Effects of sterilization methods on the physical, chemical, and biological properties of silk fibroin membranes Mariana Agostini de Moraes, Raquel Farias Weska, Marisa Masumi Beppu School of Chemical Engineering, University of Campinas, UNICAMP, 13083-852, Campinas, SP, Brazil Received 10 August 2013; accepted 13 October 2013 Published online 21 November 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33069 Abstract: Silk fibroin has been widely explored for many biomedical applications, due to its biocompatibility and biodegradability. Sterilization is a fundamental step in biomaterials processing and it must not jeopardize the functionality of medical devices. The aim of this study was to analyze the influence of different sterilization methods in the physical, chemical, and biological characteristics of dense and porous silk fibroin membranes. Silk fibroin membranes were treated by several procedures: immersion in 70% ethanol solution, ultraviolet radiation, autoclave, ethylene oxide, and gamma radiation, and were analyzed by scanning electron microscopy, Fourier-transformed infrared spectroscopy (FTIR), X-ray diffraction, tensile strength and in vitro cytotoxicity to Chinese hamster ovary cells. The results indicated that the sterilization methods did not cause perceivable morphological
changes in the membranes and the membranes were not toxic to cells. The sterilization methods that used organic solvent or an increased humidity and/or temperature (70% ethanol, autoclave, and ethylene oxide) increased the silk II content in the membranes: the dense membranes became more brittle, while the porous membranes showed increased strength at break. Membranes that underwent sterilization by UV and gamma radiation presented properties similar to the nonsterilized membranes, mainly for tensile strength and C 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part FTIR results. V B: Appl Biomater, 102B: 869–876, 2014.
Key Words: porous membranes, dense membranes, fibroin, sterilization, biomaterials
How to cite this article: de Moraes MA, Weska RF, Beppu MM. 2014. Effects of sterilization methods on the physical, chemical, and biological properties of silk fibroin membranes. J Biomed Mater Res Part B 2014:102B:869–876.
INTRODUCTION
Silk fibroin (SF) is a fibrous protein extracted from cocoons of Bombyx mori silkworm. SF exhibit two conformational forms, called silk I and II. Silk I is the water-soluble and meta-stable form, with dominating random coil and a-helix structures. Silk II conformation is the hydrophobic form, dominated by b-sheet structures that are thermodynamically stable presenting strong hydrogen bonds and Van der Waals forces.1,2 SF transition from silk I to silk II can be achieved using organic solvents or physical shear.3,4 SF presents good biocompatibility, good oxygen, and water vapor permeability, biodegradability, minimal inflammatory reaction, can be prepared in different morphologies, presents high mechanical strength, high thermal stability, and microbial resistance.5–7 It has been widely explored for biomedical applications, such as substrate for enzymes immobilization, wound dressings, scaffolds for bone, cartilage and tendons regeneration, among others 1,8. In addition, the use of SF as biomaterial is approved by Food and Drug Administration as nonabsorbable sutures, and for soft tissue repair. The sterilization process is an essential step for the development of a material or device that will be used as biomaterial. The optimum procedure should sterilize the
material without altering its functionality, mainly regarding biomechanical and biocompatible properties.9 This is a complex issue in natural polymers, since they can be easily damaged by the severe sterilization conditions. SF is a protein and has a complex structure that may be affected by heat or irradiation, resulting in changes on its secondary and tertiary structure and, consequently, on the physical and/or biological properties of the SF-based material. Thus, the investigation of the material properties after sterilization is necessary to understand the alterations that may be caused by sterilization method. Several papers report the sterilization of SF matrices by different methods, such as immersion in 70% ethanol solution,10 UV radiation for 30 min,11 autoclave,12 ethylene oxide,13 and gamma radiation,14 but none of them studied the influence of sterilization on the physical–chemical and biological properties of those matrixes. The treatment with ethanol is considered to be a disinfectant method, rather than a sterilization, since ethanol does not act on the endospores of most bacteria species, limiting its use as a surface-sterilizing agent.15 Nevertheless, the treatment with ethanol is the most used disinfecting method for several materials.15,16
Correspondence to: M. M. Beppu (E - mail: [email protected])
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UV radiation has the DNA as main target for microorganisms’ elimination. UV radiation has strong antimicrobial action, but it is not capable to penetrate solid matter, despite being highly absorbed by glasses and plastics.17 Autoclave is a well-known sterilization method that applies saturated vapor at 121 C, which is capable to destroy metabolic and structural components of the microorganisms. Autoclave is extensively used in laboratory and industrial scales because of its efficacy, easiness to process, and for not producing toxic residues. However, the high values of temperature and humidity required for autoclave are the main restrain for its use with polymeric, and mainly for biopolymeric materials. The most used method to sterilize polymers that are sensible to heat and humidity is the exposure to ethylene oxide, because this method is effective at low temperatures and has high penetration in a wide range of materials. However, ethylene oxide can chemically react with the material and it is a toxic gas, requiring repeated air washings after sterilization.18 Finally, gamma radiation is the most used method to sterilize biomaterials, because of its efficiency and price.19 Gamma rays destroy the microorganisms by ionizing cell components, especially nucleic acids. The main advantages of this method are the high penetration, low chemical reactivity, low content of residues and low temperature raise. However, some materials can be degraded when irradiated or cross links can be formed, altering the material structure.20 As we can see, each sterilization method has advantages and limitations, and the choice of a suitable method will depend on each material. In this context, the aim of this study is to investigate the influence of five different sterilization methods on the physical, chemical, and biological properties of SF membranes. The sterilization methods proposed to be used in this work were 70% ethanol, UV radiation, autoclave, ethylene oxide, and gamma radiation. We also analyzed fibroin membranes processed by different ways and, therefore, with different morphological features, such as dense and porous SF membranes. MATERIALS AND METHODS
Preparation of SF Solution Raw silk fibers of Bombyx mori silkworm (supplied by Bratac-Brazil) were degummed three times in 5 g L21 Na2CO3 solution at 85 C, for 30 min, to remove the sericin. Fibers were washed with deionized water, dried at room temperature, and dissolved in a solvent of CaCl2:CH3CH2OH:H2O (1:2:8 molar) at 85 C to a concentration of 10 wt %. Preparation of SF Dense Membranes SF dense membranes were prepared according to our previously established method.4 In brief, SF salt solution was dialyzed in distilled water for 3 days, to remove the salts of the solvent. The dialyzed solution was then cast in polystyrene Petri dishes and the membranes were formed by solvent evaporation at room temperature. The membranes were immersed in 70% ethanol for 20 min to reduce water
