Three-dimensional silk impregnated HAp/PHBV nanofibrous scaffolds for bone regeneration

Three-dimensional silk fibroin impregnated poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofibrous scaffolds with or without hydroxyapatite (HAp) were prepared by wet-electrospinning method followed by freeze-drying. Scaffolds with cotton wool-like structure have the average fiber diameter of 450–850?nm with 80–85% porosity. In-vitro cell culture tests using MG-63 osteosarcoma human cells revealed improved cell viability, alkaline phosphatase (ALP) activity and total cellular protein amount on the silk impregnated scaffolds compared to PHBV and HAp/PHBV scaffolds after 10 days of cell culture. Immunohistochemical analyses on the silk impregnated scaffolds showed that HAp triggered cell penetration and type I collagen production. Besides, HAp mineralization tendency increased with a decrease in percent crystallinity of the scaffolds comprising HAp and silk after 4 weeks of incubation in simulated body fluid. Consequently, cotton wool-like HAp/PHBV-SF scaffold would be a promising candidate as a bone-filling material for tissue regeneration.     

Copolymer‐induced silk‐based hydrogel with porous and nanofibrous structure

A novel physical blend method was developed to accelerate the self‐assembly process of silk fibroin (SF) solution into porous and nanofibrous hydrogel by temperature‐sensitive copolymer. Silk‐based hydrogel was firstly achieved through blending SF solution with copolymer aqueous solution and then removed the copolymer from blend solution by heat treatment (50°C) after 24 h hydrogelation. Copolymer molecules would interact with SF molecules resulting in reduction of copolymer micelles, which further affect the hydrogelation of SF solutions. Copolymers could be separated from blend solution by heat treatment under an acceptable temperature (50°C), especially the copolymer₂. Fourier transform infrared (FTIR) and X‐ray diffraction showed the blending of copolymer significantly accelerated the self‐assembly of SF into physically crosslinked β‐sheet crystals at room temperature which led to the sol‐gel transition. Results from DTA and X‐ray diffraction showed that the effect of copolymer on crystalline structure of SF in silk‐based hydrogel was very weak. SF molecules transformed from distributed globular nanoparticles to nanofilaments clustered during hydrogelation, resulting in the porous and nanofibrous structure of silk‐based hydrogel. Furthermore, silk‐based hydrogel was prepared in aqueous solution avoiding organic solvents and harsh processing conditions, suggesting that this silk‐based hydrogel could be a potential candidate scaffold for biomedical applications. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

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

In this study, biomimetic sodium alginate (SA)/silk fibroin (SF) scaffolds were successfully fabricated by supercritical CO₂ technology. 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.     

Electrospun three‐dimensional silk fibroin nanofibrous scaffold

Practical application to three‐dimensional (3‐D) tissue culture has been limited by the structural restriction of two‐dimensional (2‐D) nature of electrospun nanofiber mat. In this study, for constructing 3‐D nanofibrous structure as real 3‐D tissue engineering scaffold, we developed new fabrication process with silk fibroin (SF) by electrospinning and evaluated the features of this SF nanofiber scaffold (SFNS) through morphological and cell‐culture analyses. Foam type of the SFNS exhibited high porosity as well as large pores and its cell proliferation well occurred inside (inner spaces of pores), which makes this suitable for 3‐D cell‐culture scaffold. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Silk fibroin‐chondroitin sulfate‐alginate porous scaffolds: Structural properties and in vitro studies

The development of porous biodegradable scaffolds is of great interest in tissue engineering. In this regard, exploration of novel biocompatible materials is needed. Silk fibroin‐chondroitin sulfate‐sodium alginate (SF‐CHS‐SA) porous hybrid scaffolds were successfully prepared via lyophilization method and crosslinked by 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide‐ethanol treatment. According to the scanning electron microscopy studies, mean pore diameters of the scaffolds were in the range of 60–187 μm. The porosity percentage of the scaffold with SF‐CHS‐SA ratio of 70 : 15 : 15 (w/w/w %) was 92.4 ± 3%. Attenuated total reflectance Fourier transform infrared spectroscopy, X‐ray diffraction, and differential scanning calorimetry results confirmed the transition from amorphous random coil to crystalline β‐sheet in treated SF‐CHS‐SA scaffold. Compressive modulus was significantly improved in hybrid scaffold with SF‐CHS‐SA ratio of 70 : 15 : 15 (3.35 ± 0.15 MPa). Cytotoxicity assay showed that the scaffolds have no toxic effects on chondrocytes. Attachment of chondrocytes was much more improved within the SF‐CHS‐SA hybrid scaffold. Real‐time polymerase chain reaction analyses showed a significant increase in gene expression of collagen type II, aggrecan, and SOX9 and decrease in gene expression of collagen type I for SF‐CHS‐SA compared with SF scaffold. This novel hybrid scaffold can be a good candidate to be utilized as an efficient scaffold for cartilage tissue engineering. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 41048.     

