This invention is directed to electrospinning three-dimensionally (3D) and randomly oriented fibrous structures from various polymer sources, including plant proteins, animal proteins, and synthetic polymers.
Ideal tissue engineering scaffolds should be capable of closely mimicking the topographies and spatial structures of native extracellular matrices (ECMs) to facilitate cells to grow and differentiate following the patterns similar to that found in native tissues and organs. Morphologies of ECMs vary according to functions of target tissues and cell types in the tissues. For example, in skin tissue, the top layer is formed by compact packing of epithelial cells on a two-dimensional (2D) fibrous ECM basement membrane. Three-dimensional spatial spreading of fibroblasts and immune cells occurs in the interior region of the skin tissue, and correspondingly the ECMs are constructed by stereoscopically and randomly oriented ultrafine protein fibers. Fibrous structures with 3D orientation and random distribution can also be found in native ECMs in breast, liver, bladder, lung, and many other organs and tissues. It has been reported that cells cultured on flat 2D substrates may differ considerably in morphology and differentiation pattern from those cultured in more physiological 3D environments. Therefore, it is reasonable to fabricate scaffolds with particular morphologies and structures according to categories and functions of original native tissues.
Due to its simplicity and high efficiency, electrospinning has been widely employed to fabricate tissue engineering scaffolds composed of nano- or submicrometer-fibers from numerous materials. However, conventional electrospun structures typically form 2D scaffolds with fibers aligned parallel to the collector and cells cultured on the conventional electrospun scaffolds could only develop into flat shapes. The functions and differentiation of many flattened cells could not resemble the native stereoscopic cells. Furthermore, small pore sizes, owing to the close arrangement of fibers, restricted access of cells to the interior of conventional electrospun scaffolds. Thus, on conventional electrospun scaffolds, cells could mainly spread and distribute within a shallow depth beneath the surface.
To date, many 3D electrospinning techniques have been developed to fabricate electrospun scaffolds with larger pores and higher porosity to improve cell accessibility of the scaffolds. Examples of such techniques include wet electrospinning, electrospinning with integration of coarse fibers, and electrospinning with porogens (e.g., dry ice, salt, or sucrose), which are based on the concept of including a “blocking agent” to increase the distances between electrospun fibers in order to lead to deeper penetration of cells into interior of scaffolds. Nonetheless, these techniques failed to change the planar orientations of the electrospun fibers and as a result the scaffolds tended to have parallel fibrous layer-by-layer structures. Additionally, the parallel fibrous layer-by-layer structures tended to not have pores that extend very far in the thickness direction as compared to in the planar directions. As a result, there was limited improvement in scaffold porosity and cells cultured thereon tended to have flattened morphologies rather than stereoscopically developed cells in many native tissues. Still further, there have been attempts to fabricate 3D electrospun scaffolds based on the electrostatic repulsion between as-spun fibers. Despite their fluffy appearances, such scaffolds still had a layer-by-layer structure of planar mats and parallel oriented fibers.
In view of the foregoing, a need still exists for randomly-oriented 3-D fibrous structures and a method for making the same via electrospinning.
In one embodiment, the present invention is directed to a method of making a randomly-oriented 3-D fibrous structure. The method comprising electrospinning a spinning dope with an electrospinning apparatus, wherein the spinning dope comprises: a solvent; a polymer dissolved in the solvent, wherein the dissolved polymer is in subunits having molecular weights that are about 5 to about 150 kDa; and a surfactant; to form one or more fibers that comprise a polymer-surfactant complex and that arrange randomly and evenly in three dimensions when contacting a collecting board of the electrospinning apparatus thereby forming the randomly-oriented 3-D fibrous structure.
