silk fibroin nanofibrous bioactive scaffolds for tissue engineering applications

Polymer 168 (2019) 86–94

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Electrospun polycaprolactone/silk fibroin nanofibrous bioactive scaffolds for tissue engineering applications

T

Muhammad Anwaar Nazeer, Emel Yilgor, Iskender Yilgor∗ KUYTAM Surface Science and Technology Center, Chemistry Department, Koc University, Istanbul, Turkey

H I GH L IG H T S

is a mild and efficient degumming reagent for sericin removal. • Ammonia degummed fibers display smooth surfaces without any hydrolytic damage. • Ammonia acid is a good, green solvent for electrospinning of PCL/SF composite fibrous scaffolds. • Formic of SF into electrospun PCL scaffolds improves cell adhesion and proliferation significantly. • Incorporation • Electrospun PCL/SF scaffolds are promising substrates for tissue engineering applications.

A R T I C LE I N FO

A B S T R A C T

Keywords: Silk degumming Silk fibroin electrospinning Tissue engineering

Degumming of Bombyx mori silk cocoons by a novel and mild process using aqueous ammonia and fabrication of electrospun polycaprolactone/silk fibroin (PCL/SF) nanofibrous scaffolds is reported. Cocoons were degummed in 0.3% w/w solutions of boiling ammonia (28–30%) for 45 min. Degummed SF fibers were dissolved in phosphoric and formic acid (7/3 v/v) mixture, coagulated in methanol, filtered and dried. PCL solutions containing different amounts of SF were electrospun in formic acid, a green solvent. Scaffolds were characterized to confirm the successful incorporation of SF and to demonstrate formation of nanofibrous webs with good biomechanical properties. Cell viability assay was performed by seeding Human BJ fibroblast cells on scaffolds. In vitro analysis showed that the scaffolds produced were non-toxic and incorporation of SF resulted in enhanced cell proliferation. Nanofibrous PCL/SF scaffolds with good biomechanical properties developed through dialysis free processing of silk fibroin can be promising substrates for tissue engineering applications.

1. Introduction Polycaprolactone (PCL), an FDA approved aliphatic polyester [1], has considerable potential in biomedical applications due to its unique properties such as high crystallinity, excellent biocompatibility, appropriate mechanical strength and good biodegradation properties [2]. However, PCL surface is fairly hydrophobic. Poor hydrophilicity of PCL surface results in poor cells adhesion, migration, proliferation and differentiation [3]. In tissue engineering applications this reduces the cell affinity towards PCL surface and lack of cellular interactions leads to fairly slow tissue growth [4]. Hydrophilicity of PCL surfaces can be improved by various surface modification techniques [5–7] or through the incorporation of bioactive materials [8–10]. PCL scaffolds have been investigated for a wide range of biomedical uses, such as; cardiac [11], bone [12], nerve [13], skin [14–17], and drug delivery applications [18,19]. PCL with controlled molecular weight can be synthesized ∗

by the ring-opening polymerization of caprolactone monomer using various catalysts, in bulk or in solution. High molecular weight PCL are usually prepared by the coordination insertion or anionic ring opening polymerization techniques [20]. Silk fibroin (SF) is a naturally occurring protein, well known for its biocompatibility, also finds a wide range of biomedical applications [21,22] including skin repair and regeneration [23]. Bombyx mori (B. mori) silk cocoons having two proteins (light chain ∼26 kDa and heavy chain 390 kDa) are linked together through a disulfide bond at a ratio of 1:1 [24]. A hydrophilic protein, sericin (20–310 kDa), is coated on silk fibroin fibers (25–30 wt%) and needs to be removed through degumming process prior to use [25]. Degumming conditions can affect mechanical properties and cytocompatibility of SF fibers [26]. Many methods, such as treating with borax-HCl buffer, urea, enzymes and most commonly the use of 0.02 M aqueous solution of sodium carbonate and bicarbonate are reported in the literature for degumming of

Corresponding author. E-mail address: [email protected] (I. Yilgor).

https://doi.org/10.1016/j.polymer.2019.02.023 Received 18 December 2018; Received in revised form 30 January 2019; Accepted 11 February 2019 Available online 12 February 2019 0032-3861/ © 2019 Elsevier Ltd. All rights reserved.

