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Silk fibroin hydrogels and uses thereof

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Silk fibroin hydrogels and uses thereof


The present specification provides for methods for purifying fibroins, purified fibroins, methods of conjugating biological and synthetic molecules to fibroins, fibroins conjugated to such molecules, methods of making fibroin hydrogels, fibroin hydrogels and fibroin hydrogel formulations useful for a variety of medical uses, including, without limitation uses as bulking agents, tissue space fillers, templates for tissue reconstruction or regeneration, cell culture scaffolds for tissue engineering and for disease models, surface coating to improve medical device function, or drug delivery devices.
Related Terms: Scaffolds For Tissue Engineering

Browse recent Allergan, Inc. patents - Irvine, CA, US
Inventors: Gregory H. Altman, Rebecca L. Horan, Adam L. Collette, Jingsong Chen, Dennis VanEpps
USPTO Applicaton #: #20120265297 - Class: 623 8 (USPTO) - 10/18/12 - Class 623 
Prosthesis (i.e., Artificial Body Members), Parts Thereof, Or Aids And Accessories Therefor > Breast Prosthesis >Implantable



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The Patent Description & Claims data below is from USPTO Patent Application 20120265297, Silk fibroin hydrogels and uses thereof.

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CROSS REFERENCE

This patent application is a continuation that claims priority pursuant to 35 U.S.C. §120 to U.S. patent application Ser. No. 12/764,050, filed Apr. 20, 2010, a 35 U.S.C. §111 patent application that claims priority pursuant to 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No. 61/170,895 filed Apr. 20, 2009, each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present specification discloses purified silk fibroin and method for purifying silk fibroins, hydrogels comprising silk fibroin with or without an amphiphilic peptide and methods for making hydrogels comprising silk fibroin and the use of silk fibroin hydrogels in a variety of medical uses, including, without limitation fillers for tissue space, templates for tissue reconstruction or regeneration, scaffolds for cells in tissue engineering applications and for disease models, a surface coating to improve medical device function, or as a platform for drug delivery.

BACKGROUND

Silk refers to a filamentous product secreted by an organism such as a spider or silkworm. Fibroin is the primary structural component of silk. It is composed of monomeric units comprising an about 350 kDa heavy chain and an about 25 kDa light chain, and interspersed within the fibroin monomers is another about 25 kDa protein derived from the P25 gene. The ratio of heavy chain:light chain:P25 protein is about 6:6:1. Fibroin is secreted by the silk glands of the organism as a pair of complementary fibrils called “brins”. As fibroin brins leave the glands, they are coated with sericin, a glue-like substance which binds the brins together. Sericin is often antigenic and may be associated with an adverse tissue reaction when sericin-containing silk is implanted in vivo.

Silkworm silk fibers traditionally available in the commercial market are often termed “degummed”, which refers to the loosening and removal of a portion of the sericin coat surrounding the two fibroin brins through washing or extraction in hot soapy water. This degummed silk often contains or is recoated with sericin and other impurities in order to bind the plied multifilament together into a single fiber. Therefore, degummed silk, unless explicitly stated to the contrary, typically contains twenty percent to twenty-eight percent (by weight) sericin and can not be assumed to be sericin-free.

Silk fibers have historically been valued in surgery for their mechanical properties, particularly in the form of braided filaments used as a suture material. Residual sericin that may be contained in these materials stands as a potential obstacle to its use as a biomaterial as it does present the possibility for a heightened immune response. This sericin contamination may be substantially removed though, resulting in a virtually sericin-free fibroin which may be used either as fibers or dissolved and reconstituted in a number of forms. For example, natural silk from the silkworm Bombyx mori may be subjected to sericin extraction, spun into yarns then used to create a matrix with high tensile strength suitable for applications such as bioengineered ligaments and tendons. Use of regenerated silk materials has also been proposed for a number of medical purposes including wound protection, cell culture substrate, enzyme immobilization, soft contact lenses, and drug-release agents.

