Silk fibroin protein

Silk from the silkworm, Bombyx mori, has been used as biomedical suture material for centuries. The unique mechanical properties of these fibers provided important clinical repair options for many applications. During the past 20 years, some biocompatibility problems have been reported for silkworm silk; however, contamination from residual sericin (glue-like proteins) was the likely cause. More recent studies with well-defined silkworm silk fibers and films suggest that the core silk fibroin fibers exhibit comparable biocompatibility in vitro and in vivo with other commonly used biomaterials such as polylactic acid and collagen. Furthermore, the unique mechanical properties of the silk fibers, the diversity of side chain chemistries for 'decoration' with growth and adhesion factors, and the ability to genetically tailor the protein provide additional rationale for the exploration of this family of fibrous proteins for biomaterial applications. For example, in designing scaffolds for tissue engineering these properties are particularly relevant and recent results with bone and ligament formation in vitro support the potential role for this biomaterial in future applications. To date, studies with silks to address biomaterial and matrix scaffold needs have focused on silkworm silk. With the diversity of silk-like fibrous proteins from spiders and insects, a range of native or bioengineered variants can be expected for application to a diverse set of clinical needs. r

Nanoscale bioactive glasses have been gaining attention due to their superior osteoconductivity when compared to conventional (micron-sized) bioactive glass materials. The combination of bioactive glass nanoparticles or nanofibers with polymeric systems enables the production of nanocomposites with potential to be used in a series of orthopedic applications, including tissue engineering and regenerative peer-ACCEPTED MANUSCRIPT 2 medicine. This review presents the state of art of the preparation of nanoscale bioactive glasses and corresponding composites with biocompatible polymers. The recent developments in the preparation methods of nano-sized bioactive glasses are reviewed, covering sol-gel routes, microemulsion techniques, gas phase synthesis method (flame spray synthesis), laser spinning, and electro-spinning. Then, examples of the preparation and properties of nanocomposites based on such inorganic bionanomaterials are presented, obtained using various polymer matrices, including polyesters such as poly(hydroxybutyrate), poly(lactic acid) and poly(caprolactone), and natural-based polymers such as polysaccharides (starch, chitin, chitosan) or proteins (silk, collagen).

The process of electrospinning has seen a resurgence of interest in the last few decades which has led to a rapid increase in the amount of research devoted to its use in tissue engineering applications. Of this research, the area of cardiovascular tissue engineering makes up a large percentage, with substantial resources going towards the creation of bioresorbable vascular grafts composed of electrospun nanofibers of collagen and other biopolymers. These bioresorbable grafts have compositions that allow for the in situ remodeling of the structure, with the eventual replacement of the graft with completely autologous tissue. This review will highlight some of the work done in the field of electrospinning for cardiovascular applications, with an emphasis on the use of biopolymers such as collagens, elastin, gelatin, fibrinogen, and silk fibroin, as well as biopolymers used in combination with resorbable synthetic polymers.

Silk nanoparticles were prepared from silk fibroin solutions of domesticated Bombyx mori and tropical tasar silkworm Antheraea mylitta and investigated in respect to its particle size, surface charge, stability and morphology along with its cellular uptake and release of growth factors. The nanoparticles were stable, spherical, negatively charged, 150-170 nm in average diameter and exhibited mostly Silk II (␤-sheet) structure and did not impose any overt toxicity. Cellular uptake studies showed the accumulation of fluorescence isothiocyanate conjugated silk nanoparticles in the cytosol of murine squamous cell carcinoma cells. In vitro VEGF release from the nanoparticles showed a significantly sustained release over 3 weeks, signifying the potential application as a growth factor delivery system.

The amino acid sequence of the heavy chain of Bombyx mori silk fibroin was derived from the gene sequence. The 5,263-residue (391-kDa) polypeptide chain comprises 12 low-complexity "crystalline" domains made up of Gly-X repeats and covering 94% of the sequence; X is Ala in 65%, Ser in 23%, and Tyr in 9% of the repeats. The remainder includes a nonrepetitive 151-residue header sequence, 11 nearly identical copies of a 43-residue spacer sequence, and a 58-residue C-terminal sequence. The header sequence is homologous to the N-terminal sequence of other fibroins with a completely different crystalline region. In Bombyx mori, each crystalline domain is made up of subdomains of ϳ70 residues, which in most cases begin with repeats of the GAGAGS hexapeptide and terminate with the GAAS tetrapeptide. Within the subdomains, the Gly-X alternance is strict, which strongly supports the classic Pauling-Corey model, in which ␤-sheets pack on each other in alternating layers of Gly/Gly and X/X contacts. When fitting the actual sequence to that model, we propose that each subdomain forms a ␤-strand and each crystalline domain a two-layered ␤-sandwich, and we suggest that the ␤-sheets may be parallel, rather than antiparallel, as has been assumed up to now. Proteins 2001;44:119-122.

Wound dressings have experienced continuous and significant changes over the years based on the knowledge of the biochemical events associated with chronic wounds. The development goes from natural materials used to just cover and conceal the wound to interactive materials that can facilitate the healing process, addressing specific issues in non-healing wounds. These new types of dressings often relate with the proteolytic wound environment and the bacteria load to enhance the healing. Recently, the wound dressing research is focusing on the replacement of synthetic polymers by natural protein materials to delivery bioactive agents to the wounds. This article provides an overview on the novel protein-based wound dressings such as silk fibroin keratin and elastin. The improved properties of these dressings, like the release of antibiotics and growth factors, are discussed. The different types of wounds and the effective parameters of healing process will be reviewed.