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the cocoons to get clean silk fibers [27,28]. After degumming, silk fibers are usually dissolved in 9.3 M LiBr solution [29,30], CaCl2/C2H5OH/ H2O (1:2:8) solution [31,32] or other salt solutions [33,34]. The main drawback of all these methods is that, since all solutions contain inorganic salts, at the end of the dissolution process, it is necessary to perform dialysis to remove the salts in order to obtain pure silk fibroin [35]. In dialysis process, porous membranes with specific molecular weight cut off are used to remove the impurities from silk solution. After long process of dialysis, silk fibroin solution in water is obtained and can be concentrated if needed. However, even after purification, aqueous silk fibroin solution may still contain salt impurities [35,36]. Silk exhibits crystalline and amorphous regions just like all naturally occurring proteins. Crystalline regions comprise of Silk-I (α-helical), Silk-II (β-folded) and Silk-III (hexagonal type crystalline) structures [37]. B. mori silk fibroin consists of 13 ± 5% water soluble Silk-I, and 56 ± 5% water insoluble Silk-II. 60–70% of fibroin is crystalline and remaining amorphous region has disordered conformation in shape of random globules [38]. Alcohol treatment, hydration, spinning or heat treatment can convert Silk-I into Silk-II [39,40]. The bioactivity of SF is useful for cell adhesion and tissue regeneration properties of developed scaffolds [40]. In tissue engineering, scaffolds provide structural support for cells to accommodate and proliferate. Proper physical and biological characteristics is needed for cell attachment, migration, proliferation and differentiation. Surface structure and topography of the scaffold should provide proper binding sites for the cells. In addition, proper level of porosity is needed for the diffusion of nutrients and for vascularization [4,41]. Mechanical properties of the scaffolds should also be compatible with that of the surrounding tissues or organs. Mechanical properties of PCL can be combined with the bioactivity of SF to fabricate scaffolds, which possess required characteristics for optimum performance [42]. Electrospinning is a simple and useful technique for producing nano/micro fibers possessing large surface areas, which are favorable for biomedical applications in terms of cellular interaction [43]. These electrospun fibrous scaffolds can mimic native extracellular matrix (ECM) by incorporating suitable substrate and moieties. The functional environment provided by these nanofibrous scaffolds can accommodate cells and help them to grow and proliferate [44]. To control the fiber structure, topography, morphology and the overall performance of electrospun polymeric webs or scaffolds for tissue engineering applications, various electrospinning processing factors, such as; static voltage, working (tip to collector) distance, solution viscosity and feed rate need to be optimized [45]. Lee and others [8] developed PCL and PCL/ SF composite scaffolds through electrospinning by using methylene chloride and dimethylformamide mixed solvent system. SF was obtained through sodium carbonate degumming and dialysis process. They observed enhanced fibroblast cell viability on PCL/SF scaffolds, which was further increased through human umbilical cord serum since it contains various growth factors. Overall, scaffolds were highly bioactive but displayed lower ultimate tensile strength (UTS). Khosravi and co-workers [46] also prepared nanofibrous scaffolds of PCL and in order to improve the bioactivity of the scaffolds they immobilized SF on the PCL scaffold surface through chemical modification. They used the same degumming, dissolution and electrospinning solvent system, which was used as by Lee [8]. Mechanical properties of the scaffolds were compromised possibly due to ozone treatment involved in surface functionalization and solvent system used. Furthermore, Lim et al., reported development of PCL/SF nanofibrous scaffolds possessing high UTS [47]. They used sodium carbonate and sodium oleate solution for degumming process and formic acid as electrospinning solvent. In conclusion we can say that all reported studies regarding PCL/SF scaffolds either involve harsh degumming conditions and chemicals, which are difficult to remove from the system, or solvents which are not environmentally friendly and/or negatively affect the mechanical properties of the scaffolds. In this study PCL/SF nanofibrous webs were prepared by

