Pure Silk Fibroin Scaffold Research Articles

Construction of functional magnetic scaffold with temperature control switch for long-distance vascular injury

Fusion of endothelial monolayer for rapid reendothelialization is the key to vascular transplantation, which is crucial for remodeling long-distance (≥10 mm) vascular injury (LDVJ) and reducing the long-term repair period. Although silk fibroin (SF) has been widely prepared as a scaffold to repair vascular injuries, it is still challenging to remodel LDVJs. The design and manufacture of functional scaffolds with a rapid reendothelialization enhancement effect are helpful to solve this problem. Here, we developed a well-designed functional magnetic scaffold (MDP) with a temperature-controlled switch, intelligently regulating the release of magnetic nanoparticles (MNPs) to induce macrophages migration and M2 polarization. The results show that the radial support force of MDP is appropriately five times higher than that of pure silk fibroin scaffold (SFC), and the degradation of MDP is delayed, which is beneficial to maintaining the integrity of the scaffold. MDP induces the migration of macrophages, upregulates the expression of repair cytokines such as TGF-β, IL-10 and VEGF, and promotes the proliferation of mouse aortic vascular smooth muscle (MOVAS) cells. More importantly, the enhanced migration of macrophages at the phase transition temperature is more conducive to the proliferation and accumulation of vascular repair-related cells, which is conducive to rapid reendothelialization. In addition, MDP also significantly regulates the polarization of macrophages to M2 type, which is conducive to the secretion of repair cytokines to further promote vascular recovery. The evaluation of endothelial cells' adhesion and function also proved that MDP could increase the expression of vascular endothelial cadherin (VE-Cad) and collagen IV (COL IV) and has the potential for rapid reendothelialization. The replacement model in vivo animal experiments further proved that MDP exhibited a better repair effect than SFC, providing a new option for the repair of LDVJ in the future.

A flexible and biocompatible bombyx mori silk fibroin/wool keratin composite scaffold with interconnective porous structure

The paper describes the preparation of a porous bombyx mori silk fibroin (SF)/wool keratin (WK) composite scaffold with mimic structure and function for cartilage tissue engineering. A porous composite scaffold made from SF/WK in an appropriate concentration and mass ratio was prepared using a freeze-drying technique. Results showed that the composite scaffolds are water-insoluble; possess good mechanical properties, porosity above 80%, and pore size above 200 μm. Larger pore size and better connectivity of the composite scaffold than the pure SF scaffolds were contributed by the WK addition. The heat resistance and water-swelling of WK enhanced the thermal and mechanical properties of the composite scaffolds. In vitro cytotoxicity assessments showed cells with a good growth state, confirming no toxicity to the cells. The results of in vivo biocompatibility assessments exhibited that there is almost no inflammatory response in the implantation site tissue of the rats. The development of porous SF/WK composite scaffold has the potential in cartilage tissue engineering.

Supercritical carbon dioxide assisted fabrication of biomimetic sodium alginate/silk fibroin nanofibrous scaffolds

AbstractIn this study, biomimetic sodium alginate (SA)/silk fibroin (SF) scaffolds were successfully fabricated by supercritical CO2technology. The SA/SF scaffolds exhibited an interconnected porous and extracellular matrix (ECM)‐like nanofibrous structures. Moreover, the SA microparticles were embedded in the SF scaffolds. Increasing the content of SA microparticles could improve tensile strength and compressive strength of the SF scaffolds and reduce the porosity of the SF scaffolds. The addition of the SA microparticles could also regulate the degradation rate of the SA/SF scaffolds. Furthermore, the results of in vitro biocompatibility evaluation, indicated that the SA/SF scaffolds exhibited no obvious cytotoxicity and higher cell adhesion ability and were more favorable for L929 fibroblasts proliferation than pure SF scaffolds. Therefore, the SA/SF scaffolds with ECM‐like nanofibrous and interconnected porous structure have potential application in skin tissue engineering.

Pro-angiogenic decellularized vessel matrix gel modified by silk fibroin for rapid vascularization of tissue engineering scaffold.