Silk-based materials have been used in the field of bone or ligament tissue engineering. In order to explore the feasibility of using purified silk fibroin to construct artificial nerve grafts, it is necessary to evaluate the biocompatibility of silk fibroin material with peripheral nerve tissues and cells. We cultured rat dorsal root ganglia (DRG) on the substrate made up of silk fibroin fibers and observed the cell outgrowth from DRG during culture by using light and electron microscopy coupled with immunocytochemistry. On the other hand, we cultured Schwann cells from rat sciatic nerves in the silk fibroin extract fluid and examined the changes of Schwann cells after different times of culture. The results of light microscopy, MTT test and cell cycle analysis showed that Schwann cells cultured in the silk fibroin extract fluid showed no significant difference in their morphology, cell viability and proliferation as compared to that in plain L15 medium. Furthermore, no significant difference was found in expression of the factors secreted by Schwann cells, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and S-100, between Schwann cells cultured in the silk fibroin extraction fluid and in plain L15 medium by the aid of immunocytochemistry, RT-PCR and Western analysis. Collectively, these data indicate that silk fibroin has good biocompatibility with DRG and is also beneficial to the survival of Schwann cells without exerting any significant cytotoxic effects on their phenotype or functions, thus providing an experimental foundation for the development of silk fibroin as a candidate material for nerve tissue engineering applications. r

Biomineralization research on mollusc shells has mostly focused on nacre formation. Chitin, silk fibroin protein, and acidic macromolecules are important components for shell formation. Although the principle concept behind shell calcification was developed many years ago, the individual components have not been well scrutinized. Besides that, Mollusca are the second largest invertebrate phylum, but comprehensive biochemical research involving a comparison of different taxa is still rare. This study reconsiders the above three components with adding some biochemical data of aculiferans. The presence of chitin in polyplacophorans sclerites was confirmed by IR and pyrolysis GC/MS. DMMB staining data inferred that sulphated groups present in aplacophoran cuticle but not in polyplacophorans cuticle. These insight suggested importance of comparison between acuriferans and conchiferans.

This article deals with the preparation and characterization of silk fibroin(Bombyz mori)/ cellulose blend films. Following dissolution with a metal complex solution, the average .molecular weight of silk fibroin slightly decreased, while cellulose was almost unaffected. After coagulation and washing, transparent films were obtained by blending fibroin and cellulose in all proportions. The crystalline structures of regenerated fibroin and cellulose were &form and cellulose 11, respectively, as shown by the characteristic x-ray diffraction profiles. Density values increased with cellulose content, though less than expected from a pure additive behavior. Moisture regain increased following the addition of a small amount of cellulose to silk fibroin. The mechanical properties showed that both strength and elongation at break of silk fibroin films were improved by blending with cellulose. IR spectra exhibited changes in the skeletaI frequences of silk fibroin, suggesting the occurrence of intermolecular interactions between fibroin and cellulose through hydrogen bond formation.

This article deals with the characterization of blend films obtained by mixing silk fibroin (SF) and polyacrylamide (PAAm). The DSC curves of SF/PAAm blend films showed overlapping of the main thermal transitions characteristic of the individual polymers. The exothermic peak at 218°C, assigned to the ␤-sheet crystallization of silk fibroin, slightly shifted to a lower temperature by blending. The weightretention properties (TG) of the blend films were intermediate between those of the two constituents. The TMA response was indicative of a higher thermal stability of the blend films, even at low PAAm content (Յ25%), the final breaking occurring at about 300°C (100°C higher than pure SF film). The peak of dynamic loss modulus of silk fibroin at 193°C gradually shifted to lower temperature in the blend films, suggesting an enhancement of the molecular motion of the fibroin chains induced by the presence of PAAm. Changes in the NH stretching region of silk fibroin were detected by FTIR analysis of blend films. These are attributable to disturbance of the hydrogen bond pattern of silk fibroin and formation of new hydrogen bonds with PAAm. The values of strength and elongation at break of blend films slightly improved at 20 -25% PAAm content. A sea-island structure was observed by examining the air surface of the blend films by scanning electron microscopy.

Growth factor releasing scaffolds are an emerging alternative to autologous or allogenous implants, providing a biologically active template for tissue (re)-generation. The goal of this study is to evaluate the feasibility of controlled insulin-like growth factor I (IGF-I) releasing silk fibroin (SF) scaffolds in the context of cartilage repair. The impact of manufacturing parameters (pH, methanol treatment and drug load)

The present study describes a semi-interpenetrating network hydrogel fabricated using silk fibroin/ polyacrylamide for controlled drug delivery applications. Hydrogels were synthesized using varied ratios of silk fibroin/acrylamide mixtures crosslinked by N,N 0 -Methylenebisacrylamide. Fourier-Transform Infrared analysis was performed suggesting b sheet transition of silk fibroin with hydrogels. Scanning electron microscopy revealed microporous surface with maximum pore size of 50 AE 11 mm. Rheological properties along with swellability, degradation, sol fraction estimation, equilibrium water content and swelling kinetics were evaluated. Compressive strength of 241.9 AE 5.5 kPa was observed suggesting mechanically stronger gels. MTT assay showed biocompatibility and absence of deleterious effects of hydrogel on cell viability and functionality. In vitro release studies using two model compounds i.e. trypan blue dye and FITC-inulin reveal their sustained release from the fabricated hydrogel constructs.

The components of musculoskeletal system that consists of bone, ligament tendons and muscles are most vulnerable to injury from sports and related activities. Replacing the injured tissues more specifically the bone is a challenge to tissue engineers. Aiming at regeneration of bone tissues, silk fibroin from Bombyx mori, a novel biocompatible natural polymer has been explored either alone or as organic/inorganic composites for optimal growth of bone cells or differentiation of stem cells to bone cells in vitro. In the process, raw silk fibroins need to be were degummed, usually by alkali treatment method, to ameliorate the cytotoxicity from sericin. Most recently, the nanofibers of silk from electrospinning has shown a promise for bone tissue engineering. To get nanofibers, the degummed silk is usually dissolved in compatible solutions i.e. CaCl2 in H2O or LiBr in C2H5OH at different concentrations followed by dialysis to remove toxic ions. The ion free silk solution can be Electrospun alone or can be blended with various biopolymers for the fabrication of nanofibre biodegradable composite. Other approaches like crosslinking an array of biopolymers with silk or freeze drying the silk fibroins are also in pipeline to develop a novel silk based scaffold for bone tissue engineering.

