- Email: [email protected]
DEF using micro-stereolithography
Microelectronic Engineering 84 (2007) 1702–1705 www.elsevier.com/locate/mee
3D scaffold fabrication with PPF/DEF using micro-stereolithography Jin Woo Lee, Phung Xuan Lan, Byung Kim, Geunbae Lim, Dong-Woo Cho
*
Department of Mechanical Engineering, POSTECH, Pohang, Gyeongbuk 790-784, Republic of Korea Available online 15 February 2007
Abstract Current studies on scaffold fabrication have focused on overcoming the limitations imposed by the mechanical properties of existing biodegradable materials and the irregular structures they produce. Recently, several promising biodegradable materials were introduced, including poly(propylene fumarate) (PPF). In addition, the development of micro-stereolithography allows the fabrication of free-form 3D microstructures by dividing a desired shape into several slices of a given thickness. This technology, however, requires a low-viscosity resin to fabricate fine structures, which excludes the use of PPF. To fabricate precise 3D scaffolds using micro-stereolithography, we created a system in which the viscosity of PPF was reduced by adding diethyl fumarate. The fabricated scaffold was sterilized, and fibroblasts in cell culture medium were seeded onto the structure. Cells were fixed and freeze-dried after 4, 7, and 28 days of culture. Under scanning electron microscopy, we observed that the cells were able to attach to the scaffold surface and grow. Ó 2007 Elsevier B.V. All rights reserved. Keywords: Micro-stereolithography; Poly(propylene fumarate) (PPF); Diethyl fumarate (DEF); 3D scaffold fabrication; Cell culture
1. Introduction Although the concept of tissue engineering has great potential, it remains largely undeveloped due to the lack of biocompatible, biodegradable materials with suitable mechanical properties. Fortunately, new biodegradable materials were recently developed, including poly(propylene fumarate) (PPF), which may resolve these limitations. In 1988, Sanderson first produced PPF by the transesterification of diethyl fumarate (DEF) and propylene glycol using a para-toluene sulfonic acid catalyst [1]. Gerhart et al. (1989), Domb et al. (1989), Yaszemski et al. (1994), and Gresser et al. (1995) also prepared PPF using their own methods [1]. Interestingly, the mechanical properties of synthetic PPF are similar to those of trabecular bone [2]. In spite of the limitless potential of bioscaffold technology, the existing methods for fabricating scaffolds are unsatisfactory in terms of 3D free-form fabrication, precision, and pore standardization. Micro-stereolithography is a new technology that can be used to fabricate free-form *
Corresponding author. Tel.: +82 54 279 2171. E-mail address: [email protected] (D.-W. Cho).
0167-9317/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mee.2007.01.267
3D microstructures using a UV laser and a low-viscosity UV-curable liquid photopolymer. This paper describes a modified synthetic method for producing a biodegradable PPF photopolymer that can be used in micro-stereolithography. We fabricated scaffolds using this system and observed the adhesion and growth of fibroblasts seeded onto the structures. 2. Experiments 2.1. Synthesis of the materials PPF was synthesized via a condensation reaction, according to Gerhart et al. [3] with the following modifications: 1.5 mol of fumaric acid (174 g powder) and 1.65 mol of propylene glycol (155.5 g liquid) were placed in a triplenecked 1-L flask with an overhead electrical stirrer, a thermometer, and a Barrett trap beneath a condenser. During synthesis, the mixture was stirred continuously between 130 and 150 rpm. After 1.5 h, the temperature of the solution was increased from room temperature to 145 °C, and boiled vigorously. Within several minutes, water began to collect in the Barrett trap. During the 2–3 h, the mixture
J.W. Lee et al. / Microelectronic Engineering 84 (2007) 1702–1705
was maintained at this temperature, we collected about 65 ml of water. The temperature was then increased to 185 °C to remove the excess propylene glycol and lowmolecular-weight impurities. After 2–3 h at 185 °C, the reaction was terminated. The mixture was kept at room temperature overnight to prevent further polymerization. The final product was a clear, light-yellow, viscous liquid at high temperatures and a gel at room temperature. Fig. 1 shows the process of PPF synthesis. To be able to use PPF as a resin for micro-stereolithography, DEF was added to reduce its viscosity. First, the PPF was heated to approximately 60 °C to decrease its viscosity, and DEF was added in 75:25 of PPF to DEF ratio. Finally, the photoinitiator dimethoxy phenyl acetophenone (DMPA) was added at 4% and the mixture was stirred continuously for 3 h.
