In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-015-9925-8
Regulation of astrocyte activity via control over stiffness of cellulose acetate electrospun nanofiber Seul Ki Min & Sang Myung Jung & Jung Hyeon Ju & Yeo Seon Kwon & Gwang Heum Yoon & Hwa Sung Shin
Received: 26 January 2015 / Accepted: 20 May 2015 / Editor: T. Okamoto # The Society for In Vitro Biology 2015
Abstract Astrocytes are involved in neuron protection following central nervous system (CNS) injury; accordingly, engineered astrocytes have been investigated for their usefulness in cell therapy for CNS injury. Nanofibers have attracted a great deal of attention in neural tissue engineering, but their mechanical properties greatly influence physiology. Cellulose acetate (CA) has been studied for use in scaffolds owing to its biocompatibility, biodegradability, and good thermal stability. In this study, stiffness of CA nanofibers controlled by heat treatment was shown to regulate astrocyte activity. Adhesion and viability increased in culture as substrate became stiffer but showed saturation at greater than 2 MPa of tensile strength. Astrocytes became more active in terms of increasing intermediate filament glial fibrillary acidic protein (GFAP). The results of this study demonstrate the effects of stiffness alone on cellular behaviors in a three-dimensional culture and highlight the efficacy of heat-treated CA for astrocyte culture in that the simple treatment enables control of astrocyte activity.
S. K. Min : S. M. Jung : J. H. Ju : Y. S. Kwon : G. H. Yoon : H. S. Shin (*) Department of Biological Engineering, Inha University, Incheon 402-751, Korea e-mail: [email protected] S. K. Min e-mail: [email protected] S. M. Jung e-mail: [email protected] J. H. Ju e-mail: [email protected] Y. S. Kwon e-mail: [email protected] G. H. Yoon e-mail: [email protected]
Keywords Cellulose acetate . Electrospun nanofiber . Heat treatment . Stiffness . Astrocyte tissue engineering
Introduction The central nervous system (CNS) contains a large number of nerve cells supported by glial cells. When the CNS is injured, it is difficult for the neural cells to regenerate. Accordingly, many studies have investigated treatment of injured CNS with drugs and transplants. Astrocytes, the most abundant cell in the brain, are a type of glial cell that occupy approximately 30–50% of the brain and play important roles in supporting neural cells and the brain-blood barrier (BBB), regulating the concentration of ions and maintaining homeostasis for neuronal function. Implantable materials for brain disease have purpose to regenerate the damaged tissue by filling the cavity and supporting structure (Wang et al. 2014). For example, ischemic stroke obstructs cerebral blood supply, and it causes loss of brain tissue resulting in the formation of infarct cavity. The infarct area leads to a larger cavity and worsens by inflammation or immune reaction as time goes by. Therefore, bioengineered astrocytic tissue has been actively investigated for their potential as implantable tissue constructs for treatment of injured neural tissue (Yucel et al. 2010; Meng et al. 2012). Astrocytes are of particular interest because lost and healthy tissue consists primarily of astrocytes that do not divide well in the normal state and astrocytes have various functions that help regenerate injured neurons, such as the ability to release neurotrophic factors (Sofroniew and Vinters 2010). Nanofiber scaffold produced by electrospinning is now widely used for astrocyte tissue engineering because its structure and topography facilitate tissue constructs similar to the actual
MIN ET AL.
