Poly(e-caprolactone)/keratin-based composite nanofibers for biomedical applications Angela Edwards,1 David Jarvis,1 Tracy Hopkins,2 Sarah Pixley,2 Narayan Bhattarai1* 1
Department of Chemical, Biological and Bioengineering & NSF ERC for Revolutionizing Metallic Biomaterials, North Carolina A&T State University, Greensboro, North Carolina 2 Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio Received 22 November 2013; revised 25 February 2014; accepted 30 March 2014 Published online 23 April 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33172 Abstract: Keratin-based composite nanofibers have been fabricated by an electrospinning technique. Aqueous soluble keratin extracted from human hair was successfully blended with poly(e-caprolactone) (PCL) in different ratios and transformed into nanofibrous membranes. Toward the potential use of this nanofibrous membrane in tissue engineering, its physicochemical properties, such as morphology, mechanical strength, crystallinity, chemical structure, and integrity in aqueous medium were studied and its cellular compatibility was determined. Nanofibrous membranes with PCL/keratin ratios from 100/00 to 70/30 showed good uniformity in fiber morphology and suitable mechanical properties, and retained the integrity of their fibrous structure in buffered solutions.
Experimental results, using cell viability assays and scanning electron microscopy imaging, showed that the nanofibrous membranes supported 3T3 cell viability. The ability to produce blended nanofibers from protein and synthetic polymers represents a significant advancement in development of composite materials with structural and material properties that will support biomedical applications. This provides new nanofibrous materials for applications in tissue engiC 2014 Wiley Periodicals, Inc. neering and regenerative medicine. V J Biomed Mater Res Part B: Appl Biomater, 103B: 21–30, 2015.
Key Words: keratin, electrospinning, scaffold, nanofiber, poly(e-caprolactone), biomedical
How to cite this article: Edwards, A, Jarvis, D, Hopkins, T, Pixley, S, Bhattarai, N. 2015. Poly(e-caprolactone)/keratin-based composite nanofibers for biomedical applications. J Biomed Mater Res Part B 2015:103B:21–30.
INTRODUCTION
Development of biopolymer-based nanofibers is of great interest for the scientific community because of their wide range of potential applications in biomedical applications.1 In the last few years, the electrospinning technique has been recognized as an efficient technology, compared to others such as self-assembly,2 phase separation,3 drawing,4 and template-directed synthesis,5 that allows creation of polymer nanofibers, in the form of nonwoven mats, and that can be scaled up from laboratory to industrial production. In biomedical engineering, electrospun nanofibers exhibit a lot of advantages, particularly to produce scaffolds whose physicochemical properties can closely resemble the naturally occurring properties of the extracellular matrix (ECM) components found in tissues. The ECM in mammalian tissues is primarily composed of proteoglycans (glycosaminoglycan) and fibrous proteins, both of which have nanoscale structural dimensions that can be mimicked by materials produced by electrospinning techniques. Because of these similarities to ECM, other exciting biomedical applications of
electrospun nanofiber mats are as materials for wound dressings, drug delivery, and for tissue engineering complex tissues such as liver, bone, heart, muscle etc.6 To achieve these applications, electrospun nanofiber mats should be biodegradable, biocompatible, have limited cytotoxicity, and exhibit specific mechanical properties. A number of synthetic polymer nanofibers have been fabricated using electrospinning techniques to guide cells to grow in different types of tissues. These include polyglycolide, poly(L-lactic acid), and their copolymers poly(glycolideco-lactide) (PLGA), poly(e-caprolactone) (PCL), and poly (pdioxanone).7 Recently, there has been growing interest in the synthesis of natural polymer-based nanofibers because of their proven biocompatibility. These fibers are also of great interest because, in many cases, they are superior to synthetic polymers in their biocompatibility during biodegradation. Other advantages of natural polymers include high hydrophilicity and low-to-no cytotoxicity, as well as enhancement of cell adhesion and proliferation.8 Nanofibers from natural polymers, especially those derived from natu-
