Nanofeatured silk fibroin membranes for dermal wound healing applications lu,1* Batur Ercan,2* Emir B. Denkbas¸,3 Thomas J. Webster2,4 Zeynep Karahalilog 1
Nanotechnology and Nanomedicine Division, Hacettepe University, Beytepe 06800, Ankara, Turkey Chemical Engineering Department, Northeastern University, Boston 02115, Massachusetts 3 Chemistry Department, Biochemistry Division, Hacettepe University, Beytepe 06800, Ankara, Turkey 4 Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, Saudi Arabia 2
Received 5 December 2013; revised 19 February 2014; accepted 25 February 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35161 Abstract: As an effort to create the next generation of improved skin graft materials, in this study, we modified the surfaces of a previously investigated material, silk fibroin, using a NaOH alkaline treatment to obtain a biologically inspired nanofeatured surface morphology. Such surfaces were characterized for roughness, energy, and chemistry. In addition, keratinocyte (skin-forming cells) adhesion and proliferation on such nanofeatured silk fibroin wound dressings were studied in an initial attempt to determine the promotion of an epidermal cover on the wound bed to form a new epidermal barrier. Dermal fibroblast adhesion and proliferation were also studied to assess the ability of nanostructured silk fibroin to replace damaged dermal tissue in chronic wounds (i.e., for diabetic foot ulcers). Results demonstrated for the
first time that keratinocyte and fibroblast cell density was greater on nanofeatured silk fibroin membranes compared with non-treated silk fibroin surfaces. The enhancement in cellular functions was correlated with an increase in silk surface nanotopography, wettability and change in chemistry after NaOH treatment. Due to the present promising results, the newly developed nanofeatured silk fibroin membranes are exciting alternative skin graft materials which should be further studied for various skin patch and wound dressing C 2014 Wiley Periodicals, Inc. J Biomed Mater Res applications. V Part A: 00A:000–000, 2014.
Key Words: nanotechnology, wound healing, silk, fibroblasts, keratinocytes
lu Z, Ercan B, Denkbas¸ EB, Webster TJ. 2014. Nanofeatured silk fibroin membranes for How to cite this article: Karahalilog dermal wound healing applications. J Biomed Mater Res Part A 2014:00A:000–000.
INTRODUCTION
Skin is the largest organ of the body and it represents approximately one-tenth of the body mass.1,2 It plays a critical role in several bodily functions, such as protection against external factors, inhibition of microorganism invasion, sensation, temperature regulation, hydration retention, and so forth.3 Skin is composed of three structural layers: the epidermidis, dermidis, and subcutis. The epidermidis is the avascular, outermost layer of the skin consisting of a mainly stratified squamous epithelium of keratinocytes, which are responsible for the cohesion of the epidermal structure and for barrier functions. The dermidis is the supportive connective tissue between the epidermidis and subcutis that is composed of fibroblasts. The dermidis has a specialized extracellular matrix (ECM) made of collagen, interwoven with elastin, glycosaminoglycans (GAGs), elastin, fibronectin, and various other proteins and it is responsible for the mechanical properties of the skin.4,5 Importantly, the dermidis has high signaling communication with the epider-
midis, which is vital for the homeostasis of the skin.6 The subcutis is the deepest layer of the skin, composed primarily of adipose cells, fibroblasts and immune cells, separating the dermidis from the underlying muscular fascia. Several diseases require skin grafts and artificial skin substitutes, including burns, wounds (diabetic foot ulcers), infection (i.e., necrotizing fasciitis, purpura fulminans etc.), removal of skin cancers and so forth. One common mechanism for the regeneration of skin is the wound healing response that automatically starts upon injury to the skin. Dermal wound healing is a complex process which requires interactions between inflammatory cells, fibroblasts, keratinocytes and endothelial cells, all of which are regulated by an array of cytokines and growth factors.7,8 Materials currently used for dermal wound healing applications mainly utilize an organic scaffold (mostly collagen based scaffolds due to its abundant presence in the dermidis) cultured with fibroblasts or keratinocytes to help regenerate native tissue.9–11 Although they successfully
*These authors contributed equally to this work. Correspondence to: T. J. Webster; e-mail: [email protected] Contract grant sponsor: Northeastern University and Hacettepe University
