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"Three Dimensional Soy Protein-Containing Scaffolds and Methods for Their Use and Production" in Patent Application Approval Process

May 13, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- A patent application by the inventors Chien, Karen B. (Chicago, IL); Shah, Ramille N. (Chicago, IL), filed on October 18, 2013, was made available online on May 1, 2014, according to news reporting originating from Washington, D.C., by NewsRx correspondents (see also Patents).

This patent application has not been assigned to a company or institution.

The following quote was obtained by the news editors from the background information supplied by the inventors: "Rapid prototyping, such as solid free-form fabrication, is a technique used to form porous three-dimensional (3D) scaffolds for tissue engineering applications. A major advantage of this fabrication method is the ability to control pore structures and geometries. 3D-Bioplotting is a method in which a viscous or a paste-like slurry is extruded by compressed air-induced pressure, onto a surface in air or submerged in a liquid medium (1-4). The continuous injection of a material using layer-by-layer deposition generates a fully interconnected pore structure. Although solid free form fabrication is commonly applied to synthetic materials, 3D printing and plotting of natural biopolymers such as proteins and polysaccharides is not as common. One challenge in printing soft materials is that they have a wide range of intrinsic properties such as viscosity, which can vary greatly between batches during sample fabrication.

"Animal and plant-based proteins are ideal biomaterials due to inherent bioactivity, degradation properties, and natural binding sites that can be tailored to control cell adhesion and growth both in vitro and in vivo (5). Specific optimization and design of printing parameters and conditions have been developed for various proteins (6-10) and composites (11, 12). 3D printing using a powder mixed with a binding solution was used to fabricate a blend of corn starch, gelatin, and dextran (11). Gelatin alone has also been plotted at 3% and 10% concentrations in water (6, 7). Landers et al. discussed the important fabrication parameters involved in plotting soft gel materials including viscosity, swelling in plotting medium, density, and thermal behavior of the plotted material, using gelatin and agar as examples (7). Bovine collagen has been fabricated using an indirect printing technique, where the slurry is printed into a negative mold, and the mold is subsequently dissolved away after freeze-drying to leave a scaffold with a predefined structure (8-10). The 3D Bioplotter was also used to fabricate 3D collagen-chitosan-hydroxyapatite hydrogels in a study characterizing the angiogenic and inflammatory response in vivo in comparison with a plotted PLGA scaffold (12).

"Two main challenges with printing natural biopolymers include controlling strut solidification upon extrusion (6) and electrostatic interactions between the biopolymer and liquid media if plotting into a solution. Scaffolds formed in air would require that the slurry dries at an appropriate rate during extrusion to support subsequent printed layers. Plotting into a liquid medium requires that the density of the solution be the same as the material being injected to preserve strand shape and to prevent dissolution of the printed strands (2, 7). Standardizing the plotting method for individual soft materials is essential since variability in moisture content of slurries and drying as a result of variable environmental conditions can affect reproducibility during mass production."

In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventors' summary information for this patent application: "Three dimensional porous soy protein-containing scaffolds comprising denatured soy proteins are provided. The soy protein chains in the denatured soy proteins can be crosslinked with enzymatic crosslinkers or with non-enzymatic chemical crosslinkers. The scaffolds can be tailored to have pore sizes suitable for promoting cell growth and proliferation within the pores and/or robust mechanical properties, as determined by their compressive moduli.

"In one aspect, a porous soy protein-containing scaffold comprises a plurality of layers configured in a vertical stack, each layer comprising a plurality of strands comprising denatured soy proteins.

"In some embodiments, the scaffold has a porosity of at least 50% and a pore interconnectivity of at least 90%.

"In some embodiments, the scaffold is configured such that within each layer the plurality of strands are spaced apart and aligned along their longitudinal axes and the angle, .theta., defined by the longitudinal axes of the strands in adjacent layers is in the range of 0.degree..ltoreq..theta..ltoreq.90.degree., such that pores are defined by the strands in adjacent layers of the vertical stack. In some embodiments, the angle .theta. is in the range of 45.degree..ltoreq..theta..ltoreq.90.degree. or in the range of 75.degree..ltoreq..theta..ltoreq.90.degree.. In some embodiments, the angle .theta. in the scaffold is in the range of 85.degree..ltoreq..theta..ltoreq.90.degree.; the soy protein chains in the denatured soy proteins are crosslinked; the crosslinking density of the soy protein chains within the scaffolds is at least 0.3; and the scaffold has a compressive modulus of at least 3500 Pa.

"In some embodiments, the scaffold has a compressive modulus of at least 1000 Pa or a compressive modulus in the range from about 2000 Pa to about 5000 Pa.

"In some embodiments, the soy protein chains in the denatured soy proteins are crosslinked and the relative crosslinking density of the soy protein chains within the scaffolds is at least 0.1 or in the range from about 0.1 to about 0.35.

"In some embodiments, the pores have a median pore diameter in the range from about 200 .mu.m to about 1000 .mu.m or in the range from about 300 .mu.m to about 400 .mu.m.

"In some embodiments, the median x-axis strand thickness for the strands is no greater than about 600 .mu.m.

"In some embodiments in which the scaffold is crosslinked, the crosslinks comprise carbodiimide crosslinks.

"In some embodiments, the scaffold further comprises at least one of a growth factor or a drug incorporated into one or more of the strands.

"In another aspect, a tissue growth scaffold comprises any of the disclosed porous soy protein-containing scaffolds and tissue-forming cells, or cells that are precursors to tissue-forming cells, integrated within the pores of the porous soy protein-containing scaffold.

"In another aspect, a method of growing tissue on a tissue growth scaffold comprises culturing the scaffold in a cell growth culture medium, wherein the scaffold comprises a plurality of layers configured in a vertical stack, each layer comprising a plurality of strands comprising denatured soy proteins; and tissue-forming cells, or cells that are precursors to tissue-forming cells, integrated within the pores of the scaffold.

"In another aspect, a method of forming a porous soy-protein containing scaffold comprises extruding a slurry comprising denatured soy proteins in the form of a first layer, the first layer comprising a plurality of strands; and extruding the slurry in the form of one or more additional layers, each additionally layer being vertically stacked upon the previously extruded layer and comprising a plurality of strands.

"In some embodiments, the strands in each layer are spaced apart and aligned along their longitudinal axes, and the angle, .theta., defined by the longitudinal axes of the strands in adjacent layers is in the range of 0.degree..ltoreq..theta..ltoreq.90.degree.

"In some embodiments, the slurry further comprises water and a plasticizer.

"In some embodiments, the method further comprises dehydrating the scaffold in an alcohol.

"In some embodiments, the method further comprises removing water from the scaffold via a dehydrothermal treatment. In some embodiments, the method further comprises freeze-drying the scaffold, whereby water is removed via the sublimation of water frozen on the strand surfaces, prior to the dehydrothermal treatment.

"In some embodiments, the method further comprises chemically or enzymatically crosslinking the soy protein chains in the denatured soy proteins.

"In some embodiments, at least 9 additional layers are extruded.

"In some embodiments, the slurry comprises at least one of a growth factor or a drug.

"In another aspect, a method of forming a porous biopolymer-containing scaffold comprises extruding a slurry comprising a biopolymer in the form of a first layer, the first layer comprising a plurality of strands; and extruding the slurry in the form of one or more additional layers, each additionally layer being vertically stacked upon the previously extruded layer and comprising a plurality of strands; wherein the mass flow rate of the slurry is maintained at a constant rate during extrusion by adjusting one or both of the extrusion pressure and extrusion speed during extrusion.

"In some embodiments, the strands in each layer are spaced apart and aligned along their longitudinal axes, and the angle, .theta., defined by the longitudinal axes of the strands in adjacent layers is in the range of 0.degree..ltoreq..theta..ltoreq.90.degree.

"Other principal features and advantages of the invention will become apparent to those skilled in the art upon review of the following drawings, the detailed description, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

"Illustrative embodiments of the invention will hereafter be described with reference to the accompanying drawings.

"FIG. 1 shows idealized schematics of Bioplotted scaffolds. (A) Definitions of the angular placement of layers (.theta.), pore width (or strand spacing) (w), strand thickness (t), layer spacing (s), and strand thickness in the z-direction (z). s can be less than, equal to, or greater than z (s.gtoreq.z for natural biopolymers), and z=t if the strand has a perfectly circular cross-section. (B) CAD images of top and cross-section views of idealized 45.degree. (left) and 90.degree. (right) scaffolds.

"FIG. 2 shows the mass flow rate of soy protein slurries of varying soy protein and glycerol concentration measured using the BioPlotter. Mass was extruded at a pressure of 3 bars for 2 seconds (N=3 measurements per temperature). All error bars represent one standard deviation.

"FIG. 3. (A) Mass flow rate of 20% soy protein, 4% glycerol slurry with and without the addition of 7.5 mM DTT. Schematics of the protein strand packing during slurry ejection through the needle in the -DTT and +DTT cases are provided to the left of each of the data series. (B, C) Macroscopic images of slurries printed at 27.degree. C. with the addition of 2% trypan blue (circle diameter set at 10 mm) Printing pressure was adjusted so that both circles were printed with a flow rate of 0.02 g/s. (B) Slurry without DTT. (C) Slurry with the addition of DTT. (D, E) SEM images of the surfaces of the printed strands from the macroscopic images. (D) Slurry without DTT. (E) Slurry with the addition of DTT.

"FIG. 4. (E) Mass flow rate of a 20% soy protein, 4% glycerol slurry at various pressures (n=3 measurements per temperature). Letters A-D represent mass flow rates with corresponding macroscopic images of strand shape. (A) Pressure less than optimal plotting pressure. (B) Optimal plotting pressure to produce well-defined pore structures and z-pores. (C, D) Pressures over the optimal plotting pressure.

"FIG. 5 shows the effect of post treatment on scaffold surface morphology. (A) Representative SEM image of scaffold surface showing pore structure of 90.degree. scaffold. (F) Mmacroscopic views of 45.degree. scaffolds with various post-treatments. N=5 for all diameters measured. NT: no further treatment beyond 95% ethanol dehydration. Average diameter was 6.57.+-.0.19 mm. FD-DHT: scaffolds freeze-dried before dehydrothermal treatment. Average diameter was 6.57.+-.0.14 mm. DHT: dehydrothermal treatment. Average diameter was 4.97.+-.0.33 mm. EDC: carbodiimide crosslinking. Average diameter was 6.09.+-.0.04 mm. (B-D) SEM images of the strand surface after various post-treatments. (B) NT. (C) EDC. (D) DHT. (E) FD-DHT.

"FIG. 6 shows the effect of post-treatment on structural, mechanical, and degradation properties of BioPlotted soy scaffolds. All error bars represent one standard deviation. (A) Crosslink density of scaffolds (N=5-6). (B) Mass loss of scaffolds upon rinsing 3.times. in PBS (N=4). (C, D) Compressive moduli of scaffolds plotted with various angles (N=5). *: P: P*: P.ltoreq.0.001.

"FIG. 7 shows the effect of post-treated scaffolds on human mesenchymal stem cell seeding efficiency. (A) Cell seeding efficiency (%) of the scaffolds with starting seeding density of 1.times.10.sup.6 cells/scaffold. (B) Proliferation of cells on scaffolds at days 1 and 7. *: P

URL and more information on this patent application, see: Chien, Karen B.; Shah, Ramille N. Three Dimensional Soy Protein-Containing Scaffolds and Methods for Their Use and Production. Filed October 18, 2013 and posted May 1, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2371&p=48&f=G&l=50&d=PG01&S1=20140424.PD.&OS=PD/20140424&RS=PD/20140424

Keywords for this news article include: Gelatin, Patents, Glycerol, Peptides, Amino Acids, Scleroproteins, Sugar Alcohols.

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Source: Life Science Weekly


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