- Email: [email protected]
Visible light-induced crosslinkable gelatin
Acta Biomaterialia 6 (2010) 4005–4010
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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Visible light-induced crosslinkable gelatin Tae Il Son a,b, Makoto Sakuragi a, Sawa Takahashi a,c, Sei Obuse a, Jeonghwa Kang a, Masako Fujishiro a, Haruhiko Matsushita a, Jiansheng Gong a, Shigeru Shimizu c, Yusuke Tajima d, Yasuhiro Yoshida e, Kazuomi Suzuki e, Toshio Yamamoto f, Mariko Nakamura g, Yoshihiro Ito a,* a
Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Department of Bioscience and Biotechnology, Chung-Ang University, 40-1 San, Nae-Ri, Daeduck-myun, Ansung-si, Kyungki-do 456-756, Republic of Korea Department of Materials and Applied Chemistry, Graduate School of Science and Technology, Nihon University, 1-8-14 Surugadai, Kanda, Chiyoda-ku, Tokyo 101-8308, Japan d Nano-Integration Materials Research Unit, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan e Department of Biomaterials, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan f Department of Oral Morphology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan g Dental Hygiene Program, Junsei Junior College, 8 Iga-cho, Takahashi, Okayama 716-8508, Japan b c
a r t i c l e
i n f o
Article history: Received 8 December 2009 Received in revised form 14 May 2010 Accepted 19 May 2010 Available online 23 May 2010 Keywords: Gelatin Photo-crosslinking Biosealant Direct pulp capping Visible light
a b s t r a c t A novel visible light-crosslinkable porcine gelatin was prepared for gelation and micropatterning. The preparation employed a photo-oxidation-induced crosslinking mechanism. First, furfuryl groups were incorporated into the gelatin. Second, the modified gelatin was mixed in water with Rose Bengal, which is a visible light sensitizer. Irradiation by visible light solidified the aqueous solution. In addition, when the solution was cast on a plate, dried and photo-irradiated in the presence of a photomask a micropattern was formed that matched the micropattern on the photomask. The gelatin-immobilized regions enhanced cell adhesion. It was also confirmed that the gelatin incorporating furfuryl and Rose Bengal have no significant toxicity. The photo-crosslinkable gelatin was employed as a direct pulp capping material in the dental field. Considering these results, this system could be useful as a new type of visible lightinduced crosslinkable biosealant. Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Photo-induced crosslinking or polymerization is a fast and convenient way to produce gels or high molecular weight polymers. Many types of materials based on photo-triggered reactions have been developed for industry. Photo-induction is also useful for rapid curing, and it has been extensively employed for hard tissues in dental medicine [1]. Matsuda’s group developed a photo-induced polymerizable system, including poly(ethylene glycol) diacrylate, derivatized gelatin and photo-induced radical initiators [2]. Similarly, the Hubbell group’s visible light-induced photopolymerizable glue, based on hydrolytically degradable poly(ethylene glycol) diacrylate derivatives and a visible light-induced photoradical generator (eosin Y), is now commercially available [3]. Other types of polymerizable biomacromolecule derivatives have also been reported [4–16]. However, these systems usually utilize synthetic polymerizable monomers as their major component. For medical utilization it is desirable to have biological molecule-based sealants.
Some types of ultraviolet-crosslinkable biomacromolecules have also been reported [17–21]. Recently, visible light-crosslinkable gelatin [21] and fibrinogen [22] have been reported. In the present investigation we developed a new type of visible light-induced crosslinkable biosealant. Tajima et al. found that a furancontaining polymer formed a gel in the presence of fullerene by photo-oxidation polycondensation [23,24]. Here, instead of fullerene, which is insoluble in water, Rose Bengal, a food dye that conjugates with furan groups in water, was employed for formation of the gelatin gel. After visible light irradiation by a photosource for dental use, the mixture was transformed from solution to gel by a photo-oxidation crosslinking (POC) mechanism, as illustrated in Fig. 1. Rose Bengal photosensitizes the oxygen molecules to generate singlet oxygen, and the resulting singlet oxygen reacts with furan derivatives to afford crosslinking through the formation of furan endoperoxide. 2. Materials and methods 2.1. Synthesis of furan-conjugated gelatin
* Corresponding author. Tel.: +81 48 467 9302; fax: +81 48 467 9300. E-mail address: [email protected] (Y. Ito).