Comparison of three-dimensional printing for fabricating silk fibroin-blended scaffolds

In this study, two types of scaffolds were fabricated by 3D printing. Morphology, physical properties, biochemical were evaluated. Cell morphology and distribution were observed. It was found that the silk fibroin/collagen (SF/C) scaffold-based material had significantly higher values than the silk fibroin/chitosan (SF/CS) scaffold-based material. Hematoxylin and eosin staining of the scaffolds revealed that the number of cells in the SF/C scaffold was higher. Cells grew well inside the SF/C scaffold as measured by scanning electron microscope. Reverse Transcriotion-Polymerase Chain Reaction (RT-PCR) and Western blot showed that type II collagen and Sox9 can be found in SF/C scaffold. Therefore, the SF/C scaffold exhibited better overall performance compared with the SF/CS scaffold.     

Influence of Post-Treatment with 75% (v/v) Ethanol Vapor on the Properties of SF/P(LLA-CL) Nanofibrous Scaffolds

In order to improve the water-resistant ability of silk fibroin (SF) and SF/P(LLA-CL) blended nanofibrous scaffolds for tissue engineering applications, 75% (v/v) ethanol vapor was used to post-treat electrospun nanofibers. SEM indicated that the treated SF and SF/P(LLA-CL) nanofibrous scaffolds maintained a nanofibrous structure and possessed good water-resistant ability. Characterization of (13)C CP-MAS NMR clarified that 75% (v/v) ethanol vapor could induce SF conformation from random coil or α-helix to β-sheet. Although the water contact showed that treated SF/P(LLA-CL) blended nanofibrous scaffolds were hydrophobic, the water uptake demonstrated that their hydrophilicity was greatly superior to those of pure P(LLA-CL) nanofibrous scaffolds. Furthermore, the treated SF/P(LLA-CL) nanofibrous scaffolds, both in dry state and wet state, could retain good mechanical properties. Therefore, 75% (v/v) ethanol vapor treatment might be an ideal method to treat SF and SF/P(LLA-CL) nanofibrous scaffolds for biomedical applications.     

Fabrication and intermolecular interactions of silk fibroin/hydroxybutyl chitosan blended nanofibers

The native extracellular matrix (ECM) is composed of a cross-linked porous network of multifibril collagens and glycosaminoglycans. Nanofibrous scaffolds of silk fibroin (SF) and hydroxybutyl chitosan (HBC) blends were fabricated using 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and trifluoroacetic acid (TFA) as solvents to biomimic the native ECM via electrospinning. Scanning electronic microscope (SEM) showed that relatively uniform nanofibers could be obtained when 12% SF was blended with 6% HBC at the weight ratio of 50:50. Meanwhile, the average nanofibrous diameter increased when the content of HBC in SF/HBC blends was raised from 20% to 100%. Fourier transform infrared spectra (FTIR) and (13)C nuclear magnetic resonance (NMR) showed SF and HBC molecules existed in hydrogen bonding interactions but HBC did not induce conformation of SF transforming from random coil form to β-sheet structure. X-ray diffraction (XRD) confirmed the different structure of SF/HBC blended nanofibers from both SF and HBC. Thermogravimetry-Differential thermogravimetry (TG-DTG) results demonstrated that the thermal stability of SF/HBC blend nanofibrous scaffolds was improved. The results indicated that the rearrangement of HBC and SF molecular chain formed a new structure due to stronger hydrogen bonding between SF and HBC. These electrospun SF/HBC blended nanofibers may provide an ideal tissue engineering scaffold and wound dressing.     

Electrospun polylactide/silk fibroin–gelatin composite tubular scaffolds for small‐diameter tissue engineering blood vessels