electrospinning and were evaluated as scaffolds for tissue engineering applications. SF was obtained by degumming B. mori silk cocoons in boiling 0.3% w/w aqueous sodium carbonate, bicarbonate and ammonia solutions for 45 min. Fairly milder ammonia solution was used as degumming reagent and its impact on the structure and morphology of SF was compared with sodium carbonate and bicarbonate processes. To the best of our knowledge, no one has reported silk cocoons degumming through ammonia solution and evaluated its impact on SF properties. Residual ammonia can easily be removed from degummed fibers compared to sodium carbonate and bicarbonate. After washing and drying, degummed fibers were processed via dialysis-free process by dissolving in phosphoric acid/formic acid (7:3) mixture. Dissolved SF was coagulated in methanol followed by washing and drying to get pure SF without any need for dialysis process. High molecular weight PCL was synthesized in our laboratories by the ring opening polymerization of epsilon-caprolactone. PCL/SF solutions with SF contents of 20 and 40% by weight were prepared in formic acid. Nanofibrous scaffolds were produced through green electrospinning process, where less toxic Q3C class-3 solvent, formic acid, was used rather than commonly used class-2 toxic solvents, such as tetrahydrofuran and N,N-dimethylformamide [48]. Scaffolds produced were characterized by scanning electron microscopy, attenuated total reflection Fourier transform infrared spectroscopy, stress-strain analysis and static water contact angle measurements. CellTiter-Glow assay was performed to access cytotoxicity by seeding Human BJ fibroblast cells to explore skin tissue engineering applications of developed scaffolds.

2. Materials and methods 2.1. Materials Silk (Bombyx mori) cocoons were kindly supplied by Idealab, Koc University, Istanbul. Ammonia solution (28–30%), tetrahydrofuran (THF), phosphoric acid, formic acid (98%), reagent grade xylene (mixture of isomers), L-glutamine (200 mM), trypsin-EDTA (25%) and εcaprolactone were purchased from Aldrich and were used as received without further purification. Technical grade methanol was used for coagulation. 1,2-Bis(3-aminopropoxy)ethane (Jeffamine® XTJ-590) was kindly provided by Huntsman Chemicals. Stannous octoate (T-9) was obtained from Air Products. Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, phosphate buffer saline (PBS) were purchased from Gibco. CellTiter-Glo luminescent cell viability assay was purchased from Promega.

2.2. Polycaprolactone synthesis PCL was synthesized by the ring-opening polymerization of ε-caprolactone (CL) in xylene solution, at 130 °C, where a diamine was used as the initiator and stannous octoate (T-9) as the catalyst [49]. Reactions were carried out in a 500 mL, three neck round bottom Pyrex flask equipped with an overhead stirrer and thermometer. Typical procedure followed for PCL synthesis was as follows: 30 g of CL, 0.049 g of diamine initiator (Jeffamine XTJ-590) and 0.104 g of T-9 catalyst were introduced into the reaction flask. Reaction mixture was stirred at 120 rpm and system was heated to 130 ± 5 °C. As the reaction proceeded 50 mL of xylene was added slowly to reduce the viscosity. Reaction was completed in about 24 h and the system was cooled down to room temperature. THF was added to dilute the mixture to obtain a solution with about 20% by weight solids content. Polymer was coagulated twice in methanol under strong agitation, filtered and dried in air oven at 50 °C until constant weight. Number (Mn) and weight (Mw) average molecular weights of PCL determined by GPC measurement were 43 and 85 kDa respectively.

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2.3. Silk cocoons degumming and fiber dissolution

were determined using adenosine triphosphate (ATP) dependent cell viability assay, CellTiter-Glo (Promega), as per manufacturer's description. Human BJ fibroblast cells (Passage # 8–16) were cultured with DMEM supplemented with 10% FBS, 3.97 mM L-glutamine, and 1% Penicillin/streptomycin in an incubator with 5% CO2 at 37 °C. Scaffolds were washed with PBS and after drying, they were placed in 24-well plate and sterilized under UV light for 30 min. The samples were rinsed in DMEM at 37 °C and medium was changed 2 times before cell seeding. Fibroblast cells were seeded onto the scaffold's surface (15000 cells/0.2 cm2). ATP standard curve was prepared in DMEM, prior to measurement. Both samples and ATP solutions were treated with CellTiter-Glo reagent and incubated at 25 °C, 100 rpm for 15 min. Luminescence values were measured on Day 1 and Day 4, using a plate reader (Biotek, Synergy H1).

Silk cocoons have sericin layer coated on the surface of fibers, which needs to be removed prior to further processes. Outermost sericin layer can be dissolved in boiling water whereas, innermost layer need to be hydrolyzed in an alkaline solution. Alkalinity of the solution and degumming time are crucial parameters to be considered. Harsh chemicals such as sodium carbonate and severe degumming conditions may trigger hydrolytic degradation that can cause surface fibrillation and leads to a decrease in the tensile strength of SF fibers [50,51]. We introduced ammonia solution as a novel degumming reagent and compared its effect on the properties of the degummed fibers and regenerated SF with conventional processes. Degumming mechanism of sodium carbonate, bicarbonate and ammonia is the same and involves the base catalyzed hydrolysis of sericin in an alkaline solution. Silk cocoons were degummed according to previously described procedure with slight modifications [52,53]. Briefly, 0.3 w/w% solution of sodium carbonate, bicarbonate and ammonia (28–30%) prepared in double distilled water was heated and stirred. At boiling, 5 g of cut and cleaned pieces of silk cocoons were added and degumming was continued for 45–90 min. After completion of degumming process, degummed fibers were washed several times in fresh distilled water until the water became neutral. For ammonia solution, filtrate became neutral after just one wash while it was quite difficult to remove the residual sodium carbonate and bicarbonate from degummed fibers, which required repeated washing. Fibers were dried under atmospheric conditions first in a fume hood and then in a vacuum oven at room temperature until constant weight. Dried fibers were dissolved in formic acid/phosphoric acid (7:3 by volume) mixture by making 3% w/v solutions [54]. Solutions were mechanically stirred for 18 h to ensure complete dissolution. Silk fibroin was coagulated in methanol, filtered and washed with distilled water until the filtrate was neutral. Purified silk fibroin was dried in fume hood at room temperature overnight and then in a vacuum oven at 40 °C for 24 h. Purified SF was dissolved in formic acid to prepare spin coated and cast mold films. Cast films were characterized by infrared spectroscopy (ATR-FTIR), x-ray diffraction (XRD), atomic force microscopy (AFM) and static water contact angle measurements. Results obtained are provided in Supplementary data file. SF films obtained from ammonia solution degumming process displayed the highest crystallinity. As a result, silk fibroin degummed by ammonia process was used in the preparation of polycaprolactone/silk fibroin (PCL/SF) electrospun nanofibrous scaffolds.

2.6. Degradation studies Degradation of nanofibrous scaffolds of PCL and PCL/SF composites was monitored in DMEM medium containing 10% FBS over 15-weeks. Before incubation, scaffolds were pre-dried in vacuum oven at room temperature for 24 h. After weighing, samples were incubated in DMEM + FBS solution at 37 °C. Samples were taken out of the oven after 1, 3, 7, 11 and 15 weeks, washed with distilled water and then with warm water to remove any residues of incubation media. After drying in vacuum oven at room temperature until constant weight, weight loss was calculated by Eqn. (1).

Weight loss (%) =

Wf − Wi Wi

× 100

(1)

Where “Wi” and “Wf” denote the initial and final weight respectively. FBS containing DMEM medium was changed subsequently at each time interval of analysis.

2.7. Characterization techniques Fourier transform infrared (FTIR) spectra of samples were obtained on Thermo-scientific iS50 spectrometer having ATR attachment. Absorption spectra for all samples were obtained at a resolution of 2 cm−1 and 64 scans. Surface images were obtained on Zeiss EVO LS-15 scanning electron microscope (SEM). Prior to analysis samples were sputter coated with 10 nm layer of gold to make surface conductive. Fiber diameter was measured using ImageJ® software. Average of 50 values are used to calculate the standard deviation. For AFM analysis, 10% w/w solution of regenerated silk fibroin for each sample was prepared in formic acid and spin coated on a glass slide at 1000 rpm for 70 s. Spin coated glass slides before and after methanol/water mixture treatment were dried under vacuum at ambient temperature and analyzed on a Bruker Dimension Icon AFM. Phase and amplitude error images were obtained through standard tapping mode by using Bruker MPP-11120-10 (RTESPA model) tip with a force constant of 40 N/m, resonant frequency of 300 kHz and back side coated with 50 ± 10 nm aluminum. Static water contact angle measurements were performed on Dataphysics OCA 35 by using deionized and triple distilled water (10 μL droplet for each measurement). Gel permeation chromatography (GPC) was performed on a Viscotek VE 2001 series instrument equipped with Dguard, D2500, D4000 and D5000 columns and refractive index detector (VE3580) to determine the molecular weight of PCL synthesized. Mobile phase used was THF at 35 °C, at a flow rate of 1 mL/min. PCL was dissolved in THF at a concentration of 1 gm/mL. Molecular weight was determined by using polystyrene standards. Stress-strain analysis of scaffolds was performed on Instron Model 4411 universal testing machine at room temperature with a crosshead speed of 25 mm/min. Samples were cut with a dog-bone shaped die (ASTM D1708) and had an initial length of 24 mm.

2.4. Preparation of polycaprolactone/silk fibroin (PCL/SF) scaffolds by electrospinning PCL/SF solutions with different SF concentration were prepared in formic acid at a concentration of 20 wt percent. Solutions were prepared by dissolving the required amount of SF in formic acid followed by the addition of corresponding amount of PCL and stirring the solutions for 2 h. PCL/SF solutions were transferred into a 21G syringe and electrospun into a web. After electrospinning, scaffolds were first dried in a fume hood at ambient conditions overnight to evaporate any residual solvent, followed by immersion in methanol for 1 h to induce crystallinity in SF. Random coil fraction of SF is converted to silk-II crystalline structure upon methanol treatment [55]. After methanol treatment, scaffolds were rinsed with de-ionized water and dried. After drying, samples were again rinsed with ammonia solution and distilled water respectively to neutralize and wash. Sample code, solution concentrations and optimized parameters of the electrospinning process are provided in Table 1. 2.5. Cell seeding and cytotoxicity analysis Cytotoxicity of PCL and PCL/SF composite electrospun scaffolds 88

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Table 1 Sample description and process parameters used during electrospinning process. Sample code

PCL

SF

Voltage (kV)

Distance (cm)

Feed rate (μL/min)

Average fiber diameter (nm) n = 50

PCL PCL/SF-20 PCL/SF-40

100 80 60

0 20 40

17.5 17.5 17.5

12 12 12

5 5 5

153 ± 26 217 ± 51 251 ± 75

3. Results and discussions

using different reagents are reproduced in Fig. 1. For comparison, Fig. 1-a shows the SEM image of the silk cocoon fibers prior to degumming. As can be seen clearly, these fibers are fairly thick due to sericin coverage and have very rough surfaces. Upon degumming and the removal of the sericin layer, regardless of the process used, finer fibers with smoother surfaces are obtained (Fig. 1 b-f). When the SEM images of degummed fibers are closely examined (high magnification images in insets), it can be seen that the reagent type and degumming time has significant effect on the surface morphology of the silk fibroin fibers produced. Harsh chemicals and/or prolonged degumming times may cause serious damage to the fiber surfaces triggered by surface hydrolysis, as shown by the yellow arrows in Fig. 1. Sodium carbonate has already been proven to be fairly damaging to fiber's surface [51]. As can be seen in Figure, 1 there was no surface deterioration when the fibers were degummed by Ammonia-45 and Ammonia-60 processes (Fig. 1-d and 1-e insets). Less damage on the fiber surface was observed even for Bicarbonate-45 (Fig. 1-c inset) as compared to Carbonate-45 (Fig. 1-b inset) and Ammonia-90 (Fig. 1-f inset). These results clearly demonstrate the superiority of ammonia degumming process developed in this study, compared to conventional carbonate and bicarbonate

Bombyx mori silk cocoons were degummed by a novel and mild process in boiling aqueous ammonia and the silk fibroin fibers (SF) obtained were compared with those produced using conventional sodium carbonate and sodium bicarbonate processes. SF films obtained by spin coating or casting from formic acid solutions were characterized by various techniques such as FTIR, XRD, AFM and water contact angle measurements, as discussed in the Supplementary Data Files provided. Regenerated SF fibers were blended with PCL at different ratios and electrospun PCL/SF nanofibrous composite scaffolds were produced. These scaffolds were characterized by various techniques, including in vitro cell viability tests and hydrolytic degradation studies to demonstrate their potential use as biocompatible and biodegradable scaffolds in tissue engineering applications. 3.1. Scanning electron microscopy (SEM) studies on degummed silk fibroin fibers SEM images of silk fibers before and after degumming process by

Fig. 1. SEM images of cocoons and degummed fibers: (a) cocoon fibers, (b) Carbonate-45, (c) Bicarbonate-45, (d) Ammonia-45, (e) Ammonia-60, and (f) Ammonia90. 89