Porcine skin gelatin (G2500) was purchased from Sigma (St Louis, MO), and 0.25 g was dissolved in 25 ml of water at
1742-7061/$ - see front matter Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2010.05.018
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Fig. 1. Schematic drawing of the photo-oxidation crosslinking (POC) mechanism.
40 °C. An aqueous solution of NaOH was used to adjust the solution pH to 9.0. Furfuryl isocyanate (100 ll) in dimethylsulfoxide (2.5 ml) was added dropwise to the solution. After addition the solution was allowed to stand for 20 h at room temperature, after which it was neutralized with dilute HCl. Finally, the solution was dialyzed using a dialysis membrane for 2 days at 40 °C. The furfurylated gelatin is referred to as F-gelatin. Gel permeation chromatography measurements were performed on samples dissolved in pure water using a TSK-GEL a-M column (Tosoh, Tokyo, Japan) at 0.6 ml min 1. The eluted peaks were detected with a RI-2031 Plus detector (JASCO, Hachioji, Japan). The calibration was performed using a polyethylene glycol kit purchased from Polymer Laboratories (Varian Inc., Palo Alto, CA). For the NMR measurements the sample was dissolved in D2O. Measurements were performed using a JNM-AL400 spectrometer (JASCO). 2.2. Gelation by photo-irradiation An aqueous solution of F-gelatin was prepared and mixed with Rose Bengal. The mixture was cast on a substrate and irradiated after drying with a halogen lamp (higher than 400 nm) or fluorescent lamp without or with a photomask, as shown in Fig. 2. Polyester disks (Thermanox™, NalgenNunc, NY) and 15 mm diameter glass disks (Matsunami, Osaka, Japan) coated with polyethylene glycol as previously reported [25] were used as substrates for micropatterning and cell culture, respectively.
2.3. Cell culture A fusion cell of two mouse embryonic stem cells (EB3 and B6G2) was cultured in Dulbecco’s Modified Eagle Medium (high glucose, Wako Osaka, Japan) with 15% fetal bovine serum (Hyclone, Logan, UT), 1% glutamate (Sigma–Aldrich), penicillin–streptomycin stock (Sigma–Aldrich) at 10 2 dilution, 1% non-essential amino acids (Invitrogen Life Technologies, Carlsbad, CA), 0.1% 2-mercaproethanol (Invitrogen) and 0.1% Leukemia Inhibitory Factor (Wako). The cell suspension was added to a sample plate sterilized with 70% ethanol. The cells were incubated at 37 °C in an atmosphere of 5% CO2 in air for 2 days and then stained with Giemsa stain for observation by microscopy. 2.4. Cytotoxicity COS-7 cells were cultured in Dulbecco’s modified minimum essential medium (high glucose, no phenol red, GIBCO 21068) with 5% fetal bovine serum (Hyclone, Waltham, MA), and penicillin– streptomycin stock (Sigma–Aldrich). The modified gelatin was added to the culture medium. The cells (100 cells ll 1) were incubated in a 96-well plate (50 ll well 1) at 37 °C in an atmosphere of 5% CO2 in air for 2 days. The cells were counted using a cell counting kit (Dojindo Molecular Technologies, Kumamoto, Japan). 2.5. Animal experiments The use of photo-curable gelatin for direct pulp capping was investigated [26]. Cavities with an exposed pulp area were pre-
Fig. 2. Photo-crosslinking procedures: (A) photo-gelation; (B) photo-micropatterning.
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pared from the first molars in rats. The cavity was formed using a 1/4 round bar and the pulp was eroded. The prepared cavity was washed with 10% sodium hypochlorite and 3% hydrogen peroxide, consecutively. All cavities were divided into two groups: in one group the exposed pulp was covered with the photo-crosslinkable gelatin preparation; in the other it was covered with conventional calcium hydroxide preparation. The cavities covered with the photo-crosslinkable gelatin preparation were photo-irradiated. A protective liner sealed the margins of the treated areas of the molars. The animals were killed 1 week after the operation. All experiments were approved and were performed following the guidelines of the Okayama University Intramural Animal Use and Care Committee. 3. Results and discussion 3.1. Preparation of photosensitive gelatin Gel permeation chromatography (Fig. 3) was used to measure the molecular weight of F-gelatin. After dialysis no reactant furfuryl isocyanate was detected. The results indicated that dialysis of the product could remove unreacted furfuryl isocyanate. Using polyethylene glycol as a molecular weight standards, the molecular weight of F-gelatin was estimated to be 80,000 (Fig. 3). The NMR spectrum of F-gelatin had singlet peaks at 6.1, 6.2 and 7.3 p.p.m., attributed to the furan group (Fig. 4). The splitting of
Fig. 4. The NMR spectra of gelatin and F-gelatin.
