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Results for “FIBROIN”

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PubMedDate not supplied

[Secretion-fiber transformation in fibroin of the natural silk by polarization-optical methods].

Structural changes in fibroin molecules upon transformation of the secret into a fiber were studied by the methods of birefringence in a longitudinal hydrodynamic field, optical rotatory dispersion, and circular dichroism. Fibroin fibers were obtained by drawing out the secret from the silk-secreting gland of silkworm moth Bombux mori. In this process, the formation of a longitudinal hydrodynamic field inside the gland was observed. The experimental data obtained make it possible to assess the degree of orientation and unfolding of macromolecules, as well as the conditions of the alpha-beta structural transition in fibroin chains.

Animals↗

PubMed2006

[Research on preparation of silk fibroin and its biocompatibility with rat bone marrow mesenchymal stem cells].

The newly developed approach of tissue engineering has been shown to be great potential on the ligament reconstruction, however, the criterion for the scaffolding material was strict. The scaffold material must have enough strength as well as elasticity, at the same time, it should be biocompatible. As a nature protein, silk is a promising tissue engineering scaffold material for its excellent mechanical property. However, because of the contamination of sericin, the chief problem of silk's medical use is degumming. We compared three degumming reagents to choose the one which has least effect on the mechanical property of silk, and then the best degumming condition was confirmed: 0.4%NazCO3, 90 degrees C, 1 h. Rat bone morrw mesenchymal stem cells (rMSCs) were seeded on the fibroin, and scanning electron microscope (SEM) and fluorescence microscope were used to detect the biocompatibility of it. And the results showed that fibroin had outstanding biocompatibility and cell affinity, which indicated the further use of fibroin in tissue engineering.

Animals↗

PubMed2006

[Experimental study on facial nerve regeneration by porous silk fibroin conduit].

OBJECTIVE: To evaluate the feasibility of using polyporus silk fibroin as a kind of novel material for facial nerve regeneration. METHODS: The porous silk fibroin conduit was used in the reconstruction of a 5 mm facial nerve gap of SD rat. Chitosan conduit was taken as control group. General observation, electrophysiological study, histological study and image analysis were performed 2, 4, 6 and 8 weeks postoperatively. RESULTS: The facial nerve of SD rat regenerated successfully as time passed through. Mean CAP percentage of regenerated nerve in SF conduit was 24.94% +/- 5.73% 8 weeks postoperatively, which had no statistical significance with that of chitosan conduit group (P = 1.125). And the average number of myelinated myelinated nerve fibers in SF conduit was 62. 5 +/- 6. 3, which had statistical significance with that in chitosan conduit group (P = 0.016). CONCLUSIONS: The porous silk fibroin conduit could effectively repair facial nerve defect and improve peripheral nerve functional recovery.

Animals↗

PubMed1989

Complex formation with the fibroin gene enhancer through a protein-protein interaction analyzed by a modified DNA-binding assay.

The distal upstream region of the fibroin gene, -238 to -73, is known to contain the cis-acting element(s) responsible for tissue-specific transcription enhancement in vitro. To identify factor(s) trans-acting on this region, we have developed a modified nitrocellulose blot protein-DNA binding procedure that detects DNA-protein complexes via protein-protein interaction. A protein that does not bind to DNA as a monomer cannot be detected by standard Southwestern blotting. To detect protein-DNA complexes involving both protein-DNA and protein-protein interactions, the protein blot was reacted with the extract protein to form a protein complex on the filter, in the presence of the DNA probe. This new method revealed that the 75-kDa protein binds to the enhancer region of the fibroin gene in the presence of the second protein(s). The 75-kDa protein and the factor(s) mediating DNA binding activity were recovered in two different column fractions. The level of the 75-kDa protein appeared to be much higher in silk gland extracts than in nonsilk gland extracts, whereas the mediating factor(s) distributed evenly. These observations suggest that the distal upstream region of the fibroin gene can be specifically recognized by a protein complex composed of at least two distinct proteins, through protein-protein interaction.

Animals↗

PubMed1985

Enhanced transcription of fibroin gene in vitro on covalently closed circular templates.

