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Investigation on biodegradable PLGA scaffold with various pore size structure for skin tissue engineering
Current Applied Physics 7S1 (2007) e37–e40 www.elsevier.com/locate/cap www.kps.or.kr
Investigation on biodegradable PLGA scaffold with various pore size structure for skin tissue engineering Jun Jae Lee a, Sang-Gil Lee a, Jong Chul Park b, Young Il Yang c, Jeong Koo Kim b
a,*
a Department of Biomedical Engineering, Inje University, Gimhae 621-749, Republic of Korea Department of Medical Engineering, College of Medicine, Yonsei University, Seoul 120-751, Republic of Korea c Paik Institute for Clinical Research, Inje University Hospital, Busan 614-735, Republic of Korea
Available online 6 February 2007
Abstract For tissue regeneration, highly open porous polymer matrices are required for high-density cell seeding, as well as sufficient nutrient and oxygen supply to the cells in the 3-D matrices. In this study, three types of scaffolds containing three different pore sizes (uniformpore size, 2-layer pore size and multi-pore size) were prepared. We used human dermal fibroblast cells to investigate cell attachment and proliferation with the prepared specimens. Not only DNA quantity measurement of the cells but also the number of cells within the cross-sectional area of the scaffold was investigated. In the DNA quantity test, the multi-pore size scaffold contained a 1.77 times larger amount than the uniform-pore size scaffold for 14 days culture. For the cell-counting assessment, the multi-pore size scaffold contained about 2.24 times more cells than the uniform-pore size scaffold in the middle sectioned area for 14 days. To prevent the generation of a polymer skin layer on the surface of the scaffolds, PLGA scaffolds were fabricated by a two-step molding method. No skin layer was observed in the scaffold. Various pore size specimens tended to degrade more and faster than uniform-pore size specimens in PBS solution. Chemical treatment on the surface of the specimen enhances cytocompatibility of the scaffold. 2006 Published by Elsevier B.V. PACS: 61.41.+e Keywords: Multi-pore size; Two-layer pore size; Cytocompatibility; Tissue engineering
1. Introduction In recent years, biodegradable polymers have been utilized to fabricate a porous scaffold for three-dimensional (3-D) cell culture to regenerate tissue-based artificial organs [1,2]. Porous scaffolds have been prepared by numerous techniques, such as phase separation, emulsion freeze drying, fiber extrusion, gas foaming, 3-D printing, and solvent casting/particulate leaching [2–4]. Usually, in the middle stage of cell culture on the scaffolds, the pores might be narrowed toward the inner space, due to the cell proliferation. This causes interference with cell growth into the inner space of the scaffold [4–6]. *
Corresponding author. Tel.: +82 55 326 3664; fax: +82 55 327 3292. E-mail address: [email protected] (J.K. Kim).
1567-1739/$ - see front matter 2006 Published by Elsevier B.V. doi:10.1016/j.cap.2006.11.011
In this study, we investigated the attachment and proliferation of HDF (human dermal fibroblast) in porous PLGA scaffolds with various types of pore size and their surface characterizations. We prepared three types of scaffolds containing three different pore sizes (uniform-pore size, 2-layer pore size and multi-pore size) to investigate the efficacy of cell growth into the inside of the scaffold and transportation of nutrients and oxygen to the inner space of the scaffold [7–10]. Moreover, the prepared scaffold often generates skin layers which make poor interconnectivity between pores and interfere with cell proliferation in the scaffold [2,5,8]. To solve this problem, a two-step process for preparing the scaffold was performed. We investigate better interconnectivity and improvement of cell attachment and proliferation, as well as surface properties of the scaffold.
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J.J. Lee et al. / Current Applied Physics 7S1 (2007) e37–e40
2. Materials and methods 2.1. Synthesis of poly(lactic-co-glycolic) acid (PLGA) Lactide and glycolide were purchased from PURAC (Holland). PLGA (75:25 represents the molar ratio of lactide to glycolide in the copolymer) was synthesized by ringopen polymerization. Under nitrogen atmosphere, lactide and glycolide with stannous 2-ethyl hexanoate were dried in a one-necked flask under vacuum and stirring at 130 C for 10 h. The copolymer was synthesized and dissolved in chloroform. 2.2. Manufacture of PLGA scaffold PLGA (75:25) scaffolds were manufactured in composite wafers with sodium chloride crystals by a solvent casting technique (PLGA and NaCl were at 1:10 weight ratio). We strained NaCl particles through micro-sieves, and the particle size ranges were 212–250 lm, 250–300 lm, and 355–500 lm. After thoroughly mixing NaCl and the polymer solution, it was poured into a teflon-coated mold and compressed at room temperature. A two-step process for making a 3-D-structure scaffold was implemented as follows. The mixed solution was poured into a tefloncoated mold; then, NaCl was sprinkled and the mixture was compressed again. Three types of scaffolds were prepared: so-called uniform-pore size (250–300 lm), 2-layer pore size (212–250 lm, 355–500 lm), and multi-pore size (212–250 lm, 250–300 lm, 355–500 lm).
