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Silk fibroin aerogels: potential scaffolds for tissue engineering applications
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Biomed. Mater. 10 035002 (http://iopscience.iop.org/1748-605X/10/3/035002) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 61.129.42.30 This content was downloaded on 08/05/2015 at 08:35
Please note that terms and conditions apply.
Biomed. Mater. 10 (2015) 035002
doi:10.1088/1748-6041/10/3/035002
Paper
received
4 September 2014
Silk fibroin aerogels: potential scaffolds for tissue engineering applications
re vised
25 March 2015 accep ted for publication
1 April 2015 published
8 May 2015
Rajendar R Mallepally1, Michael A Marin1, Vasudha Surampudi1, Bano Subia2, Raj R Rao1, Subhas C Kundu2 and Mark A McHugh1 1 2
Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA 23284, USA Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal 721302, India
E-mail: [email protected] Keywords: silk fibroin, supercritical CO2, aerogel, cell culture, tissue engineering
Abstract Silk fibroin (SF) is a natural protein, which is derived from the Bombyx mori silkworm. SF based porous materials are extensively investigated for biomedical applications, due to their biocompatibility and biodegradability. In this work, CO2 assisted acidification is used to synthesize SF hydrogels that are subsequently converted to SF aerogels. The aqueous silk fibroin concentration is used to tune the morphology and textural properties of the SF aerogels. As the aqueous fibroin concentration increases from 2 to 6 wt%, the surface area of the resultant SF aerogels increases from 260 to 308 m2 g−1 and the compressive modulus of the SF aerogels increases from 19.5 to 174 kPa. To elucidate the effect of the freezing rate on the morphological and textural properties, SF cryogels are synthesized in this study. The surface area of the SF aerogels obtained from supercritical CO2 drying is approximately five times larger than the surface area of SF cryogels. SF aerogels exhibit distinct pore morphology compared to the SF cryogels. In vitro cell culture studies with human foreskin fibroblast cells demonstrate the cytocompatibility of the silk fibroin aerogel scaffolds and presence of cells within the aerogel scaffolds. The SF aerogels scaffolds created in this study with tailorable properties have potential for applications in tissue engineering.
1. Introduction In the past two decades, tissue repair and regeneration using synthetic scaffolds has emerged as one of the most promising approaches in tissue engineering [1]. An ideal scaffold should be (3D) with an interconnected pore network for cell growth and tissue vascularization, biocompatible and biodegradable, and the scaffold surface chemistry should promote cell attachment, proliferation, and differentiation. Natural and synthetic polymers such as collagen, gelatin, alginate, chitosan, starch, hyaluronic acid, silk fibroin, poly(lactic-coglycolic acid), and poly(ε-caprolactone) etc have been used in tissue engineering as scaffolding materials [2]. Among them, natural polymers have unique, intrinsic properties that make them suitable candidate materials for scaffold fabrication [2–4]. Silk fibroin (SF) from Bombyx mori silkworm cocoons has been a widely investigated natural polymer for numerous biomedical applications due to its versatile properties [4]. SF biomaterials have been fabricated in different forms such as hydrogels [5–7], films [8], micro/nano spheres [9, 10], scaffolds, and fibers [11] © 2015 IOP Publishing Ltd
for different applications. Among these, SF 3D scaffolds have been successfully investigated for the tissue engineering of bone, ligaments, cartilage, nerves, and blood vessels as well as support matrices for cells including osteoblasts, fibroblasts, hepatocytes, and stem cells [12, 13]. The micro-architecture is known to play a significant role in tissue repair and regeneration. For instance, in the case of bone tissue engineering, a scaffold with a bi-modal pore size distribution exhibited improved in vivo performance where pores of ~5 µm are appropriate for tissue vascularization and pores of ~100-to-400 µm are appropriate for cell adhesion and proliferation [14, 15]. Salt leaching is a widely used method for fabricating macro-porous SF scaffolds [16]. After the salt is extracted, the scaffolds are typically freeze dried yielding cryogels. Although freeze drying preserves the primary macro-pore structure, the growth of ice crystals damages the secondary micro-pore structure resulting in less surface area [17, 18]. Supercritical CO2 drying is an alternative technology used to create aerogels with high surface area and high porosity [19]. The aerogels synthesized with supercritical CO2 drying include