Current pro-angiogenic methods in the fields of tissue engineering always aim to enrich the vascular network but neglect to provide an appropriate environment for cells, which may lead to incomplete endothelium or thrombosis. Decellularized matrix gels derived from specific tissue are expected to be suitable for targeted tissue regeneration because they preserve the biochemical properties of the native tissue. Decellularized vascular matrix gel (DVMG) has shown promise for rapid vascularization. However, DVMG is difficult to directly apply due to its weak mechanical properties and rapid degradation. In this work, silk fibroin (SF) was introduced to the DVMG to improve the physical properties of the hybrid scaffolds. The performances of the SF/DVMG scaffolds were characterized, and the results showed that SF effectively improved the overall mechanical properties of the scaffold and decreased the degradation rate. SF/DVMG scaffolds also showed good cell growth promotion effects in vitro. After the scaffolds were subcutaneously implanted in the dorsa of rats, more CD34-positive endothelial cells were expressed in the DVMG-containing scaffolds, and the number of vascular loops significantly increased compared to that of the pure SF scaffold control. The development of DVMG creates more possibilities for the rapid vascular network generation of clinically engineered scaffolds.

PC12 cells proliferation and morphological aspects: Inquiry into raffinose-grafted graphene oxide in silk fibroin-based scaffold

Peripheral nerves injuries (PNIs) still associated with both clinical and social problems. Accordingly, tissue engineers’ and surgeons’ attentions have been drawn for finding efficient solutions. Herein, scaffolds based on silk fibroin (SF)/raffinose-grafted-GO (S.RafGO) nanocomposite were fabricated. Subsequently, PC12 cells growth in term of number and morphology were investigated on neat SF polymer, SF/GO (S.GO), and S.RafGO scaffolds. Characterization via scanning electron microscopy (SEM) exhibited more fibrous structures with few lamellar nanosheets for S.GO; although, S.RafGO showed extended lamellar with lower fibrous structure. Due to the incorporation of GO and raffinose-GO nanosheets into SF structure, electrical conductivity increased ~30 and 40%, respectively. Water contact angle data revealed that S.RafGO is more wettable than SF and S.GO. Real-time PCR technique detected higher expressions of the β-tubulin, MAP2 genes on S.RafGO scaffolds in comparison with S.GO and the control group. Immunocytochemistry staining studies confirmed the overexpression of neural-specific proteins including nestin, β-tubulin of S.GO, and S.RafGO nanocomposites in comparison with pure SF scaffolds.

Usnic-Acid-Functionalized Silk Fibroin Composite Scaffolds for Cutaneous Wounds Healing.

Despite the progress in chronic wound treatment, antibacterial cutaneous scaffold with high efficiency in wound healing is still the hot spot in the field. In present study, a functionalized silk fibroin (SF) cutaneous scaffold incorporated with natural medicine usnic acid (UA) is investigated, in which UA is used as an antibacterial and wound-healing reagent. Via electrospinning, UA-SF mixture is fabricated into UA-SF composite scaffold (USCS), which is composed of uniform nanofibers with average diameters of around 360 ± 10nm. The interwoven nanofibers form mesh structure providing sufficient moisture permeability for scaffold. With methanol treatment, USCS presents improved mechanical properties and stability to protease XIV. In the presence of USCS, the growth rate of both Gram-positive and Gram-negative bacteria, including Staphylococcus aureus, Streptococci pyogenes, Escherichia coli, and Pseudomonas aeruginosa, is significantly inhibited in plate culture and suspension assays. In a cutaneous excisional mouse wound model, USCS presents a significant increase of wound closure rate, compared with pure SF scaffold and commercial dressing, Tegaderm Hydrocolloid 3M . The histological assessments further prove that USCS can enhance re-epithelialization, vascularization, and collagen deposition in wound site to promote the wound-healing process, which indicates the potential application of USCS in chronic wound treatment.