C 2014 WILEY PERIODICALS, INC. V
1
form a matrix over the damaged tissue, total skin regeneration with complete functional and aesthetic recovery has not been achieved to date. Additional drawbacks to the currently used skin grafts and artificial skin substitutes include slow preparation times upon culturing cells in vitro, high production costs, variable engraftment rates, delayed vascularization, immunogenicity issues for allographs, donor site morbidity, limited donor site availability for autographs, scar tisue formation, poor integration with host tissue, as well as infection.12,13 Skin, just like the other tissue types, contains numerous components from the nanoscale to the micronscale. Therefore, an alternative potential approach (not yet widely exploited) to regenerate native skin tissue is to mimic the nanofeature size of components of skin in biomaterials. Numerous studies have demonstrated altered interactions between biomaterials and cells (including osteoblasts, chondrocytes, smooth muscle cells, endothelial cells, etc.) upon changing the biomaterial surface architecture from the micrometer to the nanometer scale.14–17 Recently, it was shown that various cell types from different tissues all positively respond to changes in biomaterial surface feature nanometer size (e.g., bone, cartilage, bladder, skin, etc.).18,19 More specifically, although limited, nanoscale surface features have been shown to affect cellular adhesion, proliferation, phenotype expression and long-term functions for skin tissue engineering applications.20 A number of natural and synthetic polymers (such as chitosan, collagen and poly-caprolactone) are currently being investigated as nanofeatured wound dressing materials for dermal wound healing applications.21–24 One such nanostructured material is silk. Silk is a natural protein which has been used for centuries as a suture material. It is typically synthesized by silkworms, the larva of Bombyx mori. Silk has two components, a double strand fibroin fiber which is surrounded by a gummy-like protein shell called sericin.25 Importantly, silk has been proposed for a wide range of tissue engineering applications due to its environmental stability, mechanical properties, biocompatibility, and the ease to modify and functionalize its surface.26 In the literature, silk fibroin has been proposed for diverse tissue engineering applications in a variety of forms (including films, sponges, hydrogels, electrospun mats, tubes, etc.).27–30 In fact, silk has been used as a suture material in biomedical applications for decades. Specifically for wound healing applications, silk-based materials enhance keratinocyte and fibroblast adhesion in vitro, while inducing less inflammation, compared with commercially-used products (such as air permeable Tegaderm (3M) tape, Sofra-tulleV, Due Active Dressing etc.).31–34 Although it is possible to improve the healing response for small wounds using silk-based materials, for deep injuries, discoloring or scarring remain when using silk-based materials, where the latter can potentially lead to undesirable skin contractions; thus, improvements in silk can be beneficial. Thus, for all of the above reasons, in this research, the aforementioned two approaches (nanotechnology and silk) were combined to create a nanofeatured surface topography R
2
KARAHALILOGLU ET AL.
which mimics the natural niche of skin. The proposed method to create nanofeatured surfaces on silk membranes is through a NaOH alkaline treatment, which was previously shown to create nanorough PLLA (poly-L-lactic acid) and poly(lactic-co-glycolic acid) (PLGA) surfaces.35,36 Briefly, the NaOH treatment was shown to break the ester bonds in the polymer chains, hence altering the nanophase topography of the surfaces. In this research, the main goal was to prepare nanorough silk membranes for dermal wound healing applications by using a similar NaOH surface treatment. Therefore, the cytocompatibility of the nanofeatured silk fibroin surfaces were evaluated in vitro using human dermal fibroblasts and epidermal keratinocytes and a potential mechanism for the observed enhancement in cellular functions was proposed. MATERIALS AND METHODS