Biomed. Mater. 10 (2015) 035002
R R Mallepally et al
those made from inorganics [20–22], organics [23], and polysaccharides [17, 24–26] (alginate, chitosan, starch, pectin, etc) used as catalysts, food additives, pharmaceutical drug carriers, and as porous synthetic bone templates. In addition to the synthesis of aerogels, supercritical or dense gas technology can be used to create other types of porous scaffolds for tissue engineering applications [27–29]. Porous scaffolds have been fabricated from both synthetic and natural polymers using supercritical CO2 foaming, phase inversion, and emulsion template approaches [30–36]. In a previous study, we reported that the molecular weight (Mw) of SF depends on the degumming method and the molecular weight of SF affects the sol–gel kinetics [37]. In another independent study, we created SF aerogels using 95 kDa Mw SF and demonstrated the potential use as drug delivery carriers [38]. The present study describes the synthesis and characterization of silk fibroin aerogels using 190 kDa Mw SF. Salt leaching is combined with supercritical fluid technology to create SF aerogel scaffolds for tissue engineering applications with bi-modal pore size distribution. The design of the scaffold is aimed towards mimicking the native 3D cell environment where cells are surrounded by supportive extra cellular matrix (ECM) that provides the necessary mechanical support. Scanning electron microscopy is used to determine the morphology of the scaffolds and nitrogen adsorption/desorption analysis is used to determine the scaffold textural properties, such as surface area, pore size, and pore volume. Mechanical compression tests are used to determine compression modulus and compression strength of the scaffolds. Finally, in vitro cell culture studies are performed to establish the biocompatibility of the synthesized SF aerogel scaffolds.
2. Materials and methods 2.1. Materials Bombyx mori silkworm cocoons are purchased from Aurora silk, USA. Sodium bicarbonate and lithium bromide are purchased from Sigma Aldrich, USA. Ethanol is purchased from Fischer Scientific, USA. CO2 (99.99%, bone dry) is purchased from Airgas, USA. Dulbecco’s Modified Eagle Medium culture medium, glucose, glutamine, penicillin/streptomycin, fetal bovine serum, Rhodamine–phalloidin, human foreskin fibroblasts, formaldehyde, AlamarBlue, and 4′,6-Diamidino-2-Phenylindole are purchased from Life Technologies, USA. 2.2. Methods 2.2.1. Preparation of silk fibroin solution Cocoons are heated in an aqueous solution of 0.02 M NaHCO3 (1% w/v) at 90 °C for 60 min. Degummed fiber is recovered and washed with boiling water to remove the sericin that coats SF. The SF fiber is dried overnight in an oven at 40 °C. Dried silk fibroin fiber is dissolved in 9.3 M aqueous LiBr solution (10% w/v) 2
at 65 ºC for four hours. The solution is cooled to 25 °C and then dialyzed (Snakeskin Dialysis Tubing, Thermo Scientific, USA, MWCO 3500) against distilled water for three days (2.5% v/v), with daily water replacement. The concentration of aqueous silk fibroin solution is measured by determining the dry weight of SF after evaporating the water at 80 °C from one gram of sample. 2.2.2. Synthesis of SF hydrogels and aerogels SF hydrogels are synthesized using CO2 as a volatile acidifying sol–gel accelerator [37–39] and using the salt-leaching method [40]. For gelation via CO 2– assisted processing, 5 mL SF solution are charged to a glass beaker (height = 35 mm and internal diameter = 18 mm) that is placed in a 300 mL stainless steel vessel, heated to 40 °C, and pressurized with CO2 to 100 bar. After two hours, the CO2 is vented over a period of 30 min and the hydrogel is recovered. For gelation via salt-leaching, 4 g of granular NaCl (particle size 300 to 400 µm) are added slowly to 2 mL of aqueous SF solution. The resulting gel is aged for ~18 h and then soaked in a series of water solutions to extract the salt. SF hydrogels from these two procedures are