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solubility, and then washed with deionized water. Finally, the membranes were dried at 25 C before sterilization and characterization procedures. Preparation of SF Porous Membranes SF porous membranes preparation method was developed by our group and it is patented.21 In brief, SF salt solution was diluted in distilled water (1:100) and foaming was induced by mechanical stirring. SF foam was compressed to remove the excess of water and promote foam agglomeration. Thickness and diameter of the membranes could be controlled by the quantity of compressed foam. Membranes were then immersed in 70% ethanol for 20 min and washed with deionized water. Finally, membranes were frozen in an ultrafreezer at 280 C, for 24 h, and lyophilized for 24 h in a freeze-drier before being submitted to sterilization. Sterilization Methods We studied the influence of five different sterilization methods in the physical, chemical, and biological characteristics of SF membranes. The studied methods were immersion in 70% ethanol solution, UV radiation, autoclave, ethylene oxide exposure, and gamma radiation, briefly described below. Nonsterilized membranes were used as control. Dense and porous SF membranes were immersed in 70 vol % ethanol, for 48 h, at 8 C. Membranes were washed in deionized water, frozen, and lyophilized before the characterizations. For the cytotoxicity test, the membranes were washed in 0.9% NaCl solution. For UV radiation, the membranes were exposed to UV light (k 5 254 nm) for 30 min on each side. Autoclave was used at 121 C for 30 min in dense and porous SF membranes. Sterilization with ethylene oxide was done by Acecil Central de Esterilizac¸~ao Comercio Ind ustria Ltda. (Campinas, SP, Brazil). Samples were exposed to Oxyfume-30 (30% ethylene oxide and 70% carbon dioxide) for 4 h at 50 C, under 400–450 mmHg vacuum and 0.5–0.6 kgf cm22 pressure. Samples were aired three times with nitrogen (N2) for removal of residual ethylene oxide. The membranes were stored at room temperature for 72 h before testing. Sterilization by gamma radiation was performed at Energy and Nuclear Research Institute (IPEN, S~ao Paulo, Brazil) by exposing the films to a constant rate of gamma radiation of 2.27 kGy h21, for 11 h, in a total dose of 25 kGy, at room temperature. Characterization The morphology of the membranes was observed by scanning electron microscopy (SEM), using a LEO 440i (Leica), with accelerating voltage of 20 kV for dense membranes and 15 kV for porous membranes. Samples were frozen in liquid nitrogen, fractured, lyophilized (Liobras, L101, Brazil) for 24 h, and coated with a gold layer before SEM observation. We investigated the secondary structure of SF membranes by Fourier transform infrared spectroscopy using a Bomem MB 102, equipped with attenuated total reflectance (FTIR-ATR) accessory and ZnSe crystal. The spectra were
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obtained in the range of 800–1800 cm21, with 4 cm21 resolution and 256 scans. X-ray diffraction (XRD) was performed in a X’PERT PW3050 Philips, using a monochromatic Cu-Ka radiation (k 5 1.54 Å), in the 2h range of 5 –35 and scanning rate of 0.6 min21. Tensile tests were performed according to ASTM D88202 using a TA.XT2 texture analyzer (Stable Microsystems SMD). Five samples of each membrane (8 3 2.5 cm) were stored under standard conditions (25 C, RH 50%) for 72 h before the tests. An initial grip separation of 50 mm and crosshead speed of 10 mm s21 were used. Cytotoxicity test was done on dense and porous SF membranes according to ISO 10993-5 (2009) using Chinese hamster ovary cell line (CHO-k1), maintained in RPMI culture medium. Extracts of porous and dense SF membranes were prepared in a proportion of 1 cm2 of membrane by 1 mL of culture medium, at 37 C for 48 h. Extracts were prepared in RPMI. Phenol 0.4% was used as positive control and high density polyethylene was used as negative control. Cell suspension and extract were added in 96-well microplate, incubated for 72 h at 37 C in a 5% CO2 humidified atmosphere. The cell viability was measured by adding MTS (supravital dye tetrazolium compound)/PMS (electron coupling agent) (20/1) solution and incubating for 2 h. The microplate was analyzed in a spectrophotometer ELISA at 495 nm. RESULTS
Morphology SF membranes presented thicknesses of about 100 lm and 3.5 mm for dense and porous membranes, respectively. Macroscopic changes in membrane morphology were not observed after sterilization (Figure 1), except for autoclaved membranes. After sterilization by autoclave, SF dense membrane became slightly yellow, while SF porous membrane had a reduction of about 3 mm in diameter, probably due to the rearrangement of SF molecular chains caused by water vapor. The structural integrity of the membranes after sterilization by the several tested methods was preserved. All the membranes remain unaltered after sterilization. SEM observations (Figure 2) did not show morphological changes in the membranes after sterilization. Dense membranes presented regular and homogeneous surface and fracture surface. Porous membrane surface was rough and a porous fracture surface could be seen by SEM. Fourier-transformed infrared spectroscopy SF molecular conformation can be detected by Fouriertransformed infrared spectroscopy (FTIR). Bands of amide I, II, and III are attributed to C@O stretch, NAH deformation and OACAN folding, respectively. Figure 3 shows the FTIR-ATR spectra for dense and porous SF membranes. For dense membranes (Figures 3-1), amide II bands were located in the region of silk II conformation (1514 cm21) and amide III bands were located in the region of silk I conformation (1235 cm21) for all samples.22–24 These results occurred probably due to the preparation
method of dense membranes. SF dense membranes prepared from SF dialyzed solution and dried at room temperature had predominant silk I conformation. To obtain hydrophobic membranes, a treatment with 70% ethanol for 20 min is done, which increase the silk II conformation.25–27 Amide I band was located in different wavelengths, depending on the sterilization method. In nonsterilized membranes and sterilized by UV or gamma radiation, amide I peaks were observed at 1645 cm21 (silk I), whilst in membranes sterilized by ethanol and autoclave the peak was located at 1622 cm21 (silk II). This indicates that after sterilization by 70% ethanol and autoclave, SF dense membrane had an increased content of b-sheet when compared with the nonsterilized membrane (control),22–24 demonstrating that in this membranes C@O groups are involved in more hydrogen bonds than in the other membranes, increasing b-sheet content.24 Lawrence et al. (2008)27 observed that autoclave increases silk II conformation in SF dense membranes, even when they were not treated with organic solvents. Our result also indicates that exposure of SF dense membranes to high temperature, humidity, and pressure induce an increase in b-sheet content, an effect similar to that of ethanol or methanol treatment.25,26 SF dense membrane sterilized by ethylene oxide showed peaks of the same intensity in 1622 and 1645 cm21, indicating a higher proportion of silk II conformation than the nonsterilized membrane, but lower than the membranes sterilized by 70% ethanol or autoclave. The treatment of membranes with ethanol, associated with the temperature of 55 C of the ethylene oxide sterilization was probably the factor that increased b-sheet content. The absence of peaks not related to SF in the membrane sterilized by ethylene oxide indicates that there were no chemical reactions in the process and that the aeration process was successful in the removal of ethylene oxide.18 The FTIR spectra of porous membranes nonsterilized and after sterilization by the several methods were similar (Figure 3-2). SF porous membranes show peaks at 1622 and 1517 cm21, attributed to silk II structure, and at 1230 cm21, indicating the presence of silk I structure.22–24 SF porous membrane nonsterilized has already a higher content of silk II than I, probably related to the way the membrane was prepared. Usually, SF salt solution (used to produce SF porous membrane) has silk I conformation due to solvation of SF molecules by the solvents. SF molecules can be stabilized (silk II conformation) by mechanical stress or treatments with heat or organic solvents.28 When preparing SF porous membrane, mechanical stress is applied and SF foam is produced due to SF precipitation. In addition, the membrane is treated with 70% ethanol for 20 min, increasing the silk II conformation. Lyophilization of SF porous membrane may be another responsible for its high content of silk II conformation. During freezing, the water presented in the membrane turns into ice crystals that sublimate during the lyophilization. During water sublimation, SF groups that were bonded to the water make new intra and intermolecular interactions, which helps in the membrane stability.29
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FIGURE 1. Photographs of dense (1) and porous (2) SF membranes: nonsterilized (a), after sterilization with 70% ethanol (b), UV radiation (c), autoclave (d), ethylene oxide (e), and gamma radiation (f).