Many synthetic scaffolds have been used as vascular substitutes for clinical use. However, many of these scaffolds may not show suitable properties when they are exposed to physiologic vascular environments, and they may fail eventually because of some unexpected conditions. Electrospinning technology offers the potential for controlling the composition, structure, and mechanical properties of scaffolds. In this study, a tubular scaffold (inner diameter = 4.5 mm) composed of a polylactide (PLA) fiber outside layer and a silk fibroin (SF)–gelatin fiber inner layer (PLA/SF–gelatin) was fabricated by electrospinning. The morphological, biomechanical, and biological properties of the composite scaffold were examined. The PLA/SF–gelatin composite tubular scaffold possessed a porous structure; the porosity of the scaffold reached 82 ± 2%. The composite scaffold achieved the appropriate breaking strength (1.28 ± 0.21 MPa) and adequate pliability (elasticity up to 41.11 ± 2.17% strain) and possessed a fine suture retention strength (1.07 ± 0.07 N). The burst pressure of the composite scaffold was 111.4 ± 2.6 kPa, which was much higher than the native vessels. A mitochondrial metabolic assay and scanning electron microscopy observations indicated that both 3T3 mouse fibroblasts and human umbilical vein endothelial cells grew and proliferated well on the composite scaffold in vitro after they were cultured for some days. The PLA/SF–gelatin composite tubular scaffolds presented appropriate characteristics to be considered as candidate scaffolds for blood vessel tissue engineering. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Biodegradable nanofibrous membrane of zein/silk fibroin by electrospinning

BACKGROUND: Electrospinning of natural polymers offers a promising approach to generate nanofibers with a similar fibrillar structure to that of native extracellular matrix. In the present work, zein/silk fibroin (SF) blends were electrospun with formic acid as solvent to fabricate bicomponent nanofibrous scaffolds for biomedical applications. RESULTS: The zein/SF electrospun nanofibers had a smaller diameter and narrower diameter distribution than pure zein nanofibers, and the average diameter gradually decreased from 265 to 230 nm with increasing SF content in the blend. The predominant presence of α‐helix zein structure and random coil form of silk I in blend fibrous membranes was confirmed from Fourier transform infrared spectral and wide‐angle X‐ray diffraction data, while conversion to the β‐sheet structure of SF was also detected. The tensile strength of the zein/SF fibrous membranes was improved as the content of SF in the blend fibers increased. A preliminary study of in vitro degradation and cytotoxicity evaluated by MTT assay indicated that biodegradable zein/SF fibrous membranes did not induce cytotoxic effects in an L929 mouse fibroblast system. CONCLUSION: Biodegradable zein/SF fibrous membranes with good mechanical properties and cytocompatibility combine the beneficial characteristics of the individual components and may be useful for biomedical applications. Copyright © 2009 Society of Chemical Industry     

Fabrication and Evaluation of Polycaprolactone–Poly(hydroxybutyrate) or Poly(3‐Hydroxybutyrate‐co‐3‐Hydroxyvalerate) Dual‐Leached Porous Scaffolds for Bone Tissue Engineering Applications

Polycaprolactone (PCL) blend with poly(hydroxybutyrate) (PHB) or poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHBV) dual‐leached scaffolds are prepared by using the solvent casting and salt–polymer‐leaching technique. The blending of the PHB and PHBV in PCL scaffolds results in decreased porosities of the scaffolds, and the water absorption capacities of the scaffolds also decrease. The compressive modulus of the PCL–PHB and PCL–PHBV dual‐leached scaffolds is greatly increased by the blending of PHB or PHBV matrix. An indirect cytotoxicity evaluation of all scaffolds with mouse fibroblastic cells (L929) and mouse calvaria‐derived preosteoblastic cell (MC3T3‐E1) indicates that all dual‐leached scaffolds are posed as nontoxic to cells. Both PCL–PHB and PCL–PHBV dual‐leached scaffolds are supported by the attachment of MC3T3‐E1 at significantly higher levels to tissue culture polystyrene plate (TCPS) and are able to support the proliferation of MC3T3‐E1 at higher levels to that cells on TCPS and PCL scaffolds. For mineralization, cells cultured on surfaces of PCL–PHB and PCL–PHBV dual‐leached scaffolds show higher mineral deposition than on TCPS and PCL scaffold.

     

Soluble‐eggshell‐membrane‐protein‐modified porous silk fibroin scaffolds with enhanced cell adhesion and proliferation properties

In this study, a porous silk fibroin (SF) scaffold was modified with soluble eggshell membrane protein (SEP) with the aim of improving the cell affinity properties of the scaffold for tissue regeneration. The pore size and porosity of the prepared scaffold were in the ranges 200–300 μm and 85–90%, respectively. The existence of SEP on the scaffold surface and the structural and thermal stability were confirmed by energy‐dispersive X‐ray spectroscopy, X‐ray diffraction, Fourier transform infrared spectroscopy, differential scanning calorimetry, and thermogravimetric analysis. The cell culture study indicated a significant improvement in the cell adhesion and proliferation of mesenchymal stem cells (MSCs) on the SF scaffold modified with SEP. The cytocompatibility of the SEP‐conjugated SF scaffold was confirmed by a 3‐(4,5‐dimethyltriazol‐2‐y1)‐2,5‐diphenyl tetrazolium assay. Thus, this study demonstrated that the biomimic properties of the scaffold could be enhanced by surface modification with SEP. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40138.     