converted to alcogels by soaking in a series of aqueous alcohol solutions of increasing alcohol concentration as described elsewhere [25]. SF aerogels are then obtained by supercritical CO 2 (scCO 2) drying at 40 °C and 100 bar. The hydrogels pretreatment method fixes the ultimate morphology of the cryogels or aerogels. A single piece of SF hydrogel is synthesized with 2 wt% SF solution using CO2 as an acidifying agent and this hydrogel is cut into three equal parts. Two parts are frozen at different freezing rates; 1) slow freeze at −20 °C and 2) fast freeze at −196 °C. After the initial freeze, the gels are freeze-dried at −20 °C and ~2 mbar pressure for 18 h. The third gel piece is dried using supercritical CO2 drying. The two scaffolds prepared from the slow freeze at −20 °C and fast freeze at −196 °C are termed cryogelslow and cryogel-fast, respectively. 2.3. Silk fibroin aerogels characterization 2.3.1. Fourier transform infrared spectroscopy (FTIR) FTIR (Smart iTR, Thermo Fischer Scientific, USA) is used for the structural characterization of SF aerogels. FTIR spectra are collected in the absorption mode as the mean of 64 scans at 4000 to 400 cm−1 with a spectral resolution of 4 cm−1. 2.3.2. Scanning electron microscope (SEM) SEM (HITACHI SU 70, USA) is used to determine the SF aerogels and SF cryogels morphology. SEM images are captured at an accelerating voltage of 5 keV. The aerogels are immersed in liquid nitrogen, crushed into a fine powder, and placed on aluminum stubs with carbon tape. A 10 nm platinum coating is then applied to the sample via spin coating (Denton Vacuum, USA, Model: Desk V TSC).
Biomed. Mater. 10 (2015) 035002
R R Mallepally et al
washed with 100% ethanol, sterilized under UV light for 30 min, washed several times with PBS (pH 7.4) and incubated with 70% alcohol for 30 min, washed with PBS several times, soaked in PBS in an incubator for 6–8 h in order to neutralize the pH, followed by incubation for three hours in cell culture medium. The 50 µl HFF cell suspensions are then added on top of the scaffold. After two hours of incubation of the scaffold with the cells in the incubator, more medium is added according to the volume of the cell culture dish containing the scaffold. For routine culture, the cell culture medium is replaced every two days with cells routinely maintained in a controlled temperature and environment incubator at 5% CO2 at 37 °C. However, in this instance the experiment is terminated after two days. Figure 1. Schematic representation of high pressure CO2–assisted SF gelation.
2.3.3. Nitrogen adsorption/desorption analysis N2 adsorption/desorption analysis is used to determine the aerogel and cryogel surface area, pore size distribution, and pore volume. Approximately 50 mg of aerogel or cryogel are weighed and then heated at 120 °C under vacuum for four hours. The nitrogen adsorption/ desorption measurements are carried out at 77 K (Nova 2200e, Quantachrome Instruments, USA). The specific surface area of the aerogel or cryogel is calculated with the multipoint Brunauer–Emmett–Teller (BET) model in the relative pressure range of 0.05 to 0.30. The pore size distribution is calculated with the Barrett–Joyner– Halenda (BJH) model using a desorption isotherm. The pore volume is calculated at a relative pressure of 0.98. 2.3.4. Mechanical properties Mechanical properties of the SF hydrogels and aerogels are determined using an Instron (Model 5543) mechanical testing instrument with a 50 N load cell. All the tested SF hydrogels and aerogels are cylindrical discs with a diameter of 15.5 ± 1.3 mm and a height of 9.0 ± 1.3 mm. Compression tests are performed at a 2 mm min−1 strain rate and the compression modulus is calculated as the tangent slope of the stress–strain curve in the linear region. All the measurements are performed in triplicate and averaged values are reported. The statistical analysis is performed using Student’s t-test and p-value
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My IOPscience
Silk fibroin aerogels: potential scaffolds for tissue engineering applications
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Biomed. Mater. 10 035002 (http://iopscience.iop.org/1748-605X/10/3/035002) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 61.129.42.30 This content was downloaded on 08/05/2015 at 08:35
Please note that terms and conditions apply.