A modified method for S1 nuclease assay has been developed to quantitate an amount of radioactive transcripts in the presence of unlabeled homologous RNA. Using this method, we have assayed in vitro transcription of fibroin gene on circular and linear templates. In a posterior silk gland cell extract, covalently closed circular (ccc) DNA forms the superhelical state and supports three to 10 times more transcription than nicked circular or linear DNA does. The efficient transcription on ccc template is also observed in a middle silk gland cell extract but never seen in a HeLa cell extract. When the posterior silk gland cell extract is first incubated with pBR322 DNA and then challenged for the fibroin gene transcription on ccc DNA, the pBR322 DNA of the ccc form, but not of the linear one, inhibits the reaction. A deletion mutant carrying only 44 base pairs of the 5'-flanking sequence reserves the preference of the ccc template, but seven single-point mutants at the TATA box and -20 region abolish it. These results suggest that some components in the silk gland extract supercoil ccc DNA and stimulate the fibroin gene transcription in the absence of the upstream enhancing sequence.

Animals↗

PubMed1981

Repeated DNA sequences near the 5'-end of the silk fibroin gene.

Four different repeated sequence elements have been found in cloned silk moth DNA, extending from 5700 base pairs upstream from the silk fibroin gene to 600 base pairs within its intervening sequence. Only 2 of the 12 overlapping restriction fragments examined, ranging from 164 to 1800 base pairs in length, do not contain a repeated sequence. The 830 base pairs adjoining the 5'-end of the fibroin gene hybridize with only one sequence in the Bombyx mori genome. Fibroin gene transcription initiates within this single-copy DNA at least 500 base pairs downstream from any repeated DNA sequence. The data show that repeated sequences comprise a major portion of the DNA flanking this developmentally regulated gene but not the DNA immediately 5' to it; that an intervening sequence can contain a repeated element; and that repeated sequences can be interspersed with one another as well as with single-copy DNA in a typical animal genome. In addition, filter transfer hybridization can be used to quantitate repetition frequency and sequence divergence and to distinguish patterns of repeated sequence organization.

Animals↗

PubMedDate not supplied

Silk fibroin-polyurethane scaffolds for tissue engineering.

Silk fibroin (SF) is a highly promising protein for its surface and structural properties, associated with a good bio- and hemo-compatibility. However, its mechanical properties and architecture cannot be easily tailored to meet the requirements of specific applications. In this work, SF was used to modify the surface properties of polyurethanes (PUs), thus obtaining 2D and 3D scaffolds for tissue regeneration. PUs were chosen for their well known advantageous properties and versatility; they can be obtained either as 2D (films) or 3D (foams) substrates. Films of a medical-grade poly-carbonate-urethane were prepared by solvent casting; PU foams were purposely designed and prepared with a morphology (porosity and cell size) adequate for cell growth. PU substrates were coated with fibroin by a dipping technique. To stabilize the coating layer, a conformational change of the protein from the alpha-form (water soluble) to the beta-form (not water soluble) was induced. Novel methodology in UV spectroscopy were developed for quantitatively analyzing the SF-concentration in dilute solutions. Pure fibroin was used as standard, as an alternative to the commonly used albumin, allowing real concentration values to be obtained. SF-coatings showed good stability in physiological-like conditions. A treatment with methanol further stabilized the coating. Preliminary results with human fibroblasts indicated that SF coating promote cell adhesion and growth, suggesting that SF-modified PUs appear to be suitable scaffolds for tissue engineering applications.

Journal Article↗

PubMed2001

Altering Fibroin Heavy Chain Gene of Silkworm Bombyx mori by Homologous Recombination.

A gene unit, which encoded fibroin-like peptide, was synthesized and constructed. The unit was multimerized to about 2 400 bp using BamHI and BglII at each end of the unit, then was fused with gfp reporter gene. The fusion gene, flanked by the 5'and 3'sequence of the fibroin heavy chain gene of silkworm Bombyx mori, was transferred into the eggs of silkworm by electroporation. After the silkworms developed and spinned silk, 73 out of about 5 400 cocoons were brighter than normal ones under UV light. The protein extracted from the brighter cocoon could react with the GFP polyclonal antibodies. Genomic DNA from these silkworms and their progenies were analyzed. The integration of gfp gene into genomic DNA of silkworm and the occurrence of expected homologous recombination event had been proved by Southern hybridization. It was shown that gfp-fibroin like fusion gene had integrated into the genomic DNA of silkworm by homologous recombination and the phenotype of "brighter cocoon" could be used to select transgenic silkworms.