In one embodiment, the present invention is directed to a method of making a randomly-oriented 3-D fibrous structure. The method comprising electrospinning a spinning dope with an electrospinning apparatus, wherein the spinning dope comprises: a solvent; a polymer dissolved in the solvent, wherein the dissolved polymer is in subunits having molecular weights that are about 5 to about 150 kDa, and wherein the polymer is selected from the group consisting of protein, synthetic polymer, and combinations thereof; and an anionic surfactant at about 5 to about 300 percent by weight of the polymer; to form one or more fibers of a fineness that is about 50 nm to about 100 μm and that comprise a polymer-surfactant complex and that arrange randomly and evenly in three dimensions when contacting a collecting board of the electrospinning apparatus thereby forming the randomly-oriented 3-D fibrous structure that further comprises interconnected pores having sizes that are about 10 to 2000 μm and that has a porosity that is about 60 to about 99.9% by volume.
In one embodiment, the present invention is directed to a randomly-oriented 3-D fibrous structure comprising: one or more fibers that comprise a polymer-surfactant complex, wherein the fiber(s) have lengths that are at least about 100 nm and finenesses that are about 50 nm to about 100 μm, and are arranged randomly and evenly in three dimensions throughout the randomly-oriented 3-D fibrous structure; and interconnected pores having sizes that are about 10 to 2,000 μm, wherein the pores comprise about 60 to about 99.9% by volume of the randomly-oriented 3-D fibrous structure.
An embodiment of the present invention is directed to a method of making a randomly-oriented 3-D fibrous structure. The method comprising electrospinning a spinning dope with an electrospinning apparatus, wherein the spinning dope comprises: a solvent; a polymer dissolved in the solvent, wherein the dissolved polymer is in subunits having molecular weights that are about 5 to about 150 kDa; and a surfactant; to form one or more fibers that comprise a polymer-surfactant complex and that arrange randomly and evenly in three dimensions when contacting a collecting board of the electrospinning apparatus thereby forming the randomly-oriented 3-D fibrous structure. Typically, the electrospinning is conducted at a temperature that is about 25 to about 70° C.
As used herein with respect to the present invention, the “randomly-oriented 3-D fibrous structure” is intended to mean fibrous structure in which the one or more fibers thereof are randomly oriented in all three dimensions rather than only being randomly oriented in two dimensions as can be found in conventional layer-by-layer, planar structures. The degree of random orientation of a fibrous structure may be quantified by, for example, identifying a cubic centimeter volume of said fibrous structure and identifying any particular imaginary plane of any orientation within said volume, identifying the number of fibers intersected or fiber intersections both of which are “intersections” with said plane, and identifying the number of said intersections in which said intersected fiber(s) are at angles greater than 30 degrees relative to said plane. In certain embodiments of the present invention, at least ½, ⅝, ⅔, and ¾ of the intersected fibers are at angles greater than 30 degrees with the plane.
As used herein the terms “scaffold” and “fibrous structure” are intended to have the same meaning and may be used interchangeably.
Without being bound to any particular theory, it is believed that the mechanisms of 2D electrospinning and an embodiment of 3D electrospinning (of the present invention produce) are depicted in
In contrast, the depicted 3D electrospinning embodiment is believed to be based on repulsive electrical force between fibers and the collector. More specifically, the method of the present invention, involves including a surfactant to decrease the surface resistivity of a fiber to thereby increase the transfer of charge from the fiber surface to the collector. When the fiber(s) strike the collector, surface static electricity transfers to the board in a faster manner, which results in less negative static electricity remaining on the fiber(s), and decreased attraction between fiber(s) and the collector. In some cases, the near portion of the fibers may even carry positive charges and may be repulsed by the collector, while the farther end of the fiber(s) is still attracted and moves towards the board. As a result, fiber(s) are collected onto the board in multiple orientations to form loose and fluffy 3D scaffolds with randomly-oriented fiber(s).
It is believed that the above-described mechanisms have been shown by experimental results shown in
As indicated above, the spinning dope comprises a polymer, which may be any appropriate polymer such as protein, synthetic polymer, and combinations thereof. Proteins may be of particular interest because they are preferred in biomedical applications due to their molecular similarity to native ECMs, and tend to be widely available at low cost. For example, plant proteins, zein and soy protein, and animal proteins, such as keratin and collagen, have been proved to be supportive to cell growth in in vitro and in vivo studies. In addition, plant and animal proteins are widely available at a low cost and they are considered to be a renewable resource. The method of the present invention may be conducted using one or more proteins selected from the group consisting of plant protein, animal protein, and combinations thereof.
