G Model
ARTICLE IN PRESS
BIOMAC 4453 1–7
International Journal of Biological Macromolecules xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Surface-modified silk hydrogel containing hydroxyapatite nanoparticle with hyaluronic acid–dopamine conjugate
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Q1
Hyung Hwan Kim, Jong Bo Park, Min Ji Kang, Young Hwan Park ∗ Department of Biosystems and Biomaterials Science and Engineering, Seoul National University, 200/5225 1 Gwanak-ro, Gwanak-gu, Seoul 151-921, Republic of Korea
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a r t i c l e
i n f o
a b s t r a c t
8 9 10 11 12 13
Article history: Received 1 April 2014 Received in revised form 25 June 2014 Accepted 26 June 2014 Available online xxx
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Keywords: Silk fibroin hydrogel Hydroxyapatite nanoparticle Secondary structure
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1. Introduction
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21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Silk fibroin/hydroxyapatite (SF/HAp) composite hydrogels were fabricated in this study, having different HAp contents (0–33 wt%) in SF matrix hydrogel. Surface modification of HAp nanoparticle with hyaluronic acid (HA)–dopamine (DA) conjugate improved a dispersibility of HAp in aqueous SF solution due to its negatively charged surface and therefore, fabrication of the SF composite hydrogel having HAp nanoparticles inside could be possible. Zeta potential of surface-modified HAP was examined by ELS. It demonstrates that surface of HAp was well modified to a negative charge with HA–DA. Morphological structure of SF hydrogel containing surface-modified HAp was examined by FE-SEM for analyzing pore structure of hydrogel and deposition of HAp nanoparticle in SF hydrogel. It was found that HAp nanoparticles were uniformly deposited on the pore wall of SF hydrogel. Structural characteristics of SF/HAp composite hydrogel was performed using X-ray diffraction and FT-IR analysis. It was found that -sheet crystal conformation of SF was significantly influenced by the HAp content during gelation of a mixture of SF and HAp. As a result of MTT assay, the SF/HAp composite hydrogel showed excellent cell proliferation ability. Therefore, it is expected that SF hydrogel containing HAp nanoparticles has a high potential as bone regeneration scaffold. © 2014 Published by Elsevier B.V.
Hydrogel is composed of three-dimensional (3D) polymeric network which retains the large amount of water. These polymeric networks can be classified according to their types of structure, chemical or physical crosslinking network. Chemical crosslinking hydrogels were crosslinked by covalent bonds. On the contrary, physical crosslinking hydrogels were crosslinked by physical interactions (e.g., ionic binding, hydrogen bonding, hydrophobic interaction) instead of chemical bonds. Due to these polymeric network structures, hydrogel is basically elastic and possesses tremendous interconnected pores in which lots of water molecules are entrapped. These properties of hydrogel allow a wide variety of applications in biomedical and pharmaceutical fields [1–5]. Especially, an application in tissue engineering scaffold is one of the promising fields for hydrogels due to highly porous 3D structure since hydrogel can provide physiologically similar environment to cells.
∗ Corresponding author. Tel.: +82 2 880 4622; fax: +82 2 873 2285. E-mail address: [email protected] (Y.H. Park).
Bone regeneration is one of the clinically most important tissue engineering fields since a large number of patients who get bone defect of critical size have huge difficulties in healing process with permanent disability. For bone regeneration, various materials have been clinically used (e.g., allograft bone, demineralized bone, bone powder, ceramic powder). However, lacks of bone replacement supplement and poor bone regeneration ability raised a problem. According to recent studies, hydrogel could be a promising material in bone tissue engineering, especially for 3D cell culture technique-based bone reconstruction [6–8]. Silk fibroin (SF) has been widely studied in tissue regeneration for its excellent biocompatibility, physical and mechanical properties, and easy processibility [6–10]. Especially, previous studies have shown that SF can be easily fabricated into hydrogel through appropriate process without additional crosslinking reagents and toxic chemicals. Therefore, the SF hydrogel fabrication process is not only physiologically safe but also biocompatible [9–12]. For the bone regeneration, a lot of studies made great effort to fabricate silk-based scaffolds (e.g., critical size cancellous defects and cartilage) [13,14]. From those studies, it has been confirmed that silk-based materials have great compatibility with osteoblast and its differentiation.
http://dx.doi.org/10.1016/j.ijbiomac.2014.06.052 0141-8130/© 2014 Published by Elsevier B.V.