Strong and biocompatible three-dimensional porous silk fibroin/graphene oxide scaffold prepared by phase separation

Silk fibroin (SF) is blended with graphene oxide (GO) to prepare the strong and biocompatible three dimensional porous SF/GO blended scaffold via phase separation. GO could be well dispersed in SF solution and GO could also be well distributed in the SF scaffold. Furthermore, the introduction of GO can lead to structural change in the bended scaffold. Higher concentration of GO resulted in more compact structure and smaller pore size of the composite scaffolds without decreasing their porosity. Scanning electron microscopy and energy dispersive spectrometry results also reveal that SF and GO are homogeneous blended together. Analysis of chemical structures of the scaffold shows that addition of GO do not affect the crystalline structure of SF and it is evenly blended with SF. The blended scaffold has significantly higher breaking strength than the pure SF scaffold. In vitro study indicates that both pure SF scaffold and SF/GO composite scaffold support growth and proliferation of MC3T3-E1 osteoprogenitor cells. However, the addition of GO contribute to the proliferation of MC3T3-E1 osteoprogenitor. The testing results show that the blended scaffold is an appropriate candidate for tissue engineering.

Electrospun silk fibroin scaffolds coated with reduced graphene promote neurite outgrowth of PC-12 cells under electrical stimulation

Novel approaches to neural research require biocompatible materials capable to act as electrode structures or scaffolds for tissue engineering in order to stimulate or restore the functionality of damaged tissues. This work offers promising results that indicate the potential use of electrospun silk fibroin (SF) scaffolds coated with reduced graphene oxide (rGO) in this sense. The coated material becomes conductor and electroactive. A complete characterisation of SF/rGO scaffolds is provided in terms of electrochemistry, mechanical behaviour and chemical conformation of fibroin. The excellent biocompatibility of this novel material is proved with cultures of PC-12 cells. The coating with rGO improved the adhesion of cells in comparison with cells growing onto the surface of pure SF scaffolds. Also, the use of SF/rGO scaffolds combined with electrical stimulation promoted the differentiation into neural phenotypes reaching comparable or even superior levels to those obtained by means of the traditional treatment with neural growth factor (NGF).

Silk scaffolds connected with different naturally occurring biomaterials for prostate cancer cell cultivation in 3D.

In the present work, different biopolymer blend scaffolds based on the silk protein fibroin from Bombyx mori (BM) were prepared via freeze-drying method. The chemical, structural, and mechanical properties of the three dimensional (3D) porous silk fibroin (SF) composite scaffolds of gelatin, collagen, and chitosan as well as SF from Antheraea pernyi (AP) and the recombinant spider silk protein spidroin (SSP1) have been systematically investigated, followed by cell culture experiments with epithelial prostate cancer cells (LNCaP) up to 14 days. Compared to the pure SF scaffold of BM, the blend scaffolds differ in porous morphology, elasticity, swelling behavior, and biochemical composition. The new composite scaffold with SSP1 showed an increased swelling degree and soft tissue like elastic properties. Whereas, in vitro cultivation of LNCaP cells demonstrated an increased growth behavior and spheroid formation within chitosan blended scaffolds based on its remarkable porosity, which supports nutrient supply matrix. Results of this study suggest that silk fibroin matrices are sufficient and certain SF composite scaffolds even improve 3D cell cultivation for prostate cancer research compared to matrices based on pure biomaterials or synthetic polymers.

Carboxymethyl cellulose enables silk fibroin nanofibrous scaffold with enhanced biomimetic potential for bone tissue engineering application

Novel silk fibroin (SF) and carboxymethyl cellulose (CMC) composite nanofibrous scaffold (SFC) were developed to investigate their ability to nucleate bioactive nanosized calcium phosphate (Ca/P) by biomineralization for bone tissue engineering application. The composite nanofibrous scaffold was prepared by free liquid surface electrospinning method. The developed composite nanofibrous scaffold was observed to control the size of Ca/P particle (≤100nm) as well as uniform nucleation of Ca/P over the surface. The obtained nanofibrous scaffolds were fully characterized for their functional, structural and mechanical property. The XRD and EDX analysis depicted the development of apatite like crystals over SFC scaffolds of nanospherical in morphology and distributed uniformly throughout the surface of scaffold. Additionally, hydrophilicity as a measure of contact angle and water uptake capacity is higher than pure SF scaffold representing the superior cell supporting property of the SF/CMC scaffold. The effect of biomimetic Ca/P on osteogenic differentiation of umbilical cord blood derived human mesenchymal stem cells (hMSCs) studied in early and late stage of differentiation shows the improved osteoblastic differentiation capability as compared to pure silk fibroin. The obtained result confirms the positive correlation of alkaline phosphatase activity, alizarin staining and expression of runt-related transcription factor 2, osteocalcin and type1 collagen representing the biomimetic property of the scaffolds. Thus, the developed composite has been demonstrated to be a potential scaffold for bone tissue engineering application.

