbon-shaped and the average width increased with increasing SF concentrations. The AFM images revealed that, after treated with methanol, there was a groove on the surface of fiber, which is conducive to cell attachment. The structure of electrospun SF fibers was characterized by NMR, WAXD, and DSC. The results displayed that SF in electrospun fibers was present in a random coil conformation, SF conformation transformed from random coil to beta-sheet when treated with methanol. Cell attachment and proliferation studies with pig iliac endothelial cells (PIECs) demonstrated that electrospun SF scaffolds significantly promoted cell attachment and proliferation in comparison with cast SF films. These results suggest electrospun SF scaffolds may be potential candidates for cardiovascular tissue engineering.

Vitamin C-reinforcing silk fibroin nanofibrous matrices for skin care application

In this work, we reported on the preparation and skin benefits of L-ascorbic acid 2-phosphate (VC-2-p)-loaded silk fibroin (SF) nanofibrous matrices for the first time. The matrices was fabricated using a facile eco-friendly electrospinning processing. With a post treatment of 75 v/v% ethanol vapor, the structure of the matrices transformed from unstable silk I form into water-stable silk II. In vitro release studies confirmed VC-2-p disassociated from SF nanofibrous matrices easily. Both neat and VC-2-p-loaded SF nanofibrous matrices were beneficial to mouse fibroblast L929 cells (L929 cells) adhering, spreading and proliferating against cover slips. Whereas compared with neat SF nanofibrous matrices, VC-2-p-loaded SF nanofibrous matrices significantly promoted the expression of collagen type I alpha 1 (Col1a1), as evidenced by real time PCR. Subsequently, the oxidative injury model further verified both matrices aided L929 cells through antioxidation to survive from tert-butyl hydroperoxide-induced oxidative stress (OS). Importantly, under severe OS, L929 cells on VC-2-p-loaded SF nanofibrous matrices maintained a higher mRNA level of Col1a1 as well as another two important functional genes, glutathione peroxidase 1 and catalase, than neat SF nanofibrous matrices. Our findings clearly indicated that the impressive skin benefits of SF nanofibrous matrices were further reinforced with the incorporation of VC-2-p, which implies the promising application of VC-2-p-loaded SF nanofibrous matrices in personal skin care and skin regeneration, including serving as wound dressings and anti-aging materials.

Electrospun Silk Fibroin–Hydroxybutyl Chitosan Nanofibrous Scaffolds to Biomimic Extracellular Matrix

Silk fibroin (SF)–hydroxybutyl chitosan (HBC) blend nanofibrous scaffolds were fabricated using 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and trifluoroacetic acid (TFA) as solvents to biomimic the native ECM by electrospinning. SEM results showed that the average nanofibrous diameter increased when the content of HBC was raised from 20% to 100%. 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. 13C-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 to β-sheet structure. Moreover, the use of genipin vapour not only induced conformation of SF to convert from random coil to β-sheet structure but also acted as a cross-linking agent for SF and HBC. Cell viability studies demonstrated that SF/HBC nanofibrous scaffolds presented good cellular compatibility. Thus, electrospun SF/HBC blended nanofibres may provide an ideal biomimic tissue-engineering scaffold.