Objective. The objective of this study was to determine the capability of silk fibroin powder as a biomaterial template for the restoration of peri-implant defects when mixed with Choukroun platelet-rich fibrin (PRF) in vivo. Study design. Ten New Zealand white rabbits were used for this study. Using a trephine bur (diameter 7.0 mm), 2 monocortical defects were prepared. Subsequently, 2 dental implants were installed into the tibia (diameter 3.0 mm, length 10.0 mm). In the experimental group, the peri-implant defect was filled with a combination graft of silk fibroin powder and Choukroun PRF. The control was left in an unfilled state. The animals were killed at 8 weeks. Subsequently, a removal torque test and a histomorphometric analysis were done. Results. The removal torque for the experimental group was 30.34 Ϯ 5.06 N⅐cm, whereas it was 21.86 Ϯ 3.39 N⅐cm for the control. The difference between the 2 groups was statistically significant (P ϭ .010). Mean new bone formation was 51.93 Ϯ 27.90% in the experimental group and 11.67 Ϯ 15.12% in the control (P ϭ .003). Mean bone-toimplant contact was 43.07 Ϯ 21.96% in the experimental group and 15.37 Ϯ 23.84% in the control (P ϭ .002).

Bombyx mori silk fibers were dissolved in N-methyl morpholine N-oxide (MMNO), an organic cyclic amine oxide used for the solvent spinning of regenerated cellulosic fibers. The commercial MMNO monohydrate used in this study as a solvent for silk is a hygroscopic compound crystalline at room temperature, which becomes an active solvent after melting at 76°C. The degree of hydration of MMNO was checked by DSC measurements. The solvation power of MMNO towards silk fibroin drastically decreased at a water content ]20-21% w/w. Dissolution of silk required both thermal and mechanical energy. The optimum temperature was 100°C. At lower temperatures dissolution proceeded very slowly. At higher temperatures, rapid depolymerization of silk fibroin occurred. The value of the Flory-Huggins interaction parameter for the MMNO -H 2 O -silk fibroin system was − 8.5, suggesting that dissolution is a thermodynamically favored process. The extent of degradation of silk fibroin was assessed by measuring the intrinsic viscosity and determining the amino acid composition of silk after regeneration with an aqueous methanol solution, which was effective in removing the solvent and coagulating silk. Regenerated silk fibroin membranes were characterized by infrared spectroscopy, differential scanning calorimetry and scanning electron microscopy. The prevailing molecular conformation of silk fibroin chains was the b-sheet structure, as shown by the intense amide I-III bands at 1704, 1627, 1515, 1260, and 1230 cm − 1 . The value of the I 1260 /I 1230 intensity ratio (crystallinity index) was 0.68, comparable to that of the fibers. The DSC thermogram was characteristic of a silk fibroin material with unoriented b-sheet crystalline structure, with an intense decomposition endotherm at 294°C. The SEM examination of fractured surfaces showed the presence of a dense microstructure with a very fine texture formed by densely packed roundish particles of about 100 -200 nm diameter.

Several different solvent systems are commonly used to dissolve Bombyx mori silk ®broin to prepare regenerated silk membranes and ®bers though differences in the behavior of these solvents have not been fully investigated. Here we compare the effects of four of these on the rheology of silk ®broin solutions and on protein secondary structure as revealed by FTIR spectroscopy of cast membranes. The results demonstrated that Ca(NO 3 ) 2 ±MeOH±H 2 O and LiBr±EtOH±H 2 O had the strongest solvation on the silk ®broin chains, which showed an almost constant viscosity (Newtonian behavior) over most of the shear rate range (0.1±500 s 21 ). In contrast, the 9.5 M aqueous LiBr appeared to have the weakest solvation with similar effects on the silk ®broin molecules to pure water as indicated by rheological behavior. It was also found that the silk ®broin membranes prepared using all four solvent system showed mainly random coil conformation with a small proportion of b-sheet by FTIR spectroscopy. We discuss the implications of these ®ndings for the preparation of regenerated silk for different applications. q

a b s t r a c t 3D in vitro model systems that are able to mimic the in vivo microenvironment are now highly sought after in cancer research. Antheraea mylitta silk fibroin protein matrices were investigated as potential biomaterial for in vitro tumor modeling. We compared the characteristics of MDA-MB-231 cells on A. mylitta, Bombyx mori silk matrices, Matrigel, and tissue culture plates. The attachment and morphology of the MDA-MB-231 cell line on A. mylitta silk matrices was found to be better than on B. mori matrices and comparable to Matrigel and tissue culture plates. The cells grown in all 3D cultures showed more MMP-9 activity, indicating a more invasive potential. In comparison to B. mori fibroin, A. mylitta fibroin not only provided better cell adhesion, but also improved cell viability and proliferation. Yield coefficient of glucose consumed to lactate produced by cells on 3D A. mylitta fibroin was found to be similar to that of cancer cells in vivo. LNCaP prostate cancer cells were also cultured on 3D A. mylitta fibroin and they grew as clumps in long term culture. The results indicate that A. mylitta fibroin scaffold can provide an easily manipulated microenvironment system to investigate individual factors such as growth factors and signaling peptides, as well as evaluation of anticancer drugs.

Structural changes of tussah ( Antheraea pernyi ) silk fibroin films induced by heat treatment were studied as a function of the treatment temperature in the range 200-250ЊC. The DSC curve of tussah films with a-helix molecular conformation displayed characteristic endo and exo peaks at 216 and 226ЊC, respectively. These peaks first weakened and then completely disappeared after heating at 230ЊC. Accordingly, the TMA thermal shrinkage at 206ЊC disappeared when the films were heated at 230ЊC. The onset of weight loss was monitored at 210ЊC by means of TG measurements. X-ray diffraction profiles gradually changed from a-helix to b-sheet crystalline structure as the treatment temperature increased from 200 to 250ЊC. On raising the heating temperature above 200ЊC, the intensity of IR and Raman bands characteristic of b-sheet conformation increased in the whole ranges of amide and skeletal modes. The sample treated at 200ЊC showed a spectral pattern intermediate between a-helix and b-sheet molecular conformation. The IR marker band for random coil structure, still detectable at 200ЊC, disappeared at higher treatment temperatures. Spectral changes attributable to the onset of thermal degradation appeared at 230ЊC.