Correspondence to: N. Bhattarai (e-mail: [email protected]) Contract grant sponsor: National Science Foundation through Engineering Research Center for Revolutionizing Metallic Biomaterials; contract grant number: ERC-0812348 Contract grant sponsor: Nanotechnology Undergraduate Education; contract grant number: NUE-1242139
C 2014 WILEY PERIODICALS, INC. V
21
ral resources, may also provide virtually unlimited resources for the development of tissue-compatible scaffolds for functional restoration of damaged or dysfunctional tissues. Collagen, gelatin, hyaluronan, silk, chitosan, alginate, keratin, fibrinogen, and elastin are the most commonly used natural polymers in tissue engineering.8,9 Among this large list of natural polymers, a few attempts have been made to prepare keratin-based nanofibrous structures by electrospinning, with some degree of success.10 These nanofibers are primarily made of polyblends of wool-based keratin and polyethylene oxide.11 Here we present the first fabrication of human hairbased keratin/PCL nanofibers by electrospinning. The design combines the technological advances in biocompatible polymers and nanotechnology to produce nanofibrous matrices with significantly improved mechanical and biological properties. Keratin, a naturally occurring polymer, is biorenewable, biodegradable, biocompatible, and biofunctional.12 Keratin refers to a broad category of proteins that assemble into what are termed “intermediate” filaments. Abundant intermediate filaments composed of keratin are characteristic of the cells that form hair, wool, horns, hooves and nails.13 These intracellular bundles of intermediate filaments provide mechanical resilience to these epithelial cells.13,14 The use of keratin in tissue engineering scaffolds (e.g., hydrated gel or sponges) has been shown to enhance cell attachment and proliferation, and to improve the biomaterial’s cellular and tissue biocompatibility, both in vitro15 and in vivo.12,16 Although these recent efforts in keratinbased biomaterials are encouraging, much remains to be explored and improved, particularly in terms of the wide range of biomedical applications where mechanical strength and integrity of nanofibers in aqueous medium are important. Keratin is mechanically weak and alone it is unable to retain its structural integrity, due to its high swelling characteristics in aqueous environments. Therefore, we sought to combine keratin with another hydrophobic polymer that would provide the enhanced mechanical strength and stability of nanofibers in aqueous medium. The complementary polymer used in this study, PCL, is commonly found in tissue engineering applications due to its good mechanical properties and its biodegradability. The issues with PCL are that it has limited cell affinity, primarily due to its hydrophobicity and lack of surface cell recognition sites. Thus PCL–keratin hybrid nanofibers, when appropriately constructed, will integrate the favorable biological properties of keratin and favorable mechanical properties of PCL. This is expected to significantly improve the resultant material properties while providing a stable, biocompatible and nurturing substrate that can support a wide variety of biomedical applications. Most synthetic and natural polyblend nanofibers, such as collagen–PCL and gelatin–PCL, require chemical crosslinking to retain their structural integrity in aqueous medium and improve mechanical strength.17 An issue with this is that most of the chemicals used to crosslink polymeric nanofibers are cytotoxic, which is a big concern in biomedical applications. Unlike the several natural/synthetic
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EDWARDS ET AL.