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Fig. 2. AFM phase images of spin coated silk fibroin films produced by different degumming processes. (a) Ammonia-45, (b) Ammonia-60, (c) Ammonia-90, (d) Bicarbonate-45, and (e) Carbonate-45.

processes. 3.2. AFM analysis of degummed silk fibroin films AFM phase images obtained on spin-coated SF films produced by different degumming processes are reproduced in Fig. 2. It is important to note that AFM is a surface sensitive technique and the results provided here describe the surface morphologies, but not necessarily the bulk. In AFM phase images darker regions show soft or amorphous structures, whereas light colored regions indicate crystalline domains. Presence of crystalline (β-sheet) domains together with amorphous silk fibroin structure is visible in all AFM images provided in Fig. 2. Ammonia-45 based film given in Fig. 2-a displays more distinct phase separated crystalline and amorphous domains compared to the others. As the duration of ammonia degumming process increases to 60 (Fig. 2-b) and 90 min (Fig. 2-c) surface morphology also changes towards more phase mixing and higher amorphous content. This is in line with the FTIR deconvolution analysis, which indicates 46% crystalline content for Ammonia-45 sample compared to 32% for Ammonia-60 as provided in Supplementary Information. Similar behavior is observed when silk fibroin produced by milder bicarbonate-45 process (Fig. 2-d) is compared with carbonate-45 (Fig. 2-e).

Fig. 3. ATR-FTIR analysis of PCL and PCL/SF composite scaffolds films.

Electrospun nanofibrous scaffolds were also characterized by ATRFTIR studies to confirm the successful incorporation of SF and its interaction with PCL matrix. Spectrum for extracted SF powder used was also obtained to compare the interactions between PCL and SF in composite fibers as provided in Fig. 3, where expanded 35002700 cm−1 and 1800-1000 cm−1 regions of the FTIR spectra are also reproduced for better resolution. Major absorption bands assigned to amide-A, (NeH stretching), amide-I (C=O stretching) and amide-II (CeN stretching and NeH in plane bending vibrations) for SF powder can be seen at 3278, 1618 and 1513 cm−1 respectively [56]. Very sharp and symmetrical H-bonded NeH absorption band at 3278 cm−1 present in SF powder is shifted to 3290 cm−1 in PCL/SF composites. Similarly, a slight shift is also detected in amide-I and amide-II regions and new bands are observed at 1628 and 1521 cm−1. These results clearly show fairly strong hydrogen bonding or dipole-dipole type intermolecular interactions between ester and amide groups [57] present in PCL and SF

3.3. ATR-FTIR analysis After demonstrating the critical effect of degumming conditions on SF structure, we decided to use product from ammonia-45 process for the preparation of PCL/SF scaffolds. Intermolecular interactions and compatibility of PCL and SF in the composite films were investigated by ATR- FTIR spectroscopy. FTIR spectra of SF and PCL/SF films prepared by casting from formic acid solution are provided in Fig. 3. Spectra were resolved using OMNIC® software to access Silk- I, Silk-II and amorphous content of the films. Details of the peak deconvolution study is provided in Supplementary Information. Peak deconvolution results confirm that, films prepared using formic acid solutions mainly consist of Silk-II type morphology. Furthermore, degumming conditions also impact the crystallinity of the films produced. Severe degradation observed in the case of carbonate and bicarbonate degumming process result in higher amorphous content. 90

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Fig. 4. SEM analysis of electrospun webs. (a) PCL, (b) PCL/ SF-20 and (c) PCL/SF-40.

to the presence of air pockets [60]. Very interestingly, incorporation of SF into PCL completely changed the wetting behavior of electrospun PCL/SF composite scaffolds. Electrospun PCL/SF-20 and PCL/SF-40 samples both displayed complete wetting, where water contact angles could not be measured, indicating the formation of superhydrophilic surfaces. This can be due the capillary effect generated by hydrophilic SF incorporation and also due to presence of micro/nano pores on the electrospun web surface. Strong interaction of the water droplet with hydrophilic SF present on the fiber surface results in very rapid wetting due to capillary effect and leads to Wenzel type wetting behavior, resulting in a superhydrophilic surface [61]. Superhydrophilic PCL/SF scaffold surfaces are expected to provide an excellent platform for cell attachment and growth.

respectively, leading to good compatibility between the two polymers. Characteristic, very strong C=O stretching peak for PCL is also present at 1722 cm−1 in pure PCL and PCL/SF composites. The presence of SF amide bands along with PCL characteristics bands, such as CH2 stretching (2943 and 2865 cm−1), C=O stretching (1722 cm−1) and CeO stretching (1293 cm−1) confirms the successful incorporation of SF in PCL matrix in electrospun composite scaffolds. These results are also in good agreement with the literature data [58,59]. 3.4. Surface analysis of electrospun PCL and PCL/SF nanofibrous scaffolds Surface morphologies of the electrospun PCL and PCL/SF nanofiber scaffolds were examined by SEM, and are presented in Fig. 4. All SEM images show formation of well-defined electrospun webs from formic acid solutions for PCL control and PCL/SF blends with 20 and 40% by weight SF respectively. As reported in Table 1, it is interesting to note a significant increase in the average fiber diameter with the incorporation of SF, from 150 nm for pure PCL to 200 nm and 280 nm for 20 and 40% SF containing blends, respectively. Wetting behavior of electrospun PCL and PCL/SF scaffolds, which plays critical roles in fiber adhesion was investigated by static water contact angle measurements. For comparison, contact angle values of spin-coated thin films on glass slides were also determined. Contact angle measurements along with the droplet images are provided in Fig. 5. Contact angle for spin coated PCL film was determined to be 89.6 ± 6.9°, indicating a fairly hydrophobic surface. Contact angle values for spin coated PCL/SF composite films display a slight decrease to 73.8 ± 1.0° and 62.8 ± 3.0° indicating improved hydrophilicity for PCL/SF-20 and PCL/SF-40 samples, respectively. Electrospun PCL web displayed a much higher contact angle of 129.1 ± 1.3°, indicating a very hydrophobic surface and Cassie-Baxter type wetting behavior due

3.5. Mechanical properties of electrospun scaffolds Ultimate tensile strength (UTS), elongation at break (EB) and elastic modulus (EM) values of electrospun scaffolds are provided in Table 2. As provided in Table 2, no significant difference was observed in the average UTS values obtained on PCL and PCL/SF composites, whereas a significant decrease was observed in the elongation at break values from 42.2 ± 2.4% (PCL) to 21.1 ± 0.4% (PCL/SF-40). On the other hand, incorporation of SF increased the EM values significantly from 21.6 ± 1.7 MPa (PCL) to 49.3 ± 6 MPa (PCL/SF-20) and 98.1 ± 23.7 MPa (PCL/SF-40). If we compare UTS, EB and EM values of natural human skin [62], then we can say that PCL/SF composite scaffolds may be a suitable option for skin tissue engineering. 3.6. In vitro cell viability analysis CellTiter-Glow reagent enables the luciferase reaction in catalytic presence of ATP. This reaction yields inactive oxyluciferin, adenosine monophosphate, CO2, and light. Light emitted is measured in plate reader. After cell lysis, ATP is released from viable cells only, so luminescence value can be directly correlated with the number of viable cells present [63]. Two-way ANOVA followed by Tukey's multiple comparison test was performed to evaluate results. As presented in Fig. 6, Day-4 viability is significantly higher than Day-1 for all samples (****p < 0.0001). Similarly, on Day-1 and Day-4, significant number of cells were present on composite scaffolds as compared to pristine PCL (****p < 0.0001). Overall, cell viability results show that all scaffolds produced are non-toxic to cells due to increase in number of cells from Day-1 to Day-4 and high proliferation rate observed in the presence of SF. PCL surface is fairly hydrophobic, while in the composite scaffolds SF acts as cell Table 2 Mechanical properties of electrospun nanofibrous scaffolds.

PCL PCL/SF-20 PCL/SF-40 Human Skin [62]

Fig. 5. Static water contact angle measurements for spin coated and electrospun PCL and PCL/SF films and scaffolds. 91

Ultimate tensile strength (MPa)

Elastic modulus (MPa)

Elongation at break (%)

6.8 ± 0.7 6.8 ± 0.1 6.4 ± 0.6 21.6 ± 8.4

21.6 49.3 98.1 83.3

42.2 ± 2.4 23.3 ± 0.2 21.1 ± 0.4 54 ± 17

± ± ± ±

1.7 6 23.7 34.9

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Fig. 6. ATP release in cell seeding analysis measured through luminescence (n = 5).