Silk fibroin devices whether native, dissolved, or reconstituted, do not typically contain cell-binding domains such as those found in collagen, fibronectin, and many other extracellular matrix (ECM) molecules. Fibroin is also strongly hydrophobic due to the β-sheet-rich crystalline network of the core fibroin protein. These two factors couple to severely limit the capacity of native host cells to bind to and interact with implanted silk devices, as neither inflammatory cells like macrophages or reparative cells like fibroblasts are able to attach strongly, infiltrate and bioresorb the silk fibroin devices. In the case of virgin silk and black braided (wax or silicone coated) silk sutures, this is typically manifested in a harsh foreign-body response featuring peripheral encapsulation. Substantially sericin-free silk experiences a similar, though substantially less vigorous response when implanted. In essence, the host cells identify silk as a foreign body and opt to wall it off rather than interact with it. This severely limits the subsequent long-term potential of the device particularly relating to tissue in-growth and remodeling and potentially, the overall utility of the device. If it is possible to provide a more effective biomaterial formulation for mediating host-device interactions whereby cells are provided with a recognizable, acceptable and hence biocompatible surface, the biological, medicinal and surgical utility of silk is dramatically improved.

One possible means of introducing this improved cell-material interaction is to alter the silk fibroin material format into a more biocompatible matrix. Manipulating the silk fibroin to make it into a silk hydrogel formulation is one particularly intriguing option because it consists of a silk protein network which is fully saturated with water, coupling the molecular resiliency of silk with the biocompatibility of a “wet” material. Generation of a silk hydrogel may be accomplished in short by breaking apart native silk fibroin polymers into its individual monomeric components using a solvent species, replacing the solvent with water, then inducing a combination of inter- and intra-molecular aggregation. It has been shown that the sol-gel transition can be selectively initiated by changing the concentration of the protein, temperature, pH and additive (e.g., ions and hygroscopic polymers such as poly(ethylene oxide) (PEO), poloxamer, and glycerol). Increasing the silk concentration and temperature may alter the time taken for silk gelation by increasing the frequency of molecular interactions, increasing the chances of polymer nucleation. Another means of accelerating silk gelation is through use of calcium ions which may interact with the hydrophilic blocks at the ends of silk molecules in solution prior to gelation. Decreasing pH and the addition of a hydrophilic polymer have been shown to enhance gelation, possibly by decreasing repulsion between individual silk molecules in solution and subsequently competing with silk fibroin molecules in solution for bound water, causing fibroin precipitation and aggregation.

Other silk fibroin gels have been produced by, for example, mixing an aqueous silk fibroin solution with protein derived biomaterials such as gelatin or chitosan. Recombinant proteins materials based on silk fibroin\'s structure have also been used to create self-assembling hydrogel structures. Another silk gel, a silk fibroin-poly-(vinyl alcohol) gel was created by freeze- or air-drying an aqueous solution, then reconstituting in water and allowing to self-assemble. Silk hydrogels have also been generated by either exposing the silk solution to temperature condition of 4° C. (Thermgel) or by adding thirty percent (v/v) glycerol (Glygel). Silk hydrogels created via a freeze-thaw process have not only been generated but also used in vitro as a cell culture scaffold.

The use of silk hydrogels as biomaterial matrices has also been explored in a number of ways. General research on hydrogels as platforms for drug delivery, specifically the release behavior of benfotiamine (a synthetic variant of vitamin B1) coupled to silk hydrogel was investigated. The study revealed both silk concentration and addition of other compounds may factor in to the eventual release profile of the material. Similarly, the release of FITC-labeled dextran from a silk hydrogel could be manipulated by altering the silk concentrations within the gel.

Further studies of silk hydrogels have been performed in vivo as well. For example, the material has been used in vivo to provide scaffolding for repair of broken bones in rabbits and showed an accelerated healing rate relative to control animals. Of particular interest, the in situ study also illustrated that the particular formulation of silk hydrogel did not elicit an extensive immune response from the host.

Despite early promise with silk hydrogel formulations in vivo, sericin contamination remains a concern in their generation and use just as with native fibroin for reasons of biocompatibility as well as the potential for sericin to alter gelation kinetics. The existence of sericin molecules in the silk solution intermediate prior to gelation may also compromise final gel structural quality, i.e., the distribution of β-sheet structure. For these reasons the removal of sericin from silk fibroin material prior to hydrogel manufacture remains a concern. The potential for disruption of gelation kinetics and structure by contaminants also presents the need for development of a process which consistently ensures structural uniformity and biocompatibility.