Material synthesis Materials. All chemicals were purchased from SigmaAldrich including disodium carbonate (Na2CO3), sodium hydroxide (NaOH), lithium bromide (LiBr), phosphate buffer saline (1X PBS), L-glutamine, and fetal bovine serum (FBS). B. mori silkworm cocoons were obtained from Kozabirlik (Bursa, Turkey). EMEM and keratinocyte serum free medium (with supplements) were purchased from ATCC and Gibco (USA), respectively. Preparation of silk solutions. A silk fibroin solution was prepared from B. mori cocoons according to previously established protocols with slight modifications.37 First, B. mori silk cocoons were cut into small pieces and boiled in 0.05M Na2HCO3 for 30 min to extract the glue-like sericin coating layer from the structural fibroin protein which was then rinsed with distilled water (diH2O) three times. The obtained silk fibroin fibers were dried overnight at 40 C, dissolved in a LiBr solution (9.3M) at 60 C for 4 h, followed by dialysis through a cellulose membrane (12–14,000, MWCO) across distilled water for 4 days. The obtained silk solutions were centrifuged twice at 13,000g, diluted to a final concentration of 2 w/v % and stored at 4 C. Nanofeatured silk fibroin membrane preparation. The aqueous silk solution (15 mL) was poured into a glass petri dish (35 mm 3 100 mm) and dried overnight to generate silk fibroin membranes with a thickness of 200 mm. Afterwards, the dried membranes were treated with a methanol solution for 1 h to make them non-water soluble. To create a nanofeatured surface topography, silk membranes were treated with 0.1, 0.5 and 1N NaOH solutions for 5 or 10 min, followed by rinsing with diH2O for four times to remove excess NaOH residues. The pH of the rinsing solution was tested and was found similar to that of the original rinsing solution, thus, suggesting that no residual NaOH remained on the silk surfaces. Specimen characterization Electron microscopy. Surface characterization of the specimens was conducted with a Hitachi S4800 Tokyo, Japan
SILK FIBROIN MEMBRANES FOR DERMAL WOUND HEALING APPLICATIONS
ORIGINAL ARTICLE
Scanning Electron Microscope (SEM, Hitachi S4800 SEM, Tokyo, Japan). A 3 kV accelerating voltage was chosen for SEM analysis and the characterization was completed using secondary electrons. Before SEM investigation, a 3 nm layer of palladium (Pd) was sputter coated (Cressington 208; Cressington Scientific Instruments, Watford, UK) onto the silk fibroin membranes to provide a conductive surface. Surface roughness. For surface roughness measurements, a Parks Scientific NX-10 Atomic Force Microscope (AFM; Suwon, Korea) was used to scan the non-treated and treated silk samples. Each sample was analyzed in ambient conditions under non-contact mode using a silicone ultrasharp cantilever (Park Systems Non-contact Cantilever). The AFM tip had a radius of curvature less than 7 nm and had a backside aluminum reflex coating approximately 30-nm thick. Tapping mode was used at 324 kHz at a scan rate of 0.5 Hz. A scan area of 2 lm 3 2 lm was investigated in these studies. Image analysis software (XEI) was used to generate AFM micrographs and to quantitatively compare the root-mean-square roughness (RMS) of the non-treated and treated silk surfaces. Surface chemistry. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) analysis was completed using a Bruker Vertex 70 FTIR spectrometer (USA) equipped with a Zinc Selenide (ZnSe) crystal to characterize surface chemistry of the samples. A spectral range of 550–4000 cm21 was investigated in these studies and each spectrum was the mean of three acquisitions. Untreated and NaOH treated silk fibroin membranes were cut into 1 3 1 cm squares, loaded onto the FTIR machine while maintaining a full contact between the Ge crystal and the sample. The background signal was subtracted from the obtained reflectance vs. wavenumber spectra. The bands in the FTIR spectra were compared with standards to determine the type of chemical bonds and possible consequence of alkaline treatment. Crystallinity. Crystallinity of the untreated and treated silk fibroin membranes was characterized using X-ray diffraction analysis (XRD, Rigaku Ultima IV, Korea) using Cu Ka radiation at 40.0 kV and a power supply of 44.0 mA. XRD spectra were analyzed for a 2u range of 10–50 . All the samples were characterized using a step size of 0.1 and the time at each step was 2 s. Water contact angle measurements. The hydrophilicity of the treated and untreated samples (untreated silk, 0.1N NaOH for 10 min, 0.5N NaOH for 10 min, and 1N NaOH for 5 and 10 min) were investigated using a drop contact angle meter (SEO Phoenix 300, Korea). Seven samples from each sample type were characterized by measuring the contact angles using double distilled water. Cytocompatibility characterization Cell culture. Human skin fibroblasts (American Type Culture Collection, CCL-110, population number 8–10) were