XRD All SF dense membranes before and after sterilization showed similar XRD patterns (Figure 4-1) with halos in 20 , related to the b-sheet crystals of 4.55 Å. In addition, other less intense halos related to b-sheet were observed at 24.5 (3.8 Å), 16.5 (5.37 Å), and 8.7 (10.1 Å). Halos related to silk I conformation were also observed at 12 and 28.5 (3.2 Å), indicating the coexistence of silks I and II conformation in SF dense membranes. SF porous membrane showed
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a main halo at 20 and other minor halos at 9.5 and 24.5 (Figure 4-2), indicating the predominance of b-sheet structure (silk II). The XRD results are in agreement with the FTIR results.22,30–32 Mechanical Properties SF dense membranes after sterilization by UV radiation and gamma radiation did not show statistically significant difference of strength at break from nonsterilized sample (Table I),
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FIGURE 2. Micrographs of surface (1) and fracture surface (2) of SF membranes: nonsterilized (a), after sterilization with 70% ethanol (b), UV radiation (c), autoclave (d), ethylene oxide (e), and gamma radiation (f).
indicating that these samples did not suffer any physical or chemical alterations or crosslinks during the sterilization process. In contrast, sterilization by 70% ethanol, autoclave, and ethylene oxide decreased the strength at break of SF dense membranes when compared with nonsterilized sample. These samples presented higher content of silk II structure than nonsterilized sample, as verified previously by
FTIR, indicating that they are more stable and, therefore, more brittle. UV radiation did not cause any change in the tensile strength of SF porous membranes as well. However, the same methods that caused a decrease in strength at break of dense membranes increased this property in porous membranes. This is probably related to the different influence that
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FIGURE 3. FTIR-ATR spectra of SF dense (1) and porous (2) membranes: nonsterilized (a), after sterilization with 70% ethanol (b), UV radiation (c), autoclave (d), ethylene oxide (e), and gamma radiation (f).
temperature, humidity, and treatment with organic solvents cause in both types of membranes. Contrarily to the dense membrane, SF porous membrane sterilized by gamma radiation had higher strength at break than nonsterilized membrane, probably due to the cross link that gamma radiation can induce in some materials, increasing their stability.20
Cytotoxicity SF dense membranes sterilized by 70% ethanol, autoclave, ethylene oxide, and gamma radiation were not toxic to cells, and cell viability was kept between 90 and 100%, comparable to the negative control (Figure 5). It was not possible to analyze the membrane sterilized by UV radiation because it
showed microbial contamination during extract preparation, indicating that the time used for UV radiation (30 min) was not sufficient to efficiently decontaminate the material. SF porous membranes sterilized by the several methods proposed in this study were not toxic to CHO cells either, and showed cell viability between 90 and 100% for all samples, as observed on Figure 5.
DISCUSSION
The process of developing a new biomaterial involves deep research and it is important to determine the most suitable sterilization method, which should not alter the biomaterials’ function and properties. Dense membranes of SF are
FIGURE 4. XRD patterns of SF dense (1) and porous (2) membranes: nonsterilized (a), after sterilization with 70% ethanol (b), UV radiation (c), autoclave (d), ethylene oxide (e), and gamma radiation (f).
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TABLE I. Results of Tensile Traction Test of SF Dense and Porous Membranes Before and After Sterilization Tensile strength at break (MPa)a
Nonsterilized 70% etanol UV radiation Autoclave Ethylene oxide Gamma radiation
Dense membrane
Porous membrane
63.5 6 5.6 52.1 6 7.3b 62.5 6 4.3 32.1 6 3.4b 49.4 6 8.1b 58.5 6 4.9
41.7 6 3.9 101.7 6 10.4b 46.3 6 7.2 82.9 6 17.6b 58.0 6 5.2b 65.3 6 3.1b
a
Mean 6 standard deviation (n 5 5). p < 0.05, mean is statistically significant different when compared with corresponding nonsterilized membrane. b
well known in the literature4,32,33 and can be used as biocompatible coatings.34,35 On the other hand, porous membranes of SF were developed by our group,21,36 the preparation process is easy and does not require timeconsuming dialysis process (as most of SF-based materials do), and are potential scaffolds for bone regeneration.37 Our study focused on the investigation of the influence of several sterilization methods on the properties of SF dense and porous membranes. We chose sterilization methods that were already used in the literature in fibroin materials, such as immersion in ethanol,10 UV radiation,11 autoclave,12 ethylene oxide,13 and gamma radiation.14 The chosen methods did not cause any morphological change of SF membranes or the formation of cytotoxic products that would decrease the cell viability. The sterilization methods had a different influence on dense and porous membranes. This means that despite of being prepared from the same material (SF extracted from Bombyx mori cocoons), the preparation method, morphology, and porosity of the membranes hardly influence their properties. SF porous membrane has a larger surface area available for interaction, when compared with the dense membrane and, therefore, reacted in different ways to the sterilization methods. For SF dense membranes, the methods that used organic solvent, high humidity and/or heat (ethanol 70%, autoclave,
and ethylene oxide), resulted in lower tensile strength, while the opposite result (increase of tensile strength) was observed for the porous membranes sterilized by the same methods. The sterilization methods cited above increased the silk II content of the dense membranes, justifying the decrease in strength, while SF porous membranes had already an elevated content of silk II. The changes observed in the membranes after sterilization by ethanol 70%, autoclave, and ethylene oxide can be used to functionalize the membranes. SF dense membranes sterilized by these methods, for example, can be used when a slow degradation rate is required, since materials with higher content of silk II degrades slowly. In contrast, for SF porous membranes, we can use these sterilization methods to obtain a stiffer material. If we desire to maintain a structure similar to the nonsterilized membranes (dense or porous), the use of UV radiation is the most adequate, despite this method is a disinfection method, not a sterilization one. Also, in our study, UV radiation for 30 min was not enough to maintain the SF dense membrane free of microorganisms, and it was not possible to evaluate the cytotoxic effect of this method. Thus, further studies on the UV radiation conditions should be developed on the future, as well as sterility and bioburden tests, to determine the efficiency of the several sterilization methods. Anyways, our main objective was to do a screening of several sterilization methods commonly used on SF-based materials, to be able to detect methods that could cause undesired alterations on the membranes. Common sterilization methods, as autoclave, cannot be used on most of natural polymers, such as chitosan, alginate or collagen, because they are sensible to high temperatures. Our results demonstrate that SF is a versatile material that can be prepared in different morphologies and submitted to several sterilization methods (including those that use severe conditions) without losing its macro-structural integrity. CONCLUSION
Sterilization of dense and porous SF membranes by 70% ethanol, UV radiation, autoclave, ethylene oxide, and gamma radiation did not cause morphological changes in the membranes and the membranes were not toxic to cells. Integrity of SF dense and porous membranes was maintained, proving that SF is a natural polymer with outstanding properties, capable to support the severe conditions imposed by sterilization methods without changing its structure. Sterilization methods that required the use of organic solvent, temperature and/or humidity (70% ethanol, autoclave, and ethylene oxide) induce SF dense membranes to a more stable structure, while porous membranes became more resistant to tensile, characteristics that can be useful for biomaterials application. Membranes sterilized by UV or gamma radiation keep properties similar to that of the nonsterilized membranes. ACKNOWLEDGMENT
FIGURE 5. Viability of CHO cells in the extract of SF dense and porous membranes after sterilization.