SUMMARY

OF THE INVENTION

The embodiments described herein provide for silk hydrogel formulations that may be useful for a variety of medical uses. More specifically, example embodiments of the present invention provide for gels including silk fibroin and peptides. Other example embodiments provide for the use of organic enhancers which improve device utility and functional peptide enhancers that may improve utility and biocompatibility of silk formulations. Silk hydrogel embodiments may be used as tissue space fillers, templates for tissue reconstruction or regeneration, cell culture scaffolds for tissue engineering and for disease models, surface coating to improve medical device function, or drug delivery devices.

One embodiment provides for an injectable silk gel comprising a gel phase and a carrier phase (which may provide additional lubricity) in which the gel phase comprises water, substantially sericin-depleted silk fibroin and an amphiphilic peptide. In another embodiment, the gel phase is about 1% to 99%, for example the gel phase is about 50% to about 99% of the total formulation volume with the carrier phase providing the remainder. For example, the gel phase is about 75% of the total formulation volume and the carrier phase is the remaining 25%. The gel phase may comprise about 0.5% to about 20% silk fibroin protein by mass, for example about 1% to about 10%, or about 4% to about 6%. In one embodiment, the silk fibroin comprises about 0.5% to about 9.9% of the total formulation mass.

In a particular embodiment, the peptide is an amphiphilic peptide consisting of a tail region, followed by a spacer region and finally the sequence arginine-glycine-aspartic acid, known as the RGD motif. For example, the total peptide is 23 amino acids in length (hereinafter, referred to as “23RGD”). The gel phase may comprise, for example, a molar ratio of about 1:100 moles to about 100:1 moles of this peptide per mole of silk fibroin.

Another example embodiment provides for an injectable gel formulation comprising silk fibroin and an amphiphilic peptide, wherein the formulation comprises from about 1% about 20%, for example about 4% to about 6% silk fibroin, and the amphiphilic peptide is 23RGD.

Yet another embodiment provides for an injectable gel formulation comprising silk fibroin and 23RGD, wherein the formulation comprises from about 4% to about 6% silk fibroin, and 23RGD concentration is 3:1 moles 23RGD/mole silk.

Another particular embodiment provides for an implantable gel formulation comprising silk fibroin and the 23RGD wherein the gel formulation comprises from about 4% to about 8% silk fibroin and the 23RGD concentration is about 1:10 to 10:1 moles of 23RGD per mole of silk fibroin.

In another embodiment, the gel phase comprises a protein structure consisting predominantly of the β-sheet conformation with components of α-helix, random coil, and unordered structures.

Another example embodiment of invention relates to a kit including a sterile silk gel formulation packaged in a 1 mL syringe with a 26 g needle and blended with a material commonly referred to as a “local anesthetic”. This anesthetic might be more specifically lidocane. Dependent upon application, the kit includes syringes sizes from 0.5 mL to 60 mL, where applications requiring larger volumes (e.g., bone fillers, disc fillers) are supplied in a larger size syringe. Additionally, needle gage is adjusted according to injection site with an acceptable range of 10 g to 30 g needles. For example, 26 g to 30 g needles are used for intradermal injections. Furthermore, the local anesthetic is not blended into the formulation for applications where the anesthetic is preferably applied separately or applications for which an anesthetic is not needed.

In another embodiment, the silk gel formulation is processed in a batch system by obtaining an 8% silk solution, adding ethanol/23RGD to generate a firm 4%-6% gel, allowing this to stand for at least 24 hours. The gel is then rinsed in water to remove residual free gelation agents (both 23RGD and ethanol), adding saline solution to the gel as a carrier phase and developing a homogeneous suspension. Suspension viscosity/injectability is then tailored by manipulating gel concentration, particle size, and saline content, milling the gel to a desired particle size that makes the gel injectable through a needle (for example a 30 g needle), loading the gel into a syringe, and sterilizing the gel with gamma irradiation.

In another aspect, the injectable formulation includes a gel comprising substantially sericin-depleted silk fibroin and an amphiphilic peptide and a carrier phase, wherein the formulation, upon injection, remains substantially at the injection site for about two weeks to about sixty months depending upon a desired application. For example, one formulation, for soft tissue filling may employ a 1%-6% silk gel with 20%-50% saline carrier at an average particle size of 20 μm-30 μm, and be deliverable through a 26 g-30 g needle with ˜5N of force while remaining substantially for one month to nine months at the injection site. One example formulation for hard tissue filling may employ a 6%-10% silk gel with 0%-25% saline carrier at a 50 μm-1000 μm particle size, and be deliverable through a 10 g-18 g needle at ˜5N of force while remaining substantially for nine to fifteen months at the injection site.