polyblend nanofibers tested to date, our PCL–keratin polyblend nanofibers produced by electrospinning have sufficient mechanical properties without chemical cross-linking. Toward the potential use of this nanofibrous membrane in tissue engineering, its physicochemical properties, such as morphology, mechanical strength, crystallinity, chemical structure, and integrity in aqueous medium were studied and its cellular compatibility was determined. Biodegradation studies of PCL/keratin nanofibers were performed and no apparent morphological changes were found when the fibers were placed for several weeks in phosphate buffer saline (PBS) solution at 37 C. PCL–keratin polyblend nanofiber mats also demonstrated excellent mechanical properties. Finally, the cellular compatibility of the nanofiber mats was studied by seeding fibroblast 3T3 cells on the mats, with cell proliferation quantified by an Alamar Blue (AB)based assay, and cell attachment characterized by scanning electron microscopy (SEM) imaging. MATERIALS AND METHODS
Materials Human hair was collected from local barber shops in Greensboro, NC. Peracetic acid, PCL (e-caprolactone polymer, Mn 70–90 kDa), trifluoroethanol (TFE), and Tris Base (TrizmaTM Base powder, 99.9% crystalline) were purchased from Sigma Aldrich (St. Louis, MO). Hydrochloric Acid (A144C-212 Lot 093601) was purchased from Fisher Scientific (Waltham, MA). Kwik-Sil was obtained from World Precision Instruments (Sarasota, FL). Keratin extraction Keratin was extracted from human hair according to a published method.18 Briefly, human hair obtained from local barber shop was thoroughly washed with warm water and soap followed by peracetic acid treatment (2%, w/v, 12 h at 25 C). Free proteins were extracted using Trizma base (1 M solution) and the solution was then neutralized to pH 7 using diluted hydrochloric acid solution (1 mM), centrifuged (VWR clinical 200 Centrifuge at 1050g for 10 min). During extraction, the solution was moderately oxidized by the oxidizing peracetic acid, which means that some disulfide bonds remain intact, while others are partially cleaved. The extracted solution was further concentrated up to onefifth of original volume by using a rotary evaporator. The concentrated solution was first dialyzed (cellulose membrane with 12–14 kDa molecular cutoff) in deionized (DI) water for 24 h and lyophilized to give keratin in a powder form. Preparation of keratin, PCL, and PCL/keratin solutions PCL and keratin solutions were prepared individually, and then mixed to create a stock solution of different ratios. PCL was dissolved in 10 wt % TFE. Keratin was dissolved in 10 wt % in DI water. Subsequently, a PCL/keratin solution was created by mixing PCL with keratin at ratios of 90:10, 80:20, 70:30, and 60:40. Solution mixtures were vortexed manually until each solution reached a homogeneous blend ready for electrospinning.
PCL/KERATIN-BASED COMPOSITE NANOFIBERS
ORIGINAL RESEARCH REPORT
Preparation of electrospun PCL/keratin fibers To electrospin blended nanofibers, approximately 8 mL of the PCL/keratin solution was placed in a 10-mL disposable syringe fitted with a 0.5-mm diameter tip. The solution was gravity fed by controlling the tilt angle of the syringe. The syringe tip was positioned 23 cm from a fiber collecting drum and 30 from the horizontal. A 25–27 kV voltage (Spellman CZE100R) was used to charge the solution. The solution was spun toward a rotating grounded drum (200 rpm) for collecting randomly oriented nanofibers. Thickness of the nanofiber membranes was obtained in the range of 100–160 mm which was measure by digital micrometer (Mitutoyo Electronic Micrometers, 293–344). Fiber morphology observation The morphology and diameter of the electrospun fibers were observed and determined with the use of an optical microscope (Olympus BX51M, Japan) and a scanning electron microscope (Hitachi SU8000, Japan). Prior to imaging with the use of SEM, a small section of the fibers on the sample holder was sputter coated with gold by using a Polaron SEM coating system for 1 min and 30 s at 15 mA. The SEM was then used to observe the samples at an accelerating voltage of 1.5 kV and 5 mA current. Triplicates of each ratio of nanofiber were analyzed by SEM. Contact-angle measurement The wettability of electrospun fiber membrane was determined by contact angle (CA) measurement. The CA measurements were carried out using a specially arranged microscope equipped with a camera (Dynamic Contact Angle Tester, Billerica, MA). The droplet used was DI water and was 0.25 mL in volume. The CA experiments were carried out at room temperature and were repeated five times. All CAs were measured within 20 s of placement of the water droplet on the electrospun fiber mat. Wettablity of nanofiber samples was analyzed by comparing the CAs of PCL/keratin nanofibers with PCL nanofibers (act as internal reference). Five samples of each ratio of nanofiber were used to measure CA. Structural characterization of nanofibers To characterize the chemical bonding between the PCL and keratin, Fourier transform