ical Co.), 100 U/ml penicillin and 100 lg/ml streptomycin in a moist atmosphere of 5% CO2 at 37 C. Cell growth was determined by the fluorometric DNA quantitative method by using PicoGreen (Molecular Probes Inc.). 2.6. Morphological analysis H&E staining was applied to the inner parts of the scaffold. The scaffold was fixed by addition of 200 ll of 3.0% formaldehyde solution for a 1-week harvest time. The samples were fixed with paraffin wax for counting cell numbers in the inner part of the scaffold. The specimen was crosssectioned horizontally by using a microtome. Five sectioned scaffolds of each group were photographed at 100 magnification, and cells were counted by light microscope. 3. Results and discussion The cross-sectioned PLGA scaffolds were observed as shown in Fig. 1. They showed an excellent interconnecting property of the porous structure. Porosity of the scaffold
2.3. Chemical treatment of the scaffolds and contact angle measurements To increase hydrophilicity of the PLGA scaffold, chemical etching with ethanol and chloric acid to PLGA films and scaffolds was performed and characterized by contact angle measurement. 2.4. Degradation rate of the PLGA scaffold A simulated biodegradation rate of the specimens was implemented with phosphate-buffered saline (PBS) solution (pH = 7.4). The scaffolds were immersed in PBS solution and stirred at 100 rpm in a shaking incubator at 37 C. The degradation rate was characterized by the change in mass at 1 week, 2 weeks, and 4 weeks. 2.5. Cell culture and proliferation Human dermal fibroblast cells were seeded in the scaffold at 1.5 · 105 cells/scaffold and cultured for 2 weeks. One group was prepared by dynamic culture, and the other group was static culture. The scaffolds were cultivated in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (FBS, Thermo Trace Ltd., Melbourne, Australia), 50 lg/ml L-ascorbic acid (Sigma Chem-
Fig. 1. Cross-section image of PLGA scaffolds: (a) 2-layer pore size structure and (b) multi-pore size structure.
J.J. Lee et al. / Current Applied Physics 7S1 (2007) e37–e40
was over 90%. Moreover, skin layers, which are a defect of the salt leaching method, were not detected throughout the scaffolds. In the 2-layer specimen, the upper layer contains bigger pores and there is a border line in the specimen. In the multi-pore size specimen, various sizes of pores were observed as shown in Fig. 1b. The surface of the scaffold treated with chloric acid showed a lower contact angle (62.2 on average; n = 5) than the control (73.2) and the ethanol-treated specimen (66.8). For the biodegradation test, the 2-layer scaffold specimen group showed a mass loss of about 20% by weight for 4 weeks. The results of DNA quantity measurements of each specimen group with dynamic culture are shown in Fig. 2a, and those with static culture in Fig. 2b. In dynamic culture, the multi-pore size specimen contained an about 1.77 times higher DNA quantity than the uniform-pore size specimens at 14-day culture. The two-layer scaffolds contained 1.33 times more cells than the uniform-pore size scaffold. Due to movement of the culture dish, the cells incompletely attached to the scaffold might be detached from the scaffold, causing a gradual decrease of the DNA quantity from the 4th day. In the dynamic test, the pore size turned out to be one of the cell culture factors and various pore sizes were effective on cell proliferation. The
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multi-pore size group was the highest-DNA content group among the three groups. In the static culture, as shown in Fig. 2b, the DNA quantity gradually increased from the initial point to the 14th day, whether the surface was treated or not. The amount of DNA content of the surface-treated group was 1.2 times higher than for the untreated group. Also, the multi-pore size group was more effective in cell proliferation than the uniform-pore size group.
Fig. 3. Number of counted cells in the vertical cross-sectioned area of the specimen (n = 5).
Fig. 2. DNA quantities of HDF seeded on the PLGA scaffolds for 14 days: (a) dynamic culture (n = 6) and (b) static culture (n = 6).