The authors thank to Acecil for the ethylene oxide sterilization and to the Energy and Nuclear Research Institute for
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gamma radiation. The authors also thank to Andrea Cecilia Dorion Rodas and Olga Higa from the Biotechnology Center of the Energy and Nuclear Research Institute (IPEN/USP) for the cytotoxicity tests and to FAPESP and CNPq for the financial support. REFERENCES 1. Vepari C, Kaplan DL. Silk as a biomaterial. Prog Polym Sci 2007; 32:991–1007. 2. Hakimi O, Knight DP, Vollrath F, Vadgama P. Spider and mulberry silkworm silks as compatible biomaterials. Composit Part B Eng 2007;38:324–337. 3. Jin HJ, Kaplan DL. Mechanism of silk processing in insects and spiders. Nature 2003;424:1057–1061. 4. Nogueira GM, Rodas ACD, Leite CAP, Giles C, Higa OZ, Polakiewicz B, Beppu MM. Preparation and characterization of ethanol-treated silk fibroin dense membranes for biomaterials application using waste silk fibers as raw material. Bioresour Technol 2010;101:8446–8451. 5. Altman GH, Diaz F, Jakuba C, Calabro T, Horan RL, Chen JS, Lu H, Richmond J, Kaplan DL. Silk-based biomaterials. Biomaterials 2003;24:401–416. 6. Um IC, Kweon HY, Park YH, Hudson S. Structural characteristics and properties of the regenerated silk fibroin prepared from formic acid. Int J Biol Macromolecules 2001;29:91–97. 7. MacIntosh AC, Kearns VR, Crawford A, Hatton PV. Skeletal tissue engineering using silk biomaterials. J Tissue Eng Regen Med 2008;2:71–80. 8. Lee KH, Ki CS, Baek DH, Kang GD, Ihm DW, Park YH. Application of electrospun silk fibroin nanofibers as an immobilization support of enzyme. Fibers Polym 2005;6:181–185. 9. Vangsness CT Jr, Wagner PP, Moore TM, Roberts MR. Overview of safety issues concerning the preparation and processing of soft-tissue allografts. Arthroscopy 2006;22:1351–1358. 10. Fuchs S, Motta A, Migliaresi C, Kirkpatrick CJ. Outgrowth endothelial cells isolated and expanded from human peripheral blood progenitor cells cells for endothelialization as a potential source of autologous of silk fibroin biomaterials. Biomaterials 2006;27: 5399–5408. 11. Gotoh Y, Tsukada M, Minoura N, Imai Y. Synthesis of poly(ethylene glycol)-silk fibroin conjugates and surface interaction between L-929 cells and the conjugates. Biomaterials 1997;18: 267–271. 12. Yang Y, Chen X, Ding F, Zhang P, Liu J, Go X. Biocompatibility evaluation of silk fibroin with peripheral nerve tissues and cells in vitro. Biomaterials 2007;28:1643–1652. 13. Panilaitis B, Altman GH, Chen JS, Jin HJ, Karageorgiou V, Kaplan DL. Macrophage responses to silk. Biomaterials 2003;24:3079– 3085. 14. Fini M, Motta A, Torricelli P, Glavaresi G, Aldini NN, Tschon M, Giardino R, Migliaresi C. The healing of confined critical size cancellous defects in the presence of silk fibroin hydrogel. Biomaterials 2005;26:3527–3536. 15. Marreco PR, da Luz Moreira P, Genari SC, Moraes AM. Effects of different sterilization methods on the morphology, #mechanical |properties, and cytotoxicity of chitosan membranes used as wound dressings. J Biomed Mater Res Part B Appl Biomater 2004;71B:268–277. 16. Siritientong T, Srichana T, Aramwit P. The effect of sterilization methods on the physical properties of silk sericin scaffolds. Aaps Pharmscitech 2011;12:771–781.
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17. von Woedtke T, Julich WD, Hartmann V, Stieber M, Abel PU. Sterilization of enzyme glucose sensors: problems and concepts. Biosens Bioelectron 2002;17:373–382. 18. Lee MH, Kim H-L, Kim CH, Lee SH, Kim JK, Lee SJ, Park J-C. Effects of low temperature hydrogen peroxide gas on sterilization and cytocompatibility of porous poly(D,L-lactic-co-glycolic acid) scaffolds. Surf Coat Technol 2008;202:5762–5767. 19. Block SS. Disinfection, Sterilization, and Preservation, Baltimore: Lippincott Williams & Wilkins; 2001. 20. Palsule AS, Clarson SJ, Widenhouse CW. Gamma irradiation of silicones. J Inorg Organomet Polym Mater 2008;18:207–221. 21. BR200601975-A, Unicamp Univ Estadual Campinas, invs.: M. M. Beppu, B. Polakiewicz, G. M. Nogueira; 2008 22. Lv Q, Cao CB, Zhang Y, Ma XL, Zhu HS. Preparation of insoluble fibroin films without methanol treatment. J Appl Polym Sci 2005; 96:2168–2173. 23. Miyaguchi Y, Hu JN. Physicochemical properties of silk fibroin after solubilization using calcium chloride with or without ethanol. Food Sci Technol Res 2005;11:37–42. 24. Rusa CC, Bridges C, Ha SW, Tonelli AE. Conformational changes induced in Bombyx mori silk fibroin by cyclodextrin inclusion complexation. Macromolecules 2005;38:5640–5646. 25. Um IC, Ki CS, Kweon HY, Lee KG, Ihm DW, Park YH. Wet spinning of silk polymer—II. Effect of drawing on the structural characteristics and properties of filament. Int J Biol Macromol 2004;34:107– 119. 26. Zuo BQ, Liu L, Wu Z. Effect on properties of regenerated silk fibroin fiber coagulated with aqueous methanol/ethanol. J Appl Polym Sci 2007;106:53–59. 27. Lawrence BD, Omenetto F, Chui K, Kaplan DL. Processing methods to control silk fibroin film biomaterial features. J Mater Sci 2008;43:6967–6985. 28. Tamada Y. New process to form a silk fibroin porous 3-D structure. Biomacromolecules 2005;6:3100–3106. 29. Moonsri P, Watanesk R, Watanesk S, Niamsup H, Deming RL. Fibroin membrane preparation and stabilization by polyethylene glycol diglycidyl ether. J Appl Polym Sci 2008;108:1402–1406. 30. Li MZ, Lu SZ, Wu ZY, Tan K, Minoura N, Kuga S. Structure and properties of silk fibroin-poly(vinyl alcohol) gel. Int J Biol Macromol 2002;30:89–94. 31. Kim SH, Nam YS, Lee TS, Park WH. Silk fibroin nanofiber. Electrospinning, properties, and structure. Polym J 2003;35:185–190. 32. Qiang L, Cao CB, Zhang Y, Man XL, Zhu HS. The preparation of insoluble fibroin films induced by degummed fibroin or fibroin microspheres. J Mater Sci Mater Med 2004;15:1193–1197. 33. Putthanarat S, Zarkoob S, Magoshi J, Chen JA, Eby RK, Stone M, Adams WW. Effect of processing temperature on the morphology of silk membranes. Polymer 2002;43:3405–3413. 34. Nogueira GM, Rodas ACD, Weska RF, Aimoli CG, Higa OZ, Maizato M, Leiner AA, Pitombo RNM, Polakiewicz B, Beppu MM. Bovine pericardium coated with biopolymeric films as an alternative to prevent calcification: In vitro calcification and cytotoxicity results. Mater Sci Eng C Mater Biol Appl 2010;30:575–582. 35. Wang XY, Kim HJ, Xu P, Matsumoto A, Kaplan DL. Biomaterial coatings by stepwise deposition of silk fibroin. Langmuir 2005;21: 11335–11341. 36. Nogueira GM, Weska RF, Vieira WC, Polakiewicz B, Rodas ACD, Higa OZ, Beppu MM. A new method to prepare porous silk fibroin membranes suitable for tissue scaffolding applications. J Appl Polym Sci 2009;114:617–623. 37. Weska RF, Nogueira GM, Vieira WC Jr, Beppu MM. Porous silk fibroin membrane as a potential scaffold for bone regeneration. Key Eng Mater 2009;396–398:187-190.
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changes in the membranes and the membranes were not toxic to cells. The sterilization methods that used organic solvent or an increased humidity and/or temperature (70% ethanol, autoclave, and ethylene oxide) increased the silk II content in the membranes: the dense membranes became more brittle, while the porous membranes showed increased strength at break. Membranes that underwent sterilization by UV and gamma radiation presented properties similar to the nonsterilized membranes, mainly for tensile strength and C 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part FTIR results. V B: Appl Biomater, 102B: 869–876, 2014.
Key Words: porous membranes, dense membranes, fibroin, sterilization, biomaterials
How to cite this article: de Moraes MA, Weska RF, Beppu MM. 2014. Effects of sterilization methods on the physical, chemical, and biological properties of silk fibroin membranes. J Biomed Mater Res Part B 2014:102B:869–876.
INTRODUCTION
Silk fibroin (SF) is a fibrous protein extracted from cocoons of Bombyx mori silkworm. SF exhibit two conformational forms, called silk I and II. Silk I is the water-soluble and meta-stable form, with dominating random coil and a-helix structures. Silk II conformation is the hydrophobic form, dominated by b-sheet structures that are thermodynamically stable presenting strong hydrogen bonds and Van der Waals forces.1,2 SF transition from silk I to silk II can be achieved using organic solvents or physical shear.3,4 SF presents good biocompatibility, good oxygen, and water vapor permeability, biodegradability, minimal inflammatory reaction, can be prepared in different morphologies, presents high mechanical strength, high thermal stability, and microbial resistance.5–7 It has been widely explored for biomedical applications, such as substrate for enzymes immobilization, wound dressings, scaffolds for bone, cartilage and tendons regeneration, among others 1,8. In addition, the use of SF as biomaterial is approved by Food and Drug Administration as nonabsorbable sutures, and for soft tissue repair. The sterilization process is an essential step for the development of a material or device that will be used as biomaterial. The optimum procedure should sterilize the
material without altering its functionality, mainly regarding biomechanical and biocompatible properties.9 This is a complex issue in natural polymers, since they can be easily damaged by the severe sterilization conditions. SF is a protein and has a complex structure that may be affected by heat or irradiation, resulting in changes on its secondary and tertiary structure and, consequently, on the physical and/or biological properties of the SF-based material. Thus, the investigation of the material properties after sterilization is necessary to understand the alterations that may be caused by sterilization method. Several papers report the sterilization of SF matrices by different methods, such as immersion in 70% ethanol solution,10 UV radiation for 30 min,11 autoclave,12 ethylene oxide,13 and gamma radiation,14 but none of them studied the influence of sterilization on the physical–chemical and biological properties of those matrixes. The treatment with ethanol is considered to be a disinfectant method, rather than a sterilization, since ethanol does not act on the endospores of most bacteria species, limiting its use as a surface-sterilizing agent.15 Nevertheless, the treatment with ethanol is the most used disinfecting method for several materials.15,16
Correspondence to: M. M. Beppu (E - mail: [email protected])
C 2013 WILEY PERIODICALS, INC. V
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UV radiation has the DNA as main target for microorganisms’ elimination. UV radiation has strong antimicrobial action, but it is not capable to penetrate solid matter, despite being highly absorbed by glasses and plastics.17 Autoclave is a well-known sterilization method that applies saturated vapor at 121 C, which is capable to destroy metabolic and structural components of the microorganisms. Autoclave is extensively used in laboratory and industrial scales because of its efficacy, easiness to process, and for not producing toxic residues. However, the high values of temperature and humidity required for autoclave are the main restrain for its use with polymeric, and mainly for biopolymeric materials. The most used method to sterilize polymers that are sensible to heat and humidity is the exposure to ethylene oxide, because this method is effective at low temperatures and has high penetration in a wide range of materials. However, ethylene oxide can chemically react with the material and it is a toxic gas, requiring repeated air washings after sterilization.18 Finally, gamma radiation is the most used method to sterilize biomaterials, because of its efficiency and price.19 Gamma rays destroy the microorganisms by ionizing cell components, especially nucleic acids. The main advantages of this method are the high penetration, low chemical reactivity, low content of residues and low temperature raise. However, some materials can be degraded when irradiated or cross links can be formed, altering the material structure.20 As we can see, each sterilization method has advantages and limitations, and the choice of a suitable method will depend on each material. In this context, the aim of this study is to investigate the influence of five different sterilization methods on the physical, chemical, and biological properties of SF membranes. The sterilization methods proposed to be used in this work were 70% ethanol, UV radiation, autoclave, ethylene oxide, and gamma radiation. We also analyzed fibroin membranes processed by different ways and, therefore, with different morphological features, such as dense and porous SF membranes. MATERIALS AND METHODS
Preparation of SF Solution Raw silk fibers of Bombyx mori silkworm (supplied by Bratac-Brazil) were degummed three times in 5 g L21 Na2CO3 solution at 85 C, for 30 min, to remove the sericin. Fibers were washed with deionized water, dried at room temperature, and dissolved in a solvent of CaCl2:CH3CH2OH:H2O (1:2:8 molar) at 85 C to a concentration of 10 wt %. Preparation of SF Dense Membranes SF dense membranes were prepared according to our previously established method.4 In brief, SF salt solution was dialyzed in distilled water for 3 days, to remove the salts of the solvent. The dialyzed solution was then cast in polystyrene Petri dishes and the membranes were formed by solvent evaporation at room temperature. The membranes were immersed in 70% ethanol for 20 min to reduce water
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solubility, and then washed with deionized water. Finally, the membranes were dried at 25 C before sterilization and characterization procedures. Preparation of SF Porous Membranes SF porous membranes preparation method was developed by our group and it is patented.21 In brief, SF salt solution was diluted in distilled water (1:100) and foaming was induced by mechanical stirring. SF foam was compressed to remove the excess of water and promote foam agglomeration. Thickness and diameter of the membranes could be controlled by the quantity of compressed foam. Membranes were then immersed in 70% ethanol for 20 min and washed with deionized water. Finally, membranes were frozen in an ultrafreezer at 280 C, for 24 h, and lyophilized for 24 h in a freeze-drier before being submitted to sterilization. Sterilization Methods We studied the influence of five different sterilization methods in the physical, chemical, and biological characteristics of SF membranes. The studied methods were immersion in 70% ethanol solution, UV radiation, autoclave, ethylene oxide exposure, and gamma radiation, briefly described below. Nonsterilized membranes were used as control. Dense and porous SF membranes were immersed in 70 vol % ethanol, for 48 h, at 8 C. Membranes were washed in deionized water, frozen, and lyophilized before the characterizations. For the cytotoxicity test, the membranes were washed in 0.9% NaCl solution. For UV radiation, the membranes were exposed to UV light (k 5 254 nm) for 30 min on each side. Autoclave was used at 121 C for 30 min in dense and porous SF membranes. Sterilization with ethylene oxide was done by Acecil Central de Esterilizac¸~ao Comercio Ind ustria Ltda. (Campinas, SP, Brazil). Samples were exposed to Oxyfume-30 (30% ethylene oxide and 70% carbon dioxide) for 4 h at 50 C, under 400–450 mmHg vacuum and 0.5–0.6 kgf cm22 pressure. Samples were aired three times with nitrogen (N2) for removal of residual ethylene oxide. The membranes were stored at room temperature for 72 h before testing. Sterilization by gamma radiation was performed at Energy and Nuclear Research Institute (IPEN, S~ao Paulo, Brazil) by exposing the films to a constant rate of gamma radiation of 2.27 kGy h21, for 11 h, in a total dose of 25 kGy, at room temperature. Characterization The morphology of the membranes was observed by scanning electron microscopy (SEM), using a LEO 440i (Leica), with accelerating voltage of 20 kV for dense membranes and 15 kV for porous membranes. Samples were frozen in liquid nitrogen, fractured, lyophilized (Liobras, L101, Brazil) for 24 h, and coated with a gold layer before SEM observation. We investigated the secondary structure of SF membranes by Fourier transform infrared spectroscopy using a Bomem MB 102, equipped with attenuated total reflectance (FTIR-ATR) accessory and ZnSe crystal. The spectra were
EFFECTS OF STERILIZATION METHODS ON THE PROPERTIES OF FIBROIN MEMBRANES
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obtained in the range of 800–1800 cm21, with 4 cm21 resolution and 256 scans. X-ray diffraction (XRD) was performed in a X’PERT PW3050 Philips, using a monochromatic Cu-Ka radiation (k 5 1.54 Å), in the 2h range of 5 –35 and scanning rate of 0.6 min21. Tensile tests were performed according to ASTM D88202 using a TA.XT2 texture analyzer (Stable Microsystems SMD). Five samples of each membrane (8 3 2.5 cm) were stored under standard conditions (25 C, RH 50%) for 72 h before the tests. An initial grip separation of 50 mm and crosshead speed of 10 mm s21 were used. Cytotoxicity test was done on dense and porous SF membranes according to ISO 10993-5 (2009) using Chinese hamster ovary cell line (CHO-k1), maintained in RPMI culture medium. Extracts of porous and dense SF membranes were prepared in a proportion of 1 cm2 of membrane by 1 mL of culture medium, at 37 C for 48 h. Extracts were prepared in RPMI. Phenol 0.4% was used as positive control and high density polyethylene was used as negative control. Cell suspension and extract were added in 96-well microplate, incubated for 72 h at 37 C in a 5% CO2 humidified atmosphere. The cell viability was measured by adding MTS (supravital dye tetrazolium compound)/PMS (electron coupling agent) (20/1) solution and incubating for 2 h. The microplate was analyzed in a spectrophotometer ELISA at 495 nm. RESULTS
Morphology SF membranes presented thicknesses of about 100 lm and 3.5 mm for dense and porous membranes, respectively. Macroscopic changes in membrane morphology were not observed after sterilization (Figure 1), except for autoclaved membranes. After sterilization by autoclave, SF dense membrane became slightly yellow, while SF porous membrane had a reduction of about 3 mm in diameter, probably due to the rearrangement of SF molecular chains caused by water vapor. The structural integrity of the membranes after sterilization by the several tested methods was preserved. All the membranes remain unaltered after sterilization. SEM observations (Figure 2) did not show morphological changes in the membranes after sterilization. Dense membranes presented regular and homogeneous surface and fracture surface. Porous membrane surface was rough and a porous fracture surface could be seen by SEM. Fourier-transformed infrared spectroscopy SF molecular conformation can be detected by Fouriertransformed infrared spectroscopy (FTIR). Bands of amide I, II, and III are attributed to C@O stretch, NAH deformation and OACAN folding, respectively. Figure 3 shows the FTIR-ATR spectra for dense and porous SF membranes. For dense membranes (Figures 3-1), amide II bands were located in the region of silk II conformation (1514 cm21) and amide III bands were located in the region of silk I conformation (1235 cm21) for all samples.22–24 These results occurred probably due to the preparation
method of dense membranes. SF dense membranes prepared from SF dialyzed solution and dried at room temperature had predominant silk I conformation. To obtain hydrophobic membranes, a treatment with 70% ethanol for 20 min is done, which increase the silk II conformation.25–27 Amide I band was located in different wavelengths, depending on the sterilization method. In nonsterilized membranes and sterilized by UV or gamma radiation, amide I peaks were observed at 1645 cm21 (silk I), whilst in membranes sterilized by ethanol and autoclave the peak was located at 1622 cm21 (silk II). This indicates that after sterilization by 70% ethanol and autoclave, SF dense membrane had an increased content of b-sheet when compared with the nonsterilized membrane (control),22–24 demonstrating that in this membranes C@O groups are involved in more hydrogen bonds than in the other membranes, increasing b-sheet content.24 Lawrence et al. (2008)27 observed that autoclave increases silk II conformation in SF dense membranes, even when they were not treated with organic solvents. Our result also indicates that exposure of SF dense membranes to high temperature, humidity, and pressure induce an increase in b-sheet content, an effect similar to that of ethanol or methanol treatment.25,26 SF dense membrane sterilized by ethylene oxide showed peaks of the same intensity in 1622 and 1645 cm21, indicating a higher proportion of silk II conformation than the nonsterilized membrane, but lower than the membranes sterilized by 70% ethanol or autoclave. The treatment of membranes with ethanol, associated with the temperature of 55 C of the ethylene oxide sterilization was probably the factor that increased b-sheet content. The absence of peaks not related to SF in the membrane sterilized by ethylene oxide indicates that there were no chemical reactions in the process and that the aeration process was successful in the removal of ethylene oxide.18 The FTIR spectra of porous membranes nonsterilized and after sterilization by the several methods were similar (Figure 3-2). SF porous membranes show peaks at 1622 and 1517 cm21, attributed to silk II structure, and at 1230 cm21, indicating the presence of silk I structure.22–24 SF porous membrane nonsterilized has already a higher content of silk II than I, probably related to the way the membrane was prepared. Usually, SF salt solution (used to produce SF porous membrane) has silk I conformation due to solvation of SF molecules by the solvents. SF molecules can be stabilized (silk II conformation) by mechanical stress or treatments with heat or organic solvents.28 When preparing SF porous membrane, mechanical stress is applied and SF foam is produced due to SF precipitation. In addition, the membrane is treated with 70% ethanol for 20 min, increasing the silk II conformation. Lyophilization of SF porous membrane may be another responsible for its high content of silk II conformation. During freezing, the water presented in the membrane turns into ice crystals that sublimate during the lyophilization. During water sublimation, SF groups that were bonded to the water make new intra and intermolecular interactions, which helps in the membrane stability.29
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FIGURE 1. Photographs of dense (1) and porous (2) SF membranes: nonsterilized (a), after sterilization with 70% ethanol (b), UV radiation (c), autoclave (d), ethylene oxide (e), and gamma radiation (f).
XRD All SF dense membranes before and after sterilization showed similar XRD patterns (Figure 4-1) with halos in 20 , related to the b-sheet crystals of 4.55 Å. In addition, other less intense halos related to b-sheet were observed at 24.5 (3.8 Å), 16.5 (5.37 Å), and 8.7 (10.1 Å). Halos related to silk I conformation were also observed at 12 and 28.5 (3.2 Å), indicating the coexistence of silks I and II conformation in SF dense membranes. SF porous membrane showed
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a main halo at 20 and other minor halos at 9.5 and 24.5 (Figure 4-2), indicating the predominance of b-sheet structure (silk II). The XRD results are in agreement with the FTIR results.22,30–32 Mechanical Properties SF dense membranes after sterilization by UV radiation and gamma radiation did not show statistically significant difference of strength at break from nonsterilized sample (Table I),
EFFECTS OF STERILIZATION METHODS ON THE PROPERTIES OF FIBROIN MEMBRANES
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FIGURE 2. Micrographs of surface (1) and fracture surface (2) of SF membranes: nonsterilized (a), after sterilization with 70% ethanol (b), UV radiation (c), autoclave (d), ethylene oxide (e), and gamma radiation (f).
indicating that these samples did not suffer any physical or chemical alterations or crosslinks during the sterilization process. In contrast, sterilization by 70% ethanol, autoclave, and ethylene oxide decreased the strength at break of SF dense membranes when compared with nonsterilized sample. These samples presented higher content of silk II structure than nonsterilized sample, as verified previously by
FTIR, indicating that they are more stable and, therefore, more brittle. UV radiation did not cause any change in the tensile strength of SF porous membranes as well. However, the same methods that caused a decrease in strength at break of dense membranes increased this property in porous membranes. This is probably related to the different influence that
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FIGURE 3. FTIR-ATR spectra of SF dense (1) and porous (2) membranes: nonsterilized (a), after sterilization with 70% ethanol (b), UV radiation (c), autoclave (d), ethylene oxide (e), and gamma radiation (f).
temperature, humidity, and treatment with organic solvents cause in both types of membranes. Contrarily to the dense membrane, SF porous membrane sterilized by gamma radiation had higher strength at break than nonsterilized membrane, probably due to the cross link that gamma radiation can induce in some materials, increasing their stability.20
Cytotoxicity SF dense membranes sterilized by 70% ethanol, autoclave, ethylene oxide, and gamma radiation were not toxic to cells, and cell viability was kept between 90 and 100%, comparable to the negative control (Figure 5). It was not possible to analyze the membrane sterilized by UV radiation because it
showed microbial contamination during extract preparation, indicating that the time used for UV radiation (30 min) was not sufficient to efficiently decontaminate the material. SF porous membranes sterilized by the several methods proposed in this study were not toxic to CHO cells either, and showed cell viability between 90 and 100% for all samples, as observed on Figure 5.
DISCUSSION
The process of developing a new biomaterial involves deep research and it is important to determine the most suitable sterilization method, which should not alter the biomaterials’ function and properties. Dense membranes of SF are
FIGURE 4. XRD patterns of SF dense (1) and porous (2) membranes: nonsterilized (a), after sterilization with 70% ethanol (b), UV radiation (c), autoclave (d), ethylene oxide (e), and gamma radiation (f).