infrared spectroscopy (FTIR) spectra were obtained at of 200 scans using a Bruker Tensor 2 instrument (Billerica, MA). The nanofiber membrane was placed in a magnetic holder and the system was purged with dry air before testing. Spectrum analysis was performed using standard Microcal Origin software (Northampton, MA). Triplicates of each ratios of nanofiber were used for FTIR analysis. The crystallinity and miscibility of PCL and keratin in nanofibers were studied by using X-ray diffraction (XRD) spectra. XRD patterns were acquired over a diffraction angle of 2h 5 10–40 at room temperature with a wide angle Xray diffractometer (Bruker AXS D8 Advance X-ray Diffractometer, Madison, WI) equipped with a position sensitive detector. Scan speed parameters were set to 1 and each
scan took approximately 20–35 min to complete. The operating voltage and current were kept at 40 kV and 20 mA, respectively, throughout the entire course of the investigation. Triplicates of each ratios of nanofiber were used for FTIR analysis. Mechanical testing The mechanical strength of the fibers was measured with a table-top Shimadzu machine (North America Analytical and Measuring Instruments AGS-X series, Columbia, MD) with a 10-N load cell at a displacement rate of 10 mm/min and data acquisition time of 500 ms. A custom-designed specimen holder (made from index cards using double-sided tape) was used for mechanical testing of nanofibers. Dimensions of each specimen were measured using a digital micrometer. Then, nanofibers were attached to specimen holders. Exact nanofiber dimensions were entered into the machine program before initiating the testing procedure. Fiber samples were strained to breakage and the ultimate tensile strength and Young’s modulus were determined from stress-strain curves. Five specimens for each ratio of nanofiber samples were used for mechanical tests. Statistical analyses of mechanical test data were performed using GraphPad Prism data analysis software was used to conduct statistical analysis. Degradation of nanofiber matrix Dried PCL/keratin nanofibrous membranes were cut into squares (30 mm 3 30 mm), sterilized in 80% alcohol (10 min incubation), and washed thoroughly with DI water. Membrane integrity was then tested by incubating samples in 15 mL PBS (pH 7.5 at 37 C). The buffer was replaced every 3 days. The membranes were taken out of the solution at specified intervals, rinsed with DI water, lyophilized, and examined for morphological and chemical changes. Triplicates of each ratios of nanofiber were used for this study. Cell culture and AB assay Nanofiber membrane samples were attached to a 12 mm square diameter coverslip using biocompatible, siliconebased elastomeric glue (i.e., Kwik-Sil). Fiber membrane was carefully glued on cover slip. The membrane was first wrapped on a coverslip and glued at the back of the glass so that front side with the porous structure was available for cell attachment and infiltration. The nanofiber samples were sterilized in 24-well plates by incubating in 80% ethanol for at least 1 h and rinsed with sterile DI water and then basal medium prior to cell seeding. Fibroblast 3T3 cells (a mouse fibroblast cell line) were purchased from the American Tissue Type Culture Collection (Manassas, VA) and, at passage 20, were plated at 62,000 cell/cm2. Triplicates of each sample were plated with cells. The growth medium was Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and antibiotics. A 1-mL aliquot of medium containing cells was seeded on nanofiber
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samples and grown in a humidified incubator (37 C, 5% CO2) for 24 h. The AB colorimetric assay (Invitrogen) was used to analyze proliferation of the 3T3 fibroblast cells grown on nanofiber samples on coverslips or, as a control, on glass coverslips, either plain or coated with the same glue (no change was observed with glue, so only control values for cells on noncoated glass are shown herein). After 24 h of culture, the coverlips were transferred to new plates, washed twice with PBS and incubated with 10 vol % (200 mL resazurin 1 1.8 mL media) AB in DMEM with 10% FBS for 2 h. A 400-mL sample of the assay solution was removed from the wells with the mats and transferred to an opaque 96-well culture plate for fluorescent measurements on a Spectra max Gemini XPS microplate reader (Molecular Devices, Sunnyvale, CA) at kex 530 nm, kem 590 nm. The relative fluorescent units were converted to a percent of the average values for cells in control wells. After the determination of cell viability with the AB assay, the cells growing on the mats were fixed and cellular morphology was examined with SEM. Cells were washed 3 times with PBS and fixed with 4% paraformaldehyde/2% glutaraldehyde (pH 7.4) for 20 min. After fixing, samples were briefly rinsed with DI water and dehydrated by sequential incubations in 50, 75, and 100% ethanol at room temperature. The samples were dried in a desiccator for 30 min and then sputter coated with Au for 1 min 30 s at 15 mA and imaged with SEM.