In one embodiment, the present invention provides a five-amino acid peptide “tail” capable of linking or conjugating a molecule X to a silk molecule or fibroin when the molecule X is attached to the tail. In one embodiment, the tail peptide comprises of hydrophobic and/or apolar amino acid residues. In another embodiment, the tail peptide comprises of amino acid residues capable of hydrogen bonding and/or covalent bonding. In other embodiments, the tail peptide comprises any of the twenty conventional standard amino acid residues.

In one embodiment, the five-amino acid peptide “tail” comprises amino acid residues that are part hydrophobic (i.e. the part of the side-chain nearest to the protein main-chain), for e.g. arginine and lysine.

In one embodiment, the five-amino acid peptide “tail” is separated from a molecule X by a spacer peptide. The length of the space peptide can be of variable length.

In one embodiment, the molecule X is any biological molecule or fragment thereof. In other embodiments, the molecule X is any recombinant, synthetic, or non-native polymeric compounds. Basically, a molecule X is any entity, natural or synthetic, that can be useful and can be use in the context of silk hydrogels.

In one embodiment, the present invention provides a synthetic molecule having the formula: (molecule X)n-(spacer peptide)0-300-(tail)-NH2 for linking with silk molecule or fibroin, wherein “n” is a whole integer ranging from 1-30, and wherein the amino acid residues of the spacer ranges from 0-300.

In one embodiment, the invention provides a method of conjugating a molecule X to a silk molecule or fibroin comprising mixing a synthetic molecule having the formula: (molecule X)n-(spacer peptide)0-300-(tail)-NH2 with a silk molecule or fibroin or silk solution, wherein “n” is a whole integer ranging from 1-30, and wherein the amino acid residues of the spacer ranges from 0-300.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the impact of 23RGD on the gelation times of silk hydrogels manufactured under various circumstances for example without enhancers or with a water/23RGD enhancer (FIG. 1A), or with an ethanol enhancer or combined ethanol-23RGD enhancers (FIG. 1B). Depending upon the ratio of 23RGD to silk used and the specific enhancer solvents, the peptide may function as either an accelerant or decelerant of the process.

FIG. 2 is a graph of HPLC data illustrating the integration of 23RGD and stability of its binding to 4% silk gel material made with an enhancer solution consisting of a 3:1 molar ratio of 23RGD:silk dissolved in 90% ethanol, 10% water when rinsed multiple times in ultra-purified water over several days. Data are shown for both total peak area and calculated 23RGD:silk molar ratio based on a 23RGD standard curve.

FIG. 3 is a graph comparing gel dry mass component at different RGD concentrations for 2% silk gels (A), 4% silk gels (B), and 6% gels (C). * Samples differ significantly, p<0.05; † sample differs significantly from all others; ‡ all samples differ significantly.

FIG. 4 illustrates the impact upon silk hydrogel water absorption and retention as identified in a gel drying assay. Data are shown as the percentage of mass retained by a silk gel sample (n=4 for each type) after being subjected to a 96-hour lyophilization process. Increasing concentrations of 23RGD enhancer caused increasing dry mass in the gel materials more substantial than the mass of the peptide itself. This phenomenon is likely due to structural differences in 23RGD-enhanced gels which do not permit a level of water entrainment equal to those of gels enhanced only with ethanol.

FIG. 5 shows a comparison of the percent mass loss over time due to bioresorption of samples cast by PG and EEG methods (A), cast from increasing silk concentrations (B), and cast using increasing RGD concentrations (C). * Samples differ significantly, p<0.05; † sample differs significantly from all others; ‡ all samples differ significantly.

FIG. 6 illustrates wet mass loss due to proteolytic bioresorption of silk hydrogels enhanced by a combination of 23RGD and ethanol at increasing concentrations of 23RGD. As a general trend, gels enhanced with 23RGD tend to be bioresorbed more quickly based upon this assay.

FIG. 7 is a second illustration of the bioresorption behavior of 23RGD-enhanced and non-23RGD-enhanced silk hydrogels when incubated in a protease solution. This bioresorption data serves to reinforce the trend, illustrated in FIG. 5, of a slightly more rapid rate of bioresorption of 23RGD-enhanced hydrogels in comparison to non-23RGD-enhanced gels. The figure also supports the more thorough removal of α-helix and random coil conformations from 23RGD-enhanced gels in FIG. 6 over four days of incubation in protease.