Many such proteins, however, are considered to be highly-linked and as a result tend to have limited solubility in water and organic or alcoholic solvents. In general, proteins with cysteine content higher than 1% in its amino acid composition are considered to be highly-crosslinked proteins. Exemplary highly-crosslinked proteins, including keratin, which has a cysteine content of about 7%, soy protein has a cysteine content of about 1.3%, and wheat glutenin a cysteine content of about 2%. In particular, electrospinning highly crosslinked proteins, such as soy protein, feather keratin and wheat glutenin, into fibrous structures via electrospinning has not been possible due to their insolubility in various solvents.
With reductant and denaturant in the spinning dope, coarse fibers from soy protein and wheat gluten via wet spinning have been produced. Reddy, N. and Y. Yang, Novel Protein Fibers from Wheat Gluten, Biomacromolecules, 2007, 8(2), p. 638-643; Reddy, N. and Y. Yang, Soy protein fibers with high strength and water stability for potential medical applications, Biotechnology Progress, 2009, 25(6), p. 1796-1802. However, electrospinning with pure protein required much better dissolution of proteins, and thus the spinning dope for wet spinning failed to be electrospun. Highly hydrolyzed soy protein with small molecules had been electrospun with PEG, which accounted for the spinability. Vega-Lugo, A. C. and L. Loong-Tak, Electrospinning of Soy Protein Isolate Nanofibers, Journal of Biobased Materials and Bioenergy, 2008, 2(3), p. 223-230. Hydrolyzed soy protein in its pure form, however, had not been electrospun.
The present invention of electrospinning may be practiced, however, with highly crosslinked protein by dissolving the protein in manner that preserves the protein subunits with appropriate molecular weights ranging from 5 to 150 kDa. Other methods of dissolving highly crosslinked proteins (e.g., U.S. Pat. Pub. No. 2006/0282958, Yang et al., entitled Process for the Production of High Quality Fibers from Wheat Proteins and Products Made From Wheat Proteins) allowed for the spinning of coarse fibers but were not suitable for electrospinning of relatively fine fibers. The electrospinning method of the present invention may be practiced with highly crosslinked proteins by using a reducing agent such a thiol, a sulfite, or a sulfide to break the disulfide bonds of the highly crosslinked proteins. For example, cysteine, an environmentally-benign thiol, may be used as a reducing agent to break the disulfide bonds and achieve dissolution of highly-crosslinked proteins. The amount of the reducing agent in the solution may be varied from about 1% to about 50% based on the weight of proteins. The solvent for the protein and cysteine was a 4-8 M urea solution with pH of 8 to 12, adjusted using 50 wt % sodium hydroxide solution. The weight ratio of protein-containing materials to urea solution may be varied within a range from, for example, 1:5 to 1:30. The temperature for the dissolution may be within a range from about 20° C. to about 90° C. Examples of additional appropriate thiols include methanethiol, ethanethiol, 1-propanethiol, 2-propanethiol, butanethiol, tert-butyl mercaptan, pentanethiols, thiophenol, thioacetic acid, coenzyme-A, glutathione, 2-mercaptoethanol, dithiothreitol, 2-mercaptoindole, 3-mercaptopropane-1,2-diol. Examples of appropriate sulfites and sulfides include sodium sulfite potassium sulfite, sodium bisulfite, potassium bisulfite, sodium sulfide, potassium sulfide, sodium metabisulfite, and potassium metabisulfite. The conditions for the reactions include the thiol/sulfite/sulfide at concentration(s) of about 0.5% to about 50% based on the weight of proteins, a pH from 3 to 12 for a duration of about 30 minutes to about 24 hours at a temperature of about 20° C. to about 90° C.
In view of the foregoing, the method of the present invention may, in certain embodiments, be conducted using appropriately dissolved highly-crosslinked plant protein, highly-crosslinked animal protein, and combinations thereof. When using proteins, including highly-crosslinked proteins, experimental results to date have shown the aging the dissolved protein at an aging temperature that is about 20° C. to about 90° C. for an aging duration that is about 0.5 to about 48 hours before conducting the electrospinning may be advantageous. For example, it has been found that such aging may provide a higher degree of disentanglement of the protein molecules to improve the spinnability of the protein spinning dope.