tissues (Ma et al. 2005; Ladd et al. 2011). Nanofibers can be produced from synthetic polymers, poly (ε-caprolactone) (PCL), poly (lactic-co-glycolic acid) (PLGA), and poly (ethylene oxide) (PEO), as well as from naturally derived polymers such as collagen, gelatin, cellulose acetate, alginate, and dextran (Huang et al. 2003; Kweon et al. 2003; Pham et al. 2006). Mechanical properties influence cell function, differentiation, morphology, and composition (Blacklock et al. 2010; Monici and Cialdai 2008; Wells 2008). Nanofiber is suitable for being used as a scaffold because it has same morphological features with extracellular matrix (ECM). Therefore, stiffness is appropriate to control not inducing morphological changes for using nanofiber. Physical and chemical reactions are required to change the stiffness of a scaffold. Nanofiber stiffness is easily modified by chemical reactions or variations in fabrication, but these methods induce differences in surface chemistry, topography, or mechanical properties among nanofibers, making analysis of the stiffness effects difficult due to contamination with other causes (Liu et al. 2011). Physical modifications that do not induce any chemical, topological, or mechanical variations other than stiffness are preferable. Stiffness of extracellular matrix generally plays a pivotal role in the formation and function of a tissue, and several studies have shown that tissues such as the bone, cartilage, brain, and stem cells detect changes in substrate stiffness via mechanical stress-sensing cascades (Mason et al. 2012). However, stiffness is known to have different effects among cell types (Tee et al. 2009). Several studies have recently investigated the effects of scaffold stiffness on astrocyte activities based on hydrogel culture systems (Jiang et al. 2007; Moshayedi et al. 2010). However, hydrogel stiffness is controlled by monomer and cross-linker densities; therefore, structural or electrochemical variations could differ. In addition, nanofiber-based studies of the effects of stiffness on cellular behaviors have rarely been performed. Cellulose is one of the most abundant natural polysaccharide polymers on earth, and cellulose acetate (CA) is a cellulose derivative that can be obtained by acetylation of the hydroxyl group of cellulose. CA has been applied to biological applications such as filters, scaffolds for artificial tissues, and dressings due to its good thermal stability, chemical resistance, and biodegradability. For example, cellulose acetate is actively used for bone and cartilage tissue engineering because of its biocompatibility and mechanical strength (Katoh and Urist 1993; Mayer‐Wagner et al. 2011). In addition, CA has been investigated as a material for vascular tissue and peripheral nervous system recovery (Han and Cheung 2011; Pooyan et al. 2012). However, few studies have employed 3D cultures of astrocytes on CA nanofiber. In this study, we fabricated an array of electrospun nanofibrous mats with cellulose acetate and manipulated their stiffness by a simple heat treatment. Structural and chemical changes were investigated
after thermal treatment to ensure that there were no chemical or physical variations among nanofibers except for stiffness, and primary astrocytes were then assessed for their physiology after cultivation on nanofibrous mats with different levels of stiffness. Therefore, it is believed that this material could be considered to implant for the regeneration of lost brain tissue.
Materials and Methods Fabrication and thermal modification of electrospun CA nanofibrous mat. CA (Mw =30,000, Sigma Aldrich) was dissolved by mixing with a 1:1 mixture of acetone and acetic acid at 15% (w/v) for 24 h. The solution was then loaded into a plastic syringe connected to an 18 gauge needle mounted on a syringe pump (KD Scientific) and electrospun onto a drum collector 17 cm from the needle tip under 17 kV at 1 ml h−1. Thermal treatment was then conducted as previously described (Wells 2008; Pooyan et al. 2012). Briefly, the CA nanofibrous mat was thermally treated in a drying oven at 207°C for 0, 1, and 2 h so that cellular behavior could be observed according to changes in the stiffness of the mat. Characterization of the electrospun CA nanofibrous mat. Contact angle was examined to compare wettability and hydrophilicity among scaffolds subjected to thermal treatment for different lengths of time. Briefly, 4-μl water droplets were placed onto the samples through a 22G syringe needle, after which the contact angle was measured using a contact angle meter (SEO, South Korea). The morphologies of the CA nanofibers were then observed via scanning electron microscopy (SEM) (Hitachi). Next, the fabricated scaffold was cut to 1×1 cm2 and sputtered with platinum for 180 s, and the samples were then observed at 15 kV under 1000× magnification. Additionally, the thickness of nanofiber strands was manually measured in the SEM images using image J (NIH, v1.47). The data were analyzed