Statistical analysis Statistical analyses of the cell culture data were performed using one-way analysis of variance. p values
Department of Chemical, Biological and Bioengineering & NSF ERC for Revolutionizing Metallic Biomaterials, North Carolina A&T State University, Greensboro, North Carolina 2 Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio Received 22 November 2013; revised 25 February 2014; accepted 30 March 2014 Published online 23 April 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33172 Abstract: Keratin-based composite nanofibers have been fabricated by an electrospinning technique. Aqueous soluble keratin extracted from human hair was successfully blended with poly(e-caprolactone) (PCL) in different ratios and transformed into nanofibrous membranes. Toward the potential use of this nanofibrous membrane in tissue engineering, its physicochemical properties, such as morphology, mechanical strength, crystallinity, chemical structure, and integrity in aqueous medium were studied and its cellular compatibility was determined. Nanofibrous membranes with PCL/keratin ratios from 100/00 to 70/30 showed good uniformity in fiber morphology and suitable mechanical properties, and retained the integrity of their fibrous structure in buffered solutions.
Experimental results, using cell viability assays and scanning electron microscopy imaging, showed that the nanofibrous membranes supported 3T3 cell viability. The ability to produce blended nanofibers from protein and synthetic polymers represents a significant advancement in development of composite materials with structural and material properties that will support biomedical applications. This provides new nanofibrous materials for applications in tissue engiC 2014 Wiley Periodicals, Inc. neering and regenerative medicine. V J Biomed Mater Res Part B: Appl Biomater, 103B: 21–30, 2015.
Key Words: keratin, electrospinning, scaffold, nanofiber, poly(e-caprolactone), biomedical
How to cite this article: Edwards, A, Jarvis, D, Hopkins, T, Pixley, S, Bhattarai, N. 2015. Poly(e-caprolactone)/keratin-based composite nanofibers for biomedical applications. J Biomed Mater Res Part B 2015:103B:21–30.
INTRODUCTION
Development of biopolymer-based nanofibers is of great interest for the scientific community because of their wide range of potential applications in biomedical applications.1 In the last few years, the electrospinning technique has been recognized as an efficient technology, compared to others such as self-assembly,2 phase separation,3 drawing,4 and template-directed synthesis,5 that allows creation of polymer nanofibers, in the form of nonwoven mats, and that can be scaled up from laboratory to industrial production. In biomedical engineering, electrospun nanofibers exhibit a lot of advantages, particularly to produce scaffolds whose physicochemical properties can closely resemble the naturally occurring properties of the extracellular matrix (ECM) components found in tissues. The ECM in mammalian tissues is primarily composed of proteoglycans (glycosaminoglycan) and fibrous proteins, both of which have nanoscale structural dimensions that can be mimicked by materials produced by electrospinning techniques. Because of these similarities to ECM, other exciting biomedical applications of
electrospun nanofiber mats are as materials for wound dressings, drug delivery, and for tissue engineering complex tissues such as liver, bone, heart, muscle etc.6 To achieve these applications, electrospun nanofiber mats should be biodegradable, biocompatible, have limited cytotoxicity, and exhibit specific mechanical properties. A number of synthetic polymer nanofibers have been fabricated using electrospinning techniques to guide cells to grow in different types of tissues. These include polyglycolide, poly(L-lactic acid), and their copolymers poly(glycolideco-lactide) (PLGA), poly(e-caprolactone) (PCL), and poly (pdioxanone).7 Recently, there has been growing interest in the synthesis of natural polymer-based nanofibers because of their proven biocompatibility. These fibers are also of great interest because, in many cases, they are superior to synthetic polymers in their biocompatibility during biodegradation. Other advantages of natural polymers include high hydrophilicity and low-to-no cytotoxicity, as well as enhancement of cell adhesion and proliferation.8 Nanofibers from natural polymers, especially those derived from natu-