The ethanol-induced conformation transition of regenerated Bombyx mori silk fibroin membrane from a poorly Ž . defined to the well ordered state was monitored by time-resolved Fourier transform infrared spectroscopy FTIR for the first time. From the analysis of FTIR difference spectra, taken on time scales as short as 6 s and up to 1 h after addition of ethanol, intensity vs. time plots of an increasing band at 1618 cm y1 were observed indicating formation of a ␤-sheet coincident with the loss of intensity of a band at 1668 cm y1 indicating decreases of random coil andror silk I structure. Both infrared markers were fitted with identical biphasic exponential decay functions, however, there was a clear burst phase occurring prior to the onset of the observed transitions. The conformation transition process is Ž . indicated to either proceed sequentially through at least two intermediate states that contain different levels of ␤-sheet structure or to have parallel pathways of initial ␤-sheet formation followed by a slower 'perfection' phase.

Physical and chemical structure, as well as thermal behavior of solution-cast regenerated films, prepared from tussah (Antherma pernyi) silk fibroin, were compared with those of solution-cast native films, in order to ascertain whether treatment (degumming, dissolution) used for preparation affected their properties. Regenerated fibroin films exhibited a higher thermal stability than native ones, as shown by differential scanning calorimetry, thermomechanical analysis, and dynamic mechanical behavior. Glass transition temperature and other relevant thermal transitions of the regenerated silk specimen shifted to higher temperatures compared with those of native specimen. Molecular conformation and crystalline structure did not show significant differences between the two kinds of silk films. Amino acid composition and molecular weight, however, distribution changed markedly after dissolving tussah silk fibroin fiber in concentrated LiSCN in polypeptide size was the main features for the regenerated silk fibroin.

When Silk fibre derived from Bombyx mori, a native biopolymer, was dissolved in highly concentrated neutral salts such as CaCl 2 , the regenerated liquid silk, a gradually degraded peptide mixture of silk fibroin, could be obtained. The silk fibroin nanoparticles were prepared rapidly from the liquid silk by using watermiscible protonic and polar aprotonic organic solvents. The nanoparticles are insoluble but well dispersed and stable in aqueous solution and are globular particles with a range of 35-125 nm in diameter by means of TEM, SEM, AFM and laser sizer. Over one half of the e-amino groups exist around the protein nanoparticles by using a trinitrobenzenesulfonic acid (TNBS) method. Raman spectra shows the tyrosine residues on the surface of the globules are more exposed than those on native silk fibers. The crystalline polymorph and conformation transition of the silk nanoparticles from random-coil and a-helix form (Silk I) into anti-parallel b-sheet form (Silk II) are investigated in detail by using infrared, fluorescence and Raman spectroscopy, DSC, 13 C CP-MAS NMR and electron diffraction. X-ray diffraction of the silk nanoparticles shows that the nanoparticles crystallinity is about four fifths of the native fiber. Our results indicate that the degraded peptide chains of the regenerated silk is gathered homogeneously or heterogeneously to form a looser globular structure in aqueous solution. When introduced into excessive organic solvent, the looser globules of the liquid silk are rapidly dispersed and simultaneously dehydrated internally and externally, resulting in the further chain-chain contact, arrangement of those hydrophobic domains inside the globules and final formation of crystalline silk nanoparticles with b-sheet configuration. The morphology and size of the nanoparticles are relative to the kinds, properties and even molecular structures of organic solvents, and more significantly to the looser globular substructure of the degraded silk fibroin in aqueous solution. It is possible that the silk protein nanoparticles are potentially useful in biomaterials such as cosmetics, anti-UV skincare products, industrial materials and surface improving materials, especially in enzyme/drug delivery system as vehicle.

Spiders spin high-performance silks through the expression and assembly of tissue-restricted fibroin proteins. Spider silks are composite protein biopolymers that have complex microstructures. Retrieval of cDNAs and genomic DNAs encoding silk fibroins has revealed an association between the protein sequences and structure-property relationships. However, before spider silks can be subject to genetic engineering for commercial applications, the complete protein sequences and their functions, as well as the details of the spinning mechanism, will require additional progress and collaborative efforts in the areas of biochemistry, molecular biology and material science. Novel approaches to reveal additional molecular constituents embedded in the spider fibers, as well as cloning strategies to manipulate the genes for expression, will continue to be important aspects of spider biology research. Here we summarize the molecular characteristics of the different spider fibroins, the mechanical properties and assembly process of spidroins and the advances in protein expression systems used for recombinant silk production. We also highlight different technical approaches being used to elucidate the molecular constituents of silk fibers.

An investigation into the influence of UV-irradiation on regenerated silk fibroin dissolved in water was carried out using UVeVis and fluorescence spectroscopy. It was found that the absorption of regenerated silk fibroin in solution increased during UV-irradiation of the sample, most notably between 250 and 400 nm. Moreover, after UV-irradiation a wide peak emerged between 290 and 340 nm with maximum at about 305 nm. The new peak suggests that new photoproducts are formed during UV-irradiation of regenerated silk fibroin.

Bone (re)-generation and bone fixation strategies utilize biomaterial implants, which are gradually replaced by autologous tissues. Ideally, these biomaterials should be biodegradable, osteoconductive, and provide mechanical strength and integrity until newly formed host tissues can maintain function. Some protein-based biomaterials such as collagens are promising because of their biological similarities to natural proteins on bone surfaces. However, their use as bone implant materials is largely hampered by poor mechanical properties. In contrast, silks offer distinguishing mechanical properties that are tailorable, along with slow degradability to permit adequate time for remodeling. To assess the suitability of silk-based biomaterials as implants for bone healing, we explored the use of novel porous silk fibroin scaffolds as templates for the engineering of bone tissues starting from human bone marrow derived stem cells cultured under osteogenic conditions for up to 5 weeks. The slowly degrading protein matrix permitted adequate temporal control of hydroxyapatite deposition and resulted in the formation of a trabecular-like bone matrix in bioreactor studies. The organic and inorganic components of the engineered bone tissues resembled those of bone, as shown by gene expression analysis, biochemical assays, and X-ray diffractometry. Implantation of the tissue-engineered bone implants (grown in bioreactors for 5 weeks prior to implantation) into calvarial critical size defects in mice demonstrated the capacity of these systems to induce advanced bone formation within 5 weeks, whereas the implantation of stem cell loaded silk scaffolds, and scaffolds alone resulted in less bone formation. These results demonstrate the feasibility of silk-based implants with engineered bone for the (re-)generation of bone tissues and expand the class of protein-based bone-implant materials with a mechanically stable and durable option.