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2.2. Scaffold fabrication method Micro-stereolithography, which was developed from a rapid prototyping process, can be used to fabricate freeform 3D microstructures by dividing a desired shape into several slices of a given thickness. During micro-stereolithography, a UV laser irradiates the free surface of a UV-curable liquid photopolymer, causing it to solidify. This differs from conventional microelectromechanical systems (MEMS) technology [4], in which a focused laser beam a few micrometers in diameter is used to solidify a very small area of liquid photopolymer. To fabricate a biodegradable scaffold, the synthesized PPF plus DEF was used as the photopolymer. A heating device was installed at the bottom of the resin reservoir to decrease the viscosity of the photopolymer. A continu-
Fig. 1. Synthesis of PPF.
Fig. 2. Schematic diagram of the micro-stereolithography system.
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J.W. Lee et al. / Microelectronic Engineering 84 (2007) 1702–1705
Fig. 3. Design of the scaffold (a) and the fabrication results (b).
ous-wave Ar ion laser (k = 351.1 nm, Spectra-Physics BeamLok 2065-4S; Newport Corp., Irvine, CA, USA) and an x–y–z stage (ATS-100; Aerotech Inc., Pittsburgh, PA, USA) with 500-nm resolution were used for scaffold fabrication. Fig. 2 shows a schematic diagram of our micro-stereolithography system [5]. 2.3. Scaffold fabrication A 3D scaffold was successfully fabricated using the synthesized PPF plus DEF at a ratio of 75:25. The feed rate of
the laser and laser power were 30 mm/min and 300, respectively. Under these conditions, we fabricated a 3D scaffold of alternating lattices and columns according to the design illustrated in Fig. 3a. The thickness of the lattice layers was 100 lm and the thickness of the column layers was 250 lm. Since two columns were stacked, the final column height was 500 lm. Thirteen layers were stacked, giving a final scaffold height of 2.5 mm. After fabrication, the structure was cleaned using isopropyl alcohol and acetone. Fig. 3b shows a scanning electron micrograph of the scaffold. The line width and pore size of the lattice structure were
Fig. 4. Cell culture using a 3D scaffold: (a) Scaffold SEM image of 4 days after seeding. (b) Scaffold SEM image of 1 week after seeding. (c) Scaffold SEM image of 4 weeks after seeding.
J.W. Lee et al. / Microelectronic Engineering 84 (2007) 1702–1705
132–143 lm and 249–260 lm, respectively. Moreover, the size difference between the fabricated pores and lines was less than 10%. Furthermore, all of the pores were connected with other pores. These results confirm that micro-stereolithography using PPF plus DEF is an effective means of scaffold fabrication.
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scanning electron microscopy (SEM). As depicted Fig. 4, the cells had adhered to the scaffold surface after 4 days, and the cells continued to grow for up to 1 week. After 4 weeks, however, cell growth decreased, the cells were packed into the scaffold pores. These results indicate that fabricated structures, like scaffolds, may be suitable surfaces for cell culture.