Biomed. Mater. 10 (2015) 035002
doi:10.1088/1748-6041/10/3/035002
Paper
received
4 September 2014
Silk fibroin aerogels: potential scaffolds for tissue engineering applications
re vised
25 March 2015 accep ted for publication
1 April 2015 published
8 May 2015
Rajendar R Mallepally1, Michael A Marin1, Vasudha Surampudi1, Bano Subia2, Raj R Rao1, Subhas C Kundu2 and Mark A McHugh1 1 2
Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA 23284, USA Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal 721302, India
E-mail: [email protected] Keywords: silk fibroin, supercritical CO2, aerogel, cell culture, tissue engineering
Abstract Silk fibroin (SF) is a natural protein, which is derived from the Bombyx mori silkworm. SF based porous materials are extensively investigated for biomedical applications, due to their biocompatibility and biodegradability. In this work, CO2 assisted acidification is used to synthesize SF hydrogels that are subsequently converted to SF aerogels. The aqueous silk fibroin concentration is used to tune the morphology and textural properties of the SF aerogels. As the aqueous fibroin concentration increases from 2 to 6 wt%, the surface area of the resultant SF aerogels increases from 260 to 308 m2 g−1 and the compressive modulus of the SF aerogels increases from 19.5 to 174 kPa. To elucidate the effect of the freezing rate on the morphological and textural properties, SF cryogels are synthesized in this study. The surface area of the SF aerogels obtained from supercritical CO2 drying is approximately five times larger than the surface area of SF cryogels. SF aerogels exhibit distinct pore morphology compared to the SF cryogels. In vitro cell culture studies with human foreskin fibroblast cells demonstrate the cytocompatibility of the silk fibroin aerogel scaffolds and presence of cells within the aerogel scaffolds. The SF aerogels scaffolds created in this study with tailorable properties have potential for applications in tissue engineering.
1. Introduction In the past two decades, tissue repair and regeneration using synthetic scaffolds has emerged as one of the most promising approaches in tissue engineering [1]. An ideal scaffold should be (3D) with an interconnected pore network for cell growth and tissue vascularization, biocompatible and biodegradable, and the scaffold surface chemistry should promote cell attachment, proliferation, and differentiation. Natural and synthetic polymers such as collagen, gelatin, alginate, chitosan, starch, hyaluronic acid, silk fibroin, poly(lactic-coglycolic acid), and poly(ε-caprolactone) etc have been used in tissue engineering as scaffolding materials [2]. Among them, natural polymers have unique, intrinsic properties that make them suitable candidate materials for scaffold fabrication [2–4]. Silk fibroin (SF) from Bombyx mori silkworm cocoons has been a widely investigated natural polymer for numerous biomedical applications due to its versatile properties [4]. SF biomaterials have been fabricated in different forms such as hydrogels [5–7], films [8], micro/nano spheres [9, 10], scaffolds, and fibers [11] © 2015 IOP Publishing Ltd
for different applications. Among these, SF 3D scaffolds have been successfully investigated for the tissue engineering of bone, ligaments, cartilage, nerves, and blood vessels as well as support matrices for cells including osteoblasts, fibroblasts, hepatocytes, and stem cells [12, 13]. The micro-architecture is known to play a significant role in tissue repair and regeneration. For instance, in the case of bone tissue engineering, a scaffold with a bi-modal pore size distribution exhibited improved in vivo performance where pores of ~5 µm are appropriate for tissue vascularization and pores of ~100-to-400 µm are appropriate for cell adhesion and proliferation [14, 15]. Salt leaching is a widely used method for fabricating macro-porous SF scaffolds [16]. After the salt is extracted, the scaffolds are typically freeze dried yielding cryogels. Although freeze drying preserves the primary macro-pore structure, the growth of ice crystals damages the secondary micro-pore structure resulting in less surface area [17, 18]. Supercritical CO2 drying is an alternative technology used to create aerogels with high surface area and high porosity [19]. The aerogels synthesized with supercritical CO2 drying include