cultured in EMEM cell culture medium containing 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin under standard cell culture conditions (5% CO2/95% air at 37 C). Human epidermal keratinocytes (Invitrogen, C0015C, population numbers 5–7) were cultured in 13 keratinocyte serum free medium supplemented with 2.5 mg human recombinant epidermal growth factor (EGF), 25 mg bovine pituitary extract and 1% penicillin-streptomycin under standard cell culture conditions (5% CO2/95% air at 37 C). Cell adhesion assay. Fibroblast and keratinocyte cell adhesion on silk fibroin membranes were investigated using an MTT [3-(4,5-dimethylthiozole-2-yl)22, 5-diphenyltetrazolium bromide] assay. Silk fibroin membranes were cut into 1cmx1cm squares followed by a 20-min EtOH soak for sterilization. For 4-h adhesion experiments, a seeding density of 3500 cells/cm2 was used for both fibroblasts and keratinocytes and after seeding the cells, the samples were cultured under standard cell culture conditions (5% CO2/95% air at 37 C). At the end of a 4 h adhesion assay, samples were transferred into clean wells and 150 mL of an MTT dye soluR 96 Non-Radioactive Proliferation Assay, Promtion (CellTiterV ega, Madison, WI) and 1 mL of cell growth medium (described in “Cell culture” section) were added and then were incubated in the dark at 37 C for 4 h. The reaction was R 96 stopped by adding 1 mL of a stop solution (CellTiterV Non-Radioactive Proliferation Assay, Promega, Madison, WI). Two hundred microliters of the final solution was placed into a 96-well plate and the absorbance values were measuered at 570 nm using a spectrophotometer (Spectramax M3 Multimode Microplate Reader, Molecular Devices). The number of cells was determined from a standard curve of absorbance versus a known number of cells run in parallel with the experimental samples. Cell proliferation assay. To evaluate cellular proliferation on silk membranes, fibroblasts (20,000 cells/cm2) or keratinocytes (40,000 cells/cm2) were seeded and were cultured in their growth media (described in “Cell culture” section) for up to 3 days under standard cell culture conditions (5% CO2/95% air at 37 C). To induce keratinocyte growth and enhance cell viability during the proliferation experiments, 10% FBS was added to the keratinocyte culture medium. At each time point (days 1, 2, and 3), samples were gently rinsed with 13 PBS three times, followed by a 4% paraformaldehyde (Sigma) fixation for 8 min. Then, cells were stained using a nuclear fluorescent dye, DAPI (6-diamidino2-phenylindole, Sigma), for 10 min. Excess DAPI solution was removed using a 1x PBS wash three times. Finally, the stained cells were counted using a fluorescence microscope (Zeiss Axio Vert.A1) to obtain cell densities on the silk fibroin membrane surfaces. Statistical analysis Numerical data were analyzed using standard analysis of variance (ANOVA) techniques and statistical differences were considered at p < 0.05. All experiments were completed in triplicate with three repeats for each experiment.
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FIGURE 1. SEM images of untreated and NaOH treated silk fibroin membranes using different NaOH concentrations and exposure times. (A) Untreated, (B) at 0.1N, (C) 0.5N, and (D) 1N for 5 min, and (E) 1N for 10 min. NaOH treatment increased silk nanofeatured surface topographies. Scale bars 5 1 mm.
Results were reported as the average 6 standard error of the mean. RESULTS AND DISCUSSION
Material characterization SEM images of the silk fibroin membranes treated with 0.1, 0.5, and 1N NaOH for 5 and 10 min are shown in Figure 1. These images reveal that the untreated silk fibroin membrane [Fig. 1(A)] had a smooth surface, whereas the mem-
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KARAHALILOGLU ET AL.
branes modified with NaOH treatment [Fig. 1(B–E)] exhibited more pronounced nanofeatures and showed a rougher surface compared with untreated silk fibroin membranes. AFM surface area and line scans Fig. 2(A–E)] confirmed the creation of a nanophase topography on silk fibroin membranes upon alkaline treatment. As demonstrated in Table I, the untreated silk fibroin membrane had the lowest rpm value and the nanophase roughness of the silk fibroin membranes increased upon NaOH treatment.
SILK FIBROIN MEMBRANES FOR DERMAL WOUND HEALING APPLICATIONS
ORIGINAL ARTICLE
FIGURE 2. AFM scans (2 3 2 mm) of the untreated and NaOH treated silk fibroin membranes using different NaOH concentrations and exposure times. (A) Untreated, (B) at 0.1N, (C) 0.5N and (D) 1N for 5 min, and (E) 1N for 10 min treated silk fibroin membranes. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
To understand the effect of the NaOH treatment on the surface chemistry, untreated and NaOH treated silk fibroin membranes were investigated using FT-IR spectroscopy
(Fig. 3). Typically, the silk fibroin structure showed three characteristic bands, that is, amide I (1700–1600 cm21), amide II (1540–1520 cm21), and amide III (1300–1220 cm21).