peaks was considered to indicate heterogenicity of the gelatin. This confirmed that the polymer contained furan groups, as designed. From the peak area it was demonstrated that the F-gelatin contained 2.66% furan groups. It is known that the content of lysine residues in gelatin is 2.69%. Considering this content, almost all the gelatin amino groups (2.66/2.69) were calculated to be coupled with furan groups. 3.2. Photo-crosslinking Aqueous solutions of F-gelatin with Rose Bengal were cast on a glass plate and the solution irradiated by visible light. Rose Bengal has an absorbance range of 500–590 nm, with a maximum at 549 nm. Therefore, light at wavelengths close to 549 nm was used to irradiate the cast sample. After a prescribed time of irradiation on each cast sample the plate was raised vertically. The solidified sample did not flow downwards but unsolidified sample did. The distance between the point at which the sample was spotted and the end point of flow was measured. In the case of no Rose Bengal or visible light irradiation no solidification occurred. This flow distance was referred to as 100%. In the presence of Rose Bengal solidification was observed after irradiation by visible light and the flow distance was dependent on irradiation time. When the
Fig. 3. Gel permeation chromatography to analyse the molecular weight distribution. The measurements were performed using a TSK-GEL a-M column, model No. 018344 (Tosoh, Tokyo, Japan) with MilliQ water used as the eluent at 0.6 ml min 1. The calibration line was obtained using a polyethylene glycol kit (No. 2080–0200) purchased from Polymer Laboratories (Varian Inc., Palo Alto, CA).
Fig. 5. The time course of solidification. The number of experiments was three and typical data are shown.
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Fig. 6. (A) Photomask for micropatterning and (B) micropatterned gelatin on a polyester disk. Scale bar 100 lm.
concentrations of F-gelatin and Rose Bengal were 3% and 1%, respectively, the solidification time was less than 3 min, as shown in Fig. 5. After drying the solution the sample was photo-irradiated in the presence of a photomask. Micropattern immobilization, as shown in Fig. 6, was observed, matching the micropattern of the photomask. Photo-irradiation through the transparent section led to a crosslinking reaction, resulting in the formation of micropatterns. Microscale patterning was useful for convenient confirmation by optical microscopy. This result indicates that solidification occurred only in the irradiated areas. Tajima et al. [23,24] reported that furan resin was produced by polycondensation of methyl 2-furoate (MFA) via oxygenation by singlet oxygen generated with fullerene as a photosensitizer. They surmised that the endoperoxides of MFA underwent polycondensation to form the conjugated polymer, the furan resin. Similarly, they reported photo-excited, fullerene-generated singlet oxygen, which migrated to the furan units of poly(2-fuoic acid [2-(methacryloyl-oxy)-ethyl]ester) (PFMA) which were oxygenated. The oxygenated furan units combined with the endoperoxides to construct a network as a result of intermolecular bonding between the furan units on different PFMA chains. In this study crosslinking occurred according to the same mechanism, but using Rose Bengal instead of fullerene, as shown in Fig. 1. 3.3. Cell adhesion When mammalian cells were seeded on the micropatterned surface they only adhered to the surface immobilized with F-gelatin, as shown in Fig. 7. This result indicates that the modified gelatin had cell adhesive activity.
Fig. 7. Cell adhesion on the F-gelatin micropatterned glass disk observed by phase contrast microscopy. Scale bar 100 lm.
We previously prepared ultraviolet-curable gelatin, which was derivatized with phenylazido groups, and micropatterned the surface by lithography [27,28]. This study reveals that the micropatterning can be produced by visible light irradiation. Photoimmobilization of biological molecules is very important for the development of various types of biomaterials [29,30]. It is known that ultraviolet irradiation causes damage, including DNA damage, such as thymine dimer formation, resulting in mutagenesis, carcinogenesis and ageing [31]. Considering that less damage is caused by visible light than by ultraviolet, this methodology is considered of importance for future progress.