Fig. 8. Degradation studies on PCL and PCL/SF nanofibrous composite scaffolds.

adhesion molecule (protein) and induces hydrophilicity due to the presence of amide (-CONH) and hydroxyl (-OH) groups as discussed in section 3.4. Studies suggest that high surface energy promotes early cell adhesion, its differentiation and proliferation. Hydrophilicity induced proliferation was also observed for human adipose-derived stem cells [64], pheochromocytoma [65], human fetal osteoblast [66], Human

umbilical vein endothelial, smooth muscle [67] and fibroblast cells [8]. After 4 days of incubation, cells on the scaffold surfaces were fixed with 2.5% glutaraldehyde solution, followed by dehydration with increasing concentration gradient of ethanol in water (30%, 50%, 70%,

Fig. 7. SEM images of cells on scaffold surfaces on Day-4. (a, b) PCL, (c, d) PCL/SF-20 and (e, f) PCL/SF-40. 92

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90% and 100% for 5 min each). Scaffolds were first dried at ambient conditions and then in a vacuum oven at room temperature. Cell containing samples were mounted on double sided carbon tape attached stubs and coated with 15 nm gold layer through sputter coating. SEM images showing the morphology of the cells on electrospun scaffolds after 4 days are provided in Fig. 7 in two different magnifications. Presence of cells on all scaffolds can clearly be seen. Furthermore, as expected, incorporation of SF into PCL dramatically increases the cell adhesion and proliferation. When the cell membranes collapsed due to ethanol dehydration, cells adhered and accumulated on each other causing larger area of spreading.

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3.7. In vitro degradation studies on PCL and PCL/SF nanofibrous scaffolds After incubation in DMEM for specific time period, samples were taken out, washed, dried and subsequently weighed. The results obtained after degradation analysis, are provided in Fig. 8. As expected, pristine PCL scaffolds exhibited almost negligible degradation over 15weeks of incubation, while SF containing scaffolds showed considerable decrease in weight as compared to PCL. After 15 weeks, mass percent remained for PCL/SF-20 and PCL/SF-40 samples were 86.6 ± 4.7% and 77.3 ± 1.2% respectively. With the increase in SF content, mass loss was higher. Slow degradation of PCL and rapid degradation of SF can be a good combination for various applications. SF can be utilized for initial attachment of cells and their growth and degrade before PCL. In the meanwhile, natural ECM will be developed by cells and ultimate support provided by PCL will be slowly diminishing as it degrades. 4. Conclusions Fabrication of polycaprolactone (PCL) and PCL/silk fibroin (SF) nanofibrous scaffolds through electrospinning, which can possibly be used in tissue engineering applications is reported. PCL was synthesized through the ring-opening polymerization of ε-caprolactone using an organic diamine as initiator. SF was obtained through novel degumming process by using milder ammonia solution as a degumming reagent, which exhibited less degradation of silk fibers when compared with conventionally used sodium bicarbonate or sodium carbonate processes. Further, influence of regenerated SF incorporation into PCL was evaluated through biomechanical properties of the electrospun composite scaffolds. SEM analysis showed nanofibrous scaffold development. Successful incorporation of SF in composite scaffolds was confirmed by ATR-FTIR analysis. Wetting behavior of electrospun PCL scaffolds displayed a dramatic change from a fairly hydrophobic surface with a water contact angle of 129.1 ± 1.3° to superhydrophilic (0° contact angle) upon the incorporation of SF. Scaffolds developed were analyzed through cell seeding, using Human BJ Fibroblast cells. ATP assay showed significant improvement in cell proliferation rate in PCL/ SF composite scaffolds, compared with pure PCL. Stress-strain analysis of electrospun scaffolds indicated fairly good tensile strength and elasticity. Degradation studies in DMEM+10% FBS solution at 37 °C indicated increased degradation rate for PCL/SF scaffolds compared to pure PCL. In conclusion, we can say that electrospun PCL/SF nanofibrous composite scaffolds developed through dialysis free processing of silk fibroin are promising substrates for tissue engineering applications. Acknowledgement We would like to thank BIOMICELL Lab., at Koc University for their help in cell culture studies. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.polymer.2019.02.023. 93

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