FIG. 8 shows structural features observed by Fourier-Transform Infrared (FTIR) spectroscopy of 4% silk fibroin hydrogel devices which are enhanced by ethanol alone, and two 23RGD-ethanol enhancers. The full spectra (FIG. 9A) of the materials are compared and the Amide I Band (1700-1600 cm−1) highlighted for particular attention (FIG. 9B) because of its relevance to secondary protein structure. Of specific interest is the commonality between all gels in their rich β-sheet structure (1700 cm−1 and 1622 cm−1 respectively, highlighted in FIGS. 9C and 9E) at all time points. These peaks become more pronounced after bioresorption, and begin to differentiate 23RGD-enhanced materials from materials enhanced with ethanol alone. This is evidenced in 23RGD-enhanced gels by a peak shift to lower wave numbers by the 1622 cm−1 peak and dramatically increased prominence of the 1700 cm−1 peak. Additional differences between bioresorbed and non-bioresorbed gels may be seen in regions of the spectrum known to correlate to α-helix and random coil conformations (1654 cm−1 and 1645 cm−1 respectively highlighted in FIG. 9D). These conformations are extensively digested in all gel types, but most completely in gels enhanced by 23RGD. This suggests that 23RGD-enhanced gels tend to bioresorb to a very β-sheet rich secondary structure in a more rapid fashion than non-23RGD-enhanced gels. Spectra shown were collected on a Bruker Equinox 55 FTIR unit using a compilation of 128 scans with a resolution of 4 cm−1.

FIG. 9 shows a comparative FTIR spectra illustrating the effects of differing gelation techniques on gel protein structure before (Day 0) and after (Day 4) proteolytic bioresorption. Groups assessed included samples cast by PG and EEG methods (A), cast from increasing silk concentrations (B), and cast using increasing RGD concentrations (C).

FIG. 10 shows representative micrographs of H&E-stained histological sections collected from silk gels implanted subcutaneously in rats. Samples of 4% silk fibroin hydrogel formed by passive gelation (4P), 4% silk fibroin hydrogel formed by ethanol-enhanced gelation (4E), and 6% silk fibroin hydrogel formed by ethanol-enhanced gelation (6E) were compared at 7 days (A, B, and E respectively) with 4E and 6E samples compared again at days 28 (C and F) and 57 (D and G).

FIG. 11 shows representative gross photographs of 8% silk fibroin hydrogel devices both unmodified (A) and 23RGD-enhanced (D) after a two-week subcutaneous incubation in Lewis rats. Also shown are micrographs resultant from H & E stains of the unmodified (B and C) and 23RGD-coupled (E and F) samples at 10× and 20× magnification. These gross images coupled with the histological micrographs provide evidence of a less extensive inflammatory response during early device integration being associated with 23RGD-enhanced gel than non-23RGD-enhanced gel.

FIG. 12 shows representative histology collected from a thirteen-week study of 4% 3:1 23RGD-enhanced silk hydrogel blended with 25% saline (left panels, H&E stain Trichrome stain) and ZYPLAST™ (right panels H&E stain, Trichrome stain) and injected into the intradermis of guinea pig. Each material type exhibited some clear evidence of implanted device in 75% of their respective implant sites. These micrographs indicate strong similarities not only between the long-term bioresorption characteristics but also long-term host tissue response between collagen-derived biomaterials and this particular 23RGD-enhanced silk hydrogel formulation.

FIG. 13 shows representative micrographs of H&E-stained histological sections collected from Day 28 explants of 4% silk fibroin, 10% saline (A); 4% silk fibroin, 1:1 23RGD, 10% saline (B); 6% silk fibroin, 1:1 23RGD, 10% saline (C); ZYPLAST™ (D); 4% silk fibroin, 25% saline (E); 4% silk fibroin, 1:1 23RGD, 25% saline (F); 6% silk fibroin, 10% saline (G); HYLAFORM™ (H); 6% silk fibroin, 25% saline (I); 4% silk fibroin, 3:1 23RGD, 25% saline (J); and 6% silk fibroin, 1:1 23RGD, 25% saline (K).

FIG. 14 shows representative micrographs of Day 92 histological sections of 4% silk fibroin, 3:1 23RGD, 25% saline (A-D) and ZYPLAST™ samples (E-H) stained with H&E at 4× (A and E), 10× (B and F), stained with Masson\'s Trichrome at 10× (C and G) and under polarized light at 10× (D and H).