In this work, silk fibroin/cellulose (SF/CE) blend films were regenerated from hydrophilic ionic liquid, 1-butyl-3-methylimidazolium chloride (BmimCl). The structure of the blend films was characterised by Fourier transform infrared spectroscopy (FTIR). The thermal and mechanical properties and the surface morphology of the blend films were also investigated. In addition, moisture content, swelling index, total water absorption and film weight loss were measured. The results indicate that with the introduction of CE, the interactions between SF and CE in the blend films induced the conformation transition of SF from random coil form or silk I to ␤-sheet structure. At the same time, the physical properties of the blend films were improved. When the ratio of SF to CE is 25:75, the stronger interactions in the matrices contribute to the higher tensile strength, higher thermal stability and higher water stability. However, the blend film with half CE illustrates higher elongation at break, a more homogeneous surface and higher miscibility.

The capability of Agaricus bisporus tyrosinase to catalyze the oxidation of tyrosine residues of silk sericin was studied under homogeneous reaction conditions, by using sericin peptides purified from industrial wastewater as the substrate. Tyrosinase was able to oxidize about 57% of sericin-bound tyrosine residues. The reaction rate was higher than with silk fibroin, but lower than with other silk-derived model peptides, i.e. tryptic and chymotryptic soluble peptide fractions of silk fibroin, suggesting that the size and the molecular conformation of the substrate influenced the kinetics of the reaction. The concentration of tyrosine in oxidized sericin samples decreased gradually with increasing the enzyme-to-substrate ratio. The average molecular weight of sericin peptides significantly increased by oxidation, indicating that cross-linking occurred via self-condensation of o-quinones and/or coupling with the free amine groups of lysine and, probably, with sulfhydryl groups of cysteine. The high temperature shift of the main thermal transitions observed in the differential scanning calorimetry curves confirmed the formation of peptide species with higher molecular weight and higher thermal stability. Fourier transform-infrared spectra of oxidized sericin samples showed slight changes related to the loss of tyrosine and formation of oxidation products. Oxidized sericin peptides were able to undergo non-enzymatic coupling with chitosan. Infrared spectra provided clear evidence of the formation of sericin-chitosan bioconjugates under homogeneous reaction conditions. Spectral changes in the NH stretching region seem to support the formation of bioconjugates via the Michael addition mechanism.

In this study, three-dimensional silk fibroin/chitosan (SF/CTS) composite sponges were successfully prepared with an interconnected porous structure and proper mechanical properties. Silk fibroin and chitosan have the potential to mimic a natural extra-cellular matrix (ECM). For the sponge preparation, silk fibroin from the Bombyx mori silkworm and chitosan were used, in different proportions, as composites. Sponges were prepared by means of freezing and lyophylization of corresponding composite solutions. The properties of composite sponges, such as the microstructure as well as the chemical and physical properties were studied. In the present investigation, silk fibroin, chitosan and their mixtures were characterized by attenuated total reflection Fourier transform infra-red spectroscopy (ATR-FTIR), swelling properties, thermogravimetric analysis and scanning electron microscopy (SEM). The ATR-FTIR analysis indicated a shift of band corresponding to C_O, C\N, C\O\C. Moreover, NH groups of SF and C_O and NH 2 groups of chitosan might have participated in a specific intermolecular interaction among themselves. Thermogravimetric results suggested a higher temperature of decomposition for SF/CTS sponges than sponges made of pure silk fibroin or chitosan. Scanning electron microscopy observations showed the sponges of SF/CTS with pore size from 20 to 150 μm, which is appropriate for cell growth. Biocompatibility of sponges was confirmed by in vitro test. SF/CTS sponges have sufficient mechanical integrity to resist handling during implantation and in vivo loading. Both the compressive modulus and compressive strength for the sponge decrease with the increase of silk fibroin content in the sponge. Sponges made of silk fibroin/chitosan mixtures can be interesting materials for tissue engineering as scaffold for temporary supporting the formation of new tissue and organs.

Nanocomposite fibers of Bombyx mori silk and single wall carbon nanotubes (SWNT) were produced by the electrospinning process. Regenerated silk fibroin dissolved in a dispersion of carbon nanotubes in formic acid was electrospun into nanofibers. The morphology, structure, and mechanical properties of the electrospun nanofibers were examined by field emission environmental scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and microtensile testing. TEM of the reinforced fibers shows that the single wall carbon nanotubes are embedded in the fibers. The mechanical properties of the SWNT reinforced fiber show an increase in Young's modulus up to 460% in comparison with the un-reinforced aligned fiber, but at the expense of the strength and strain to failure.

Time-resolved FTIR analysis was used to monitor the conformation transition induced by treating regenerated Bombyx mori silk fibroin films and solutions with different concentrations of ethanol. The resulting curves showing the kinetics of the transition for both films and fibroin solutions were influenced by the ethanol concentration. In addition, for silk fibroin solutions the protein concentration also had an effect on the kinetics. At low ethanol concentrations (for example, less than 40% v/v in the case of film), films and fibroin solutions showed a phase in which b-sheets slowly formed at a rate dependent on the ethanol concentration. Reducing the concentration of the fibroin in solutions also slowed the formation of b-sheets. These observations suggest that this phase represents a nucleation step. Such a nucleation phase was not seen in the conformation transition at ethanol concentrations >40% in films or >50% in silk fibroin solutions. Our results indicate that the ethanol-induced conformation transition of silk fibroin in films and solutions is a threephase process. The first phase is the initiation of bsheet structure (nucleation), the second is a fast phase of b-sheet growth while the third phase represents a slow perfection of previously formed b-sheet structure. The nucleation step can be very fast or relatively slow, depending on factors that influence protein chain mobility and intermolecular hydrogen bond formation. The findings give support to the previous evidence that natural silk spinning in silkworms is nucleation-dependent, and that silkworms (like spiders) use concentrated silk protein solutions, and careful control of the pH value and metallic ion content of the processing environment to speed up the nucleation step to produce a rapid conformation transition to convert the water soluble spinning dope to a tough solid silk fiber. Proteins 2007;68:223-231. V V C 2007 Wiley-Liss, Inc.