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The number of cells in the inner part of each scaffold in the groups is shown in Fig. 3. The multi-pore size group showed the highest number of cells in the middle of the scaffold. Statistical analysis shows that each group has a significantly different number of cells (p < 0.01). This means that the multi-pore size specimen could make better spaces for cell growth towards the inside of the scaffold than the other groups.
4. Conclusion Boss the multi-pore size and the 2-layer scaffold showed better environments for cell culture, compared with the uniform-pore size scaffold. The multi-pore size PLGA scaffold could make room for cells to move inside of the scaffold and transport nutrients and oxygen efficiently to the inside of the scaffold. Various pore size distributions if the scaffold could offer an improved environment for culturing human dermal fibroblast cells and proliferation. The surface-modified multi-pore size PLGA scaffold was most effective for cell culture in this study.
Acknowledgement This work was supported by the 2005 Inje University Research Grant. References [1] T.G. Park, J.J. Yoon, Polymer Sci. Technol. 10 (1999) 722. [2] G.S. Khang, J.H. Jeon, J.C. Cho, Polymer (Korea) 23 (1999) 861. [3] Tony G. Van Tienen, Ralf G.J.C. Heijkants, Pieter Bumaa, Biomaterials 23 (2002) 1731. [4] T.M. Freyman, I.V. Yannas, L.J. Gibson, Prog. Mater. Sci. 46 (2001) 273. [5] H.R. Lin, C.J. Kuo, C.Y. Yang, et al., J. Biomed. Mater. Res. 63 (2002) 271. [6] Y.S. Nam, J.J. Yoon, T.G. Park, J. Biomed. Mater. Res. (Appl. Biomater.) 53 (2000) 1. [7] L.D. Harris, Byung-Soo Kim, David J. Mooney, J. Biomed. Mater. Res. 42 (1998) 396. [8] C.J. Liao, C.F. Chen, J.H. Chen, S.F. Chiang, Y.J. Lin, K.Y. Chang, J. Biomed. Mater. Res. 59 (2002) 676. [9] Z. Ma, C. Gao, J. Yuan, J. Ji, Y. Gong, J. Shen, J. Appl. Polym. Sci. 85 (2002) 2163. [10] S.F. El-Amin, H.H. Lu, Y. Khan, J. Burems, J. Mitchell, R.S. Tuan, C.T. Laurencin, Biomaterials 24 (2003) 1213.
Investigation on biodegradable PLGA scaffold with various pore size structure for skin tissue engineering Jun Jae Lee a, Sang-Gil Lee a, Jong Chul Park b, Young Il Yang c, Jeong Koo Kim b
a,*
a Department of Biomedical Engineering, Inje University, Gimhae 621-749, Republic of Korea Department of Medical Engineering, College of Medicine, Yonsei University, Seoul 120-751, Republic of Korea c Paik Institute for Clinical Research, Inje University Hospital, Busan 614-735, Republic of Korea
Available online 6 February 2007
Abstract For tissue regeneration, highly open porous polymer matrices are required for high-density cell seeding, as well as sufficient nutrient and oxygen supply to the cells in the 3-D matrices. In this study, three types of scaffolds containing three different pore sizes (uniformpore size, 2-layer pore size and multi-pore size) were prepared. We used human dermal fibroblast cells to investigate cell attachment and proliferation with the prepared specimens. Not only DNA quantity measurement of the cells but also the number of cells within the cross-sectional area of the scaffold was investigated. In the DNA quantity test, the multi-pore size scaffold contained a 1.77 times larger amount than the uniform-pore size scaffold for 14 days culture. For the cell-counting assessment, the multi-pore size scaffold contained about 2.24 times more cells than the uniform-pore size scaffold in the middle sectioned area for 14 days. To prevent the generation of a polymer skin layer on the surface of the scaffolds, PLGA scaffolds were fabricated by a two-step molding method. No skin layer was observed in the scaffold. Various pore size specimens tended to degrade more and faster than uniform-pore size specimens in PBS solution. Chemical treatment on the surface of the specimen enhances cytocompatibility of the scaffold. 2006 Published by Elsevier B.V. PACS: 61.41.+e Keywords: Multi-pore size; Two-layer pore size; Cytocompatibility; Tissue engineering
1. Introduction In recent years, biodegradable polymers have been utilized to fabricate a porous scaffold for three-dimensional (3-D) cell culture to regenerate tissue-based artificial organs [1,2]. Porous scaffolds have been prepared by numerous techniques, such as phase separation, emulsion freeze drying, fiber extrusion, gas foaming, 3-D printing, and solvent casting/particulate leaching [2–4]. Usually, in the middle stage of cell culture on the scaffolds, the pores might be narrowed toward the inner space, due to the cell proliferation. This causes interference with cell growth into the inner space of the scaffold [4–6]. *
Corresponding author. Tel.: +82 55 326 3664; fax: +82 55 327 3292. E-mail address: [email protected] (J.K. Kim).