Please cite this article in press as: H.H. Kim, et al., Int. J. Biol. Macromol. (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.06.052
37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
G Model
ARTICLE IN PRESS
BIOMAC 4453 1–7
H.H. Kim et al. / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
2 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94
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Despite of such great advantages, SF hydrogel still has lack in terms of bone regeneration capability and it is not fully sufficient as a bone regenerative scaffold. There have been many trials to improve the functionality and performance of SF hydrogel for bone regeneration by blending SF hydrogel with bioactive additives (e.g., VEGF, BMP-2, and hydroxyapatite) or other biopolymers (e.g., gelatin and polyurethane) [9,10,15–18]. Among various additives for modification of silk-based scaffold, hydroxyapatite (HAp), known as a major bone ingredient in human body, has a prominent bone cells proliferation and differentiation promotion abilities that several researches were progressed in SF hydrogel incorporating with HAp [19,20]. However, HAp generally shows a low dispersibility in aqueous phase, which leads to easy aggregation in SF aqueous solution due to low compatibility between HAp nanoparticles and SF. Therefore, it is a huge obstacle in incorporation of HAp nanoparticles and SF. To overcome such a limitation, Kim et al. suggested that chemical modification of HAp nanoparticles surface using hyaluronic acid (HA)–dopamine (DA) conjugate could improve the dispersibility of HAp nanoparticles in aqueous solution [21]. HA has a negative charge (pH < 7) in aqueous solution and DA has a catechol functional group that it plays a critical role as a linker molecule between HA and HAp nanoparticle surface. In this study, silk fibroin (SF) hydrogel containing hydroxyapatite (HAp) nanoparticles (SF/HAp composite hydrogel) was prepared and its structural characteristics were studied for bone tissue engineering. In general, gelation of polymeric materials and its properties can be affected by preparation conditions (i.e., concentration, temperature, time, pH, physical and mechanical treatment, additives). Therefore, we investigated the fabrication method for SF hydrogel containing HAp nanoparticles, in which the HAp nanoparticles are uniformly deposited, and morphological structure was examined. Structural characteristics of the SF/HAp composite hydrogel were also examined in addition to evaluate proliferation ability of this composite hydrogel. 2. Materials and methods 2.1. Preparation of SF aqueous solution
105
To remove silk sericin, Bombyx mori cocoons were boiled in sodium oleate 0.3% (w/v) and sodium carbonate 0.2% (w/v) cocktail solution at 100 ◦ C for 1 h. Then, the degummed SF was dissolved in 9.3 M LiBr solution at 60 ◦ C for 30 min and the solution was subsequently dialyzed against de-ionized water using cellulose acetate dialysis tube (MWCO: 12,000–14,000 Da) for 3 days. Final concentration of SF solution was 7.5% (w/v) and the solution was stored at 4 ◦ C until it was used. Before the gelation, the solution was diluted to 5% (w/v) concentration using de-ionized water.
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2.2. Surface modification of HAp
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In order to modify the surface of HAp nanoparticle, HA–DA conjugate was prepared first. Reaction of the conjugate was conducted following the previously reported method [21]. Briefly, after sodium hyaluronan (2 mg/ml) was dissolved completely, dopamine hydrochloride (0.95 mg/ml) and N-(3-dimethyl aminopropyl)-N ethylcarbodiimide hydrochloride (EDC, 0.87 mg/ml) were added and the solution pH was adjusted to 4.7 by adding 0.1 N hydrochloric acid, followed by the incubation at room temperature for 2 h. After the incubation, the unreacted EDC was removed by dialysis tube against 100 mM of NaCl solution for 1 day. Finally, residual chemicals and salts were thoroughly removed by dialysis against de-ionized water for 2 days. Reaction of HAp with HA–DA conjugate was carried out according to following method. HAp nanoparticle
Table 1 Preparation conditions of SF hydrogel containing HAp nanoparticle. Sample
Concentration of SF (% (w/v))
Concentration of HAp (% (w/v))
SF:HAp weight ratio in mixed solution
SF100HAp0 SF90HAp10 SF80HAp20 SF67HAp33
5 5 5 5
0 0.55 1.25 2.50
100:0 90:10 80:20 67:33
(particle size:
ARTICLE IN PRESS
BIOMAC 4453 1–7
International Journal of Biological Macromolecules xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Surface-modified silk hydrogel containing hydroxyapatite nanoparticle with hyaluronic acid–dopamine conjugate