after measuring 200 nanofibers. The tensile strength and viscoelasticity of the CA nanofibrous mat was measured using a Universal Testing Machine (Instron). All tensile testing samples were in the form of a rectangle 30 mm long, 16 mm wide, and 140 μm thick, and tests were carried out at a constant drawing speed of 10 mm min−1 in the length direction at room temperature. To confirm the structural changes in the nanofiber component after heat treatment, the infrared from the FT-IR (Bruker) was reflected off the samples and analyzed. Each graph was plotted using data obtained from at least 30 scans and peaks and was compared with reference peaks. Primary astrocyte cell culture. Primary astrocytes were obtained from the cerebrum of neonatal Sprague-Dawley rats
HEAT-TREATED CA NANOFIBER FOR THE ASTROCYTE CULTURE
(Samtako, South Korea) using a previously described protocol (Fedoroff and Richardson 2001). The brain was then isolated from the skull, and its meninges were gently removed from the cerebrum. The cerebrum was then minced with a Pasteur pipette and passed through a 0.7-mm pore cell strainer. For astrocyte isolation, various cells with culture media were poured into a T-75 polystyrene-coated tissue culture flask (5×106 cells) and then incubated under 5% CO2 at 37°C for 1 wk. The culture medium consisted of Dulbecco-modified eagle’s medium (DMEM-high glucose, GIBCO) with 10% FBS and 1% antibiotic-antimycotic solution. After 1 wk, samples were cultured on a shaker at 200 rpm in a CO2 incubator for 48 h to remove the other glial cells. Astrocytes were strongly adhered and therefore remained on the T-75 cell culture flask. Adhesion and viability of astrocytes onto the CA nanofiber. To measure the adhesion and viability, astrocytes were seeded onto a CA nanofiber mat in a 24-well cell culture plate at a concentration of 2×104 cells/well and 1×104 cells/well, respectively. The adhesion and viability of astrocytes were then measured at 2, 8, and 24 h and 1, 4, and 7 d, respectively. To investigate the viability and adhesion, a WST-1 assay was conducted according to the manufacturer’s instructions. Western blotting analysis of GFAP. Western blotting was conducted to measure the level of glial fibrillary acidic protein (GFAP). First, astrocytes were seeded on a CA nanofiber mat that had been thermally treated. Proteins were then extracted from the astrocytes using RIPA buffer, after which they were quantified with a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific). The same amount of proteins from each sample was separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), after which separated proteins were transferred to a poly (vinylidene fluoride) (PVDF) membrane (Sigma Aldrich). The membrane was then blocked by incubation in blocking buffer (5% skim milk in triethanolamine-buffered saline (TBS)) at 4°C overnight. Next, the PVDF membrane was treated with rabbit antiGFAP polyclonal IgG antibody as primary antibody in TBS containing 5% skim milk overnight at 4°C followed by goat anti-rabbit IgG-HRP as the secondary antibody for 1 h. The PVDF membrane was washed with TBST three times for 5 min each between antibody treatment steps. And then, antibodies attached on the PVDF membrane reacted with ECL solution (RPN2232, GE healthcare, UK) and the membrane developed on the film in a dark room. Statistical analysis. All experiments were carried out three times for statistical analyses and expressed as the means±standard deviation (S.D.). A Student’s t test was then used to identify significant differences among groups with a p value
Regulation of astrocyte activity via control over stiffness of cellulose acetate electrospun nanofiber Seul Ki Min & Sang Myung Jung & Jung Hyeon Ju & Yeo Seon Kwon & Gwang Heum Yoon & Hwa Sung Shin
Received: 26 January 2015 / Accepted: 20 May 2015 / Editor: T. Okamoto # The Society for In Vitro Biology 2015
Abstract Astrocytes are involved in neuron protection following central nervous system (CNS) injury; accordingly, engineered astrocytes have been investigated for their usefulness in cell therapy for CNS injury. Nanofibers have attracted a great deal of attention in neural tissue engineering, but their mechanical properties greatly influence physiology. Cellulose acetate (CA) has been studied for use in scaffolds owing to its biocompatibility, biodegradability, and good thermal stability. In this study, stiffness of CA nanofibers controlled by heat treatment was shown to regulate astrocyte activity. Adhesion and viability increased in culture as substrate became stiffer but showed saturation at greater than 2 MPa of tensile strength. Astrocytes became more active in terms of increasing intermediate filament glial fibrillary acidic protein (GFAP). The results of this study demonstrate the effects of stiffness alone on cellular behaviors in a three-dimensional culture and highlight the efficacy of heat-treated CA for astrocyte culture in that the simple treatment enables control of astrocyte activity.