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TABLE I. RMS Values of the Silk Fibroin Membranes Sample
RMS Value (nm)
Untreated 0.1N25 min 0.5N25 min 1N25 min 1N210 min
5.17 6 0.22a 14.02 6 2.84 14.88 6 3.13 8.5 6 0.51 13.05 6 0.85
NaOH treatment increased root mean square roughness of the silk fibroin membranes. Values are mean6SEM; n53. a p
Nanotechnology and Nanomedicine Division, Hacettepe University, Beytepe 06800, Ankara, Turkey Chemical Engineering Department, Northeastern University, Boston 02115, Massachusetts 3 Chemistry Department, Biochemistry Division, Hacettepe University, Beytepe 06800, Ankara, Turkey 4 Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, Saudi Arabia 2
Received 5 December 2013; revised 19 February 2014; accepted 25 February 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35161 Abstract: As an effort to create the next generation of improved skin graft materials, in this study, we modified the surfaces of a previously investigated material, silk fibroin, using a NaOH alkaline treatment to obtain a biologically inspired nanofeatured surface morphology. Such surfaces were characterized for roughness, energy, and chemistry. In addition, keratinocyte (skin-forming cells) adhesion and proliferation on such nanofeatured silk fibroin wound dressings were studied in an initial attempt to determine the promotion of an epidermal cover on the wound bed to form a new epidermal barrier. Dermal fibroblast adhesion and proliferation were also studied to assess the ability of nanostructured silk fibroin to replace damaged dermal tissue in chronic wounds (i.e., for diabetic foot ulcers). Results demonstrated for the
first time that keratinocyte and fibroblast cell density was greater on nanofeatured silk fibroin membranes compared with non-treated silk fibroin surfaces. The enhancement in cellular functions was correlated with an increase in silk surface nanotopography, wettability and change in chemistry after NaOH treatment. Due to the present promising results, the newly developed nanofeatured silk fibroin membranes are exciting alternative skin graft materials which should be further studied for various skin patch and wound dressing C 2014 Wiley Periodicals, Inc. J Biomed Mater Res applications. V Part A: 00A:000–000, 2014.
Key Words: nanotechnology, wound healing, silk, fibroblasts, keratinocytes
lu Z, Ercan B, Denkbas¸ EB, Webster TJ. 2014. Nanofeatured silk fibroin membranes for How to cite this article: Karahalilog dermal wound healing applications. J Biomed Mater Res Part A 2014:00A:000–000.
INTRODUCTION
Skin is the largest organ of the body and it represents approximately one-tenth of the body mass.1,2 It plays a critical role in several bodily functions, such as protection against external factors, inhibition of microorganism invasion, sensation, temperature regulation, hydration retention, and so forth.3 Skin is composed of three structural layers: the epidermidis, dermidis, and subcutis. The epidermidis is the avascular, outermost layer of the skin consisting of a mainly stratified squamous epithelium of keratinocytes, which are responsible for the cohesion of the epidermal structure and for barrier functions. The dermidis is the supportive connective tissue between the epidermidis and subcutis that is composed of fibroblasts. The dermidis has a specialized extracellular matrix (ECM) made of collagen, interwoven with elastin, glycosaminoglycans (GAGs), elastin, fibronectin, and various other proteins and it is responsible for the mechanical properties of the skin.4,5 Importantly, the dermidis has high signaling communication with the epider-
midis, which is vital for the homeostasis of the skin.6 The subcutis is the deepest layer of the skin, composed primarily of adipose cells, fibroblasts and immune cells, separating the dermidis from the underlying muscular fascia. Several diseases require skin grafts and artificial skin substitutes, including burns, wounds (diabetic foot ulcers), infection (i.e., necrotizing fasciitis, purpura fulminans etc.), removal of skin cancers and so forth. One common mechanism for the regeneration of skin is the wound healing response that automatically starts upon injury to the skin. Dermal wound healing is a complex process which requires interactions between inflammatory cells, fibroblasts, keratinocytes and endothelial cells, all of which are regulated by an array of cytokines and growth factors.7,8 Materials currently used for dermal wound healing applications mainly utilize an organic scaffold (mostly collagen based scaffolds due to its abundant presence in the dermidis) cultured with fibroblasts or keratinocytes to help regenerate native tissue.9–11 Although they successfully
*These authors contributed equally to this work. Correspondence to: T. J. Webster; e-mail: [email protected] Contract grant sponsor: Northeastern University and Hacettepe University