1567-1739/$ - see front matter 2006 Published by Elsevier B.V. doi:10.1016/j.cap.2006.11.011
In this study, we investigated the attachment and proliferation of HDF (human dermal fibroblast) in porous PLGA scaffolds with various types of pore size and their surface characterizations. We prepared three types of scaffolds containing three different pore sizes (uniform-pore size, 2-layer pore size and multi-pore size) to investigate the efficacy of cell growth into the inside of the scaffold and transportation of nutrients and oxygen to the inner space of the scaffold [7–10]. Moreover, the prepared scaffold often generates skin layers which make poor interconnectivity between pores and interfere with cell proliferation in the scaffold [2,5,8]. To solve this problem, a two-step process for preparing the scaffold was performed. We investigate better interconnectivity and improvement of cell attachment and proliferation, as well as surface properties of the scaffold.
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J.J. Lee et al. / Current Applied Physics 7S1 (2007) e37–e40
2. Materials and methods 2.1. Synthesis of poly(lactic-co-glycolic) acid (PLGA) Lactide and glycolide were purchased from PURAC (Holland). PLGA (75:25 represents the molar ratio of lactide to glycolide in the copolymer) was synthesized by ringopen polymerization. Under nitrogen atmosphere, lactide and glycolide with stannous 2-ethyl hexanoate were dried in a one-necked flask under vacuum and stirring at 130 C for 10 h. The copolymer was synthesized and dissolved in chloroform. 2.2. Manufacture of PLGA scaffold PLGA (75:25) scaffolds were manufactured in composite wafers with sodium chloride crystals by a solvent casting technique (PLGA and NaCl were at 1:10 weight ratio). We strained NaCl particles through micro-sieves, and the particle size ranges were 212–250 lm, 250–300 lm, and 355–500 lm. After thoroughly mixing NaCl and the polymer solution, it was poured into a teflon-coated mold and compressed at room temperature. A two-step process for making a 3-D-structure scaffold was implemented as follows. The mixed solution was poured into a tefloncoated mold; then, NaCl was sprinkled and the mixture was compressed again. Three types of scaffolds were prepared: so-called uniform-pore size (250–300 lm), 2-layer pore size (212–250 lm, 355–500 lm), and multi-pore size (212–250 lm, 250–300 lm, 355–500 lm).
ical Co.), 100 U/ml penicillin and 100 lg/ml streptomycin in a moist atmosphere of 5% CO2 at 37 C. Cell growth was determined by the fluorometric DNA quantitative method by using PicoGreen (Molecular Probes Inc.). 2.6. Morphological analysis H&E staining was applied to the inner parts of the scaffold. The scaffold was fixed by addition of 200 ll of 3.0% formaldehyde solution for a 1-week harvest time. The samples were fixed with paraffin wax for counting cell numbers in the inner part of the scaffold. The specimen was crosssectioned horizontally by using a microtome. Five sectioned scaffolds of each group were photographed at 100 magnification, and cells were counted by light microscope. 3. Results and discussion The cross-sectioned PLGA scaffolds were observed as shown in Fig. 1. They showed an excellent interconnecting property of the porous structure. Porosity of the scaffold
2.3. Chemical treatment of the scaffolds and contact angle measurements To increase hydrophilicity of the PLGA scaffold, chemical etching with ethanol and chloric acid to PLGA films and scaffolds was performed and characterized by contact angle measurement. 2.4. Degradation rate of the PLGA scaffold A simulated biodegradation rate of the specimens was implemented with phosphate-buffered saline (PBS) solution (pH = 7.4). The scaffolds were immersed in PBS solution and stirred at 100 rpm in a shaking incubator at 37 C. The degradation rate was characterized by the change in mass at 1 week, 2 weeks, and 4 weeks. 2.5. Cell culture and proliferation Human dermal fibroblast cells were seeded in the scaffold at 1.5 · 105 cells/scaffold and cultured for 2 weeks. One group was prepared by dynamic culture, and the other group was static culture. The scaffolds were cultivated in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (FBS, Thermo Trace Ltd., Melbourne, Australia), 50 lg/ml L-ascorbic acid (Sigma Chem-
Fig. 1. Cross-section image of PLGA scaffolds: (a) 2-layer pore size structure and (b) multi-pore size structure.
