Enhancing surface properties of breast implants by using electrospun silk fibroin A. A. Valencia–Lazcano A. Rodrıguez–Ortega4
n–Doval,2 E. De La Cruz–Burelo,1,3 E. J. Milla n–Casarrubias,2 ,1 R. Roma
1
n y de Estudios Avanzados del IPN, Department of Science and Technology Development for Society, Centro de Investigacio Ave. IPN 2508, San Pedro Zacatenco 07360, Mexico City, Mexico 2 n y de Estudios Avanzados del IPN, Ave. IPN 2508, Department of Nanoscience and Nanotechnology, Centro de Investigacio San Pedro Zacatenco, Mexico City, Mexico 3 n y de Estudios Avanzados del IPN, Ave. IPN 2508, San Pedro Zacatenco 07360, Physics Department, Centro de Investigacio Mexico City, Mexico 4 cnica Francisco I. Madero, Tepatepec, Hidalgo, Mexico Agrotechnology Engineering Department, Universidad Polite Received 17 April 2017; revised 18 July 2017; accepted 4 August 2017 Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33973 Abstract: In the present study, a new electrospun silk fibroin coating of silicone breast implants with improved biocompatibility and mechanical properties was obtained. Fibrous scaffolds were produced by electrospinning a solution containing silk fibroin, derived from Bombyx mori cocoons, and polyethylene oxide (PEO) to be used as a coating of breast implants. A randomly oriented structure of fibroin/PEO was electrospun on implants as assessed by SEM analysis, roughness measurements and ATR-FTIR spectroscopy. The scaffold showed 0.25 mm diameter fibres, 0.76 mm size superficial pores, arithmetic roughness of 0.632 6 0.12 mm and texture aspect ratio of 0.893 6 0.04. ATR-FTIR spectroscopy demonstrates the presence of PEO and fibroin in the coating. The mechanical characterisation of the implants before and after being coated with fibroin/PEO demonstrated that
the fibroin/PEO scaffold contributes to the increase in the elastic modulus from 0.392 6 0.02 to 0.560 6 0.03 MPa and to a more elastic behaviour of the breast implants. Using the fibroin/PEO coating, human fibroblasts seeded on this matrix increased viability up to 30% compared to conventional breast implants. Electrospun silk fibroin could represent a clinically compatible, viable form to coat breast implants. Low cytotoxicity by the fibroin coating and its physico-chemical and mechanical properties may find application in improving breast implants bioC 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: compatibility. V Appl Biomater 00B: 000–000, 2017.
Key Words: breast implants, silk fibroin, electrospinning, physico-chemical and mechanical characterisation
n–Doval R, De La Cruz–Burelo E, Milla n–Casarrubias EJ, Rodrıguez–Ortega How to cite this article: Valencia–Lazcano AA, Roma A. 2017. Enhancing surface properties of breast implants by using electrospun silk fibroin. J Biomed Mater Res Part B 2017:00B:000–000.
INTRODUCTION
Notwithstanding the developments on breast implants designed for reconstruction following mastectomy or for cosmetic breast augmentation, around 30% of women with breast implants report complications related with infection, capsular contracture, prosthesis rupture and gel bleed.1,2 There are many factors related to these complications that go from the ones attributed to the implant, (silicone composition, filler material, and surface texture), the surgical handling, manufacturing artefacts and the silicone response in the body.3 To face these complications is necessary a multidisciplinary approach that takes into account the mechanical properties and the creation of biocompatible surfaces of the breast implants.