Study on the properties of the electrospun silk fibroin/gelatin blend nanofibers for scaffolds

Silk fibroin (SF)/gelatin blend nanofibers membranes as scaffolds were fabricated successfully via electrospinning with different composition ratios in formic acid. The formation of intermolecular hydrogen bonds and the conformational transition of SF provided scaffolds with excellent mechanical properties. FTIR and DTA analysis showed the SF/gelatin nanofibers had more β‐sheet structures than the pure SF nanofibers. The former's breaking tenacity increased from 0.95 up to 1.60 MPa, strain at break was 7.6%, average fiber diameter was 89.2 nm, porosity was 87%, and pore diameter was 142 nm. MTT, H&E stain, and SEM results showed that the adhesion, spreading, and proliferation of human umbilic vein endothelium cells (HUVECs) and mouse fibroblasts on the SF/gelatin nanofibers scaffolds were definitely better than that on the SF nanofibers scaffolds. The scaffolds could replace the natural ECM proteins, support long‐term cell growth, form three‐dimensional networks of the nanofibrous structure, and grow in the direction of fiber orientation. Our results prove that the addition of gelatin improved the mechanical and biological properties of the pure SF nanofibers, these SF/gelatin blend nanofiber membranes are desirable for the scaffolds and may be a good candidate for blood vessel engineering scaffolds. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

A novel silk fibroin/sodium alginate hybrid scaffolds

Porous silk fibroin/sodium alginate hybrid scaffolds were prepared through lyophilization method. Hybrid scaffolds were characterized for morphological and functional properties related to different mixture ratios between silk fibroin and sodium alginate. The silk fibroin/sodium alginate hybrid scaffolds showed a thin‐layer structure and much more irregular rod‐like structure appeared at the layer surface after adding 50% sodium alginate. The results of wide‐angle X‐ray diffraction and Fourier transform infrared analysis confirmed that the crystal structure of silk fibroin was not influenced by adding the different contents of sodium alginate, exhibiting the random coil structure in the hybrid scaffolds. The thermal behavior of the hybrid scaffolds exhibited major change with containing 30% sodium alginate or more. The porosity of the scaffolds varied between 92 and 94% with a favorable compressive modulus and stress. The mechanical properties results depicted the hybrid scaffolds containing 10% sodium alginate, with a porosity of 94.0 ± 0.10%, attained the highest compressive modulus and stress for 41 ± 6 and 44 ± 3 kPa, respectively. In addition, mineralization results showed hydroxyapatite crystal growing on the surface of the scaffold. This hybrid biomaterial should offer new and important options to the needs related to biomineralization and tissue engineering, in general. POLYM. ENG. SCI., 54:129–136, 2014. © 2013 Society of Plastics Engineers     

Rapid formation of flexible silk fibroin gel‐like films

Flexible silk fibroin gel‐like films with microporous morphology were prepared from B. mori silk fibroin fibers directly solubilized in formic acid/CaCl₂ solvent. These films were characterized by several analysis techniques to determine the structure and properties of films. The pore size of gel‐like films can be adjusted through SF concentration and Ca ions concentration. The controllable pore size in gel‐like films was grew from 3–5 μm to 100 μm under the increase of fibroin concentration from 1.0 wt % to 8.0 wt %. At the same time, the water content of silk fibroin gel‐like film decreased from 83.5 ± 3.4% to 68.2 ± 2.6%. With increasing Ca ions contents from 2.0 wt % to 10.0 wt % in dissolution process, the pore size and water content of silk fibroin gel‐like films grew larger, especially its water content values reached 86.2 ± 4.0% at 10.0 wt % Ca ions concentration. At wet condition, the gel‐like film with β‐sheet structure showed higher breaking stress (4.26 ± 0.31 MPa) and elongation (45.45 ± 15.79%) at 8.0 wt % concentration. With the preparation method, the membrane is hydrophilic and the pore size is adjustable, which contributes to high toughness and favorable cell growth environment, suggesting that these silk fibroin gel‐like films can be a potential candidate scaffold for biomedical applications, such as wound dressing, facial mask, contact lenses, etc. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41842.     