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EFFECTS OF STERILIZATION METHODS ON THE PROPERTIES OF FIBROIN MEMBRANES
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TABLE I. Results of Tensile Traction Test of SF Dense and Porous Membranes Before and After Sterilization Tensile strength at break (MPa)a
Nonsterilized 70% etanol UV radiation Autoclave Ethylene oxide Gamma radiation
Dense membrane
Porous membrane
63.5 6 5.6 52.1 6 7.3b 62.5 6 4.3 32.1 6 3.4b 49.4 6 8.1b 58.5 6 4.9
41.7 6 3.9 101.7 6 10.4b 46.3 6 7.2 82.9 6 17.6b 58.0 6 5.2b 65.3 6 3.1b
a
Mean 6 standard deviation (n 5 5). p < 0.05, mean is statistically significant different when compared with corresponding nonsterilized membrane. b
well known in the literature4,32,33 and can be used as biocompatible coatings.34,35 On the other hand, porous membranes of SF were developed by our group,21,36 the preparation process is easy and does not require timeconsuming dialysis process (as most of SF-based materials do), and are potential scaffolds for bone regeneration.37 Our study focused on the investigation of the influence of several sterilization methods on the properties of SF dense and porous membranes. We chose sterilization methods that were already used in the literature in fibroin materials, such as immersion in ethanol,10 UV radiation,11 autoclave,12 ethylene oxide,13 and gamma radiation.14 The chosen methods did not cause any morphological change of SF membranes or the formation of cytotoxic products that would decrease the cell viability. The sterilization methods had a different influence on dense and porous membranes. This means that despite of being prepared from the same material (SF extracted from Bombyx mori cocoons), the preparation method, morphology, and porosity of the membranes hardly influence their properties. SF porous membrane has a larger surface area available for interaction, when compared with the dense membrane and, therefore, reacted in different ways to the sterilization methods. For SF dense membranes, the methods that used organic solvent, high humidity and/or heat (ethanol 70%, autoclave,
and ethylene oxide), resulted in lower tensile strength, while the opposite result (increase of tensile strength) was observed for the porous membranes sterilized by the same methods. The sterilization methods cited above increased the silk II content of the dense membranes, justifying the decrease in strength, while SF porous membranes had already an elevated content of silk II. The changes observed in the membranes after sterilization by ethanol 70%, autoclave, and ethylene oxide can be used to functionalize the membranes. SF dense membranes sterilized by these methods, for example, can be used when a slow degradation rate is required, since materials with higher content of silk II degrades slowly. In contrast, for SF porous membranes, we can use these sterilization methods to obtain a stiffer material. If we desire to maintain a structure similar to the nonsterilized membranes (dense or porous), the use of UV radiation is the most adequate, despite this method is a disinfection method, not a sterilization one. Also, in our study, UV radiation for 30 min was not enough to maintain the SF dense membrane free of microorganisms, and it was not possible to evaluate the cytotoxic effect of this method. Thus, further studies on the UV radiation conditions should be developed on the future, as well as sterility and bioburden tests, to determine the efficiency of the several sterilization methods. Anyways, our main objective was to do a screening of several sterilization methods commonly used on SF-based materials, to be able to detect methods that could cause undesired alterations on the membranes. Common sterilization methods, as autoclave, cannot be used on most of natural polymers, such as chitosan, alginate or collagen, because they are sensible to high temperatures. Our results demonstrate that SF is a versatile material that can be prepared in different morphologies and submitted to several sterilization methods (including those that use severe conditions) without losing its macro-structural integrity. CONCLUSION
Sterilization of dense and porous SF membranes by 70% ethanol, UV radiation, autoclave, ethylene oxide, and gamma radiation did not cause morphological changes in the membranes and the membranes were not toxic to cells. Integrity of SF dense and porous membranes was maintained, proving that SF is a natural polymer with outstanding properties, capable to support the severe conditions imposed by sterilization methods without changing its structure. Sterilization methods that required the use of organic solvent, temperature and/or humidity (70% ethanol, autoclave, and ethylene oxide) induce SF dense membranes to a more stable structure, while porous membranes became more resistant to tensile, characteristics that can be useful for biomaterials application. Membranes sterilized by UV or gamma radiation keep properties similar to that of the nonsterilized membranes. ACKNOWLEDGMENT
FIGURE 5. Viability of CHO cells in the extract of SF dense and porous membranes after sterilization.
The authors thank to Acecil for the ethylene oxide sterilization and to the Energy and Nuclear Research Institute for
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gamma radiation. The authors also thank to Andrea Cecilia Dorion Rodas and Olga Higa from the Biotechnology Center of the Energy and Nuclear Research Institute (IPEN/USP) for the cytotoxicity tests and to FAPESP and CNPq for the financial support. REFERENCES 1. Vepari C, Kaplan DL. Silk as a biomaterial. Prog Polym Sci 2007; 32:991–1007. 2. Hakimi O, Knight DP, Vollrath F, Vadgama P. Spider and mulberry silkworm silks as compatible biomaterials. Composit Part B Eng 2007;38:324–337. 3. Jin HJ, Kaplan DL. Mechanism of silk processing in insects and spiders. Nature 2003;424:1057–1061. 4. Nogueira GM, Rodas ACD, Leite CAP, Giles C, Higa OZ, Polakiewicz B, Beppu MM. Preparation and characterization of ethanol-treated silk fibroin dense membranes for biomaterials application using waste silk fibers as raw material. Bioresour Technol 2010;101:8446–8451. 5. Altman GH, Diaz F, Jakuba C, Calabro T, Horan RL, Chen JS, Lu H, Richmond J, Kaplan DL. Silk-based biomaterials. Biomaterials 2003;24:401–416. 6. Um IC, Kweon HY, Park YH, Hudson S. Structural characteristics and properties of the regenerated silk fibroin prepared from formic acid. Int J Biol Macromolecules 2001;29:91–97. 7. MacIntosh AC, Kearns VR, Crawford A, Hatton PV. Skeletal tissue engineering using silk biomaterials. J Tissue Eng Regen Med 2008;2:71–80. 8. Lee KH, Ki CS, Baek DH, Kang GD, Ihm DW, Park YH. Application of electrospun silk fibroin nanofibers as an immobilization support of enzyme. Fibers Polym 2005;6:181–185. 9. Vangsness CT Jr, Wagner PP, Moore TM, Roberts MR. Overview of safety issues concerning the preparation and processing of soft-tissue allografts. Arthroscopy 2006;22:1351–1358. 10. Fuchs S, Motta A, Migliaresi C, Kirkpatrick CJ. Outgrowth endothelial cells isolated and expanded from human peripheral blood progenitor cells cells for endothelialization as a potential source of autologous of silk fibroin biomaterials. Biomaterials 2006;27: 5399–5408. 11. Gotoh Y, Tsukada M, Minoura N, Imai Y. Synthesis of poly(ethylene glycol)-silk fibroin conjugates and surface interaction between L-929 cells and the conjugates. Biomaterials 1997;18: 267–271. 12. Yang Y, Chen X, Ding F, Zhang P, Liu J, Go X. Biocompatibility evaluation of silk fibroin with peripheral nerve tissues and cells in vitro. Biomaterials 2007;28:1643–1652. 13. Panilaitis B, Altman GH, Chen JS, Jin HJ, Karageorgiou V, Kaplan DL. Macrophage responses to silk. Biomaterials 2003;24:3079– 3085. 14. Fini M, Motta A, Torricelli P, Glavaresi G, Aldini NN, Tschon M, Giardino R, Migliaresi C. The healing of confined critical size cancellous defects in the presence of silk fibroin hydrogel. Biomaterials 2005;26:3527–3536. 15. Marreco PR, da Luz Moreira P, Genari SC, Moraes AM. Effects of different sterilization methods on the morphology, #mechanical |properties, and cytotoxicity of chitosan membranes used as wound dressings. J Biomed Mater Res Part B Appl Biomater 2004;71B:268–277. 16. Siritientong T, Srichana T, Aramwit P. The effect of sterilization methods on the physical properties of silk sericin scaffolds. Aaps Pharmscitech 2011;12:771–781.
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