Biopolymers, such as silk fibroin, collagen, and chitosan, are promising candidates for a variety of applications that merge the fields of biomedical optics and biomaterials. Biocompatible silk fibroin, in particular, shows promise as a biomaterial, based on a number of attributes. Silk fibroin is the strongest and toughest natural fiber known and is easily formed into robust films of thermodynamically stable beta-sheets of a controllable range of thicknesses (between tens of nanometers and hundreds of micrometers). These films have excellent (ca. 95%) optical transparency across the visible range and can be easily characterized and biochemically functionalized because of the all-aqueous processing, broadening their overall value.

Silk fibroin (SF) was regenerated using three of the most common protocols described in the bibliography for the dissolution of raw SF (LiBr 9.3 M, CaCl 2 50 wt.% or CaCl 2 :EtOH:H 2 O 1:2:8 in molar ratio). The integrity of regenerated SF in aqueous solution was analyzed by SDS-PAGE and different profiles of degradation were observed depending on the protocol used. This fact was found to affect also the aqueous solubility of the freeze dried protein. These different SFs were used to produce electrospun mats using SF solutions of SF 17 wt.% in 1,1,1,1′,1′,1′-hexafluoro-2-propanol (HFIP) and significant differences in fiber sizes, elongation and ultimate strength values were found. This work provides a global overview of the manner that different methods of SF extraction can affect the properties of electrospun SF-mats and consequently it should be considered depending on the use they are going to be made for.

We design, fabricate, and characterize split-ring resonator ͑SRR͒ based planar terahertz metamaterials ͑MMs͒ on ultrathin silicon nitride substrates for biosensing applications. Proof-of-principle demonstration of increased sensitivity in thin substrate SRR-MMs is shown by detection of doped and undoped protein thin films ͑silk fibroin͒ of various thicknesses and by monitoring transmission changes using terahertz time-domain spectroscopy. SRR-MMs fabricated on thin film substrates show significantly better performance than identical SRR-MMs fabricated on bulk silicon substrates paving the way for improved biological and chemical sensing applications.

Different hydrogel materials have been prepared to investigate the effects of culture substrate on the behaviour of pluripotent cells. In particular, genipin-crosslinked gelatin–silk fibroin hydrogels of different compositions have been prepared, physically characterized and used as substrates for the culture of pluripotent cells. Pluripotent cells cultured on hydrogels remained viable and proliferated. Gelatin and silk fibroin promoted the proliferation of cells in the short and long term, respectively. Moreover, cells cultured on genipin-crosslinked gelatin–silk fibroin blended hydrogels were induced to an epithelial ectodermal differentiation fate, instead of the neural ectodermal fate obtained by culturing on tissue culture plates. This work confirms that specific culture substrates can be used to modulate the behaviour of pluripotent cells and that our genipin-crosslinked gelatin–silk fibroin blended hydrogels can induce pluripotent cells differentiation to an epithelial ectode...

Silk fibroin (SF) was dissolved in N-methyl morpholine N-oxide (NMMO) at a polymer concentration of 13% (w/w); thermal and rheological solution properties were characterized. The melting/crystallization behaviour of NMMO was influenced by SF presence. Melting of NMMO hydrate decreased to 71 • C and a cold crystallization peak appeared at 35 • C on heating. None crystallization occurred on cooling. Quenching at a temperature of 50 • C or higher did not induce any crystallization on heating. Viscosity of SF-NMMO solutions decreased as a function of temperature. At 75 • C, viscosity remained constant for 360 min. SF-NMMO dope was spun by using a lab-scale wet spinning line. The extruded filament was coagulated in an ethanol bath. Regenerated SF fibres were collected at different draw ratios and their morphological, physical, and mechanical properties were characterized. Fibre diameters ranged from 133 to 19 m, cross-section was regularly circular, and surface was generally smooth, with a very fine granular aspect. Birefringence increased with increasing the draw ratio, especially when take up and post-spinning draw were coupled. FT-IR spectra and DSC thermograms confirmed that SF fibres crystallized into Silk II structure. The IR crystallinity index did not change as a function of drawing. Regenerated SF fibres undrawn or drawn only during the coagulation step showed the mechanical behaviour typical of a brittle material. However, when both take up and post-spinning draw were applied, fibres displayed a ductile-stable behaviour. Typical values of the mechanical parameters of regenerated SF fibres were: E = 8.7 GPa, σ b = 120 MPa and ε b = 35%. (E. Marsano). sue engineering . To this aim, not only native SF in fibre form, but also various kinds of regenerated SF materials (gel, sponge, powder, film) exert a strong attractiveness owing to their biological properties and to the ability to support cell adhesion and proliferation . Currently, efforts are aimed at adjusting the structure of biomaterials to their required functions in order to address a broad range of biomedical needs. In this context, the possibility of engineering silk-based biomaterials by regenerating the native biopolymer in other forms, including regenerated SF fibres, pure or blended with other polymers [9], with the inclusion of new useful functionalities [10] is a crucial scientific and technological challenge.

An investigation into the influence of UV-irradiation on regenerated silk fibroin dissolved in water was carried out using UVeVis and fluorescence spectroscopy. It was found that the absorption of regenerated silk fibroin in solution increased during UV-irradiation of the sample, most notably between 250 and 400 nm. Moreover, after UV-irradiation a wide peak emerged between 290 and 340 nm with maximum at about 305 nm. The new peak suggests that new photoproducts are formed during UV-irradiation of regenerated silk fibroin.

Silk fibroin has been successfully used as a biomaterial for tissue regeneration. In order to prepare silk fibroin biomaterials for human implantation a series of processing steps are required to purify the protein. Degumming to remove inflammatory sericin is a crucial step related to biocompatibility and variability in the material. Detailed characterization of silk fibroin degumming is reported. The degumming conditions significantly affected cell viability on the silk fibroin material and the ability to form three-dimensional porous scaffolds from the silk fibroin, but did not affect macrophage activation or β-sheet content in the materials formed. Methods are also provided to determine the content of residual sericin in silk fibroin solutions and to assess changes in silk fibroin molecular weight. Amino acid composition analysis was used to detect sericin residuals in silk solutions with a detection limit between 1.0% and 10% wt/wt, while fluorescence spectroscopy was used to reproducibly distinguish between silk samples with different molecular weights. Both methods are simple and require minimal sample volume, providing useful quality control tools for silk fibroin preparation processes.