C 2014 WILEY PERIODICALS, INC. V
1
form a matrix over the damaged tissue, total skin regeneration with complete functional and aesthetic recovery has not been achieved to date. Additional drawbacks to the currently used skin grafts and artificial skin substitutes include slow preparation times upon culturing cells in vitro, high production costs, variable engraftment rates, delayed vascularization, immunogenicity issues for allographs, donor site morbidity, limited donor site availability for autographs, scar tisue formation, poor integration with host tissue, as well as infection.12,13 Skin, just like the other tissue types, contains numerous components from the nanoscale to the micronscale. Therefore, an alternative potential approach (not yet widely exploited) to regenerate native skin tissue is to mimic the nanofeature size of components of skin in biomaterials. Numerous studies have demonstrated altered interactions between biomaterials and cells (including osteoblasts, chondrocytes, smooth muscle cells, endothelial cells, etc.) upon changing the biomaterial surface architecture from the micrometer to the nanometer scale.14–17 Recently, it was shown that various cell types from different tissues all positively respond to changes in biomaterial surface feature nanometer size (e.g., bone, cartilage, bladder, skin, etc.).18,19 More specifically, although limited, nanoscale surface features have been shown to affect cellular adhesion, proliferation, phenotype expression and long-term functions for skin tissue engineering applications.20 A number of natural and synthetic polymers (such as chitosan, collagen and poly-caprolactone) are currently being investigated as nanofeatured wound dressing materials for dermal wound healing applications.21–24 One such nanostructured material is silk. Silk is a natural protein which has been used for centuries as a suture material. It is typically synthesized by silkworms, the larva of Bombyx mori. Silk has two components, a double strand fibroin fiber which is surrounded by a gummy-like protein shell called sericin.25 Importantly, silk has been proposed for a wide range of tissue engineering applications due to its environmental stability, mechanical properties, biocompatibility, and the ease to modify and functionalize its surface.26 In the literature, silk fibroin has been proposed for diverse tissue engineering applications in a variety of forms (including films, sponges, hydrogels, electrospun mats, tubes, etc.).27–30 In fact, silk has been used as a suture material in biomedical applications for decades. Specifically for wound healing applications, silk-based materials enhance keratinocyte and fibroblast adhesion in vitro, while inducing less inflammation, compared with commercially-used products (such as air permeable Tegaderm (3M) tape, Sofra-tulleV, Due Active Dressing etc.).31–34 Although it is possible to improve the healing response for small wounds using silk-based materials, for deep injuries, discoloring or scarring remain when using silk-based materials, where the latter can potentially lead to undesirable skin contractions; thus, improvements in silk can be beneficial. Thus, for all of the above reasons, in this research, the aforementioned two approaches (nanotechnology and silk) were combined to create a nanofeatured surface topography R
2
KARAHALILOGLU ET AL.
which mimics the natural niche of skin. The proposed method to create nanofeatured surfaces on silk membranes is through a NaOH alkaline treatment, which was previously shown to create nanorough PLLA (poly-L-lactic acid) and poly(lactic-co-glycolic acid) (PLGA) surfaces.35,36 Briefly, the NaOH treatment was shown to break the ester bonds in the polymer chains, hence altering the nanophase topography of the surfaces. In this research, the main goal was to prepare nanorough silk membranes for dermal wound healing applications by using a similar NaOH surface treatment. Therefore, the cytocompatibility of the nanofeatured silk fibroin surfaces were evaluated in vitro using human dermal fibroblasts and epidermal keratinocytes and a potential mechanism for the observed enhancement in cellular functions was proposed. MATERIALS AND METHODS