Exemplary plant proteins include wheat gluten, wheat gliadin, wheat glutenin, soy protein, camelina protein, peanut protein, canola protein, sorghum protein, rice protein, millet protein, sunflower seed protein, pumpkin seed protein, mung bean protein, red bean protein, chickpea protein, green pea protein, and combinations thereof;
Exemplary animal proteins include chicken feather, egg white, wool keratin, casein, silk, fibrin, collagen, gelatin, hair keratin, horn keratin, nail keratin, whey protein, and combinations thereof.
Exemplary synthetic polymers include polyethylene glycol (PEG), poly lactic acid (PLA), poly glycolic acid (PGA), polyhydroxyalkanoates (PHAs), poly(lactic-co-glycolic acid) (PLGA), poly-3-hydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and combinations thereof.
As indicated above, the solution may comprise one, two, or even more polymers. In certain embodiments with more than a single polymer, the polymer that is in the largest amount (usually more than half of the total polymer content) is often referred to as the “primary” polymer, component, or material and the additional polymers are often referred to as “secondary” polymer(s), component(s), or material(s) with concentrations that are from about 0.5% to about 50% by weight of the primary polymer.
As indicated above, the spinning dope comprises a surfactant, which may be any appropriate surfactant. The hydrophobic portions of surfactant bond with polymers through hydrophobic interaction to form a protein-surfactant complex. Without being bound to a particular theory, it is believed that the hydrophilic portions, including functional groups that carry positive or negative charges and polar uncharged groups, gather on the fiber surface and thus effectively increase the surface conductivity (or lower the surface resistivity) by introducing surface water layer, which facilitates delivery of charges on the surface.
The surfactant(s) may be of different hydrocarbon chain lengths and different electrical properties, including anionic surfactants, cationic surfactants, nonionic surfactants, zwitterionic surfactants. In an embodiment, combinations of surfactants may be used, as appropriate. In another embodiment, the surfactant is one or more anionic surfactants.
Typically, the spinning dope has a concentration of the surfactant that is about 5 to about 300 percent by weight of the polymer. In another embodiment, the spinning dope has a concentration of the surfactant that is about 50 to about 150 percent by weight of the polymer.
Exemplary anionic surfactants include sodium dodecyl sulfate, sodium dodecyl benzenesulfonate, sodium lauryl sarcosinate, perflourobutanesulfonic acid, ammonium lauryl sulfate, sodium stearate, sodium pareth sulfate, dioctyl sodium sulfosuccinate, potassium lauryl sulfate, sodium laureth sulfate, sodium myreth sulfate, and combinations thereof.
Exemplary cationic surfactants include benzalkonium chloride, cetrimonium bromide, tetramethylammonium hydroxide, octenidine dihydrochloride cetyl trimethylammonium bromide, hexadecyl trimethyl ammonium bromide, cetyl trimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), 5-bromo-5-nitro-1,3-dioxane, dimethyldioctadecylammonium chloride, cetrimonium bromide, dioctadecyldimethylammonium bromide (DODAB), and combinations thereof.
Exemplary nonionic surfactants include decyl glucoside, octyl phenol ethoxylated, polysorbate 80, polysorbate 20, and combinations thereof.
Exemplary zwitterionic surfactants include cocamidopropyl hydroxysultaine, cocamidopropyl betaine, lecithin, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and combinations thereof.
As indicated above, the spinning dope comprises a solvent, which may be any appropriate solvent. Exemplary solvents include water, phosphate buffered saline (PBS), carbonate buffer, tris-glycine buffer, borate buffer, acetate buffer, n-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer, citric buffer, ethanol, chloroform, 1,4-dioxane, methanol, ethylene glycol, acetone, ethyl acetate, methyl acetate, hexane, petrol ether, citrus terpenes, diethyl ether, dichloromethane, dimethylformamide (DMF), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), formic acid, n-butanol, isopropanol (IPA), n-propanol, acetic acid, nitromethane, dichloromethane, and combinations thereof. In another embodiment, the solvent is selected from the group consisting of water, phosphate buffered saline (PBS), carbonate buffer, tris-glycine buffer, borate buffer, acetate buffer, n-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer, citric buffer, chloroform, and combinations thereof.