J.J. Lee et al. / Current Applied Physics 7S1 (2007) e37–e40
was over 90%. Moreover, skin layers, which are a defect of the salt leaching method, were not detected throughout the scaffolds. In the 2-layer specimen, the upper layer contains bigger pores and there is a border line in the specimen. In the multi-pore size specimen, various sizes of pores were observed as shown in Fig. 1b. The surface of the scaffold treated with chloric acid showed a lower contact angle (62.2 on average; n = 5) than the control (73.2) and the ethanol-treated specimen (66.8). For the biodegradation test, the 2-layer scaffold specimen group showed a mass loss of about 20% by weight for 4 weeks. The results of DNA quantity measurements of each specimen group with dynamic culture are shown in Fig. 2a, and those with static culture in Fig. 2b. In dynamic culture, the multi-pore size specimen contained an about 1.77 times higher DNA quantity than the uniform-pore size specimens at 14-day culture. The two-layer scaffolds contained 1.33 times more cells than the uniform-pore size scaffold. Due to movement of the culture dish, the cells incompletely attached to the scaffold might be detached from the scaffold, causing a gradual decrease of the DNA quantity from the 4th day. In the dynamic test, the pore size turned out to be one of the cell culture factors and various pore sizes were effective on cell proliferation. The
e39
multi-pore size group was the highest-DNA content group among the three groups. In the static culture, as shown in Fig. 2b, the DNA quantity gradually increased from the initial point to the 14th day, whether the surface was treated or not. The amount of DNA content of the surface-treated group was 1.2 times higher than for the untreated group. Also, the multi-pore size group was more effective in cell proliferation than the uniform-pore size group.
Fig. 3. Number of counted cells in the vertical cross-sectioned area of the specimen (n = 5).
Fig. 2. DNA quantities of HDF seeded on the PLGA scaffolds for 14 days: (a) dynamic culture (n = 6) and (b) static culture (n = 6).
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The number of cells in the inner part of each scaffold in the groups is shown in Fig. 3. The multi-pore size group showed the highest number of cells in the middle of the scaffold. Statistical analysis shows that each group has a significantly different number of cells (p < 0.01). This means that the multi-pore size specimen could make better spaces for cell growth towards the inside of the scaffold than the other groups.
4. Conclusion Boss the multi-pore size and the 2-layer scaffold showed better environments for cell culture, compared with the uniform-pore size scaffold. The multi-pore size PLGA scaffold could make room for cells to move inside of the scaffold and transport nutrients and oxygen efficiently to the inside of the scaffold. Various pore size distributions if the scaffold could offer an improved environment for culturing human dermal fibroblast cells and proliferation. The surface-modified multi-pore size PLGA scaffold was most effective for cell culture in this study.
Acknowledgement This work was supported by the 2005 Inje University Research Grant. References [1] T.G. Park, J.J. Yoon, Polymer Sci. Technol. 10 (1999) 722. [2] G.S. Khang, J.H. Jeon, J.C. Cho, Polymer (Korea) 23 (1999) 861. [3] Tony G. Van Tienen, Ralf G.J.C. Heijkants, Pieter Bumaa, Biomaterials 23 (2002) 1731. [4] T.M. Freyman, I.V. Yannas, L.J. Gibson, Prog. Mater. Sci. 46 (2001) 273. [5] H.R. Lin, C.J. Kuo, C.Y. Yang, et al., J. Biomed. Mater. Res. 63 (2002) 271. [6] Y.S. Nam, J.J. Yoon, T.G. Park, J. Biomed. Mater. Res. (Appl. Biomater.) 53 (2000) 1. [7] L.D. Harris, Byung-Soo Kim, David J. Mooney, J. Biomed. Mater. Res. 42 (1998) 396. [8] C.J. Liao, C.F. Chen, J.H. Chen, S.F. Chiang, Y.J. Lin, K.Y. Chang, J. Biomed. Mater. Res. 59 (2002) 676. [9] Z. Ma, C. Gao, J. Yuan, J. Ji, Y. Gong, J. Shen, J. Appl. Polym. Sci. 85 (2002) 2163. [10] S.F. El-Amin, H.H. Lu, Y. Khan, J. Burems, J. Mitchell, R.S. Tuan, C.T. Laurencin, Biomaterials 24 (2003) 1213.