Material synthesis Materials. All chemicals were purchased from SigmaAldrich including disodium carbonate (Na2CO3), sodium hydroxide (NaOH), lithium bromide (LiBr), phosphate buffer saline (1X PBS), L-glutamine, and fetal bovine serum (FBS). B. mori silkworm cocoons were obtained from Kozabirlik (Bursa, Turkey). EMEM and keratinocyte serum free medium (with supplements) were purchased from ATCC and Gibco (USA), respectively. Preparation of silk solutions. A silk fibroin solution was prepared from B. mori cocoons according to previously established protocols with slight modifications.37 First, B. mori silk cocoons were cut into small pieces and boiled in 0.05M Na2HCO3 for 30 min to extract the glue-like sericin coating layer from the structural fibroin protein which was then rinsed with distilled water (diH2O) three times. The obtained silk fibroin fibers were dried overnight at 40 C, dissolved in a LiBr solution (9.3M) at 60 C for 4 h, followed by dialysis through a cellulose membrane (12–14,000, MWCO) across distilled water for 4 days. The obtained silk solutions were centrifuged twice at 13,000g, diluted to a final concentration of 2 w/v % and stored at 4 C. Nanofeatured silk fibroin membrane preparation. The aqueous silk solution (15 mL) was poured into a glass petri dish (35 mm 3 100 mm) and dried overnight to generate silk fibroin membranes with a thickness of 200 mm. Afterwards, the dried membranes were treated with a methanol solution for 1 h to make them non-water soluble. To create a nanofeatured surface topography, silk membranes were treated with 0.1, 0.5 and 1N NaOH solutions for 5 or 10 min, followed by rinsing with diH2O for four times to remove excess NaOH residues. The pH of the rinsing solution was tested and was found similar to that of the original rinsing solution, thus, suggesting that no residual NaOH remained on the silk surfaces. Specimen characterization Electron microscopy. Surface characterization of the specimens was conducted with a Hitachi S4800 Tokyo, Japan
SILK FIBROIN MEMBRANES FOR DERMAL WOUND HEALING APPLICATIONS
ORIGINAL ARTICLE
Scanning Electron Microscope (SEM, Hitachi S4800 SEM, Tokyo, Japan). A 3 kV accelerating voltage was chosen for SEM analysis and the characterization was completed using secondary electrons. Before SEM investigation, a 3 nm layer of palladium (Pd) was sputter coated (Cressington 208; Cressington Scientific Instruments, Watford, UK) onto the silk fibroin membranes to provide a conductive surface. Surface roughness. For surface roughness measurements, a Parks Scientific NX-10 Atomic Force Microscope (AFM; Suwon, Korea) was used to scan the non-treated and treated silk samples. Each sample was analyzed in ambient conditions under non-contact mode using a silicone ultrasharp cantilever (Park Systems Non-contact Cantilever). The AFM tip had a radius of curvature less than 7 nm and had a backside aluminum reflex coating approximately 30-nm thick. Tapping mode was used at 324 kHz at a scan rate of 0.5 Hz. A scan area of 2 lm 3 2 lm was investigated in these studies. Image analysis software (XEI) was used to generate AFM micrographs and to quantitatively compare the root-mean-square roughness (RMS) of the non-treated and treated silk surfaces. Surface chemistry. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) analysis was completed using a Bruker Vertex 70 FTIR spectrometer (USA) equipped with a Zinc Selenide (ZnSe) crystal to characterize surface chemistry of the samples. A spectral range of 550–4000 cm21 was investigated in these studies and each spectrum was the mean of three acquisitions. Untreated and NaOH treated silk fibroin membranes were cut into 1 3 1 cm squares, loaded onto the FTIR machine while maintaining a full contact between the Ge crystal and the sample. The background signal was subtracted from the obtained reflectance vs. wavenumber spectra. The bands in the FTIR spectra were compared with standards to determine the type of chemical bonds and possible consequence of alkaline treatment. Crystallinity. Crystallinity of the untreated and treated silk fibroin membranes was characterized using X-ray diffraction analysis (XRD, Rigaku Ultima IV, Korea) using Cu Ka radiation at 40.0 kV and a power supply of 44.0 mA. XRD spectra were analyzed for a 2u range of 10–50 . All the samples were characterized using a step size of 0.1 and the time at each step was 2 s. Water contact angle measurements. The hydrophilicity of the treated and untreated samples (untreated silk, 0.1N NaOH for 10 min, 0.5N NaOH for 10 min, and 1N NaOH for 5 and 10 min) were investigated using a drop contact angle meter (SEO Phoenix 300, Korea). Seven samples from each sample type were characterized by measuring the contact angles using double distilled water. Cytocompatibility characterization Cell culture. Human skin fibroblasts (American Type Culture Collection, CCL-110, population number 8–10) were