S. K. Min : S. M. Jung : J. H. Ju : Y. S. Kwon : G. H. Yoon : H. S. Shin (*) Department of Biological Engineering, Inha University, Incheon 402-751, Korea e-mail: [email protected] S. K. Min e-mail: [email protected] S. M. Jung e-mail: [email protected] J. H. Ju e-mail: [email protected] Y. S. Kwon e-mail: [email protected] G. H. Yoon e-mail: [email protected]
Keywords Cellulose acetate . Electrospun nanofiber . Heat treatment . Stiffness . Astrocyte tissue engineering
Introduction The central nervous system (CNS) contains a large number of nerve cells supported by glial cells. When the CNS is injured, it is difficult for the neural cells to regenerate. Accordingly, many studies have investigated treatment of injured CNS with drugs and transplants. Astrocytes, the most abundant cell in the brain, are a type of glial cell that occupy approximately 30–50% of the brain and play important roles in supporting neural cells and the brain-blood barrier (BBB), regulating the concentration of ions and maintaining homeostasis for neuronal function. Implantable materials for brain disease have purpose to regenerate the damaged tissue by filling the cavity and supporting structure (Wang et al. 2014). For example, ischemic stroke obstructs cerebral blood supply, and it causes loss of brain tissue resulting in the formation of infarct cavity. The infarct area leads to a larger cavity and worsens by inflammation or immune reaction as time goes by. Therefore, bioengineered astrocytic tissue has been actively investigated for their potential as implantable tissue constructs for treatment of injured neural tissue (Yucel et al. 2010; Meng et al. 2012). Astrocytes are of particular interest because lost and healthy tissue consists primarily of astrocytes that do not divide well in the normal state and astrocytes have various functions that help regenerate injured neurons, such as the ability to release neurotrophic factors (Sofroniew and Vinters 2010). Nanofiber scaffold produced by electrospinning is now widely used for astrocyte tissue engineering because its structure and topography facilitate tissue constructs similar to the actual
MIN ET AL.
tissues (Ma et al. 2005; Ladd et al. 2011). Nanofibers can be produced from synthetic polymers, poly (ε-caprolactone) (PCL), poly (lactic-co-glycolic acid) (PLGA), and poly (ethylene oxide) (PEO), as well as from naturally derived polymers such as collagen, gelatin, cellulose acetate, alginate, and dextran (Huang et al. 2003; Kweon et al. 2003; Pham et al. 2006). Mechanical properties influence cell function, differentiation, morphology, and composition (Blacklock et al. 2010; Monici and Cialdai 2008; Wells 2008). Nanofiber is suitable for being used as a scaffold because it has same morphological features with extracellular matrix (ECM). Therefore, stiffness is appropriate to control not inducing morphological changes for using nanofiber. Physical and chemical reactions are required to change the stiffness of a scaffold. Nanofiber stiffness is easily modified by chemical reactions or variations in fabrication, but these methods induce differences in surface chemistry, topography, or mechanical properties among nanofibers, making analysis of the stiffness effects difficult due to contamination with other causes (Liu et al. 2011). Physical modifications that do not induce any chemical, topological, or mechanical variations other than stiffness are preferable. Stiffness of extracellular matrix generally plays a pivotal role in the formation and function of a tissue, and several studies have shown that tissues such as the bone, cartilage, brain, and stem cells detect changes in substrate stiffness via mechanical stress-sensing cascades (Mason et al. 2012). However, stiffness is known to have different effects among cell types (Tee et al. 2009). Several studies have recently investigated the effects of scaffold stiffness on astrocyte activities based on hydrogel culture systems (Jiang et al. 2007; Moshayedi et al. 2010). However, hydrogel stiffness is controlled by monomer and cross-linker densities; therefore, structural or electrochemical variations could differ. In addition, nanofiber-based studies of the effects of stiffness on cellular behaviors have rarely been performed. Cellulose is one of the most abundant natural polysaccharide polymers on earth, and cellulose acetate (CA) is a cellulose derivative that can be obtained by acetylation of the hydroxyl group of cellulose. CA has been applied to biological applications such as filters, scaffolds for artificial tissues, and dressings due to its good thermal stability, chemical resistance, and biodegradability. For example, cellulose acetate is actively used for bone and cartilage tissue engineering because of its biocompatibility and mechanical strength (Katoh and Urist 1993; Mayer‐Wagner et al. 2011). In addition, CA has been investigated as a material for vascular tissue and peripheral nervous system recovery (Han and Cheung 2011; Pooyan et al. 2012). However, few studies have employed 3D cultures of astrocytes on CA nanofiber. In this study, we fabricated an array of electrospun nanofibrous mats with cellulose acetate and manipulated their stiffness by a simple heat treatment. Structural and chemical changes were investigated
after thermal treatment to ensure that there were no chemical or physical variations among nanofibers except for stiffness, and primary astrocytes were then assessed for their physiology after cultivation on nanofibrous mats with different levels of stiffness. Therefore, it is believed that this material could be considered to implant for the regeneration of lost brain tissue.