For highly porous form such as sponges or scaffolds, the induction of the β-sheet formation of silk fibroin to make the water-stable materials usually results in their high shrinkage leading to a difficulty in controlling shape and size of materials. Thus, the objective of this study was to improve dimensional stability of silk fibroin sponge by incorporating chitin whiskers as nanofiller. Chitin whiskers exhibited the average length and width of 427 and 43 nm, respectively. Nanocomposite sponges at chitin whiskers to silk fibroin weight ratio (C/S ratio) of 0, 1/8, 2/8, or 4/8 were prepared by using a freeze-drying technique. The dispersion of chitin whiskers embedded in the silk fibroin matrix was found to be homogeneous. The presence of chitin whiskers embedded into silk fibroin sponge not only improved its dimensional stability but also enhanced its compression strength. Regardless of the chitin whisker content, SEM micrographs showed that all samples possessed an interconnected pore network with an average pore size of 150 μm. To investigate the feasibility of the nanocomposites for tissue engineering applications, L929 cells were seeded onto their surfaces, the results indicated that silk fibroin sponges both with and without chitin whiskers were cytocompatible. Moreover, when compared to the neat silk fibroin sponge, the incorporation of chitin whiskers into the silk fibroin matrix was found to promote cell spreading.

Observation of the oxidation–reduction processes occurring at the nanoelectrode–solution interface demonstrates how electrochemical behavior depends upon nanoelectrode size. The use of a modified form of pulsed laser ablation as an improved method to synthesize nanometer-scaled electrode materials easily and consistently is reported. This method of fabrication enables platinum metal nanoparticles averaging 3 nm in diameter and approximately 5.0 × 1011 particles/cm2 to be deposited onto silicon substrates using optimum ablation parameters. A platinum silicide phase exists at the interface of the platinum and silicon as a result of the ablation process. Electrochemical results demonstrate the presence of a large number of isolated platinum particles (1.1 × 107 particles/cm2), separated by an average edge to edge distance of 14 nm, which are electrochemically active nanoelectrodes.► Fabrication of Pt nanoparticles using the method of through thin film ablation results in an isolated array of nanoelectrodes. ► Characterization of the array is consistent for isolated nanoelectrodes where the diffusional profiles do not overlap. ► Enhancements of the limits of detection as an electrochemical sensor are amplified due to the large number of isolated nanoelectrodes in the array.

PEMBENTUKAN SPONTAN KOMPLEK POLIELEKTROLIT FIBROIN SUTERA DENGAN ALGINAT SEBAGAI MODEL PENGHANTARAN OBAT (Gita Cahya Eka Darma) Fibroin Sutera (SF) dan Alginat (ALG) merupakan biomaterial yang memiliki efek terapeutik dalam pengobatan luka yaitu hemostatik, dan kemampuannya dalam menyerap eksudat luka. Agen terapetik alami memiliki banyak keunggulan daripada penggunaan obat sintetik kimiawi, sehingga menjadikannya sebagai alternatif pilihan yang baik dalam pengobatan. Penelitian ini bertujuan membuat model penghantaran untuk pengobatan luka yaitu berupa membran serat hasil kompleksasi polielektrolit (PEC) melalui metode koaservasi. SF dari kepompong ulat sutera (Bombyx mori Linn) digunakan sebagai agen polikation, ALG sebagai agen polianion dan Kloramfenikol (CHL) sebagai model obatnya. Karakterisasi membran serat PEC yang dilakukan meliputi analisis gugus fungsi (FTIR), derajat kristalinitas (XRD), sifat termal (DSC), mikroskopik (SEM). Kemudian, dilakukan perhitungan perolehan koaservat (coacervation yield), uji keabuhan (swelling), dan uji pelepasan CHL secara in‒vitro. SDS PAGE digunakan untuk mengidentifikasi protein. Penelitian ini telah menghasilkan SF dengan karakteristik baru yang lebih baik, sehingga diperoleh membran serat PEC yang sesuai sebagai model penghantaran untuk pengobatan luka pada kulit. Membran serat PEC merupakan hasil modifikasi dari koaservat SF‒ALG berbentuk hidrogel. SF dalam membran berada pada konformasi strukturnya yang stabil yaitu Silk II dengan derajat kristalinitas dari SF maupun ikat‒silang elektrostatik SF‒ALG yang baik, sehingga membran serat ini dapat mengembang atau larut bergantung pada pH sistem. Analisis karakteristik membran telah membuktikan bahwa terjadi ikat‒silang elektrostatik antara SF dengan ALG, bentuk polimorf serta derajat kristalinitas SF dan membran dapat dikendalikan sehingga menentukan hasil akhir membran. Membran serat PEC responsif terhadap kondisi keasaman sistem, pada pH < 3 membran mengembang sementara pH > 3‒5,5 membran melarut sehingga dapat disesuaikan dengan kondisi pH tempat pelepasannya. Profil keabuhan meningkat dari pH 1 menuju pH 3 dengan persentase paling rendah adalah 414,3% (F3) dan paling tinggi adalah 844,4% (F2). Profil pelepasan CHL dari F1‒F3 berturut-turut adalah 91,33; 98,88; 81,94% selama 30 menit. Pelepasan paling cepat diperoleh dari formula dengan jumlah ALG lebih besar, sebaliknya pada jumlah SF lebih besar maka pelepasannya lebih lama sehingga rasio ini dapat menentukan jenis pelepasan yang diinginkan. Kata kunci: Fibroin sutera, natrium alginat, koaservasi, komplek polielektrolit, membran serat -------------------------------------------------------------------------------------------------------------------------------------------
SELF-ASSEMBLED POLYELECTROLYTE COMPLEX OF SILK FIBROIN WITH ALGINATE AS DRUG DELIVERY MODEL (Gita Cahya Eka Darma) Silk Fibroin (SF) and Alginate (ALG) are biomaterial that have therapeutic efect in wound dressing which is hemostatic, and the ability upon absorption of wound exudates. Natural therapeutic agents has many advantages than the use of chemical synthetic drugs, so that making it a good alternative in the medication. This research aims to make delivery model for wounds dressing in the form of fiber membrane results from polyelectrolyte complexation (PEC) through coacervation method. SF from silkworm cocoons (Bombyx mori Linn) used as polycations agent, ALG as polyanions agent and Chloramphenicol (CHL) as drug model. Characterization of PEC fiber membranes are conducted with analysis of the functional groups (FTIR), crystallinity degree (XRD), thermal properties (DSC), microscopic (SEM). Then, the study continued to the measurement of coacervation yield, swelling test, and in-vitro drug release test. SDS PAGE was used to verify the protein. This research has gained SF with better new characteristics, in order to obtain the appropriate PEC fiber membrane as delivery models for the treatment of skin wounds. PEC fiber membrane is a modified version from hydrogel-forming of SF‒ALG coacervate. SF in the membrane is in the stable conformation structure (Silk II) with a proper cystallinity degrees of SF and the SF‒ALG electrostatic crosslinking, so this fiber membrane can swelling or dissolve depending on the pH of the system. Characterization analysis of the membrane shown that the electrostatic bonds occurs between SF with ALG, polymorphic forms of the protein fibroin and crystallinity degree of SF and membrane can be controlled to determine the final result of the membrane. PEC fiber membrane is responsive to pH, at pH < 3 swelling occurs while at pH > 3‒5,5 it dissolved, this condition could be adjusted to the pH condition of the site released. The swelling profile increased from pH 1 upto pH 3 with the lowest percentage is 414,3% (F3) and the highest is 844,4% (F2). The CHL release profile of F1‒F3 respectively are 91,33; 98,88; 81,94% during 30 minute. The fastest drug release profile obtained from the formula with a higher amount of ALG, and otherwise on the formula that SF amounts is higher then the release is slower, this ratio can determine the type of release as required. Keywords: Silk fibroin, sodium alginate, coacervation, polyelectrolyte complex, fiber membrane