Mammary epithelial cell adhesion, viability, and infiltration on blended or coated silk fibroin–collagen type I electrospun scaffolds

Interactions between cells and the extracellular matrix (ECM) play a crucial role in regulating biological tissue function. Silk biomaterials from Bombyx mori (B. mori) silkworm silk are widely used in tissue engineering. As this silk fibroin (SF) contains no strong adhesion sites, we assessed whether the blending or coating of SF with collagen would further improve SF biocompatibility, in part through the addition of the specific integrin recognition sequences. In the present study, electrospun scaffolds were developed by blending 7% SF and 7% type I collagen solutions at ratios of 100:0 (pure SF), 95:5, 90:10, and 85:15 (SF:collagen, v/v) prior to electrospinning. Pure SF scaffolds were further coated with collagen type I. The physical and mechanical properties of these scaffolds and MCF10A mammary epithelial cell adhesion, viability, and infiltration into these blended or coated SF–collagen (SF–C) scaffolds were determined. The blending of SF with collagen decreased average pore sizes and fiber diameters of the electrospun scaffolds regardless of the ratio (p<0.01). The mechanical strength of these scaffolds did not change in their hydrated state (ns), but was decreased for 85:15 SF–C blended scaffolds in the dry state (p<0.05). The adhesion of MCF10A cells was significantly increased in SF–C blended or coated scaffolds compared to pure SF scaffolds (p<0.01). MCF10A cell viability and infiltration on SF–C coated scaffolds were significantly higher compared to all other conditions tested (p<0.01).

Effect of hyaluronan molecular weight on structure and biocompatibility of silk fibroin/hyaluronan scaffolds

The structure of scaffolds is known to play a key role in tissue engineering as it provides structural support and physical environment allowing cells to reside and rebuild the target tissue. In this work we investigated the effects of hyaluronan (HA) molecular weight (MW: 0.6, 1.6 and 2.6×106Da) on the pore structure, secondary structure, and biocompatibility of lyophilized silk fibroin (SF)/HA composite scaffolds. The results showed that HA promoted the pore structure formation and restrained the formation of separate sheet like structures in the SF/HA blend scaffolds, which was dependent on HA MW. The 3D pore structure maintained the scaffold shape during the process of 75% ethanol annealing. Structural studies indicated that HA did not induce but hinder SF conformation transition from random coil to β-sheet before and after treatment. In addition, SF/HA scaffolds showed an increase in cell proliferation compared to pure SF scaffold. These findings demonstrated the important role of HA MW in preparing SF/HA blend scaffolds suitable for application in tissue engineering.

Calcium Ion Treatment Behavior of Silk Fibroin/Sodium Alginate Scaffolds

The silk fibroin/sodium alginate scaffolds were prepared using lyophilization method. And then, the blend scaffolds were treated with calcium ions. The morphology of the blend scaffolds exhibited a thin layer structure before calcium ions treatment, and much more rod-like structure appeared at the layer surface with adding the increase content of sodium alginate in the blend scaffolds. After calcium ions treatment, much more rod-like structure disappeared after adding 30% sodium alginate or more in the blend scaffolds. Wide angle X-ray diffraction and Fourier transform infrared analysis results confirmed the crystal structure of silk fibroin was not influenced by adding the different content of sodium alginate, exhibiting the silk I and silk II structure co-existed in the blend scaffolds. And the same time, the average mass loss value of the blend scaffolds was higher than the pure silk fibroin scaffold, reaching 9.884%, 11.2%, and 8.626%, respectively, when the blend scaffolds contained 10%, 30%, and 50% sodium alginate, respectively. Thus, the silk fibroin/sodium alginate scaffolds should be a useful biomaterial applicable for a wide range of tissue engineering.