3.4. Cytotoxicity The cytotoxicity of Rose Bengal was compared with that of other compounds, such as 2,2-bis[(p-2-hydroxy-3-methacryloxy-propoxy)phenyl]propane (Bis-GMA) and 2-hydroxy methacrylate (HEMA), which are used as standards in the field of dental materials (Fig. 8). As Ratanasathie et al. [32] reported, the TC50 (50% toxic concentration) value of HEMA was about 1000 times that of Bis-GMA. The cytotoxicity of Rose Bengal is less than that of Bis-GMA and similar to that of HEMA. This result indicates that Rose Bengal could be applicable in the medical field. F-gelatin showed no cytotoxicity, even with more than 20% Rose Bengal. Cell culture was performed in the presence of samples irradiated for different times. When the concentration of Rose Bengal in the sample was lower than the cytotoxic concentration the irradiation time did not affect the growth of cells.
Fig. 8. Cytotoxicities of F-gelatin, Rose Bengal, HEMA and G-BIS. The number of experiments was four for each concentration.
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Fig. 9. (A) Histological changes after pulp capping with the photo-crosslinkable gelatin preparation. Hematoxylin–eosin (H&E) staining. Slight inflammation (star) was seen. (B) Histological changes after pulp capping with calcium hydroxide preparation. Severe inflammation (star) was seen. The number of experiments was 30 for each sample and typical photos are shown.
3.5. Animal experiments In both groups the capped, treated teeth were observed 1 week after the operation. Although at 1 week after cavity preparation coalescence of inflammatory cells was observed subjacent to the site of the exposed dental pulp for teeth treated with both the calcium hydroxide preparation and the photo-crosslinkable gelatin preparation, the inflammatory response was significantly weaker when the cavities had been treated with the photo-crosslinkable gelatin preparation (Fig. 9). In this animal test we did not observe any remaining gelatin in the cross-section.
[5]
[6] [7]
[8]
[9] [10]
4. Conclusion [11]
A new type of visible light-curable gelatin derivative was prepared by conjugation with furfuryl groups and mixing with Rose Bengal, which is a visible light sensitizer. When the solution was cast on a plate, dried and photo-irradiated in the presence of a photomask a micropattern was formed that matched the micropattern on the photomask. The gelatin-immobilized regions enhanced cell adhesion. The photo-crosslinkable gelatin was useful as a direct pulp capping material in the dental field. Considering that all the materials are non-toxic, the system could be applicable for bioadhesives or biosealants in the future.
[12] [13]
[14]
[15]
[16]
Acknowledgement The authors thank Mr. M. Kawashima at Kuraray Medical Co. for his helpful suggestions.
[17]
[18]
Appendix A. Figures with essential color discrimination [19]
Certain figures in this article, particularly Figures 3, 5, 6, 7, 8, and 9, are difficult to interpret in black and white. The full color images can be found in the on-line version, at doi:10.1016/ j.actbio.2010.05.018. References [1] Suri S, Singh A, Schmidt E. Photofunctionalization of materials to promote protein and cell interactions for tissue-engineering applications. In: Puleo DA, Bizios R, editors. Biological Interactions on Materials Surfaces. New York: Springer; 2009. p. 297–318. [2] Ito T, Matsuda T. Photocurable surgical tissue adhesive glues composed of photoreactive gelatin and poly(ethylene glycol) diacrylate. J Artif Organs 1989;18:132–6. [3] Sawhney AS, Pathak CP, Hubbell JA. Bioerodible hydrogels based on photopolymerized poly(ethylene glycol)–co-poly(alpha-hydroxy acid) diacrylate macromers. Macromolecules 1993;26:581–7. [4] Li C, Sajiki T, Nakayama Y, Fukui M, Matsuda T. Novel visible-light-induced photocurable tissue adhesive composed of multiply styrene-derivatized