FIG. 15 is a photograph of a custom-built testing jig used in conjunction with an Instron 8511 (Instron Corporation, Canton Mass.) in conjunction with Series IX software and a 100 N load cell for characterizing the injection forces associated with forcing silk gel through a 30 g needle.

FIG. 16 illustrates the average extrusion force data from mechanical testing of various silk gel formulations illustrating the effects of changing comminution method (A), saline concentration (B), silk concentration (C), and RGD content (D). Values are reported as an average of n=4 tests at each displacement rate with standard deviation illustrated as error bars. * Samples differ significantly, p<0.05; † sample differs significantly from all others in group at same strain rate; ‡ all samples in group differ significantly from all others in group at same strain rate.

FIG. 17 shows representative ESEM micrographs of selected RGD/ethanol-induced silk precipitates generated from the previously mentioned formulations. BASE (A), SCVLO (B), RHI (C), RLO (D), AVHI (E), ECLO (F), AVLO (G), and 3R 6.7:1 (H) are shown at 200× magnification.

FIG. 18 shows a comparison of the total dry mass of precipitate recovered from each silk precipitate formulation (n=4 for each type) after being subjected to a 96-hour lyophilization process. Data are grouped to compare the effects of changing volume ratio of accelerant added (A), concentration of 23RGD in the accelerant (B), changing the initial silk concentration (C), and changing the concentration of ethanol in the accelerant (D). It was shown that increasing any of these volumes or concentrations resulted in greater quantities of precipitate, though none appear to have substantially greater impact than another. This phenomenon is likely due to basic kinetics of the assembly reaction, with each reagent in turn appearing both as an excess and as limiting dependent upon the specific formulation. *—significant difference, p<0.05; †—Group differs significantly from all others.

FIG. 19 shows a comparison of the percentage of dry mass in each of precipitate recovered (n=4 for each type) after being subjected to a 96-hour lyophilization process. Data are grouped to compare the effects of changing volume ratio of accelerant added (A), concentration of 23RGD in the accelerant (B), changing the initial silk concentration (C), and changing the concentration of ethanol in the accelerant (D). Increasing the concentration of 23RGD used increased the dry mass percentage of precipitates, while increasing the ethanol percentage in the accelerant decreased dry mass. These changes may stem from formation of altered gel network structures caused by manipulation of these variables, likely more crystalline in the case of 23RGD increases and less crystalline in the case of ethanol concentration increases. *—significant difference, p<0.05; †—Group differs significantly from all others.

FIG. 20 shows representative FTIR spectra of the Amide I band for 23RGD/ethanol-induced silk precipitates immediately after processing (D0). Spectra are grouped to compare the effects of changing volume ratio of accelerant added (A), concentration of 23RGD in the accelerant (B), changing the initial silk concentration (C), and changing the concentration of ethanol in the accelerant (D). These spectra illustrate that similarities exist between all groups although changing 23RGD concentrations and ethanol concentrations may substantially impact precipitate structure. Increasing concentrations of decreased β-sheet seen in a peak shift from 1621 cm−1 in RVLO to ˜1624 cm−1 in RLO. A further increase in 23RGD concentration in both BASE and RHI caused this weakened β-sheet again along with increased signal values in the 1654 cm−1 and 1645 cm−1 range, correlating to increased random coil and α-helical content. An increased percentage of ethanol decreased the content of α-helical and random coil shown by decreased signal between 1670 cm−1 and 1630 cm−1 in both ECLO and BASE samples relative to ECVLO. This decrease in α-helical and random coil is accompanied by an increase in β-sheet structure. The findings relating to 23RGD and ethanol concentrations reinforce the trends observed in the percent dry mass of the precipitates, supposing that α-helical and random coil motifs entrain more water than β-sheet regions.

FIG. 21 is a representative micrograph of Congo red-stained 23RGD/ethanol-induced silk precipitates under polarized light at 20× magnification. A lack of emerald-green birefringence indicates a negative result in testing for amyloid fibril formation.



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Key IP Translations - Patent Translations


stats Patent Info
Application #
US 20120265297 A1
Publish Date
10/18/2012
Document #
13420356
File Date
03/14/2012
USPTO Class
623/8
Other USPTO Classes
514 212
International Class
/
Drawings
25


Scaffolds For Tissue Engineering


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