Silk fibroin (SF) and alginate (AA) have been proved to be invaluable natural materials in the field of biomedical engineering. This study was designed to compare the wound healing effect of SF, AA and SF/AA-blended sponge (SF/AA) with clinically used Nu Gauze TM (CONT) in a rat full thickness wound model. Two circular skin wounds on the back of rat were covered with either of CONT, SF, AA or SF/AA. On the postoperative days of 3, 7, 10 and 14, residual wound area was calculated, and skin wound tissues were biopsied to measure the area of regenerated epithelium and collagen deposition as well as the number of proliferating cell nuclear antigen (PCNA)-immunoreactive cells. Half healing time (HT 50 ) of SF/AA was dramatically reduced as compared with that of SF, AA or CONT. Furthermore, SF/AA significantly increased the size of re-epithelialization and the number of PCNA positive cells, whereas the effect of SF/AA on collagen deposition was not significantly different as compared with that of SF or AA. These results demonstrate that the wound healing effect of SF/AA is the best among other treatments including SF and AA, and this synergic effect is mediated by re-epithelialization via rapid proliferation of epithelial cell.

The potential of silk protein is increased because of its importance as natural biopolymer for biotechnological and biomedical applications. The main disadvantage of silk fibroin films is their high brittleness. Thus, we studied blends of fibroin with other polymers to improve the film properties. Considering the possible applications of films in biomedical applications, we used a natural and biodegradable polymer as the second component. This study reports the fabrication and characterization of mulberry silk protein fibroin and sodium carboxymethylcellulose (NaCMC) blended films as potential substrates for in vitro cell culture. The blended films are investigated of their chemical interactions, morphologies, thermal, mechanical properties in addition to its swelling properties and biocompatibility. The addition of NaCMC improves the elasticity of fibroin films and its thermal properties. The change of morphology, swelling behavior and increase of surface roughness of the films were also observed in the blended films. The films become insoluble on alcohol treatment and are stable for longer duration in hydrolytic medium. The blended films are cytocompatible and supported adhesion and growth of mouse fibroblast cells. The results suggest that NaCMC blended silk fibroin films are found to be potential substratum for supporting cell adhesion and proliferation.

Silk fibroin (SF) and elastin (EL) scaffolds were successfully produced for the first time for the treatment of burn wounds. The self-assembly properties of SF, together with the excellent chemical and mechanical stability and biocompatibility, were combined with elastin protein to produce scaffolds with the ability to mimic the extracellular matrix (ECM). Porous scaffolds were obtained by lyophilization and were further crosslinked with genipin (GE). Genipin crosslinking induces the conformational transition from random coil to b-sheet of SF chains, yielding scaffolds with smaller pore size and reduced swelling ratios, degradation and release rates. All results indicated that the composition of the scaffolds had a significant effect on their physical properties, and that can easily be tuned to obtain scaffolds suitable for biological applications. Wound healing was assessed through the use of human full-thickness skin equivalents (Epi-dermFT). Standardized burn wounds were induced by a cautery and the best re-epithelialization and the fastest wound closure was obtained in wounds treated with 50SF scaffolds; these contain the highest amount of elastin after 6 days of healing in comparison with other dressings and controls. The cytocompatibility demonstrated with human skin fibroblasts together with the healing improvement make these SF/EL scaffolds suitable for wound dressing applications.

Silk fibroin from silk gland of Bombyx mori 5th instar larvae was utilized to fabricate films, which may find possible applications as two-dimensional matrices for tissue engineering. Bombyx mori cocoon fibroin is well characterized as potential biomaterial by virtue of its good mechanical strength, water stability, thermal properties, surface roughness and biocompatibility. The present study aims to characterize the biophysical, thermal, mechanical, rheological, swelling properties along with spectroscopic analysis, surface morphology and biocompatibility of the silk gland fibroin films compared with cocoon fibroin. Fibroin solutions showed increased turbidity and shear thinning at higher concentration. The films after methanol treatment swelled moderately and were less hydrophilic compared to the untreated. The spectroscopic analysis of the films illustrated the presence of various amide peaks and conformational transition from random coil to b sheet on methanol treatment. X-ray diffraction studies also confirmed the secondary structure. Thermogravimetric analysis showed distinct weight loss of the films. The films were mechanically stronger and AFM studies showed surfaces were rougher on methanol treatment. The matrices were biocompatible and supported L929 mouse fibroblast cell growth and proliferation. The results substantiate the silk gland fibroin films as potential biomaterial matrices.