Chondrogenic differentiation of rat MSCs on porous scaffolds of silk fibroin/chitosan blends

Adult bone marrow derived mesenchymal stem cells are undifferentiated, multipotential cells and have the potential to differentiate into multiple lineages like bone, cartilage or fat. In this study, polyelectrolyte complex silk fibroin/chitosan blended porous scaffolds were fabricated and examined for its ability to support in vitro chondrogenesis of mesenchymal stem cells. Silk fibroin matrices provide suitable substrate for cell attachment and proliferation while chitosan are promising biomaterial for cartilage repair due to it’s structurally resemblance with glycosaminoglycans. We compared the formation of cartilaginous tissue in the silk fibroin/chitosan blended scaffolds with rat mesenchymal stem cells and cultured in vitro for 3 weeks. Additionally, pure silk fibroin scaffolds of non-mulberry silkworm, Antheraea mylitta and mulberry silkworm, Bombyx mori were also utilized for comparative studies. The constructs were analyzed for cell attachment, proliferation, differentiation, histological and immunohistochemical evaluations. Silk fibroin/chitosan blended scaffolds supported the cell attachment and proliferation as indicated by SEM observation, Confocal microscopy and metabolic activities. Alcian Blue and Safranin O histochemistry and expression of collagen II indicated the maintenance of chondrogenic phenotype in the constructs after 3 weeks of culture. Glycosaminoglycans and collagen accumulated in all the scaffolds and was highest in silk fibroin/chitosan blended scaffolds and pure silk fibroin scaffolds of A. mylitta. Chondrogenic differentiation of MSCs in the silk fibroin/chitosan and pure silk fibroin scaffolds was evident by real-time PCR analysis for cartilage-specific ECM gene markers. The results represent silk fibroin/chitosan blended 3D scaffolds as suitable scaffold for mesenchymal stem cells-based cartilage repair.

Preparation, characterization and biocompatibility of electrospinning heparin-modified silk fibroin nanofibers

In this study, the electrospun silk fibroin nanofibrous scaffolds were modified with heparin by grafting after plasma treatment and blending electrospinning. Morphology, microstructure, chemical composition and grafting efficiency of the heparin-modified silk fibroin nanofibrous scaffolds were characterized to evaluate the effect of modification by means of scanning electron microscopy (SEM), Fourier transform infrared spectra (FTIR) and X-ray photoelectron spectrometer (XPS). The results showed that the heparin was successfully introduced to the silk fibroin nanofibrous scaffolds by both the two kinds of modification, and there was a hydrogen bonding between the silk fibroin and heparin. Moreover, the hydrophilicity, O-containing groups and negative charge density of the heparin-modified scaffolds were enhanced. In vitro coagulation time tests showed that the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) of the heparin-modified scaffolds were much higher than those of the pure silk fibroin scaffolds. L929 fibroblasts and EVCs spread and proliferated better on the heparin-modified scaffolds than on the pure silk fibroin scaffolds. Macrophages, neutrophils and lymphocytes were not observed in the heparin-modified scaffolds, which indicated that the modified scaffolds could induce minor inflammation in vivo. The results indicated that the electrospun heparin-modified silk fibroin nanofibrous scaffolds could be considered as ideal candidates for tissue engineering scaffolds.

Fabrication and characterization of bioactive silk fibroin/wollastonite composite scaffolds

Composite scaffolds of silk fibroin (SF) with bioactive wollastonite were prepared by freeze-drying. X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy analysis showed that random coil and β-sheet structure co-existed in the SF scaffold. The mechanical performance, surface hydrophilicity and water-uptake capacity of the composite scaffolds were improved compared with those of pure SF scaffold. The bioactivity of the composite scaffold was evaluated by soaking in a simulated body fluid (SBF), and formation of a hydroxycarbonate apatite (HCA) layer was determined by FT-IR and XRD. The results showed that the SF/wollastonite composite scaffold was bioactive as it induced the formation of HCA on the surface of the composite scaffold after soaking in SBF for 5 days. In vitro cell attachment and proliferation tests showed that the composite scaffold was a good matrix for the growth of L929 mouse fibroblast cells. Consequently, the incorporation of wollastonite into the SF scaffold can enhance both the mechanical strength and bioactivity of the scaffold, which suggests that the SF/wollastonite composite scaffold may be a potential biomaterial for tissue engineering.