1
2
3 4 5
Q1
Hyung Hwan Kim, Jong Bo Park, Min Ji Kang, Young Hwan Park ∗ Department of Biosystems and Biomaterials Science and Engineering, Seoul National University, 200/5225 1 Gwanak-ro, Gwanak-gu, Seoul 151-921, Republic of Korea
6
7 19
a r t i c l e
i n f o
a b s t r a c t
8 9 10 11 12 13
Article history: Received 1 April 2014 Received in revised form 25 June 2014 Accepted 26 June 2014 Available online xxx
14
18
Keywords: Silk fibroin hydrogel Hydroxyapatite nanoparticle Secondary structure
20
1. Introduction
15 16 17
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Silk fibroin/hydroxyapatite (SF/HAp) composite hydrogels were fabricated in this study, having different HAp contents (0–33 wt%) in SF matrix hydrogel. Surface modification of HAp nanoparticle with hyaluronic acid (HA)–dopamine (DA) conjugate improved a dispersibility of HAp in aqueous SF solution due to its negatively charged surface and therefore, fabrication of the SF composite hydrogel having HAp nanoparticles inside could be possible. Zeta potential of surface-modified HAP was examined by ELS. It demonstrates that surface of HAp was well modified to a negative charge with HA–DA. Morphological structure of SF hydrogel containing surface-modified HAp was examined by FE-SEM for analyzing pore structure of hydrogel and deposition of HAp nanoparticle in SF hydrogel. It was found that HAp nanoparticles were uniformly deposited on the pore wall of SF hydrogel. Structural characteristics of SF/HAp composite hydrogel was performed using X-ray diffraction and FT-IR analysis. It was found that -sheet crystal conformation of SF was significantly influenced by the HAp content during gelation of a mixture of SF and HAp. As a result of MTT assay, the SF/HAp composite hydrogel showed excellent cell proliferation ability. Therefore, it is expected that SF hydrogel containing HAp nanoparticles has a high potential as bone regeneration scaffold. © 2014 Published by Elsevier B.V.
Hydrogel is composed of three-dimensional (3D) polymeric network which retains the large amount of water. These polymeric networks can be classified according to their types of structure, chemical or physical crosslinking network. Chemical crosslinking hydrogels were crosslinked by covalent bonds. On the contrary, physical crosslinking hydrogels were crosslinked by physical interactions (e.g., ionic binding, hydrogen bonding, hydrophobic interaction) instead of chemical bonds. Due to these polymeric network structures, hydrogel is basically elastic and possesses tremendous interconnected pores in which lots of water molecules are entrapped. These properties of hydrogel allow a wide variety of applications in biomedical and pharmaceutical fields [1–5]. Especially, an application in tissue engineering scaffold is one of the promising fields for hydrogels due to highly porous 3D structure since hydrogel can provide physiologically similar environment to cells.
∗ Corresponding author. Tel.: +82 2 880 4622; fax: +82 2 873 2285. E-mail address: [email protected] (Y.H. Park).
Bone regeneration is one of the clinically most important tissue engineering fields since a large number of patients who get bone defect of critical size have huge difficulties in healing process with permanent disability. For bone regeneration, various materials have been clinically used (e.g., allograft bone, demineralized bone, bone powder, ceramic powder). However, lacks of bone replacement supplement and poor bone regeneration ability raised a problem. According to recent studies, hydrogel could be a promising material in bone tissue engineering, especially for 3D cell culture technique-based bone reconstruction [6–8]. Silk fibroin (SF) has been widely studied in tissue regeneration for its excellent biocompatibility, physical and mechanical properties, and easy processibility [6–10]. Especially, previous studies have shown that SF can be easily fabricated into hydrogel through appropriate process without additional crosslinking reagents and toxic chemicals. Therefore, the SF hydrogel fabrication process is not only physiologically safe but also biocompatible [9–12]. For the bone regeneration, a lot of studies made great effort to fabricate silk-based scaffolds (e.g., critical size cancellous defects and cartilage) [13,14]. From those studies, it has been confirmed that silk-based materials have great compatibility with osteoblast and its differentiation.
http://dx.doi.org/10.1016/j.ijbiomac.2014.06.052 0141-8130/© 2014 Published by Elsevier B.V.