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gelatin and poly(ethylene glycol) diacrylate. J Biomed Mater Res B Appl Biomater 2003;66:439–46. Mahoney MJ, Anseth KS. Three-dimensional growth and function of neural tissue in degradable polyethylene glycol hydrogels. Biomaterials 2006;27: 2265–74. Lee HJ, Lee JS, Chansakul T, Yu C, Elisseeff JH, Yu SM. Collagen mimetic peptideconjugated photopolymerizable PEG hydrogel. Biomaterials 2006;27:5268–76. Yeh J, Ling Y, Karp JM, Gantz J, Chandawakar A, Eng G, et al. Micromolding of shape-controlled, harvestable cell-laden hydrogels. Biomaterials 2006;27: 5391–8. Gonen-Wadmany M, Oss-Ronen L, Sekiktar D. Protein–polymer conjugates for forming photopolymerizable biomimetic hydrogels for tissue engineering. Biomaterials 2007;28:3876–86. Grinstaff MW. Designing hydrogel adhesives for corneal wound repair. Biomaterials 2007;28:5205–14. Weiner AA, Shuck DM, Bush JR, Shastri VP. In vitro degradation characteristics of photocrosslinked anhydride systems for bone augmentation applications. Biomaterials 2007;28:5259–70. Weiner AA, Bock EA, Gipson ME, Shastri VP. Photocrosslinked anhydride systems for long-term protein release. Biomaterials 2008;29:2400–7. Ding KFC, Hsu SH, Chiang YW. Synthesis of a new photoreactive gelatin with BTDA and HEMA derivatives. J Apply Polym Sci 2008;109:589–96. Degoricija L, Bansal PN, Soentjens SHM, Joshi NS, Takahashi M, Snyder B, et al. Hydrogels for osteochondral repair based on photocrosslinkable carbamate dendrimers. Biomacromolecules 2008;9:2863–72. Sanabria-DeLong N, Crosby AJ, Tew GN. Photo-cross-linked PLA–PEO–PLA hydrogels from self-assembled physical networks: mechanical properties and influence of assumed constitutive relationships. Biomacromolecules 2008;9: 2784–91. Chien HW, Chang TY, Tsai WB. Spatial control of cellular adhesion using photocrosslinked micropatterned polyelectrolyte multilayer films. Biomaterials 2009;30:2209–18. Pourcelle V, Freichels H, Stoffelbach F, Auzely-Velty R, Jerome C, MarchandBrynaert J. Light induced functionalization of PCL–PEG block copolymers for the covalent immobilization of biomolecules. Biomacromolecules 2009;10: 966–74. Miyamoto K, Sasaki M, Minamisawa Y, Kurahashi Y, Kano H, Ishikawa S. Evaluation of in vivo biocompatibility and biodegradation of photocrosslinked hyaluronate hydrogels (HADgels). J Biomed Mater Res A 2004;70:550–9. Fukuda J, Khademhosseini A, Yeo Y, Yang X, Yeh J, Eng G, et al. Micromolding of photocrosslinkable chitosan hydrogel for spheroid microarray and co-cultures. Biomaterials 2006;27:5259–67. Jeon O, Bouhadir KH, Mansour JM, Alsberg E. Photocrosslinked alginate hydrogels with tunable biodegradation rates and mechanical properties. Biomaterials 2009;30:2724–34. Vesperinasm A, Eastoe J, Wyatt P, Grilo I, Heenan RK. Photosensitive gelatin. Chem Commun 2006;42:4407–9. Nakayama Y, Kameo T, Ohtaka A, Hirano Y. Enhancement of visible lightinduced gelation of photocurable gelatin by addition of polymeric amine. J. Photochem Photobiol A Chem 2006;177:205–11. Elvin CM, Brownlee AG, Huson MG, Tebb TA, Kim M, Lyons RE, et al. The development of photochemically crosslinked native fibrinogen as a rapidly formed and mechanically strong surgical tissue sealant. Biomaterials 2009;30:2059–65. Tajima Y, Tezuka Y, Ishii T, Takeuchi K. Application of a furan-containing polymer/fullerene system to photolithography. Polym J 1997;29:1016–9. Shigemitsu Y, Tajima Y, Hoshino M, Tezuka Y, Takeuchi T. Laser flash photolysis studies on the model system for photo-oxidation-induced polycondensation polyimides with fullerene C60. Jpn J App Phys 2004;43: 1172–7. Ito Y, Hasuda H, Sakuragi M, Tsuzuki S. Surface modification of plastic, glass and titanium by photoimmobilization of polyethylene glycol for antibiofouling. Acta Biomater 2007;3:1024–32. Yamaji K, Horikawa G, Nishitani Y, Izawa S, Yoshiyama M. Dentin regeneration by direct pulp capping using rhBMP-2. J Oral Tissue Eng 2008;5:145–9.
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[27] Ito Y, Hasuda H, Yamauchi T, Komatsu N, Ikebuchi K. Immobilization of erythropoietin to culture erythropoietin-dependent human leukemia cell line. Biomaterials 2004;25:2293–8. [28] Heydari M, Hasuda H, Sakuragi M, Yoshida Y, Suzuki K, Ito Y. Modification of the titan surface with photoreactive gelatin to regulate cell attachment. J Biomed Mater Res A 2007;83:906–14. [29] Ito Y. Photoimmobilization for microarrays. Biotechnol Prog 2006;22:924–32.