Materials and Methods Fabrication and thermal modification of electrospun CA nanofibrous mat. CA (Mw =30,000, Sigma Aldrich) was dissolved by mixing with a 1:1 mixture of acetone and acetic acid at 15% (w/v) for 24 h. The solution was then loaded into a plastic syringe connected to an 18 gauge needle mounted on a syringe pump (KD Scientific) and electrospun onto a drum collector 17 cm from the needle tip under 17 kV at 1 ml h−1. Thermal treatment was then conducted as previously described (Wells 2008; Pooyan et al. 2012). Briefly, the CA nanofibrous mat was thermally treated in a drying oven at 207°C for 0, 1, and 2 h so that cellular behavior could be observed according to changes in the stiffness of the mat. Characterization of the electrospun CA nanofibrous mat. Contact angle was examined to compare wettability and hydrophilicity among scaffolds subjected to thermal treatment for different lengths of time. Briefly, 4-μl water droplets were placed onto the samples through a 22G syringe needle, after which the contact angle was measured using a contact angle meter (SEO, South Korea). The morphologies of the CA nanofibers were then observed via scanning electron microscopy (SEM) (Hitachi). Next, the fabricated scaffold was cut to 1×1 cm2 and sputtered with platinum for 180 s, and the samples were then observed at 15 kV under 1000× magnification. Additionally, the thickness of nanofiber strands was manually measured in the SEM images using image J (NIH, v1.47). The data were analyzed after measuring 200 nanofibers. The tensile strength and viscoelasticity of the CA nanofibrous mat was measured using a Universal Testing Machine (Instron). All tensile testing samples were in the form of a rectangle 30 mm long, 16 mm wide, and 140 μm thick, and tests were carried out at a constant drawing speed of 10 mm min−1 in the length direction at room temperature. To confirm the structural changes in the nanofiber component after heat treatment, the infrared from the FT-IR (Bruker) was reflected off the samples and analyzed. Each graph was plotted using data obtained from at least 30 scans and peaks and was compared with reference peaks. Primary astrocyte cell culture. Primary astrocytes were obtained from the cerebrum of neonatal Sprague-Dawley rats
HEAT-TREATED CA NANOFIBER FOR THE ASTROCYTE CULTURE
(Samtako, South Korea) using a previously described protocol (Fedoroff and Richardson 2001). The brain was then isolated from the skull, and its meninges were gently removed from the cerebrum. The cerebrum was then minced with a Pasteur pipette and passed through a 0.7-mm pore cell strainer. For astrocyte isolation, various cells with culture media were poured into a T-75 polystyrene-coated tissue culture flask (5×106 cells) and then incubated under 5% CO2 at 37°C for 1 wk. The culture medium consisted of Dulbecco-modified eagle’s medium (DMEM-high glucose, GIBCO) with 10% FBS and 1% antibiotic-antimycotic solution. After 1 wk, samples were cultured on a shaker at 200 rpm in a CO2 incubator for 48 h to remove the other glial cells. Astrocytes were strongly adhered and therefore remained on the T-75 cell culture flask. Adhesion and viability of astrocytes onto the CA nanofiber. To measure the adhesion and viability, astrocytes were seeded onto a CA nanofiber mat in a 24-well cell culture plate at a concentration of 2×104 cells/well and 1×104 cells/well, respectively. The adhesion and viability of astrocytes were then measured at 2, 8, and 24 h and 1, 4, and 7 d, respectively. To investigate the viability and adhesion, a WST-1 assay was conducted according to the manufacturer’s instructions. Western blotting analysis of GFAP. Western blotting was conducted to measure the level of glial fibrillary acidic protein (GFAP). First, astrocytes were seeded on a CA nanofiber mat that had been thermally treated. Proteins were then extracted from the astrocytes using RIPA buffer, after which they were quantified with a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific). The same amount of proteins from each sample was separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), after which separated proteins were transferred to a poly (vinylidene fluoride) (PVDF) membrane (Sigma Aldrich). The membrane was then blocked by incubation in blocking buffer (5% skim milk in triethanolamine-buffered saline (TBS)) at 4°C overnight. Next, the PVDF membrane was treated with rabbit antiGFAP polyclonal IgG antibody as primary antibody in TBS containing 5% skim milk overnight at 4°C followed by goat anti-rabbit IgG-HRP as the secondary antibody for 1 h. The PVDF membrane was washed with TBST three times for 5 min each between antibody treatment steps. And then, antibodies attached on the PVDF membrane reacted with ECL solution (RPN2232, GE healthcare, UK) and the membrane developed on the film in a dark room. Statistical analysis. All experiments were carried out three times for statistical analyses and expressed as the means±standard deviation (S.D.). A Student’s t test was then used to identify significant differences among groups with a p value