As is well known a capsule surrounds de breast implant once it is inserted in the body. According to Wolfram,4 this capsule consists of three layers, the tissue in contact with the implant contains fibroblasts, the next layer is composed of loosely arranged connective tissue and internal vascular supply and the last layer is dense connective tissue with external vascular supply. In an attempt to disrupt capsule formation, changes in the surface of the shell of breast implants has been modified.5 As Maxwell and Gabriel describes how the main breast implants manufacturers in the United States modify the surface texture of their implants: Mentor Corporation creates the Siltex surface by imprint stamping, Sientra produces the TRUE Texture by making small hollow pores and Allergan creates the BioCell texture using the loss-salt technique.6
Disclosure: We confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. Correspondence to: A. A. Valencia-Lazcano; e-mail: [email protected] n y de Estudios Avanzados del Instituto Polite cnico Nacional; Consejo Nacional de Ciencia y Contract grant sponsors: Centro de Investigacio Tecnologıa
C 2017 WILEY PERIODICALS, INC. V
1
Therefore, it is important to mimic the natural environment for fibroblasts on the surface of the breast implant. The typical diameter of protein fibril/fibres in native extracellular matrix (ECM) is from several tens to 300 nm.7 In this regard, a technique used to create tissue-engineering scaffolds is electrospinning,8 which produces nano and micro-fibres from a solution by accelerating a charged polymer jet in a very high electric field toward a collecting target.9,10 Electrospinning tailors a network of fibres that mimic the structure of the ECM with great biocompatibility.11 In addition to the physical support for cells that electrospun fibres provide, a scaffold should promote tissue regeneration in vitro as native ECM does in vivo.7 Despite the silicone used to make breast implant shells is considered inert; some silicones have clear biologic effects so a chemical composition necessary to minimize these effects should be proposed.12 Natural polymers such as fibroin isolated from Bombyx mori cocoons has been studied for biomedical applications due to its biological activity, controlled degradability,13 and capability of turning to varied structural formats.14 Since its ancient use as suture, silk fibroin has showed to be widely used biomaterial due to its low immunogenicity and mechanical stability.15 Native Bombyx mori silk is composed of silk fibroin protein coated by sericin proteins. Sericins are adhesive proteins that account for 25–30% of the total silkworm cocoon by weight.16 The silk fibroin consists of a light chain (Mw approximately 26 kDa) and a heavy chain (Mw 390 kDa) linked by a disulfide bond.14 In terms of improving the mechanical properties, due to inserting an implant into the breast pocket involves a mechanical challenge considering the use of surgical instruments and hands employed in. In comparison to other polymers silk fibroin showed to be noncytotoxic and lower antigenicity.17 Meinel et al. showed similar or less in vivo inflammatory reaction and foreign body response to silk and collagen films.18 Silk fibroin has been showed to combine high strength and extensibility19 so it can protect the breast implant against mechanical damage and allowed to be manipulated during the surgery. In the present study, an electrospun silk fibroin scaffold on textured breast implants was evaluated as a potential coating for breast implants. Fundamental results are shown for understanding the performance, and potential as a candidate coating are discussed in terms of tensile properties, chemical properties, and microscopic observations to protect the breast implant against mechanical damage due to surgery and prevent breast implant puncture. In vitro biocompatibility of fibroin/PEO was evaluated by methyl thiazolyl tetrazolium (MTT) assay using breast-derived fibroblasts. The results support the utility of the silk fibroin matrix as potential coating for breast implants. MATERIALS AND METHODS
The breast prostheses studied correspond to commercially available implants used in clinical practice. The type of breast implant used in this research was a cohesive silicone gel-filled round implant of midrange profile. Breast implant shells of textured surface are made of poly (dimethylsiloxane) (PDMS),
2
VALENCIA–LAZCANO ET AL.
whose composition of amorphous silica is 21–27% in the elastomer for the shell and in shell patches, and 16.5% in barrier coats. Samples for the different assays were removed from each breast implant shell. They were sonicated in water with detergent for 10 min, and rinsed in distilled water. This procedure was repeated using acetone and then ethanol. Samples were air dried and kept in petri dishes until used. Fibroin/PEO coating preparation Bombyx mori cocoon shells were supplied by the Polytechnic University Francisco I. Madero in Mexico.20 Cocoons were cut in thirds and worm and debris were discarded. Degumming process was utilized to remove sericin coating from silk fibre by boiling 5.0 g of cocoons in 2.0 L of distilled water for 30 min; cocoons were washed thoroughly before and after being boiled. Fibres were allowed to dry overnight in a flow cabin. Silk fibres were dissolved in 9.3 M LiBr for a 15% (w/v) solution of silk. Silk fibroin/LiBr solution was incubated at 608C for 4 h, stirring gently. Lithium bromide was removed followed by silk fibroin concentration in a stirred cell for 7 h (Stirred Cell Model 8010, Amicon). To obtain the required viscosity for electrospinning, 2.5 wt % PEO (900 kDa, Sigma) solution was added to fibroin. Solution was 8–8.5pH. The electrospinning process was carried on a commercial electrospinning instrument (Standard unit, NEU-01). The fibroin/PEO solution was transferred to a 10 mL syringe. An electrical field of 10 kV was applied to the fibroin/PEO solution to attain a stable Taylor cone. The solution was feed into the needle at a flow rate of 1 mL h21 by a syringe pump. An electrospun fibrous mat was collected on a grounded 10 3 10 cm2 aluminium plate with the breast implant attached to it. The collecting device was placed at a distance of 10 cm from the needle and the temperature of the system was at 258C. The x-axial sliding device moved the base of the needle at 2.5 RPS. Characterisation The morphology of the electrospun silk fibroin/PEO on breast implants was observed by high-resolution scanning electron microscopy. The chemical characterisation of the scaffold was analysed by attenuated total reflectance Fourier transforms infrared (ATR-FTIR). The mechanical characterisation was performed by puncture tensile tests. Roughness of the surface of the coating was imaged by an optical microscope and measured by Gwyddion Software. In addition, cell viability was tested by MTT assays. Morphology analysis: Scanning electron microscopy The morphology of the electrospun scaffolds of fibroin was characterised by high–resolution scanning electron microscopy (HRSEM-AURIGA) at 1.0 kV. The samples were analysed at different magnifications (80, 500, 1000, 3000, and 50003). Diameter of 600 fibres was measured at random on images of magnification of 30003 using the Image J software to obtain the mean fibre diameter, superficial pore size and standard deviation.