Correspondence to: N. Bhattarai (e-mail: [email protected]) Contract grant sponsor: National Science Foundation through Engineering Research Center for Revolutionizing Metallic Biomaterials; contract grant number: ERC-0812348 Contract grant sponsor: Nanotechnology Undergraduate Education; contract grant number: NUE-1242139
C 2014 WILEY PERIODICALS, INC. V
21
ral resources, may also provide virtually unlimited resources for the development of tissue-compatible scaffolds for functional restoration of damaged or dysfunctional tissues. Collagen, gelatin, hyaluronan, silk, chitosan, alginate, keratin, fibrinogen, and elastin are the most commonly used natural polymers in tissue engineering.8,9 Among this large list of natural polymers, a few attempts have been made to prepare keratin-based nanofibrous structures by electrospinning, with some degree of success.10 These nanofibers are primarily made of polyblends of wool-based keratin and polyethylene oxide.11 Here we present the first fabrication of human hairbased keratin/PCL nanofibers by electrospinning. The design combines the technological advances in biocompatible polymers and nanotechnology to produce nanofibrous matrices with significantly improved mechanical and biological properties. Keratin, a naturally occurring polymer, is biorenewable, biodegradable, biocompatible, and biofunctional.12 Keratin refers to a broad category of proteins that assemble into what are termed “intermediate” filaments. Abundant intermediate filaments composed of keratin are characteristic of the cells that form hair, wool, horns, hooves and nails.13 These intracellular bundles of intermediate filaments provide mechanical resilience to these epithelial cells.13,14 The use of keratin in tissue engineering scaffolds (e.g., hydrated gel or sponges) has been shown to enhance cell attachment and proliferation, and to improve the biomaterial’s cellular and tissue biocompatibility, both in vitro15 and in vivo.12,16 Although these recent efforts in keratinbased biomaterials are encouraging, much remains to be explored and improved, particularly in terms of the wide range of biomedical applications where mechanical strength and integrity of nanofibers in aqueous medium are important. Keratin is mechanically weak and alone it is unable to retain its structural integrity, due to its high swelling characteristics in aqueous environments. Therefore, we sought to combine keratin with another hydrophobic polymer that would provide the enhanced mechanical strength and stability of nanofibers in aqueous medium. The complementary polymer used in this study, PCL, is commonly found in tissue engineering applications due to its good mechanical properties and its biodegradability. The issues with PCL are that it has limited cell affinity, primarily due to its hydrophobicity and lack of surface cell recognition sites. Thus PCL–keratin hybrid nanofibers, when appropriately constructed, will integrate the favorable biological properties of keratin and favorable mechanical properties of PCL. This is expected to significantly improve the resultant material properties while providing a stable, biocompatible and nurturing substrate that can support a wide variety of biomedical applications. Most synthetic and natural polyblend nanofibers, such as collagen–PCL and gelatin–PCL, require chemical crosslinking to retain their structural integrity in aqueous medium and improve mechanical strength.17 An issue with this is that most of the chemicals used to crosslink polymeric nanofibers are cytotoxic, which is a big concern in biomedical applications. Unlike the several natural/synthetic
22
EDWARDS ET AL.
polyblend nanofibers tested to date, our PCL–keratin polyblend nanofibers produced by electrospinning have sufficient mechanical properties without chemical cross-linking. Toward the potential use of this nanofibrous membrane in tissue engineering, its physicochemical properties, such as morphology, mechanical strength, crystallinity, chemical structure, and integrity in aqueous medium were studied and its cellular compatibility was determined. Biodegradation studies of PCL/keratin nanofibers were performed and no apparent morphological changes were found when the fibers were placed for several weeks in phosphate buffer saline (PBS) solution at 37 C. PCL–keratin polyblend nanofiber mats also demonstrated excellent mechanical properties. Finally, the cellular compatibility of the nanofiber mats was studied by seeding fibroblast 3T3 cells on the mats, with cell proliferation quantified by an Alamar Blue (AB)based assay, and cell attachment characterized by scanning electron microscopy (SEM) imaging. MATERIALS AND METHODS
Materials Human hair was collected from local barber shops in Greensboro, NC. Peracetic acid, PCL (e-caprolactone polymer, Mn 70–90 kDa), trifluoroethanol (TFE), and Tris Base (TrizmaTM Base powder, 99.9% crystalline) were purchased from Sigma Aldrich (St. Louis, MO). Hydrochloric Acid (A144C-212 Lot 093601) was purchased from Fisher Scientific (Waltham, MA). Kwik-Sil was obtained from World Precision Instruments (Sarasota, FL). Keratin extraction Keratin was extracted from human hair according to a published method.18 Briefly, human hair obtained from local barber shop was thoroughly washed with warm water and soap followed by peracetic acid treatment (2%, w/v, 12 h at 25 C). Free proteins were extracted using Trizma base (1 M solution) and the solution was then neutralized to pH 7 using diluted hydrochloric acid solution (1 mM), centrifuged (VWR clinical 200 Centrifuge at 1050g for 10 min). During extraction, the solution was moderately oxidized by the oxidizing peracetic acid, which means that some disulfide bonds remain intact, while others are partially cleaved. The extracted solution was further concentrated up to onefifth of original volume by using a rotary evaporator. The concentrated solution was first dialyzed (cellulose membrane with 12–14 kDa molecular cutoff) in deionized (DI) water for 24 h and lyophilized to give keratin in a powder form. Preparation of keratin, PCL, and PCL/keratin solutions PCL and keratin solutions were prepared individually, and then mixed to create a stock solution of different ratios. PCL was dissolved in 10 wt % TFE. Keratin was dissolved in 10 wt % in DI water. Subsequently, a PCL/keratin solution was created by mixing PCL with keratin at ratios of 90:10, 80:20, 70:30, and 60:40. Solution mixtures were vortexed manually until each solution reached a homogeneous blend ready for electrospinning.
PCL/KERATIN-BASED COMPOSITE NANOFIBERS
ORIGINAL RESEARCH REPORT
Preparation of electrospun PCL/keratin fibers To electrospin blended nanofibers, approximately 8 mL of the PCL/keratin solution was placed in a 10-mL disposable syringe fitted with a 0.5-mm diameter tip. The solution was gravity fed by controlling the tilt angle of the syringe. The syringe tip was positioned 23 cm from a fiber collecting drum and 30 from the horizontal. A 25–27 kV voltage (Spellman CZE100R) was used to charge the solution. The solution was spun toward a rotating grounded drum (200 rpm) for collecting randomly oriented nanofibers. Thickness of the nanofiber membranes was obtained in the range of 100–160 mm which was measure by digital micrometer (Mitutoyo Electronic Micrometers, 293–344). Fiber morphology observation The morphology and diameter of the electrospun fibers were observed and determined with the use of an optical microscope (Olympus BX51M, Japan) and a scanning electron microscope (Hitachi SU8000, Japan). Prior to imaging with the use of SEM, a small section of the fibers on the sample holder was sputter coated with gold by using a Polaron SEM coating system for 1 min and 30 s at 15 mA. The SEM was then used to observe the samples at an accelerating voltage of 1.5 kV and 5 mA current. Triplicates of each ratio of nanofiber were analyzed by SEM. Contact-angle measurement The wettability of electrospun fiber membrane was determined by contact angle (CA) measurement. The CA measurements were carried out using a specially arranged microscope equipped with a camera (Dynamic Contact Angle Tester, Billerica, MA). The droplet used was DI water and was 0.25 mL in volume. The CA experiments were carried out at room temperature and were repeated five times. All CAs were measured within 20 s of placement of the water droplet on the electrospun fiber mat. Wettablity of nanofiber samples was analyzed by comparing the CAs of PCL/keratin nanofibers with PCL nanofibers (act as internal reference). Five samples of each ratio of nanofiber were used to measure CA. Structural characterization of nanofibers To characterize the chemical bonding between the PCL and keratin, Fourier transform infrared spectroscopy (FTIR) spectra were obtained at of 200 scans using a Bruker Tensor 2 instrument (Billerica, MA). The nanofiber membrane was placed in a magnetic holder and the system was purged with dry air before testing. Spectrum analysis was performed using standard Microcal Origin software (Northampton, MA). Triplicates of each ratios of nanofiber were used for FTIR analysis. The crystallinity and miscibility of PCL and keratin in nanofibers were studied by using X-ray diffraction (XRD) spectra. XRD patterns were acquired over a diffraction angle of 2h 5 10–40 at room temperature with a wide angle Xray diffractometer (Bruker AXS D8 Advance X-ray Diffractometer, Madison, WI) equipped with a position sensitive detector. Scan speed parameters were set to 1 and each
scan took approximately 20–35 min to complete. The operating voltage and current were kept at 40 kV and 20 mA, respectively, throughout the entire course of the investigation. Triplicates of each ratios of nanofiber were used for FTIR analysis. Mechanical testing The mechanical strength of the fibers was measured with a table-top Shimadzu machine (North America Analytical and Measuring Instruments AGS-X series, Columbia, MD) with a 10-N load cell at a displacement rate of 10 mm/min and data acquisition time of 500 ms. A custom-designed specimen holder (made from index cards using double-sided tape) was used for mechanical testing of nanofibers. Dimensions of each specimen were measured using a digital micrometer. Then, nanofibers were attached to specimen holders. Exact nanofiber dimensions were entered into the machine program before initiating the testing procedure. Fiber samples were strained to breakage and the ultimate tensile strength and Young’s modulus were determined from stress-strain curves. Five specimens for each ratio of nanofiber samples were used for mechanical tests. Statistical analyses of mechanical test data were performed using GraphPad Prism data analysis software was used to conduct statistical analysis. Degradation of nanofiber matrix Dried PCL/keratin nanofibrous membranes were cut into squares (30 mm 3 30 mm), sterilized in 80% alcohol (10 min incubation), and washed thoroughly with DI water. Membrane integrity was then tested by incubating samples in 15 mL PBS (pH 7.5 at 37 C). The buffer was replaced every 3 days. The membranes were taken out of the solution at specified intervals, rinsed with DI water, lyophilized, and examined for morphological and chemical changes. Triplicates of each ratios of nanofiber were used for this study. Cell culture and AB assay Nanofiber membrane samples were attached to a 12 mm square diameter coverslip using biocompatible, siliconebased elastomeric glue (i.e., Kwik-Sil). Fiber membrane was carefully glued on cover slip. The membrane was first wrapped on a coverslip and glued at the back of the glass so that front side with the porous structure was available for cell attachment and infiltration. The nanofiber samples were sterilized in 24-well plates by incubating in 80% ethanol for at least 1 h and rinsed with sterile DI water and then basal medium prior to cell seeding. Fibroblast 3T3 cells (a mouse fibroblast cell line) were purchased from the American Tissue Type Culture Collection (Manassas, VA) and, at passage 20, were plated at 62,000 cell/cm2. Triplicates of each sample were plated with cells. The growth medium was Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and antibiotics. A 1-mL aliquot of medium containing cells was seeded on nanofiber
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samples and grown in a humidified incubator (37 C, 5% CO2) for 24 h. The AB colorimetric assay (Invitrogen) was used to analyze proliferation of the 3T3 fibroblast cells grown on nanofiber samples on coverslips or, as a control, on glass coverslips, either plain or coated with the same glue (no change was observed with glue, so only control values for cells on noncoated glass are shown herein). After 24 h of culture, the coverlips were transferred to new plates, washed twice with PBS and incubated with 10 vol % (200 mL resazurin 1 1.8 mL media) AB in DMEM with 10% FBS for 2 h. A 400-mL sample of the assay solution was removed from the wells with the mats and transferred to an opaque 96-well culture plate for fluorescent measurements on a Spectra max Gemini XPS microplate reader (Molecular Devices, Sunnyvale, CA) at kex 530 nm, kem 590 nm. The relative fluorescent units were converted to a percent of the average values for cells in control wells. After the determination of cell viability with the AB assay, the cells growing on the mats were fixed and cellular morphology was examined with SEM. Cells were washed 3 times with PBS and fixed with 4% paraformaldehyde/2% glutaraldehyde (pH 7.4) for 20 min. After fixing, samples were briefly rinsed with DI water and dehydrated by sequential incubations in 50, 75, and 100% ethanol at room temperature. The samples were dried in a desiccator for 30 min and then sputter coated with Au for 1 min 30 s at 15 mA and imaged with SEM.
Statistical analysis Statistical analyses of the cell culture data were performed using one-way analysis of variance. p values