Biomed. Mater. 10 (2015) 035002
R R Mallepally et al
those made from inorganics [20–22], organics [23], and polysaccharides [17, 24–26] (alginate, chitosan, starch, pectin, etc) used as catalysts, food additives, pharmaceutical drug carriers, and as porous synthetic bone templates. In addition to the synthesis of aerogels, supercritical or dense gas technology can be used to create other types of porous scaffolds for tissue engineering applications [27–29]. Porous scaffolds have been fabricated from both synthetic and natural polymers using supercritical CO2 foaming, phase inversion, and emulsion template approaches [30–36]. In a previous study, we reported that the molecular weight (Mw) of SF depends on the degumming method and the molecular weight of SF affects the sol–gel kinetics [37]. In another independent study, we created SF aerogels using 95 kDa Mw SF and demonstrated the potential use as drug delivery carriers [38]. The present study describes the synthesis and characterization of silk fibroin aerogels using 190 kDa Mw SF. Salt leaching is combined with supercritical fluid technology to create SF aerogel scaffolds for tissue engineering applications with bi-modal pore size distribution. The design of the scaffold is aimed towards mimicking the native 3D cell environment where cells are surrounded by supportive extra cellular matrix (ECM) that provides the necessary mechanical support. Scanning electron microscopy is used to determine the morphology of the scaffolds and nitrogen adsorption/desorption analysis is used to determine the scaffold textural properties, such as surface area, pore size, and pore volume. Mechanical compression tests are used to determine compression modulus and compression strength of the scaffolds. Finally, in vitro cell culture studies are performed to establish the biocompatibility of the synthesized SF aerogel scaffolds.
2. Materials and methods 2.1. Materials Bombyx mori silkworm cocoons are purchased from Aurora silk, USA. Sodium bicarbonate and lithium bromide are purchased from Sigma Aldrich, USA. Ethanol is purchased from Fischer Scientific, USA. CO2 (99.99%, bone dry) is purchased from Airgas, USA. Dulbecco’s Modified Eagle Medium culture medium, glucose, glutamine, penicillin/streptomycin, fetal bovine serum, Rhodamine–phalloidin, human foreskin fibroblasts, formaldehyde, AlamarBlue, and 4′,6-Diamidino-2-Phenylindole are purchased from Life Technologies, USA. 2.2. Methods 2.2.1. Preparation of silk fibroin solution Cocoons are heated in an aqueous solution of 0.02 M NaHCO3 (1% w/v) at 90 °C for 60 min. Degummed fiber is recovered and washed with boiling water to remove the sericin that coats SF. The SF fiber is dried overnight in an oven at 40 °C. Dried silk fibroin fiber is dissolved in 9.3 M aqueous LiBr solution (10% w/v) 2
at 65 ºC for four hours. The solution is cooled to 25 °C and then dialyzed (Snakeskin Dialysis Tubing, Thermo Scientific, USA, MWCO 3500) against distilled water for three days (2.5% v/v), with daily water replacement. The concentration of aqueous silk fibroin solution is measured by determining the dry weight of SF after evaporating the water at 80 °C from one gram of sample. 2.2.2. Synthesis of SF hydrogels and aerogels SF hydrogels are synthesized using CO2 as a volatile acidifying sol–gel accelerator [37–39] and using the salt-leaching method [40]. For gelation via CO 2– assisted processing, 5 mL SF solution are charged to a glass beaker (height = 35 mm and internal diameter = 18 mm) that is placed in a 300 mL stainless steel vessel, heated to 40 °C, and pressurized with CO2 to 100 bar. After two hours, the CO2 is vented over a period of 30 min and the hydrogel is recovered. For gelation via salt-leaching, 4 g of granular NaCl (particle size 300 to 400 µm) are added slowly to 2 mL of aqueous SF solution. The