ENHANCING SURFACE PROPERTIES OF BREAST IMPLANTS
ORIGINAL RESEARCH REPORT
FIGURE 1. SEM images of the fibroin/PEO electrospun on breast implants. SEM image at 380 (left hand side) and SEM image at 33000 (right hand side).
ATR-FTIR spectroscopy. The electrospun scaffolds were analysed by Attenuated total reflectance Fourier transforms infrared (ATR-FTIR) spectroscopic (VARIAN 640-IR) to observe the functional groups of PEO and fibroin incorporated in the scaffolds. Mechanical characterisation. The mechanical characteristics of the fibroin-PEO coated silicone breast implants were analysed to evaluate the development of the fibroin-PEO coating on the breast implants. Tensile tests were performed to determine the mechanical development of the coating and the implant while being stretched, as breast implants are manipulated at the time of being inserted into the body. The tensile tests were performed on a Texture Analyser (Stable Micro Systems, UK) equipped with a 25 kg load cell. Breast implant discs of 38 mm diameter uncoated and coated with electrospun fibroin/PEO were tested in quintuplicate each in order to see if the coating provides more resistance to puncture. A sample was mounted horizontally on the holder. During the tensile test, a cylindrical flat probe penetrated the sample at a constant speed of 1 mm s21. Force versus deformation data was recorded and the mechanical parameters were determined using Exponent software (Stable Micro Systems, UK). Roughness. Roughness of the surface of five samples was measured before and after coating the breast implants using the Contour EliteTM 3 D optical microscope (BRUKER, USA). Gwyddion software was used for data visualization and analysis. Two parameters of roughness were selected to evaluate the surface topography; arithmetic roughness (Ra) and texture aspect ratio (Str), of the coated and uncoated breast implants to evaluate the effects of the electrospun fibroin on the surface of the implants. Cell viability Normal human breast fibroblasts were purchased from Sigma (Sigma, U.S.A.). All cultures were passage 2 and were grown to confluence in 125-mL culture flasks in Dulbecco’s Culture Medium (DMEM) (Sigma–Aldrich, UK) substituted with 10% foetal bovine serum (Sigma–Aldrich, USA), 1%
L-glutamine (PAA laboratories, Austria), 1% non-essential amino acid solution (Sigma–Aldrich, USA) and 1% penicillin/ streptomycin (PAA laboratories, Austria) at 378C in a 5% CO2 atmosphere. Prior to seeding the fibroblasts on the surfaces, fibroblasts were arrested to take them to the G0/G1 phase. Breast implant discs of 15 mm diameter uncoated and coated with the electrospun silk fibroin/PEO were placed on each of the wells of a 24-well-plate. About 2 mL of 80% ethanol was poured into each well, after 20 min the ethanol was discarded, and then samples were washed three times with PBS and allowed to dry in a culture hood. Fibroblasts were seeded at a concentration of 10,000 cells per well in 300 mL culture medium. After 24-h incubation in a humidified atmosphere of 378C and 5% CO2, 30 ml of MTT (Roche, USA) was added to each well. The plate was incubated for 4 h in a humidified atmosphere of 378C and 5% CO2. After the incubation period, medium is carefully removed and 300 mL of the solubilisation solution was added into each well. The plate was covered with tinfoil and placed on an orbital shaker for 15 min. The spectrophotometric absorbance was measured at 590 nm with a reference filter of 620 nm. Statistical analysis Statistical analysis was performed using the Prism v.5.0 software (GraphPad Software, USA), applying the one-way ANOVA test; when the p values was
n–Doval,2 E. De La Cruz–Burelo,1,3 E. J. Milla n–Casarrubias,2 ,1 R. Roma
1
n y de Estudios Avanzados del IPN, Department of Science and Technology Development for Society, Centro de Investigacio Ave. IPN 2508, San Pedro Zacatenco 07360, Mexico City, Mexico 2 n y de Estudios Avanzados del IPN, Ave. IPN 2508, Department of Nanoscience and Nanotechnology, Centro de Investigacio San Pedro Zacatenco, Mexico City, Mexico 3 n y de Estudios Avanzados del IPN, Ave. IPN 2508, San Pedro Zacatenco 07360, Physics Department, Centro de Investigacio Mexico City, Mexico 4 cnica Francisco I. Madero, Tepatepec, Hidalgo, Mexico Agrotechnology Engineering Department, Universidad Polite Received 17 April 2017; revised 18 July 2017; accepted 4 August 2017 Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33973 Abstract: In the present study, a new electrospun silk fibroin coating of silicone breast implants with improved biocompatibility and mechanical properties was obtained. Fibrous scaffolds were produced by electrospinning a solution containing silk fibroin, derived from Bombyx mori cocoons, and polyethylene oxide (PEO) to be used as a coating of breast implants. A randomly oriented structure of fibroin/PEO was electrospun on implants as assessed by SEM analysis, roughness measurements and ATR-FTIR spectroscopy. The scaffold showed 0.25 mm diameter fibres, 0.76 mm size superficial pores, arithmetic roughness of 0.632 6 0.12 mm and texture aspect ratio of 0.893 6 0.04. ATR-FTIR spectroscopy demonstrates the presence of PEO and fibroin in the coating. The mechanical characterisation of the implants before and after being coated with fibroin/PEO demonstrated that
the fibroin/PEO scaffold contributes to the increase in the elastic modulus from 0.392 6 0.02 to 0.560 6 0.03 MPa and to a more elastic behaviour of the breast implants. Using the fibroin/PEO coating, human fibroblasts seeded on this matrix increased viability up to 30% compared to conventional breast implants. Electrospun silk fibroin could represent a clinically compatible, viable form to coat breast implants. Low cytotoxicity by the fibroin coating and its physico-chemical and mechanical properties may find application in improving breast implants bioC 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: compatibility. V Appl Biomater 00B: 000–000, 2017.
Key Words: breast implants, silk fibroin, electrospinning, physico-chemical and mechanical characterisation
n–Doval R, De La Cruz–Burelo E, Milla n–Casarrubias EJ, Rodrıguez–Ortega How to cite this article: Valencia–Lazcano AA, Roma A. 2017. Enhancing surface properties of breast implants by using electrospun silk fibroin. J Biomed Mater Res Part B 2017:00B:000–000.
INTRODUCTION
Notwithstanding the developments on breast implants designed for reconstruction following mastectomy or for cosmetic breast augmentation, around 30% of women with breast implants report complications related with infection, capsular contracture, prosthesis rupture and gel bleed.1,2 There are many factors related to these complications that go from the ones attributed to the implant, (silicone composition, filler material, and surface texture), the surgical handling, manufacturing artefacts and the silicone response in the body.3 To face these complications is necessary a multidisciplinary approach that takes into account the mechanical properties and the creation of biocompatible surfaces of the breast implants.