2.4. Fibroblast isolation Fibroblasts were isolated from human nasal septum cartilage removed during a nasal cavity biopsy. The specimen was washed three times in PBS and chopped as finely as possible using a dissecting knife. The cells were then dissociated by overnight digestion at 37 °C in a 5% CO2 incubator using 0.1% collagenase type II (Gibco, Grand Island, NY, USA). The digest was strained through a cell strainer (Falcon, Franklin Lakes, NJ, USA) and centrifuged at 1000 rpm for 7 min to isolate the fibroblasts, which were then plated on a /100 tissue-culture plate and incubated in 10 ml of Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin, and 100 lg/ml streptomycin (Gibco). 2.5. Cell culture on the 3D scaffold The fibroblasts were cultured in high-glucose DMEM with 10% FBS, 100 U/ml penicillin, and 100 lg/ml streptomycin at 37 °C in a humidified atmosphere with 5% CO2. The medium was changed 2–3 days after seeding. The cells were harvested at 80–90% confluence using trypsin–EDTA (Gibco), and 105 cells were then seeded onto a scaffold and cultured in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 lg/ml streptomycin. 3. Results At time points between 4 and 28 days of culture, cell adhesion and growth on each scaffold were evaluated under
4. Conclusions In this study, we successfully fabricated 3D scaffolds using micro-stereolithography. All of the pores in the scaffold were connected with other pores. Moreover, the shapes of the solidified lines and pores in the scaffold were very clear and regular. Fibroblasts were cultured on the scaffolds to observe cell adhesion and biocompatibility. Cells were fixed after 4 days, 1 week, and 4 weeks of culture and observed under SEM. Initially, the cells adhered to the scaffold, and over time, we observed additional cells. We conclude that fabricated 3D scaffolds are capable of supporting cell culture. Acknowledgements This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the National Research Laboratory Program funded by the Ministry of Science and Technology (No. M10500000042-06J000004210). References [1] A.J. Domb, J. Kost, D.M. Wiseman (Eds.), Handbook of Biodegradable Polymers, Harwood Academic Publishers, London, 1997. [2] J.P. Fisher, D. Dean, A.G. Mikos, Biomaterials 23 (2002) 4333. [3] D.D. Frazier, V.K. Lathi, T.N. Gerhart, W.C. Hayes, J. Biomed. Mat. Res. 35 (1997) 383. [4] H. Zhang, D.W. Hutmacher, F. Chollet, A.N. Poo, E. Burdet, Macromol. Biosci. 5 (2005) 477. [5] I.H. Lee, D.-W. Cho, Microsys. Technol. 10 (2004) 592.
3D scaffold fabrication with PPF/DEF using micro-stereolithography Jin Woo Lee, Phung Xuan Lan, Byung Kim, Geunbae Lim, Dong-Woo Cho
*
Department of Mechanical Engineering, POSTECH, Pohang, Gyeongbuk 790-784, Republic of Korea Available online 15 February 2007
Abstract Current studies on scaffold fabrication have focused on overcoming the limitations imposed by the mechanical properties of existing biodegradable materials and the irregular structures they produce. Recently, several promising biodegradable materials were introduced, including poly(propylene fumarate) (PPF). In addition, the development of micro-stereolithography allows the fabrication of free-form 3D microstructures by dividing a desired shape into several slices of a given thickness. This technology, however, requires a low-viscosity resin to fabricate fine structures, which excludes the use of PPF. To fabricate precise 3D scaffolds using micro-stereolithography, we created a system in which the viscosity of PPF was reduced by adding diethyl fumarate. The fabricated scaffold was sterilized, and fibroblasts in cell culture medium were seeded onto the structure. Cells were fixed and freeze-dried after 4, 7, and 28 days of culture. Under scanning electron microscopy, we observed that the cells were able to attach to the scaffold surface and grow. Ó 2007 Elsevier B.V. All rights reserved. Keywords: Micro-stereolithography; Poly(propylene fumarate) (PPF); Diethyl fumarate (DEF); 3D scaffold fabrication; Cell culture
1. Introduction Although the concept of tissue engineering has great potential, it remains largely undeveloped due to the lack of biocompatible, biodegradable materials with suitable mechanical properties. Fortunately, new biodegradable materials were recently developed, including poly(propylene fumarate) (PPF), which may resolve these limitations. In 1988, Sanderson first produced PPF by the transesterification of diethyl fumarate (DEF) and propylene glycol using a para-toluene sulfonic acid catalyst [1]. Gerhart et al. (1989), Domb et al. (1989), Yaszemski et al. (1994), and Gresser et al. (1995) also prepared PPF using their own methods [1]. Interestingly, the mechanical properties of synthetic PPF are similar to those of trabecular bone [2]. In spite of the limitless potential of bioscaffold technology, the existing methods for fabricating scaffolds are unsatisfactory in terms of 3D free-form fabrication, precision, and pore standardization. Micro-stereolithography is a new technology that can be used to fabricate free-form *
Corresponding author. Tel.: +82 54 279 2171. E-mail address: [email protected] (D.-W. Cho).