resulting gel is aged for ~18 h and then soaked in a series of water solutions to extract the salt. SF hydrogels from these two procedures are converted to alcogels by soaking in a series of aqueous alcohol solutions of increasing alcohol concentration as described elsewhere [25]. SF aerogels are then obtained by supercritical CO 2 (scCO 2) drying at 40 °C and 100 bar. The hydrogels pretreatment method fixes the ultimate morphology of the cryogels or aerogels. A single piece of SF hydrogel is synthesized with 2 wt% SF solution using CO2 as an acidifying agent and this hydrogel is cut into three equal parts. Two parts are frozen at different freezing rates; 1) slow freeze at −20 °C and 2) fast freeze at −196 °C. After the initial freeze, the gels are freeze-dried at −20 °C and ~2 mbar pressure for 18 h. The third gel piece is dried using supercritical CO2 drying. The two scaffolds prepared from the slow freeze at −20 °C and fast freeze at −196 °C are termed cryogelslow and cryogel-fast, respectively. 2.3. Silk fibroin aerogels characterization 2.3.1. Fourier transform infrared spectroscopy (FTIR) FTIR (Smart iTR, Thermo Fischer Scientific, USA) is used for the structural characterization of SF aerogels. FTIR spectra are collected in the absorption mode as the mean of 64 scans at 4000 to 400 cm−1 with a spectral resolution of 4 cm−1. 2.3.2. Scanning electron microscope (SEM) SEM (HITACHI SU 70, USA) is used to determine the SF aerogels and SF cryogels morphology. SEM images are captured at an accelerating voltage of 5 keV. The aerogels are immersed in liquid nitrogen, crushed into a fine powder, and placed on aluminum stubs with carbon tape. A 10 nm platinum coating is then applied to the sample via spin coating (Denton Vacuum, USA, Model: Desk V TSC).
Biomed. Mater. 10 (2015) 035002
R R Mallepally et al
washed with 100% ethanol, sterilized under UV light for 30 min, washed several times with PBS (pH 7.4) and incubated with 70% alcohol for 30 min, washed with PBS several times, soaked in PBS in an incubator for 6–8 h in order to neutralize the pH, followed by incubation for three hours in cell culture medium. The 50 µl HFF cell suspensions are then added on top of the scaffold. After two hours of incubation of the scaffold with the cells in the incubator, more medium is added according to the volume of the cell culture dish containing the scaffold. For routine culture, the cell culture medium is replaced every two days with cells routinely maintained in a controlled temperature and environment incubator at 5% CO2 at 37 °C. However, in this instance the experiment is terminated after two days. Figure 1. Schematic representation of high pressure CO2–assisted SF gelation.
2.3.3. Nitrogen adsorption/desorption analysis N2 adsorption/desorption analysis is used to determine the aerogel and cryogel surface area, pore size distribution, and pore volume. Approximately 50 mg of aerogel or cryogel are weighed and then heated at 120 °C under vacuum for four hours. The nitrogen adsorption/ desorption measurements are carried out at 77 K (Nova 2200e, Quantachrome Instruments, USA). The specific surface area of the aerogel or cryogel is calculated with the multipoint Brunauer–Emmett–Teller (BET) model in the relative pressure range of 0.05 to 0.30. The pore size distribution is calculated with the Barrett–Joyner– Halenda (BJH) model using a desorption isotherm. The pore volume is calculated at a relative pressure of 0.98. 2.3.4. Mechanical properties Mechanical properties of the SF hydrogels and aerogels are determined using an Instron (Model 5543) mechanical testing instrument with a 50 N load cell. All the tested SF hydrogels and aerogels are cylindrical discs with a diameter of 15.5 ± 1.3 mm and a height of 9.0 ± 1.3 mm. Compression tests are performed at a 2 mm min−1 strain rate and the compression modulus is calculated as the tangent slope of the stress–strain curve in the linear region. All the measurements are performed in triplicate and averaged values are reported. The statistical analysis is performed using Student’s t-test and p-value