As is well known a capsule surrounds de breast implant once it is inserted in the body. According to Wolfram,4 this capsule consists of three layers, the tissue in contact with the implant contains fibroblasts, the next layer is composed of loosely arranged connective tissue and internal vascular supply and the last layer is dense connective tissue with external vascular supply. In an attempt to disrupt capsule formation, changes in the surface of the shell of breast implants has been modified.5 As Maxwell and Gabriel describes how the main breast implants manufacturers in the United States modify the surface texture of their implants: Mentor Corporation creates the Siltex surface by imprint stamping, Sientra produces the TRUE Texture by making small hollow pores and Allergan creates the BioCell texture using the loss-salt technique.6
Disclosure: We confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. Correspondence to: A. A. Valencia-Lazcano; e-mail: [email protected] n y de Estudios Avanzados del Instituto Polite cnico Nacional; Consejo Nacional de Ciencia y Contract grant sponsors: Centro de Investigacio Tecnologıa
C 2017 WILEY PERIODICALS, INC. V
1
Therefore, it is important to mimic the natural environment for fibroblasts on the surface of the breast implant. The typical diameter of protein fibril/fibres in native extracellular matrix (ECM) is from several tens to 300 nm.7 In this regard, a technique used to create tissue-engineering scaffolds is electrospinning,8 which produces nano and micro-fibres from a solution by accelerating a charged polymer jet in a very high electric field toward a collecting target.9,10 Electrospinning tailors a network of fibres that mimic the structure of the ECM with great biocompatibility.11 In addition to the physical support for cells that electrospun fibres provide, a scaffold should promote tissue regeneration in vitro as native ECM does in vivo.7 Despite the silicone used to make breast implant shells is considered inert; some silicones have clear biologic effects so a chemical composition necessary to minimize these effects should be proposed.12 Natural polymers such as fibroin isolated from Bombyx mori cocoons has been studied for biomedical applications due to its biological activity, controlled degradability,13 and capability of turning to varied structural formats.14 Since its ancient use as suture, silk fibroin has showed to be widely used biomaterial due to its low immunogenicity and mechanical stability.15 Native Bombyx mori silk is composed of silk fibroin protein coated by sericin proteins. Sericins are adhesive proteins that account for 25–30% of the total silkworm cocoon by weight.16 The silk fibroin consists of a light chain (Mw approximately 26 kDa) and a heavy chain (Mw 390 kDa) linked by a disulfide bond.14 In terms of improving the mechanical properties, due to inserting an implant into the breast pocket involves a mechanical challenge considering the use of surgical instruments and hands employed in. In comparison to other polymers silk fibroin showed to be noncytotoxic and lower antigenicity.17 Meinel et al. showed similar or less in vivo inflammatory reaction and foreign body response to silk and collagen films.18 Silk fibroin has been showed to combine high strength and extensibility19 so it can protect the breast implant against mechanical damage and allowed to be manipulated during the surgery. In the present study, an electrospun silk fibroin scaffold on textured breast implants was evaluated as a potential coating for breast implants. Fundamental results are shown for understanding the performance, and potential as a candidate coating are discussed in terms of tensile properties, chemical properties, and microscopic observations to protect the breast implant against mechanical damage due to surgery and prevent breast implant puncture. In vitro biocompatibility of fibroin/PEO was evaluated by methyl thiazolyl tetrazolium (MTT) assay using breast-derived fibroblasts. The results support the utility of the silk fibroin matrix as potential coating for breast implants. MATERIALS AND METHODS
The breast prostheses studied correspond to commercially available implants used in clinical practice. The type of breast implant used in this research was a cohesive silicone gel-filled round implant of midrange profile. Breast implant shells of textured surface are made of poly (dimethylsiloxane) (PDMS),
2
VALENCIA–LAZCANO ET AL.
whose composition of amorphous silica is 21–27% in the elastomer for the shell and in shell patches, and 16.5% in barrier coats. Samples for the different assays were removed from each breast implant shell. They were sonicated in water with detergent for 10 min, and rinsed in distilled water. This procedure was repeated using acetone and then ethanol. Samples were air dried and kept in petri dishes until used. Fibroin/PEO coating preparation Bombyx mori cocoon shells were supplied by the Polytechnic University Francisco I. Madero in Mexico.20 Cocoons were cut in thirds and worm and debris were discarded. Degumming process was utilized to remove sericin coating from silk fibre by boiling 5.0 g of cocoons in 2.0 L of distilled water for 30 min; cocoons were washed thoroughly before and after being boiled. Fibres were allowed to dry overnight in a flow cabin. Silk fibres were dissolved in 9.3 M LiBr for a 15% (w/v) solution of silk. Silk fibroin/LiBr solution was incubated at 608C for 4 h, stirring gently. Lithium bromide was removed followed by silk fibroin concentration in a stirred cell for 7 h (Stirred Cell Model 8010, Amicon). To obtain the required viscosity for electrospinning, 2.5 wt % PEO (900 kDa, Sigma) solution was added to fibroin. Solution was 8–8.5pH. The electrospinning process was carried on a commercial electrospinning instrument (Standard unit, NEU-01). The fibroin/PEO solution was transferred to a 10 mL syringe. An electrical field of 10 kV was applied to the fibroin/PEO solution to attain a stable Taylor cone. The solution was feed into the needle at a flow rate of 1 mL h21 by a syringe pump. An electrospun fibrous mat was collected on a grounded 10 3 10 cm2 aluminium plate with the breast implant attached to it. The collecting device was placed at a distance of 10 cm from the needle and the temperature of the system was at 258C. The x-axial sliding device moved the base of the needle at 2.5 RPS. Characterisation The morphology of the electrospun silk fibroin/PEO on breast implants was observed by high-resolution scanning electron microscopy. The chemical characterisation of the scaffold was analysed by attenuated total reflectance Fourier transforms infrared (ATR-FTIR). The mechanical characterisation was performed by puncture tensile tests. Roughness of the surface of the coating was imaged by an optical microscope and measured by Gwyddion Software. In addition, cell viability was tested by MTT assays. Morphology analysis: Scanning electron microscopy The morphology of the electrospun scaffolds of fibroin was characterised by high–resolution scanning electron microscopy (HRSEM-AURIGA) at 1.0 kV. The samples were analysed at different magnifications (80, 500, 1000, 3000, and 50003). Diameter of 600 fibres was measured at random on images of magnification of 30003 using the Image J software to obtain the mean fibre diameter, superficial pore size and standard deviation.