cultured in EMEM cell culture medium containing 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin under standard cell culture conditions (5% CO2/95% air at 37 C). Human epidermal keratinocytes (Invitrogen, C0015C, population numbers 5–7) were cultured in 13 keratinocyte serum free medium supplemented with 2.5 mg human recombinant epidermal growth factor (EGF), 25 mg bovine pituitary extract and 1% penicillin-streptomycin under standard cell culture conditions (5% CO2/95% air at 37 C). Cell adhesion assay. Fibroblast and keratinocyte cell adhesion on silk fibroin membranes were investigated using an MTT [3-(4,5-dimethylthiozole-2-yl)22, 5-diphenyltetrazolium bromide] assay. Silk fibroin membranes were cut into 1cmx1cm squares followed by a 20-min EtOH soak for sterilization. For 4-h adhesion experiments, a seeding density of 3500 cells/cm2 was used for both fibroblasts and keratinocytes and after seeding the cells, the samples were cultured under standard cell culture conditions (5% CO2/95% air at 37 C). At the end of a 4 h adhesion assay, samples were transferred into clean wells and 150 mL of an MTT dye soluR 96 Non-Radioactive Proliferation Assay, Promtion (CellTiterV ega, Madison, WI) and 1 mL of cell growth medium (described in “Cell culture” section) were added and then were incubated in the dark at 37 C for 4 h. The reaction was R 96 stopped by adding 1 mL of a stop solution (CellTiterV Non-Radioactive Proliferation Assay, Promega, Madison, WI). Two hundred microliters of the final solution was placed into a 96-well plate and the absorbance values were measuered at 570 nm using a spectrophotometer (Spectramax M3 Multimode Microplate Reader, Molecular Devices). The number of cells was determined from a standard curve of absorbance versus a known number of cells run in parallel with the experimental samples. Cell proliferation assay. To evaluate cellular proliferation on silk membranes, fibroblasts (20,000 cells/cm2) or keratinocytes (40,000 cells/cm2) were seeded and were cultured in their growth media (described in “Cell culture” section) for up to 3 days under standard cell culture conditions (5% CO2/95% air at 37 C). To induce keratinocyte growth and enhance cell viability during the proliferation experiments, 10% FBS was added to the keratinocyte culture medium. At each time point (days 1, 2, and 3), samples were gently rinsed with 13 PBS three times, followed by a 4% paraformaldehyde (Sigma) fixation for 8 min. Then, cells were stained using a nuclear fluorescent dye, DAPI (6-diamidino2-phenylindole, Sigma), for 10 min. Excess DAPI solution was removed using a 1x PBS wash three times. Finally, the stained cells were counted using a fluorescence microscope (Zeiss Axio Vert.A1) to obtain cell densities on the silk fibroin membrane surfaces. Statistical analysis Numerical data were analyzed using standard analysis of variance (ANOVA) techniques and statistical differences were considered at p < 0.05. All experiments were completed in triplicate with three repeats for each experiment.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00
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FIGURE 1. SEM images of untreated and NaOH treated silk fibroin membranes using different NaOH concentrations and exposure times. (A) Untreated, (B) at 0.1N, (C) 0.5N, and (D) 1N for 5 min, and (E) 1N for 10 min. NaOH treatment increased silk nanofeatured surface topographies. Scale bars 5 1 mm.
Results were reported as the average 6 standard error of the mean. RESULTS AND DISCUSSION
Material characterization SEM images of the silk fibroin membranes treated with 0.1, 0.5, and 1N NaOH for 5 and 10 min are shown in Figure 1. These images reveal that the untreated silk fibroin membrane [Fig. 1(A)] had a smooth surface, whereas the mem-
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KARAHALILOGLU ET AL.
branes modified with NaOH treatment [Fig. 1(B–E)] exhibited more pronounced nanofeatures and showed a rougher surface compared with untreated silk fibroin membranes. AFM surface area and line scans Fig. 2(A–E)] confirmed the creation of a nanophase topography on silk fibroin membranes upon alkaline treatment. As demonstrated in Table I, the untreated silk fibroin membrane had the lowest rpm value and the nanophase roughness of the silk fibroin membranes increased upon NaOH treatment.
SILK FIBROIN MEMBRANES FOR DERMAL WOUND HEALING APPLICATIONS
ORIGINAL ARTICLE
FIGURE 2. AFM scans (2 3 2 mm) of the untreated and NaOH treated silk fibroin membranes using different NaOH concentrations and exposure times. (A) Untreated, (B) at 0.1N, (C) 0.5N and (D) 1N for 5 min, and (E) 1N for 10 min treated silk fibroin membranes. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
To understand the effect of the NaOH treatment on the surface chemistry, untreated and NaOH treated silk fibroin membranes were investigated using FT-IR spectroscopy
(Fig. 3). Typically, the silk fibroin structure showed three characteristic bands, that is, amide I (1700–1600 cm21), amide II (1540–1520 cm21), and amide III (1300–1220 cm21).
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00
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TABLE I. RMS Values of the Silk Fibroin Membranes Sample
RMS Value (nm)
Untreated 0.1N25 min 0.5N25 min 1N25 min 1N210 min
5.17 6 0.22a 14.02 6 2.84 14.88 6 3.13 8.5 6 0.51 13.05 6 0.85
NaOH treatment increased root mean square roughness of the silk fibroin membranes. Values are mean6SEM; n53. a p