Fabrication and evaluation of nanofibrous polyhydroxybutyrate valerate scaffolds containing hydroxyapatite particles for bone tissue engineering

Tissue engineering is a new approach for regeneration of damaged tissues. The current clinical methods such as autograft and allograft transplantation are not effective for repairing bone damages, mainly due to the limited available sources and the donor-site side effects. In this research, the nanocomposite poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/nano hydroxyapatite (nHA) scaffolds with different nHA ratios for bone regeneration were utilized. The diameter and porosity of scaffolds were approximately 200?nm and 74%, respectively. The degradability test of the scaffolds suggests a low degradation rate with total degradation of 30% after 3 months. Cytotoxicity result showed that cultured osteoblast cells (MC3T3) on nanocomposite scaffolds had superiority in terms of higher proliferation and attachment in comparison with PHBV scaffold. The protein expression of alkaline phosphatase illustrated that nanofibrous scaffold containing hydroxyapatite had the highest alkaline phosphatase activities as a result of better proliferation. These results recommend that PHBV/nHA scaffolds are suitable candidates for bone tissue engineering.     

Interaction of human smooth muscle cells on random and aligned nanofibrous scaffolds of PHBV and PHBV-gelatin

Tissue-engineered scaffolds with nanofibrous morphology have been shown to be effective in regeneration of tissues because nanofibers mimic the native architecture of the extracellular matrix. The unique alignment in the native tissue motivated the authors to fabricate aligned nanofibers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and PHBV-gelatin. The in vitro potential of the scaffolds was evaluated using human smooth muscle cells. MTS study confirmed that PHBV aligned nanofibrous scaffold promotes better cell proliferation as well as gene expression of key contractile and extracellular matrix markers than their PHBV-gelatin counterparts. Hence, the PHBV aligned nanofibers can be used as a biomimetic scaffold for the regeneration of esophagus. Electrospinning system for aligned nanofibers fabrication (A) and interaction of human smooth muscle cells on aligned nanofibers (B).     

Study on secondary structural transition of nano‐TiO2 modified silk fibroin composite films by two‐dimensional Raman correlation spectroscopy and solid‐state 13C‐NMR spectroscopy

By a sol–gel processing, the nano‐TiO₂/silk fibroin (SF) composite films were prepared. One‐dimensional (1D) Raman, two‐dimensional (2D) correlation Raman spectroscopy, and ¹³C cross‐polarization magic‐angle‐spinning nuclear magnetic resonance (¹³C CP‐MAS NMR) were used to characterize the structural evolution of SF as the nano‐TiO₂ content increased from 0 to 0.4 wt%. The experimental data demonstrated that the secondary structures in the pure SF film and nano‐TiO₂/silk fibroin (SF) composite films were random coil, α‐helix and β‐sheet structures. The nano‐TiO₂ particles formed in the SF films might induce partial structural transitions from random coil and Silk I (α‐helix) to Silk II (β‐sheet). The transition identified by 2D‐Raman correlation spectra was the following order: silk I‐like structure, silk I (α‐helical structure), Silk II‐like structure, and Silk II (β‐sheet structure). POLYM. COMPOS., 36:121–127, 2015. © 2014 Society of Plastics Engineers     

Chitosan/silk fibroin composite scaffolds for wound dressing

Three‐dimensional (3D) chitosan/silk fibroin (CS/SF) porous composite scaffolds have been prepared by simply coating a thin layer of CS onto spunlaced SF scaffolds via hydrogen‐bonding assembly technique, and they were characterized by Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), X‐ray diffraction (XRD), and mechanical property measurements. The results show that porous scaffolds have a pore diameter around 50–200 μm, and improved mechanical property compared with SF, resulting from strong intermolecular hydrogen bonding interactions between CS and SF, together with the maintained β‐sheet structure of SF. The medical and biological properties of the composite scaffolds were further evaluated. The results demonstrate that they possess good biocompatibility and a broad spectrum of antimicrobial properties. The in vivo animal experiments show that the composite scaffolds promote skin regeneration of rats without any teratogenic effect and inflection, thus they are very promising in the application of wound dressings. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42503.     

In vitro biocompatibility of electrospun silk fibroin mats with Schwann cells

In this study, electrospinning was used to fabricate silk‐fibroin (SF)‐based mats, which served as substrates for the culturing of rat Schwann cells. Microscopic observation and physical parameter measurements revealed that the electrospun SF mats had a nanofibrous structure with favorable physical properties. Fourier transform infrared analysis provided chemical characterization of the molecular confirmation of the SF proteins in the mats. The morphology and immunocytochemistry showed that the mats supported the survival and growth of the cultured Schwann cells, and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide analysis indicated that the electrospun SF mat extract had no cytotoxic effects on Schwann cell proliferation. Collectively, all of the results suggest that the electrospun SF mats might become a candidate scaffold for tissue‐engineered nerve grafts to promote peripheral nerve regeneration. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011