In addition to the above-described components, the spinning dope may comprise one or more additional components such as a cross-linker, a sacrificial component, or a combination thereof. Alternatively or in addition to including such additional components in the spinning dope, the method may further comprise contacting the fibers with a second solution that comprises a second cross-linker, a surface decorator, or a combination thereof.
Cross-linker(s)
Exemplary cross-linkers include polycarboxylic acid which contains at least three carboxylic acid groups (such as citric acid, iso-citric acid, propane-1,2,3-tricarboxylic acid, trimesic acid, aconitic acid, mellitic acid, 1,2,3,4-Butane tetracarboxylic acid (BTCA)), oxysucrose, genepin, glutaraldehyde, oxaldehyde, NHS esters, maleimides, carbodiimide, isocyanate, and combinations thereof. If present in the spinning dope and/or in a cross-linking solution, the concentration of cross-linker is typically about 0.01 mol/L to about 10 mol/L. In an embodiment in which the spinning dope comprises a first cross-linker and the method further comprises contacting the fibers with a cross-linking solution that comprises a second cross-linker, the first and second cross-linkers may be independently selected and as such may be identical or different (e.g., in terms of material, concentration, or both). In an embodiment of the present invention, the first and second cross-linkers are independently selected from the group consisting citric acid, 1,2,3,4-Butane tetracarboxylic acid (BTCA), oxysucrose, genepin, glutaraldehyde, oxaldehyde, and combinations thereof.
Sacrificial Component
Exemplary sacrificial components include PEG, egg white protein, zein, wheat gliadin, and other materials that may be dissolved with the above-described polymer(s) before electrospinning at concentrations of about 0.5% to about 50% based on the weight of the polymer. The sacrificial component(s) may be removed from the spun fibrous structure via rinsing with water, organic solvents or alcoholic solvents.
Surface Decorator
Exemplary surface decorators of fibers include peptides such as Arg-Gly-Asp (RGD), Ile-Lys-Val-Ala-Val (IKVAV), Asn-Ser-Gly-Ala-Ile-Thr-Ile-Gly (NSGAITIG), and combinations thereof at concentrations of about 0.5% to about 10% based on the weight of the polymer.
The method may further comprise contacting the fibers with a coagulation solution that comprises a coagulant to modify the water stability and mechanical properties of the fibers (e.g., tensile properties, compressive properties, abrasion properties, and bending properties). Exemplary coagulants include methanol, ethanol, sodium sulfate and acetic acid, acetone, sulfuric acid, hydrochloric acid, and combinations thereof. In an embodiment of the present invention, the coagulant is selected from the group consisting of methanol, ethanol, sodium sulfate and acetic acid, and combinations thereof. The amount of coagulant in the coagulant solution is such that the concentration of coagulant is typically about 1% to about 100%. Typically, the fibers are contacted with the coagulation solution for a duration of about 5 minutes to about 24 hours. Also, the coagulation solution is typically at a temperature that is from about 20° C. to about 100° C.
The above described method may be performed to make a randomly-oriented 3-D fibrous structure. Such a fibrous structure comprises: one or more fibers that comprise a polymer-surfactant complex, wherein the fiber(s) have lengths that are at least about 100 nm and finenesses that are about 50 nm to about 100 μm, and are arranged randomly and evenly in three dimensions throughout the randomly-oriented 3-D fibrous structure; and interconnected pores having sizes that are about 10 to 2,000 μm, wherein the pores comprise about 60 to about 99.9% by volume of the randomly-oriented 3-D fibrous structure. Advantageously, experimental results to date indicated that such randomly-oriented, three-dimensionally fibrous structures may be used to facilitate cells to grow and spread in three dimensions in a manner similar to that seen in native ECMs.