ENHANCING SURFACE PROPERTIES OF BREAST IMPLANTS
ORIGINAL RESEARCH REPORT
FIGURE 1. SEM images of the fibroin/PEO electrospun on breast implants. SEM image at 380 (left hand side) and SEM image at 33000 (right hand side).
ATR-FTIR spectroscopy. The electrospun scaffolds were analysed by Attenuated total reflectance Fourier transforms infrared (ATR-FTIR) spectroscopic (VARIAN 640-IR) to observe the functional groups of PEO and fibroin incorporated in the scaffolds. Mechanical characterisation. The mechanical characteristics of the fibroin-PEO coated silicone breast implants were analysed to evaluate the development of the fibroin-PEO coating on the breast implants. Tensile tests were performed to determine the mechanical development of the coating and the implant while being stretched, as breast implants are manipulated at the time of being inserted into the body. The tensile tests were performed on a Texture Analyser (Stable Micro Systems, UK) equipped with a 25 kg load cell. Breast implant discs of 38 mm diameter uncoated and coated with electrospun fibroin/PEO were tested in quintuplicate each in order to see if the coating provides more resistance to puncture. A sample was mounted horizontally on the holder. During the tensile test, a cylindrical flat probe penetrated the sample at a constant speed of 1 mm s21. Force versus deformation data was recorded and the mechanical parameters were determined using Exponent software (Stable Micro Systems, UK). Roughness. Roughness of the surface of five samples was measured before and after coating the breast implants using the Contour EliteTM 3 D optical microscope (BRUKER, USA). Gwyddion software was used for data visualization and analysis. Two parameters of roughness were selected to evaluate the surface topography; arithmetic roughness (Ra) and texture aspect ratio (Str), of the coated and uncoated breast implants to evaluate the effects of the electrospun fibroin on the surface of the implants. Cell viability Normal human breast fibroblasts were purchased from Sigma (Sigma, U.S.A.). All cultures were passage 2 and were grown to confluence in 125-mL culture flasks in Dulbecco’s Culture Medium (DMEM) (Sigma–Aldrich, UK) substituted with 10% foetal bovine serum (Sigma–Aldrich, USA), 1%
L-glutamine (PAA laboratories, Austria), 1% non-essential amino acid solution (Sigma–Aldrich, USA) and 1% penicillin/ streptomycin (PAA laboratories, Austria) at 378C in a 5% CO2 atmosphere. Prior to seeding the fibroblasts on the surfaces, fibroblasts were arrested to take them to the G0/G1 phase. Breast implant discs of 15 mm diameter uncoated and coated with the electrospun silk fibroin/PEO were placed on each of the wells of a 24-well-plate. About 2 mL of 80% ethanol was poured into each well, after 20 min the ethanol was discarded, and then samples were washed three times with PBS and allowed to dry in a culture hood. Fibroblasts were seeded at a concentration of 10,000 cells per well in 300 mL culture medium. After 24-h incubation in a humidified atmosphere of 378C and 5% CO2, 30 ml of MTT (Roche, USA) was added to each well. The plate was incubated for 4 h in a humidified atmosphere of 378C and 5% CO2. After the incubation period, medium is carefully removed and 300 mL of the solubilisation solution was added into each well. The plate was covered with tinfoil and placed on an orbital shaker for 15 min. The spectrophotometric absorbance was measured at 590 nm with a reference filter of 620 nm. Statistical analysis Statistical analysis was performed using the Prism v.5.0 software (GraphPad Software, USA), applying the one-way ANOVA test; when the p values was