Journal Article↗

PubMed1982

Structural analysis of sericin genes. Homologies with fibroin gene in the 5' flanking nucleotide sequences.

Two closely related mRNAs (11.0 and 9.6 kilobases long) for sericin, a cocoon protein other than fibroin, were isolated from the middle silk gland of Bombyx mori. They were shown to be specifically accumulated in this particular part of the gland. Nine genomic clones complementary to the mRNAs were isolated from the recombinant phage library of Bombyx DNA. They represent two structurally similar genomic segments, and contain the 5' end and its surrounding region of sericin gene. We have identified at least 5 exons in then, and the intervening coding junctions were determined at nucleotide level. The two genomic segments differ mainly in the 5' flanking as well as in the intervening sequences and may represent alleles of a sericin locus. DNA sequence analysis of an exon has revealed an internally repetitive unit of 114 base pairs. From an internally repetitive unit of 114 base pairs. From an open frame of the repetitive DNA sequence, a peptide consisting of 38 amino acids abundant in serine was deduced. We propose the peptide represents a repetitive unit of a sericin protein. The probable site of initiation of sericin gene transcription in vivo has been identified at the nucleotide level that also turned out to be the 5' end of mature sericin mRNA. There exist strong homologous regions in the sericin and fibroin genes at their corresponding 5' flanking sequences. A possible involvement of the homologous sequences around -111 and -186 for the concerted expression of the sericin and fibroin genes in the silk glands is discussed.

Amino Acid Sequence↗

PubMed2003

Structural characterization and artificial fiber formation of Bombyx mori silk fibroin in hexafluoro-iso-propanol solvent system.

High-resolution solution (13)C-NMR and CD studies of Bombyx mori silk fibroin revealed the presence of an ordered secondary structure 3(10)-helix, in hexafluoro-iso-propanol (HFIP). The solid-state structure of the silk fibroin film prepared by drying it gently from the HFIP solution still keep the structure, 3(10)-helix, which was studied with high-resolution solid state (13)C-NMR. The structural transition from the 3(10)-helix to silk II structure, heterogeneous structure including antiparallel beta-sheet, occurred during the artificial spinning from the HFIP solution. The wide-angle x-ray diffraction and differential scanning calorimetry thermograms of the artificial spinning fiber after postspinning treatments were observed together with the stress-strain curves. The results emphasize that the molecular structures, controlled morphology, and mechanical properties of the protein-based synthetic polymers can be modulated for enhancing biocompatibility.

Animals↗

PubMed2003

Vibrational 13C-cross-polarization/magic angle spinning NMR spectroscopic and thermal characterization of poly(alanine-glycine) as model for silk I Bombyx mori fibroin.

This study focuses on the conformational characterization of poly(alanine-glycine) II (pAG II) as a model for a Bombyx mori fibroin silk I structure. Raman, IR, and 13C-cross-polarization/magic angle spinning NMR spectra of pAG II are discussed in comparison with those of the crystalline fraction of B. mori silk fibroin (chymotryptic precipitate, Cp) with a silk I (silk I-Cp) structure. The spectral data give evidence that silk I-Cp and the synthetic copolypeptide pAG II have similar conformations. Moreover, the spectral findings reveal that silk I-Cp is more crystalline than pAG II; consequently, the latter contains a larger amount of the random coil conformation. Differential scanning calorimetry measurements confirm this result. N-Deuteration experiments on pAG II allow us to attribute the Raman component at 1320 cm(-1) to the amide III mode of a beta-turn type II conformation, thus confirming the results of those who propose a repeated beta-turn type II structure for silk I. The analysis of the Raman spectra in the nuNH region confirms that the silk I structure is characterized by the presence of different types of H-bonding arrangements, in agreement with the above model.

Animals↗

PubMed2004

Structural evolution of regenerated silk fibroin under shear: combined wide- and small-angle x-ray scattering experiments using synchrotron radiation.

The structural evolution of regenerated Bombyx mori silk fibroin during shearing with a Couette cell has been studied in situ by synchrotron radiation small- and wide-angle x-ray scattering techniques. An elongation of fibroin molecules was observed with increasing shear rate, followed by an aggregation phase. The aggregates were found to be amorphous with beta-conformation according to infrared spectroscopy. Scanning x-ray microdiffraction with a 5 microm beam on aggregated material, which had solidified in air, showed silk II reflections and a material with equatorial reflections close to the silk I structure reflections, but with strong differences in reflection intensities. This silk I type material shows up to two low-angle peaks suggesting the presence of water molecules that might be intercalated between hydrogen-bonded sheets.

Fibroins↗

PubMed2004

Raman study of poly(alanine-glycine)-based peptides containing tyrosine, valine, and serine as model for the semicrystalline domains of Bombyx mori silk fibroin.

For a deeper insight into the structure of Bombyx mori silk fibroin, some model peptides containing tyrosine (Y), valine (V), and serine (S) in the basic (AG)n sequence were synthesized by the solid-phase method and analyzed by Raman spectroscopy in order to clarify their conformation and to evaluate the formation and/or disruption of the ordered structure typical of B. mori silk fibroin upon incorporation of Y, V, and S residues into the basic (AG)n sequence. The Raman results indicated that the silk I structure remains stable only when the Y residue is positioned near the chain terminus; otherwise, a silk I --> silk II conformational transition occurs. The peptides AGVGAGYGAGVGAGYGAGVGAGYG(AG)3 and (AG)3YG(AG)2VGYG(AG)3YG(AG)3 treated with LiBr revealed a prevalent silk II conformation; moreover, the former contained a higher amount of random coil than the latter. This result was explained in relation to the different degrees of interruption of the (AG)n sequence. The Raman analysis of the AGSGAG-containing samples confirmed that the AGSGAG hexapeptide is a good model for the silk II crystalline domain. As the number of AGSGAG repeating units decreased, the random coil content increased. The study of the Y domain (I850/I830 intensity ratio) allowed us to hypothesize that in the packing characteristic of Silk I and Silk II conformations the Y residues experience different environments and hydrogen-bonding arrangements; the packing typical of silk I structure traps the tyrosyl side chains in environments more unfavorable to phenoxyl hydrogen-bonding interactions.

Animals↗

PubMed2005

Vibrational infrared conformational studies of model peptides representing the semicrystalline domains of Bombyx mori silk fibroin.

The structural organization of Bombyx mori silk fibroin was investigated by infrared (IR) spectroscopy. To this aim, (AG)15 and other model peptides of varying chain length, containing tyrosine (Y), valine (V), and serine (S) in the basic (AG)n sequence were synthesized by the solid phase method and their spectroscopic properties were determined. Both the position and the relative content of Y, V, and S residues in the (AG)n model system appeared critical in determining the preferred conformation, i.e., silk I, silk II, and unordered structures. Curve fitting analysis in the amide I range showed that the model peptides with prevailing silk II structure displayed different beta-sheet content, which was dependent on the degree of interruption of the (AG)n sequence. In this regard, the bands at about 1000 and 980 cm(-1), specifically assigned to the AG sequence of the B. mori silk fibroin chain, were identified as marker of the degree of interruption of the (AG)n sequence.A stable silk I structure was observed only when the Y residue was located near the chain terminus, while a silk I --> silk II conformational transition occurred when it was positioned in the central region of the peptide. Analysis of the second-derivative spectra in the amide I range allowed us to identify a band at 1639 cm(-1) (4 --> 1 hydrogen-bonded type II beta-turns), which is characteristic of the silk I conformation.

Animals↗

PubMed1989

CD and small-angle x-ray scattering of silk fibroin in solution.

We investigated the structure of silk fibroin dissolved in water and in water-organic solvent mixtures by CD and small-angle x-ray scattering (SAXS). CD spectra indicated a disordered secondary structure in water and a beta-sheet conformation in aqueous organic solvents, such as methanol, dioxane, and trifluoroethanol (in trifluoroethanol a transient form evolving toward beta-sheet conformation was seen just after dissolution). The SAXS technique indicated the presence of fibroin particles of lamellar shape. The molecular weight was 188,000 daltons in water and 302,000 daltons in aqueous methanol.

Animals↗

PubMed2004

Bone morphogenetic protein-2 decorated silk fibroin films induce osteogenic differentiation of human bone marrow stromal cells.

Bone morphogenetic protein (BMP)-2 has a critical role in bone formation and regeneration. Therefore, the ability to immobilize this molecule in certain matrices may be crucial in bone tissue engineering. Using carbodiimide chemistry, BMP-2 was directly immobilized on silk fibroin films. Whereas human bone marrow stromal cells cultured on unmodified silk fibroin films in the presence of osteogenic stimulants exhibited little if any osteogenesis, the same cells cultured on BMP-2 decorated films in the presence of osteogenic stimulants differentiated into an osteoblastic lineage as assessed by their significantly elevated alkaline phosphatase activity, calcium deposition, and higher transcript levels of collagen type I, bone sialoprotein, osteopontin, osteocalcin, BMP-2, and cbfa1. Using cell culture inserts, it was demonstrated that differentiation was induced by the immobilized protein and not by protein released into the culture medium. Comparison with a similar amount of medium-supplemented BMP-2, where no additional protein was added with medium changes, showed that delivery of BMP-2 immobilized on the biomaterial surface was more efficient than soluble delivery. The results illustrate that BMP-2 covalently coupled on silk biomaterial matrices retains biological function in vitro based on the induction of osteogenic markers in seeded bone marrow stromal cells.

Alkaline Phosphatase↗

PubMed2005

Structural and mechanical characteristics of silk fibroin and chitosan blend scaffolds for tissue regeneration.

The expanding field of tissue engineering has required the necessity of developing biomaterials that are tissue compatible, biodegradable, and comparable in mechanical properties to that of native tissue. We propose that the blending of two natural polymers, silk fibroin (SF) and chitosan (CS), into a 3D scaffold will provide unique chemical, structural, and mechanical properties that can be utilized for in vivo tissue regeneration. SF is an attractive material for biomedical applications because it is a fibrous protein that has high permeability to oxygen and water, relatively low thrombogenicity, low inflammatory response, protease susceptibility, supports cell adhesion and growth, and, foremost, high tensile strength with flexibility. CS is a crystalline polysaccharide, with structure similar to glycosaminoglycans, that has good wound healing properties, is nontoxic, and has minimal foreign body reactions. We hypothesized that increasing the SF-to-CS ratio would increase the ultimate tensile strength and elastic modulus and decrease the water capacity of the SFCS scaffolds. With increasing content of silk fibroin, it is observed that the ultimate tensile strength and elastic modulus increase significantly. The ultimate tensile strength and the elastic modulus were significantly higher in the short axis direction for 25:75 and 50:50 SFCS blends as compared to the long axis (p<0.05), while they were similar for the 75:25 SFCS blend. However, no differences were observed in the strain at failure among blends or due to directionality of applied strain. Increasing the chitosan content resulted in an increased water capacity of SFCS blends.

Biocompatible Materials↗

PubMed2006

Porous silk fibroin 3-D scaffolds for delivery of bone morphogenetic protein-2 in vitro and in vivo.

Bone morphogenetic protein-2 (BMP-2) plays a key role in osteogenesis. Biomaterials used for the sustained delivery of BMP-2 in vivo have shown therapeutic benefits. In the present study, BMP-2 was loaded in porous silk fibroin scaffolds derived from silkworm cocoons (2.4 +/- 0.14 microg per scaffold). The release profile of BMP-2 under dynamic culture conditions (spinner flasks) showed that after 1 week in culture 25% of the initial BMP-2 was retained adsorbed to the scaffold; up to 4 weeks no additional BMP-2 was released. BMP-2 induced human bone marrow stromal cells (hMSCs) to undergo osteogenic differentiation when the seeded scaffolds were cultured in medium supplemented with osteogenic stimulants for 4 weeks, based on elevated alkaline phosphatase activity, calcium deposition, and transcript levels for bone sialoprotein, osteopontin, osteocalcin, BMP-2, and cbfa-1. Micro-computed tomography revealed densely deposited mineral at the center of the scaffolds. In contrast, hMSCs cultured in control scaffolds (no BMP-2) exhibited limited osteogenesis. When implanted in critical sized cranial defects in mice, scaffolds loaded with BMP-2 and seeded with hMSCs resulted in significant bone ingrowth. These results were qualitatively similar to scaffolds loaded with BMP-2 but no hMSCs or with BMP-2 and hMSCs but not pregrown into bone-like tissue. Bone-related outcomes were improved when compared with the scaffold controls implanted without BMP-2. These studies illustrate the potential use of slow degrading silk fibroin 3-D scaffolds loaded with BMP-2, in combination with hMSCs, in osteogenesis studies in vitro and in vivo, and provide a new range of material properties for these applications.

Adsorption↗