In another embodiment, the fibers have a fineness that is about 50 nm to about 100 μm and the randomly-oriented 3-D fibrous structure further comprises interconnected pores having sizes that are about 10 to 2000 μm and has a porosity that is about 60 to about 99.9% by volume.
In yet another embodiment, the fibers have a fineness that is about 50 nm to about 20 μm and the randomly-oriented 3-D fibrous structure further comprises interconnected pores having sizes that are about 10 to 1000 μm and has a porosity that is about 90 to about 99.9% by volume of the structure.
In an embodiment, the one or more fibers consist of the polymer-surfactant complex. In another embodiment, the randomly-oriented 3-D fibrous structure consists of the one or more fibers and the interconnected pores. In yet another embodiment, the randomly-oriented 3-D fibrous structure consists of one or more fibers and the interconnected pores, wherein the one or more fibers consist of the polymer-surfactant complex.
After the fibers/fibrous structure are formed it may be heat treated at a temperature that is about 70 to 150° C. to modify the water stability and mechanical properties of the fibers (e.g., tensile properties, compressive properties, abrasion properties, and bending properties).
Scaffold Preparation
Thirty 2D zein scaffolds were prepared by electrospinning 25 wt % zein (Freeman Industries LLC, Tuckahoe, N.Y.) in 70% v/v aqueous ethanol (EMD Chemicals Inc., Gibbstown, N.J.) solution. Three dimensional zein scaffolds were prepared by electrospinning aqueous solution containing 25 wt % zein and 25 wt % SDS. A concentration of 9 wt % (based on the weight of zein) citric acid (EMD Chemicals Inc., Gibbstown, N.J.) was added into both 2D and 3D spinning dopes for cross-linking. Different solvent systems were utilized since zein could not be dissolved in water. The 2D PEG scaffold was prepared by electrospinning 10 wt % PEG (50 kDa, Sigma-Aldrich, St. Louis, Mo.) aqueous solution. The 3D PEG scaffold was prepared by electrospinning 10 wt % PEG and 10 wt % SDS in aqueous solution. All the electrospinning parameters, including the extrusion speed of 2 mL/hr, voltage of 42 kV, and distance from the needle to the collecting board of 25 cm, were kept the same for all the samples. The needle was negatively charged, and the collecting board was positively charged.
Morphologies and Structures of Scaffolds
The 2D and 3D scaffolds were observed using a scanning electron microscope (S3000N, Hitachi Inc. Schaumburg, Ill.) and a Nikon A1 confocal laser scanning microscope (Nikon Inc., Melville, N.Y.).
Specific Pore Volume
Specific pore volume indicating volume of pore in unit mass of scaffolds as shown in the following equation was selected to evaluate fluffiness of the scaffolds:
where Vsp is the specific pore volume, Vpore is the volume of pores encompassed in the scaffolds, mscaffold is the mass of scaffolds, Vscaffold is the volume of the scaffolds after precise measurement of the length, width, and thickness of scaffolds, and ρmaterial is the density of the material.
Surface Resistivity
Since the surface resistivity of ultrafine fibers is very difficult to test, films containing same polymer to surfactant/salt ratio with relevant electrospun fibers were prepared to measure the surface resistivity. The films were casted onto Teflon coated plates and dried at 20° C. and 65% relative humidity. Surface resistivity was measured by employing a surface resistivity tester (Monroe Electronics Inc., Lyndonville, N.Y.) according to ASTM D-257 standard.
Fiber Deposition Process
A CCD camera with a longworking-distance lens was used in capturing the moment photographs of fiber deposition and scaffold formation. The time interval for each consequential photograph was 0.125 seconds.
Cell Attachment and Proliferation
NIH 3T3 mouse fibroblast cells (ATCC CRL-1658, Manassas, Va.) were cultured to quantitatively estimate effects of 2D and 3D structures of zein scaffolds on cell attachment and proliferation. Cells were cultured in culture medium at 37° C. in a humidified 5% CO2 atmosphere. Electrospun 2D and 3D zein scaffolds were first rinsed in 60 wt % acetone (BDH, West Chester, Pa.) aqueous solution containing 5 wt % potassium chloride (Fisher Scientific, Fair Lawn, N.J.) to remove SDS, washed in distilled water three times, and then lyophilized. MTS assays were performed to quantitatively investigate cell viability at attachment and proliferation stages. Samples were prepared with same weight and then were subjected to sterilization at 120° C. for 1 hour. After sterilization, the scaffolds were placed in 48-well culture plates (TPP Techno Plastic Products, Switzerland). Fibroblast cells were seeded onto the scaffolds (1×105 cells mL−1, 500 μL well−1) and then cultured at 37° C. in a humidified 5% CO2 atmosphere for different time intervals. At each time point, the samples were washed with PBS, placed in new 48-well plates containing 450 μL well−1 20% MTS reagent (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promenade) in Dulbecco's modified Eagle's medium (DMEM) and incubated at 37° C. in a humidified 5% CO2 atmosphere for 3 hours. After incubation, 150 μL of the solution from each well was pipetted into a 96-well plate and the optical densities were measured at 490 nm using a UV/vis multiplate spectrophotometer (Multiskan Spectrum, Thermo Scientific). The MTS solution in DMEM without cells served as the blank.
Cell Penetration and Spreading
To compare penetration ability of cells on 2D and 3D scaffolds, cells were stained by Phalloidin 633 solution (1:200 Alexa Fluor 633 Phalloidin, Invitrogen, Grand Island, N.Y.) and observed using a Nikon A1 confocal laser scanning microscope (Nikon Inc., Melville, N.Y.). Alexa Fluor 633 Phalloidin is a far red fluorescent dye that specifically bonds to F-actin in cells. This dye was selected since zein shows fluorescence across the full spectrum with the weakest signal in the far red range. To observe the spreading behaviors and stereoscopic morphologies of cells in 2D and 3D scaffolds, cells were stained by Phalloidin 633 solution for F-actin and Hoechst 33342 solution (Invitrogen, Grand Island, N.Y.) for the nuclei of cells.
Statistical Analysis
One-way analysis of variance with Tukey's pairwise multiple comparisons was employed to analyze the data. The confidence interval was set at 95%, and a P value less than 0.05 was considered to be a statistically significant difference. In the results, data labeled with different symbols were significantly different from each other.
Ex. 1: Morphology of 3D Electrospun Zein Scaffolds
A piece of 3D zein scaffold was prepared by electrospinning aqueous solution containing 25 wt % of zein and 25 wt % of sodium dodecyl sulfate (SDS), and a piece of 2D zein scaffold was prepared by electrospinning 25 wt % zein in 70% v/v aqueous ethanol solution; 9 wt % of citric acid based on the weight of zein was added into both 2D and 3D spinning dopes for crosslinking. Different solvent systems were utilized because the zein, itself, could not be dissolved in water. The electrospinning process was conducted at an extrusion speed of 2 mL/hr, a voltage of 42 kV, and a distance from the needle to the collecting board of 25 cm. The needle was negatively charged and the collecting board was positively charged.
As is shown in
Ex. 2: In Vitro Cell Culture Study of 3D and 2D Electrospun Zein Scaffolds
NIH 3T3 fibroblast cells were cultured on both 2D and 3D electrospun zein scaffolds to evaluate the effects of scaffold architecture on cellular attachment, penetration and proliferation. Cell penetration was evaluated 48 hours after culturing the 2D and 3D scaffolds. Significantly higher cell accessibility of 3D zein electrospun scaffold compared with 2D scaffold is shown in
Methanethiosulfonate (MTS) assays were conducted to quantitatively investigate cell attachment and proliferation, the results of which are shown in
3D zein scaffolds showed a great potential for tissue engineering applications. For example,
Ex. 3: Morphology of 3D Electrospun PEG Scaffolds Using Anionic Surfactant
PEG is a water soluble synthetic polymer, and could also be electrospun into 3D stereoscopic architecture. PEG (25 wt %) and SDS (25 wt %) were dissolved in water to prepare spinning dopes. The electrospinning process was conducted at an extrusion speed of 2 mL/hr, a voltage of 42 kV, and a distance from the needle to the collecting board of 25 cm. The needle was negatively charged and the collecting board was positively charged. As is shown in
Ex. 4: Morphology of 3D Electrospun PEG Scaffolds Using Nonionic Surfactant
PEG (25 wt %) and TRITON X-100 (25 wt %), a nonionic surfactant, was dissolved in water for spinning. The electrospinning process was conducted at an extrusion speed of 2 mL/hr, a voltage of 42 kV, and a distance from the needle to the collecting board of 25 cm. The needle was negatively charged and the collecting board was positively charged. As is shown in
Ex. 5: Extraction of Spinable Proteins from Soy Protein and Wheat Glutenin
Soy protein and wheat glutenin represent two types of highly crosslinked proteins that are high in molecular weights and insoluble in water. These proteins were treated in 8 M urea under mild alkaline condition with existence of cysteine, a common amino acid, which is also a nontoxic and environmentally benign reductant. After being treated at 70° C. for 24 hours, the soluble proteins were collected. SDS PAGE results of these extracted proteins showing molecular weights are shown in
Ex. 6: Morphology of 3D Electrospun Scaffold from Soy Protein
Soy protein extracted using protocol as mentioned in Example 4 (25 wt %) and 25 wt % of SDS were dissolved in water and electrospun into 3D structures as shown in
Ex. 7: Water Stability of 3D Electrospun Soy Protein Scaffold
After post treatment of coagulation bath of 10% Na2SO4 and 10% acetic acid, the 3D electrospun soy protein scaffold was soaked in PBS at 50° C. for 3 days. As shown in
Ex. 8 Morphology of 3D Electrospun Scaffold from Feather Keratin
Feather keratin extracted using protocol as mentioned in Example 4 (25 wt %), 25 wt % of SDS and cysteine (10 wt % based on keratin) were dissolved in water and electrospun into 3D structures as shown in
Ex. 9 Morphology of 3D Electrospun Scaffold from Wheat Glutenin
Wheat glutenin extracted using protocol as mentioned in Example 4 (25 wt %), 25 wt % of SDS and cysteine (10 wt % based on wheat glutenin) were dissolved in water and electrospun into 3D structures as shown in
Ex. 10: Morphology of 3D Electrospun Scaffold from Wheat Gliadin
Wheat gliadin (25 wt %), a prolamin in wheat, and 25 wt % of SDS were dissolved in water and electrospun into 3D structures as shown in
Ex. 11: Morphology of 3D Electrospun Scaffold from Casein
Casein (25 wt %), the family of proteins commonly found in mammalian milk, and 25 wt % of SDS were dissolved in water and electrospun into 3D structures as shown in
Ex. 12: Morphology of 3D Electrospun Scaffold from Peanut Protein
Peanut protein (25 wt %) and 25 wt % of SDS were dissolved in water and electrospun into 3D structures as shown in
Relationship between Specific Pore Volume and Surface Resistivity
A power function was used to simulate the relationship between the specific pore volume of electrospun scaffolds and corresponding polymer surface resistivity. As shown in
To further investigate the effect of electron transference on formation of 3D architectures, a solution with 25 wt % zein and 25 wt % SDS was electrospun onto the positively charged collecting board covered by a layer of insulator. Delivery of electrons was interrupted though positive potential still existed. Zein fibers with electrons on the surface were attracted by the positive collector and then hit the board vertically as shown in
Having illustrated and described the principles of the present invention, it should be apparent to persons skilled in the art that the invention can be modified in arrangement and detail without departing from such principles.
Although the materials and methods of this invention have been described in terms of various embodiments and illustrative examples, it will be apparent to those of skill in the art that variations can be applied to the materials and methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
The present application is a non-provisional application claiming the benefit of U.S. Provisional Patent Application 61/668,269, filed Jul. 5, 2012, which is incorporated herein by reference in its entirety.
This invention was made with Government Support under a grant from the U.S. Dept. of Agriculture (NEB 37-037). The government has certain rights to this invention.
Number | Date | Country | |
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61668269 | Jul 2012 | US |