Please cite this article in press as: H.H. Kim, et al., Int. J. Biol. Macromol. (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.06.052
37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
G Model
ARTICLE IN PRESS
BIOMAC 4453 1–7
H.H. Kim et al. / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
2 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94
95
96
Despite of such great advantages, SF hydrogel still has lack in terms of bone regeneration capability and it is not fully sufficient as a bone regenerative scaffold. There have been many trials to improve the functionality and performance of SF hydrogel for bone regeneration by blending SF hydrogel with bioactive additives (e.g., VEGF, BMP-2, and hydroxyapatite) or other biopolymers (e.g., gelatin and polyurethane) [9,10,15–18]. Among various additives for modification of silk-based scaffold, hydroxyapatite (HAp), known as a major bone ingredient in human body, has a prominent bone cells proliferation and differentiation promotion abilities that several researches were progressed in SF hydrogel incorporating with HAp [19,20]. However, HAp generally shows a low dispersibility in aqueous phase, which leads to easy aggregation in SF aqueous solution due to low compatibility between HAp nanoparticles and SF. Therefore, it is a huge obstacle in incorporation of HAp nanoparticles and SF. To overcome such a limitation, Kim et al. suggested that chemical modification of HAp nanoparticles surface using hyaluronic acid (HA)–dopamine (DA) conjugate could improve the dispersibility of HAp nanoparticles in aqueous solution [21]. HA has a negative charge (pH < 7) in aqueous solution and DA has a catechol functional group that it plays a critical role as a linker molecule between HA and HAp nanoparticle surface. In this study, silk fibroin (SF) hydrogel containing hydroxyapatite (HAp) nanoparticles (SF/HAp composite hydrogel) was prepared and its structural characteristics were studied for bone tissue engineering. In general, gelation of polymeric materials and its properties can be affected by preparation conditions (i.e., concentration, temperature, time, pH, physical and mechanical treatment, additives). Therefore, we investigated the fabrication method for SF hydrogel containing HAp nanoparticles, in which the HAp nanoparticles are uniformly deposited, and morphological structure was examined. Structural characteristics of the SF/HAp composite hydrogel were also examined in addition to evaluate proliferation ability of this composite hydrogel. 2. Materials and methods 2.1. Preparation of SF aqueous solution
105
To remove silk sericin, Bombyx mori cocoons were boiled in sodium oleate 0.3% (w/v) and sodium carbonate 0.2% (w/v) cocktail solution at 100 ◦ C for 1 h. Then, the degummed SF was dissolved in 9.3 M LiBr solution at 60 ◦ C for 30 min and the solution was subsequently dialyzed against de-ionized water using cellulose acetate dialysis tube (MWCO: 12,000–14,000 Da) for 3 days. Final concentration of SF solution was 7.5% (w/v) and the solution was stored at 4 ◦ C until it was used. Before the gelation, the solution was diluted to 5% (w/v) concentration using de-ionized water.
106
2.2. Surface modification of HAp
97 98 99 100 101 102 103 104
107 108 109 110 111 112 113 114 115 116 117 118 119
In order to modify the surface of HAp nanoparticle, HA–DA conjugate was prepared first. Reaction of the conjugate was conducted following the previously reported method [21]. Briefly, after sodium hyaluronan (2 mg/ml) was dissolved completely, dopamine hydrochloride (0.95 mg/ml) and N-(3-dimethyl aminopropyl)-N ethylcarbodiimide hydrochloride (EDC, 0.87 mg/ml) were added and the solution pH was adjusted to 4.7 by adding 0.1 N hydrochloric acid, followed by the incubation at room temperature for 2 h. After the incubation, the unreacted EDC was removed by dialysis tube against 100 mM of NaCl solution for 1 day. Finally, residual chemicals and salts were thoroughly removed by dialysis against de-ionized water for 2 days. Reaction of HAp with HA–DA conjugate was carried out according to following method. HAp nanoparticle
Table 1 Preparation conditions of SF hydrogel containing HAp nanoparticle. Sample
Concentration of SF (% (w/v))
Concentration of HAp (% (w/v))
SF:HAp weight ratio in mixed solution
SF100HAp0 SF90HAp10 SF80HAp20 SF67HAp33
5 5 5 5
0 0.55 1.25 2.50
100:0 90:10 80:20 67:33
(particle size:
