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Development of cylindrical microfibrous scaffold using melt-spinning method for vascular tissue engineering
Materials Letters 228 (2018) 334–338
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/mlblue
Development of cylindrical microfibrous scaffold using melt-spinning method for vascular tissue engineering Azizah Pangesty a, Mitsugu Todo b,⇑ a b
Department of Molecular and Material Science, Interdisciplinary Graduate School of Engineering Science, Kyushu University, Kasuga, Fukuoka 816-8580, Japan Research Institute for Applied Mechanics, Kyushu University, Kasuga, Fukuoka 816-8580, Japan
a r t i c l e
i n f o
Article history: Received 27 March 2018 Received in revised form 1 June 2018 Accepted 13 June 2018
Keywords: Microfibrous scaffold Cotton candy machine Vascular tissue engineering
a b s t r a c t In this report, we demonstrated fabrication of cylindrical scaffold made of poly (lactide-co-caprolactone) (PLCL) fibers for vascular tissue engineering by the melt-spinning method with use of a commercially available cotton candy machine. This method was able to generate microfibrous cylindrical scaffold with the fiber diameter ranged of 1–17 mm. It was found that the microfibrous scaffold showed greater mechanical properties including elastic modulus and ring tensile strength than those of cylindrical microporous PLCL scaffold prepared by the phase separation method. Biological test using hMSCs also demonstrated that the microfibrous scaffold exhibited better cells growth behavior including larger cell area and aspect ratio than the microporous scaffold after 3 h culture. Cell proliferation on the microfibrous scaffold was significantly higher than that on the microporous scaffold during 7 days culture. In conclusion, microfibrous scaffold with improved mechanical and biological properties was successfully fabricated using the melt-spinning method. Ó 2018 Published by Elsevier B.V.
1. Introduction Vascular Tissue Engineering (VTE) is projected as an alternative graft for treatment of vascular diseases. Scaffold plays an important role as a structural support for cell adhesion and tissue development in VTE [1]. Among various methods [2], phase separation and electrospinning have widely been used to fabricate scaffolds. The phase separation method has widely been used to prepare porous scaffold with pore sizes in the order of a few to hundreds of microns. However, the pores formed using this technique are usually not uniformly distributed which may affect the mechanical properties and cell binding and spreading [3]. To mimic the fibrous structure of the natural extracellular matrix in tissue, electrospinning method is developed to engineer fibrous scaffold. Micro/nanofibrous structures obtained by electrospinning have large surface area, providing more surface anchorage for cell binding and proliferation [3]. Although the electrospinning have extensively been studied, its low production rate and complexity of control parameters of electrospinning method may limit its clinical application [4]. In addition, the electrospinning often uses toxic
⇑ Corresponding author. E-mail address: [email protected] (M. Todo). https://doi.org/10.1016/j.matlet.2018.06.046 0167-577X/Ó 2018 Published by Elsevier B.V.
organic solvents during the fabrication process and therefore, their residue in the products may harm the human body [5]. To overcome those problems, other alternative techniques must be developed to fabricate fibrous scaffolds without toxic solvents. On the other hand, the melt-spinning method, known as the production method of cotton candy, has been utilized to fabricate scaffolds with cotton-like fibrous structures [6]. One of the main advantages of this technique is that commercially available cotton-candy machines may be directly used to fabricate scaffolds for medical applications. In the cotton-candy machines, melted polymer is centrifugally forced through a small hole to make fibers. Although this technique allows us to fabricate polymeric fibers in a simple cost-effective way and free from toxic solvents, to the best of our knowledge, few attempts have been made to fabricate fibrous cylindrical scaffolds using the melt-spinning method [7]. The aim of this study is therefore to produce fibrous cylindrical scaffolds for VTE using the melt-spinning method with biocompatible and biodegradable polymer. A commercially available cottoncandy machine was utilized to fabricate microfibrous cylindrical structures. As the fundamental properties, tensile mechanical properties and cellular activities were examined. Furthermore, the fabricated microfibrous cylindrical scaffold was compared with the microporous cylindrical scaffold previously developed using the phase-separation and freeze-drying method in our laboratory [8].
A. Pangesty, M. Todo / Materials Letters 228 (2018) 334–338
2. Methods 2.1. Fabrication of cylindrical scaffolds Microfibrous cylindrical scaffolds were fabricated from PLCL (75/25) (BMG Co., Japan) with the melting temperature of 163 °C
335
using a commercial cotton candy machine (EA-WA2805, Azuma Engineering Co., Japan). Two gram of PLCL granules were put into the spinning head of the machine. The spinning head has a fixed rotating speed of 1800 rpm and a heating temperature of 180 °C. The temperature was measured using a digital thermometer (Custom, Japan) during fabrication process of PLCL fibers. They
Fig. 1. The microfibrous cylindrical scaffold (a–d). (a) Fabrication process of microfibrous scaffold using cotton candy machine. (b) Photograph of the microfibrous scaffold. (c) Distribution of fibers diameter. (d) SEM images of microstructure. The microporous cylindrical scaffold (e–g). (e) Photograph of the microporous scaffold. (f) Distribution of pore diameter. (g) SEM images of microstructure.
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A. Pangesty, M. Todo / Materials Letters 228 (2018) 334–338
were immediately collected on the surface of a Teflon rod (/ 5 mm) and then dried for one night. The fabrication process is illustrated in Fig. 1a. Microporous cylindrical scaffolds were also fabricated by the solid–liquid phase separation and freeze-drying method with use of solution of PLCL 75/25 and dioxane as the solvent (6% w/v) [9]. Briefly, a Teflon rod (/ 5 mm) prepared at 80 °C was dipped into the polymer solution at a constant rate, and dried using a freeze-drying machine. 2.2. Characterization Ring tensile test was performed at a displacement rate of 1 mm/ min by a conventional testing machine (EZ Test, Shimadzu Co., Japan), as illustrated in Fig. 2a [8]. Force and displacement were recorded in a personal computer. The circumferential strain, e, was calculated from the elongation, DC, of the internal circumference:
e¼
DC C0
ð1Þ
F 2Lt
2.3. Cell culture study Human mesenchymal stem cells (hMSCs) (Riken BRC, Japan) were seeded into the microfibrous and microporous scaffolds, respectively, with cell density of 12,000 cells/cm2. They were then cultured for 7 days with cell growth medium containing MEM-a, 10% Fetal bovine serum and 1% penicillin streptomycin at 37 °C/5% CO2. The number of viable cells was monitored using a cell counting kit (Dojindo, Japan). The cell morphology was observed using FE-SEM and analyzed using Image-J. Statistical analysis was performed using ANOVA with Fischer’s LSD post hoc test. Each data represented as mean ± SD and P < 0.05. 3. Results and discussion 3.1. Morphology
where C0 is the initial length of the internal circumference. The circumferential stress, r, was calculated from the force using the following formula:
r¼
FE-SEM images obtained, fiber diameter and pore size were measured by an image analysis software Image-J (NIH). The density of the scaffold was calculated by the ratio of the mass to the total of the geometric volume, according to Chung et al. [7].
ð2Þ
where L and t were the length and thickness of cylindrical scaffolds, respectively. The elastic modulus was obtained as the initial slope of the stress–strain relation. The tensile strength was also evaluated as the maximum value of stress. Microstructures of the specimens were observed by a fieldemission electron microscope (FE-SEM, Hitachi, Japan). Using the
The microfibrous scaffold with thickness of 0.5 mm and the density of 0.37 g/cm3 could be fabricated from two gram of PLCL (Fig. 1b). The fibers were randomly oriented and overlapped each other three-dimensionally to form a fiber mesh with high porosity and large surface area, suitable for cell binding and spreading. In the internal wall, the diameter of fibers ranged from 2.0 mm to 17.0 mm (Fig. 1c and d). On the other hand, the fiber diameter ranged from 1.0 mm to 5.0 mm in the external wall. Viscosity of the melted polymer is considered to be one of the key factors that affect the fiber diameter and in general, the lower viscosity results in the higher diameter. The increased temperature likely caused
Fig. 2. (a) Typical stress–strain curve, (b) Elastic moduli, and (c) circumferential tensile strength. Each data represent as mean ± SD (n = 3), * indicates P < 0.05.
A. Pangesty, M. Todo / Materials Letters 228 (2018) 334–338
less viscosity of the melted polymer, therefore, the diameter decreased during fabrication process. An overview of the microporous scaffold is shown in Fig. 1e. The area of pores was in the range of square micro-meter as shown in Fig. 1f. A typical porous structure obtained from phase separation method was observed in this scaffold (Fig. 1g). This type of porous structure was created by the phase separation between the solvent and the polymer-rich phase. After solvent evaporation, the pores were formed and the polymer-rich phase solidified to form strut. 3.2. Mechanical properties The results of the mechanical tests are shown in Fig. 2. The microfibrous scaffold demonstrated steeper stress–strain curve with higher stress and larger strain compared to the microporous scaffold (Fig. 2a). The elastic modulus (Fig. 2b) and the circumferential tensile strength (Fig. 2c) of the microfibrous scaffold were approximately 1.5 times higher than those of the microporous scaffold. These results clearly demonstrated that the microfibrous structure resulted in greater tensile mechanical properties than the microporous structure constructed with strut structures. 3.3. Biological properties The initial cellular responses to the microfibrous and microporous scaffolds are shown in Fig. 3. Three hours after culture, cells on the microfibrous scaffold were more flattened, stretched and intimately adhered compared with those attached on the microp-
337
orous scaffold (Fig. 3a). Furthermore, cells on the microfibrous scaffold have developed filopodia, while no filapodia was found in the cells attached on microporous scaffold. Cells further grew on both the scaffolds. Twenty four hours after culture, no distinctive cell features were observed on both scaffolds (Fig. 3a). Cells on the microfibrous scaffold had significantly larger cell area and aspect ratio compared to those on the microporous scaffold (Fig. 3b). It was reported that larger cell area and aspect ratio improved cell proliferation [10]. As the result, cells proliferation on the microfibrous scaffold was significantly higher than that on the microporous scaffold (Fig. 3b). 4. Conclusions In this preliminary study, we demonstrated the use of cotton candy machine to fabricate microfibrous cylindrical scaffold for vascular tissue engineering. The microfibrous scaffold showed improved tensile mechanical properties such as elastic modulus and tensile strength, approximately 1.5 times higher than those of the microporous scaffold obtained by the phase separation. The initial cell study using hMSCs suggested that the microfibrous scaffold provided more favorable environment for cells growth than the microporous cylindrical scaffold evidenced from better cell morphology and higher proliferation. The use of commercially available cotton candy machines allowed us to fabricate microfibrous cylindrical scaffolds without using toxic solvent. Furthermore, the simplicity of our method provides an opportunity for scale-up in a cost-effective way.
Fig. 3. (a) SEM images of cell attachment on microfibrous and microporous scaffold at 3 and 24 h culture (cell, red arrow). (b) Quantification of cell area (left), aspect ratio (middle) and cell proliferation (right). Cell area and aspect ratio at 3 h culture were quantified by Image-J (n 159). Cell proliferation (n = 6) at 4 and 7 days. * indicates P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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A. Pangesty, M. Todo / Materials Letters 228 (2018) 334–338
References [1] S. Tara, K.A. Rocco, N. Hibino, T. Sugiura, H. Kurobe, C.K. Breuer, T. Shinoka, Vessel bioengineering, Circ. J. 12–19 (2014). [2] P.X. Ma, Scaffolds for tissue fabrication, Mater. Today 7 (2004) 30–40. [3] M. Stevens, J. George, Exploring and engineering the cell surface interface, Science 80 (2005) 1135–1139. [4] K. Sarkar, C. Gomez, S. Zambrano, M. Ramirez, E. De Hoyos, H. Vasquez, K. Lozano, Electrospinning to forcespinning, Mater. Today 13 (2010) 12–14. [5] S. Khorshidi, A. Solouk, H. Mirzadeh, S. Mazinani, J.M. Lagaron, S. Sharifi, S. Ramakrishna, A review of key challenges of electrospun scaffolds for tissueengineering applications, J. Tissue Eng. Regener. Med. 10 (2016) 715–738. [6] J.B. Lee, X. Wang, S. Faley, B. Baer, D.A. Balikov, H. Sung, L.M. Bellan, Development of 3D microvascular networks within gelatin hydrogels using
[7]
[8]
[9] [10]
thermoresponsive sacrificial microfibers, Adv. Healthcare Mater. 5 (2016) 781– 785. S. Chung, N.P. Ingle, G.A. Montero, S.H. Kim, M.W. King, Bioresorbable elastomeric vascular tissue engineering scaffolds via melt spinning and electrospinning, Acta Biomater. 6 (2010) 1958–1967. A.I. Pangesty, T. Arahira, M. Todo, Development and characterization of hybrid tubular structure of PLCL porous scaffold with hMSCs/ECs cell sheet, J. Mater. Sci. Mater. Med. 28 (2017) 165. I.A. Pangesty, T. Arahira, M. Todo, Characterization of tensile mechanical behavior of MSCs/PLCL hybrid layered sheet, J. Funct. Biomater. 7 (2016). R. Sunarso, K. Toita, K. Tsuru, Ishikawa, Immobilization of calcium and phosphate ions improves the osteoconductivity of titanium implants, Mater. Sci. Eng., C 68 (2016) 291–298.
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/mlblue
Development of cylindrical microfibrous scaffold using melt-spinning method for vascular tissue engineering Azizah Pangesty a, Mitsugu Todo b,⇑ a b
Department of Molecular and Material Science, Interdisciplinary Graduate School of Engineering Science, Kyushu University, Kasuga, Fukuoka 816-8580, Japan Research Institute for Applied Mechanics, Kyushu University, Kasuga, Fukuoka 816-8580, Japan
a r t i c l e
i n f o
Article history: Received 27 March 2018 Received in revised form 1 June 2018 Accepted 13 June 2018
Keywords: Microfibrous scaffold Cotton candy machine Vascular tissue engineering
a b s t r a c t In this report, we demonstrated fabrication of cylindrical scaffold made of poly (lactide-co-caprolactone) (PLCL) fibers for vascular tissue engineering by the melt-spinning method with use of a commercially available cotton candy machine. This method was able to generate microfibrous cylindrical scaffold with the fiber diameter ranged of 1–17 mm. It was found that the microfibrous scaffold showed greater mechanical properties including elastic modulus and ring tensile strength than those of cylindrical microporous PLCL scaffold prepared by the phase separation method. Biological test using hMSCs also demonstrated that the microfibrous scaffold exhibited better cells growth behavior including larger cell area and aspect ratio than the microporous scaffold after 3 h culture. Cell proliferation on the microfibrous scaffold was significantly higher than that on the microporous scaffold during 7 days culture. In conclusion, microfibrous scaffold with improved mechanical and biological properties was successfully fabricated using the melt-spinning method. Ó 2018 Published by Elsevier B.V.
1. Introduction Vascular Tissue Engineering (VTE) is projected as an alternative graft for treatment of vascular diseases. Scaffold plays an important role as a structural support for cell adhesion and tissue development in VTE [1]. Among various methods [2], phase separation and electrospinning have widely been used to fabricate scaffolds. The phase separation method has widely been used to prepare porous scaffold with pore sizes in the order of a few to hundreds of microns. However, the pores formed using this technique are usually not uniformly distributed which may affect the mechanical properties and cell binding and spreading [3]. To mimic the fibrous structure of the natural extracellular matrix in tissue, electrospinning method is developed to engineer fibrous scaffold. Micro/nanofibrous structures obtained by electrospinning have large surface area, providing more surface anchorage for cell binding and proliferation [3]. Although the electrospinning have extensively been studied, its low production rate and complexity of control parameters of electrospinning method may limit its clinical application [4]. In addition, the electrospinning often uses toxic
⇑ Corresponding author. E-mail address: [email protected] (M. Todo). https://doi.org/10.1016/j.matlet.2018.06.046 0167-577X/Ó 2018 Published by Elsevier B.V.
organic solvents during the fabrication process and therefore, their residue in the products may harm the human body [5]. To overcome those problems, other alternative techniques must be developed to fabricate fibrous scaffolds without toxic solvents. On the other hand, the melt-spinning method, known as the production method of cotton candy, has been utilized to fabricate scaffolds with cotton-like fibrous structures [6]. One of the main advantages of this technique is that commercially available cotton-candy machines may be directly used to fabricate scaffolds for medical applications. In the cotton-candy machines, melted polymer is centrifugally forced through a small hole to make fibers. Although this technique allows us to fabricate polymeric fibers in a simple cost-effective way and free from toxic solvents, to the best of our knowledge, few attempts have been made to fabricate fibrous cylindrical scaffolds using the melt-spinning method [7]. The aim of this study is therefore to produce fibrous cylindrical scaffolds for VTE using the melt-spinning method with biocompatible and biodegradable polymer. A commercially available cottoncandy machine was utilized to fabricate microfibrous cylindrical structures. As the fundamental properties, tensile mechanical properties and cellular activities were examined. Furthermore, the fabricated microfibrous cylindrical scaffold was compared with the microporous cylindrical scaffold previously developed using the phase-separation and freeze-drying method in our laboratory [8].
A. Pangesty, M. Todo / Materials Letters 228 (2018) 334–338
2. Methods 2.1. Fabrication of cylindrical scaffolds Microfibrous cylindrical scaffolds were fabricated from PLCL (75/25) (BMG Co., Japan) with the melting temperature of 163 °C
335
using a commercial cotton candy machine (EA-WA2805, Azuma Engineering Co., Japan). Two gram of PLCL granules were put into the spinning head of the machine. The spinning head has a fixed rotating speed of 1800 rpm and a heating temperature of 180 °C. The temperature was measured using a digital thermometer (Custom, Japan) during fabrication process of PLCL fibers. They
Fig. 1. The microfibrous cylindrical scaffold (a–d). (a) Fabrication process of microfibrous scaffold using cotton candy machine. (b) Photograph of the microfibrous scaffold. (c) Distribution of fibers diameter. (d) SEM images of microstructure. The microporous cylindrical scaffold (e–g). (e) Photograph of the microporous scaffold. (f) Distribution of pore diameter. (g) SEM images of microstructure.
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A. Pangesty, M. Todo / Materials Letters 228 (2018) 334–338
were immediately collected on the surface of a Teflon rod (/ 5 mm) and then dried for one night. The fabrication process is illustrated in Fig. 1a. Microporous cylindrical scaffolds were also fabricated by the solid–liquid phase separation and freeze-drying method with use of solution of PLCL 75/25 and dioxane as the solvent (6% w/v) [9]. Briefly, a Teflon rod (/ 5 mm) prepared at 80 °C was dipped into the polymer solution at a constant rate, and dried using a freeze-drying machine. 2.2. Characterization Ring tensile test was performed at a displacement rate of 1 mm/ min by a conventional testing machine (EZ Test, Shimadzu Co., Japan), as illustrated in Fig. 2a [8]. Force and displacement were recorded in a personal computer. The circumferential strain, e, was calculated from the elongation, DC, of the internal circumference:
e¼
DC C0
ð1Þ
F 2Lt
2.3. Cell culture study Human mesenchymal stem cells (hMSCs) (Riken BRC, Japan) were seeded into the microfibrous and microporous scaffolds, respectively, with cell density of 12,000 cells/cm2. They were then cultured for 7 days with cell growth medium containing MEM-a, 10% Fetal bovine serum and 1% penicillin streptomycin at 37 °C/5% CO2. The number of viable cells was monitored using a cell counting kit (Dojindo, Japan). The cell morphology was observed using FE-SEM and analyzed using Image-J. Statistical analysis was performed using ANOVA with Fischer’s LSD post hoc test. Each data represented as mean ± SD and P < 0.05. 3. Results and discussion 3.1. Morphology
where C0 is the initial length of the internal circumference. The circumferential stress, r, was calculated from the force using the following formula:
r¼
FE-SEM images obtained, fiber diameter and pore size were measured by an image analysis software Image-J (NIH). The density of the scaffold was calculated by the ratio of the mass to the total of the geometric volume, according to Chung et al. [7].
ð2Þ
where L and t were the length and thickness of cylindrical scaffolds, respectively. The elastic modulus was obtained as the initial slope of the stress–strain relation. The tensile strength was also evaluated as the maximum value of stress. Microstructures of the specimens were observed by a fieldemission electron microscope (FE-SEM, Hitachi, Japan). Using the
The microfibrous scaffold with thickness of 0.5 mm and the density of 0.37 g/cm3 could be fabricated from two gram of PLCL (Fig. 1b). The fibers were randomly oriented and overlapped each other three-dimensionally to form a fiber mesh with high porosity and large surface area, suitable for cell binding and spreading. In the internal wall, the diameter of fibers ranged from 2.0 mm to 17.0 mm (Fig. 1c and d). On the other hand, the fiber diameter ranged from 1.0 mm to 5.0 mm in the external wall. Viscosity of the melted polymer is considered to be one of the key factors that affect the fiber diameter and in general, the lower viscosity results in the higher diameter. The increased temperature likely caused
Fig. 2. (a) Typical stress–strain curve, (b) Elastic moduli, and (c) circumferential tensile strength. Each data represent as mean ± SD (n = 3), * indicates P < 0.05.
A. Pangesty, M. Todo / Materials Letters 228 (2018) 334–338
less viscosity of the melted polymer, therefore, the diameter decreased during fabrication process. An overview of the microporous scaffold is shown in Fig. 1e. The area of pores was in the range of square micro-meter as shown in Fig. 1f. A typical porous structure obtained from phase separation method was observed in this scaffold (Fig. 1g). This type of porous structure was created by the phase separation between the solvent and the polymer-rich phase. After solvent evaporation, the pores were formed and the polymer-rich phase solidified to form strut. 3.2. Mechanical properties The results of the mechanical tests are shown in Fig. 2. The microfibrous scaffold demonstrated steeper stress–strain curve with higher stress and larger strain compared to the microporous scaffold (Fig. 2a). The elastic modulus (Fig. 2b) and the circumferential tensile strength (Fig. 2c) of the microfibrous scaffold were approximately 1.5 times higher than those of the microporous scaffold. These results clearly demonstrated that the microfibrous structure resulted in greater tensile mechanical properties than the microporous structure constructed with strut structures. 3.3. Biological properties The initial cellular responses to the microfibrous and microporous scaffolds are shown in Fig. 3. Three hours after culture, cells on the microfibrous scaffold were more flattened, stretched and intimately adhered compared with those attached on the microp-
337
orous scaffold (Fig. 3a). Furthermore, cells on the microfibrous scaffold have developed filopodia, while no filapodia was found in the cells attached on microporous scaffold. Cells further grew on both the scaffolds. Twenty four hours after culture, no distinctive cell features were observed on both scaffolds (Fig. 3a). Cells on the microfibrous scaffold had significantly larger cell area and aspect ratio compared to those on the microporous scaffold (Fig. 3b). It was reported that larger cell area and aspect ratio improved cell proliferation [10]. As the result, cells proliferation on the microfibrous scaffold was significantly higher than that on the microporous scaffold (Fig. 3b). 4. Conclusions In this preliminary study, we demonstrated the use of cotton candy machine to fabricate microfibrous cylindrical scaffold for vascular tissue engineering. The microfibrous scaffold showed improved tensile mechanical properties such as elastic modulus and tensile strength, approximately 1.5 times higher than those of the microporous scaffold obtained by the phase separation. The initial cell study using hMSCs suggested that the microfibrous scaffold provided more favorable environment for cells growth than the microporous cylindrical scaffold evidenced from better cell morphology and higher proliferation. The use of commercially available cotton candy machines allowed us to fabricate microfibrous cylindrical scaffolds without using toxic solvent. Furthermore, the simplicity of our method provides an opportunity for scale-up in a cost-effective way.
Fig. 3. (a) SEM images of cell attachment on microfibrous and microporous scaffold at 3 and 24 h culture (cell, red arrow). (b) Quantification of cell area (left), aspect ratio (middle) and cell proliferation (right). Cell area and aspect ratio at 3 h culture were quantified by Image-J (n 159). Cell proliferation (n = 6) at 4 and 7 days. * indicates P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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A. Pangesty, M. Todo / Materials Letters 228 (2018) 334–338
References [1] S. Tara, K.A. Rocco, N. Hibino, T. Sugiura, H. Kurobe, C.K. Breuer, T. Shinoka, Vessel bioengineering, Circ. J. 12–19 (2014). [2] P.X. Ma, Scaffolds for tissue fabrication, Mater. Today 7 (2004) 30–40. [3] M. Stevens, J. George, Exploring and engineering the cell surface interface, Science 80 (2005) 1135–1139. [4] K. Sarkar, C. Gomez, S. Zambrano, M. Ramirez, E. De Hoyos, H. Vasquez, K. Lozano, Electrospinning to forcespinning, Mater. Today 13 (2010) 12–14. [5] S. Khorshidi, A. Solouk, H. Mirzadeh, S. Mazinani, J.M. Lagaron, S. Sharifi, S. Ramakrishna, A review of key challenges of electrospun scaffolds for tissueengineering applications, J. Tissue Eng. Regener. Med. 10 (2016) 715–738. [6] J.B. Lee, X. Wang, S. Faley, B. Baer, D.A. Balikov, H. Sung, L.M. Bellan, Development of 3D microvascular networks within gelatin hydrogels using
[7]
[8]
[9] [10]
thermoresponsive sacrificial microfibers, Adv. Healthcare Mater. 5 (2016) 781– 785. S. Chung, N.P. Ingle, G.A. Montero, S.H. Kim, M.W. King, Bioresorbable elastomeric vascular tissue engineering scaffolds via melt spinning and electrospinning, Acta Biomater. 6 (2010) 1958–1967. A.I. Pangesty, T. Arahira, M. Todo, Development and characterization of hybrid tubular structure of PLCL porous scaffold with hMSCs/ECs cell sheet, J. Mater. Sci. Mater. Med. 28 (2017) 165. I.A. Pangesty, T. Arahira, M. Todo, Characterization of tensile mechanical behavior of MSCs/PLCL hybrid layered sheet, J. Funct. Biomater. 7 (2016). R. Sunarso, K. Toita, K. Tsuru, Ishikawa, Immobilization of calcium and phosphate ions improves the osteoconductivity of titanium implants, Mater. Sci. Eng., C 68 (2016) 291–298.