0167-9317/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mee.2007.01.267
3D microstructures using a UV laser and a low-viscosity UV-curable liquid photopolymer. This paper describes a modified synthetic method for producing a biodegradable PPF photopolymer that can be used in micro-stereolithography. We fabricated scaffolds using this system and observed the adhesion and growth of fibroblasts seeded onto the structures. 2. Experiments 2.1. Synthesis of the materials PPF was synthesized via a condensation reaction, according to Gerhart et al. [3] with the following modifications: 1.5 mol of fumaric acid (174 g powder) and 1.65 mol of propylene glycol (155.5 g liquid) were placed in a triplenecked 1-L flask with an overhead electrical stirrer, a thermometer, and a Barrett trap beneath a condenser. During synthesis, the mixture was stirred continuously between 130 and 150 rpm. After 1.5 h, the temperature of the solution was increased from room temperature to 145 °C, and boiled vigorously. Within several minutes, water began to collect in the Barrett trap. During the 2–3 h, the mixture
J.W. Lee et al. / Microelectronic Engineering 84 (2007) 1702–1705
was maintained at this temperature, we collected about 65 ml of water. The temperature was then increased to 185 °C to remove the excess propylene glycol and lowmolecular-weight impurities. After 2–3 h at 185 °C, the reaction was terminated. The mixture was kept at room temperature overnight to prevent further polymerization. The final product was a clear, light-yellow, viscous liquid at high temperatures and a gel at room temperature. Fig. 1 shows the process of PPF synthesis. To be able to use PPF as a resin for micro-stereolithography, DEF was added to reduce its viscosity. First, the PPF was heated to approximately 60 °C to decrease its viscosity, and DEF was added in 75:25 of PPF to DEF ratio. Finally, the photoinitiator dimethoxy phenyl acetophenone (DMPA) was added at 4% and the mixture was stirred continuously for 3 h.
1703
2.2. Scaffold fabrication method Micro-stereolithography, which was developed from a rapid prototyping process, can be used to fabricate freeform 3D microstructures by dividing a desired shape into several slices of a given thickness. During micro-stereolithography, a UV laser irradiates the free surface of a UV-curable liquid photopolymer, causing it to solidify. This differs from conventional microelectromechanical systems (MEMS) technology [4], in which a focused laser beam a few micrometers in diameter is used to solidify a very small area of liquid photopolymer. To fabricate a biodegradable scaffold, the synthesized PPF plus DEF was used as the photopolymer. A heating device was installed at the bottom of the resin reservoir to decrease the viscosity of the photopolymer. A continu-
Fig. 1. Synthesis of PPF.
Fig. 2. Schematic diagram of the micro-stereolithography system.
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J.W. Lee et al. / Microelectronic Engineering 84 (2007) 1702–1705
Fig. 3. Design of the scaffold (a) and the fabrication results (b).
ous-wave Ar ion laser (k = 351.1 nm, Spectra-Physics BeamLok 2065-4S; Newport Corp., Irvine, CA, USA) and an x–y–z stage (ATS-100; Aerotech Inc., Pittsburgh, PA, USA) with 500-nm resolution were used for scaffold fabrication. Fig. 2 shows a schematic diagram of our micro-stereolithography system [5]. 2.3. Scaffold fabrication A 3D scaffold was successfully fabricated using the synthesized PPF plus DEF at a ratio of 75:25. The feed rate of
the laser and laser power were 30 mm/min and 300, respectively. Under these conditions, we fabricated a 3D scaffold of alternating lattices and columns according to the design illustrated in Fig. 3a. The thickness of the lattice layers was 100 lm and the thickness of the column layers was 250 lm. Since two columns were stacked, the final column height was 500 lm. Thirteen layers were stacked, giving a final scaffold height of 2.5 mm. After fabrication, the structure was cleaned using isopropyl alcohol and acetone. Fig. 3b shows a scanning electron micrograph of the scaffold. The line width and pore size of the lattice structure were
Fig. 4. Cell culture using a 3D scaffold: (a) Scaffold SEM image of 4 days after seeding. (b) Scaffold SEM image of 1 week after seeding. (c) Scaffold SEM image of 4 weeks after seeding.
J.W. Lee et al. / Microelectronic Engineering 84 (2007) 1702–1705
132–143 lm and 249–260 lm, respectively. Moreover, the size difference between the fabricated pores and lines was less than 10%. Furthermore, all of the pores were connected with other pores. These results confirm that micro-stereolithography using PPF plus DEF is an effective means of scaffold fabrication.
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scanning electron microscopy (SEM). As depicted Fig. 4, the cells had adhered to the scaffold surface after 4 days, and the cells continued to grow for up to 1 week. After 4 weeks, however, cell growth decreased, the cells were packed into the scaffold pores. These results indicate that fabricated structures, like scaffolds, may be suitable surfaces for cell culture.
2.4. Fibroblast isolation Fibroblasts were isolated from human nasal septum cartilage removed during a nasal cavity biopsy. The specimen was washed three times in PBS and chopped as finely as possible using a dissecting knife. The cells were then dissociated by overnight digestion at 37 °C in a 5% CO2 incubator using 0.1% collagenase type II (Gibco, Grand Island, NY, USA). The digest was strained through a cell strainer (Falcon, Franklin Lakes, NJ, USA) and centrifuged at 1000 rpm for 7 min to isolate the fibroblasts, which were then plated on a /100 tissue-culture plate and incubated in 10 ml of Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin, and 100 lg/ml streptomycin (Gibco). 2.5. Cell culture on the 3D scaffold The fibroblasts were cultured in high-glucose DMEM with 10% FBS, 100 U/ml penicillin, and 100 lg/ml streptomycin at 37 °C in a humidified atmosphere with 5% CO2. The medium was changed 2–3 days after seeding. The cells were harvested at 80–90% confluence using trypsin–EDTA (Gibco), and 105 cells were then seeded onto a scaffold and cultured in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 lg/ml streptomycin. 3. Results At time points between 4 and 28 days of culture, cell adhesion and growth on each scaffold were evaluated under
4. Conclusions In this study, we successfully fabricated 3D scaffolds using micro-stereolithography. All of the pores in the scaffold were connected with other pores. Moreover, the shapes of the solidified lines and pores in the scaffold were very clear and regular. Fibroblasts were cultured on the scaffolds to observe cell adhesion and biocompatibility. Cells were fixed after 4 days, 1 week, and 4 weeks of culture and observed under SEM. Initially, the cells adhered to the scaffold, and over time, we observed additional cells. We conclude that fabricated 3D scaffolds are capable of supporting cell culture. Acknowledgements This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the National Research Laboratory Program funded by the Ministry of Science and Technology (No. M10500000042-06J000004210). References [1] A.J. Domb, J. Kost, D.M. Wiseman (Eds.), Handbook of Biodegradable Polymers, Harwood Academic Publishers, London, 1997. [2] J.P. Fisher, D. Dean, A.G. Mikos, Biomaterials 23 (2002) 4333. [3] D.D. Frazier, V.K. Lathi, T.N. Gerhart, W.C. Hayes, J. Biomed. Mat. Res. 35 (1997) 383. [4] H. Zhang, D.W. Hutmacher, F. Chollet, A.N. Poo, E. Burdet, Macromol. Biosci. 5 (2005) 477. [5] I.H. Lee, D.-W. Cho, Microsys. Technol. 10 (2004) 592.