[30] Ito Y. Covalently immobilized biosignal molecule materials for tissue engineering. Soft Matter 2008;4:46–51. [31] Lindhal T. Instability and decay of primary structure of DNA. Nature 1993;362:709–15. [32] Ratanasathie S, Wataha JC, Hanks CT, Dennison JB. Cytotoxic interactive effects of dentin bonding components on mouse fibroblasts. J Dent Res 1995;74: 1602–6.
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Visible light-induced crosslinkable gelatin Tae Il Son a,b, Makoto Sakuragi a, Sawa Takahashi a,c, Sei Obuse a, Jeonghwa Kang a, Masako Fujishiro a, Haruhiko Matsushita a, Jiansheng Gong a, Shigeru Shimizu c, Yusuke Tajima d, Yasuhiro Yoshida e, Kazuomi Suzuki e, Toshio Yamamoto f, Mariko Nakamura g, Yoshihiro Ito a,* a
Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Department of Bioscience and Biotechnology, Chung-Ang University, 40-1 San, Nae-Ri, Daeduck-myun, Ansung-si, Kyungki-do 456-756, Republic of Korea Department of Materials and Applied Chemistry, Graduate School of Science and Technology, Nihon University, 1-8-14 Surugadai, Kanda, Chiyoda-ku, Tokyo 101-8308, Japan d Nano-Integration Materials Research Unit, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan e Department of Biomaterials, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan f Department of Oral Morphology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan g Dental Hygiene Program, Junsei Junior College, 8 Iga-cho, Takahashi, Okayama 716-8508, Japan b c
a r t i c l e
i n f o
Article history: Received 8 December 2009 Received in revised form 14 May 2010 Accepted 19 May 2010 Available online 23 May 2010 Keywords: Gelatin Photo-crosslinking Biosealant Direct pulp capping Visible light
a b s t r a c t A novel visible light-crosslinkable porcine gelatin was prepared for gelation and micropatterning. The preparation employed a photo-oxidation-induced crosslinking mechanism. First, furfuryl groups were incorporated into the gelatin. Second, the modified gelatin was mixed in water with Rose Bengal, which is a visible light sensitizer. Irradiation by visible light solidified the aqueous solution. In addition, when the solution was cast on a plate, dried and photo-irradiated in the presence of a photomask a micropattern was formed that matched the micropattern on the photomask. The gelatin-immobilized regions enhanced cell adhesion. It was also confirmed that the gelatin incorporating furfuryl and Rose Bengal have no significant toxicity. The photo-crosslinkable gelatin was employed as a direct pulp capping material in the dental field. Considering these results, this system could be useful as a new type of visible lightinduced crosslinkable biosealant. Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Photo-induced crosslinking or polymerization is a fast and convenient way to produce gels or high molecular weight polymers. Many types of materials based on photo-triggered reactions have been developed for industry. Photo-induction is also useful for rapid curing, and it has been extensively employed for hard tissues in dental medicine [1]. Matsuda’s group developed a photo-induced polymerizable system, including poly(ethylene glycol) diacrylate, derivatized gelatin and photo-induced radical initiators [2]. Similarly, the Hubbell group’s visible light-induced photopolymerizable glue, based on hydrolytically degradable poly(ethylene glycol) diacrylate derivatives and a visible light-induced photoradical generator (eosin Y), is now commercially available [3]. Other types of polymerizable biomacromolecule derivatives have also been reported [4–16]. However, these systems usually utilize synthetic polymerizable monomers as their major component. For medical utilization it is desirable to have biological molecule-based sealants.
Some types of ultraviolet-crosslinkable biomacromolecules have also been reported [17–21]. Recently, visible light-crosslinkable gelatin [21] and fibrinogen [22] have been reported. In the present investigation we developed a new type of visible light-induced crosslinkable biosealant. Tajima et al. found that a furancontaining polymer formed a gel in the presence of fullerene by photo-oxidation polycondensation [23,24]. Here, instead of fullerene, which is insoluble in water, Rose Bengal, a food dye that conjugates with furan groups in water, was employed for formation of the gelatin gel. After visible light irradiation by a photosource for dental use, the mixture was transformed from solution to gel by a photo-oxidation crosslinking (POC) mechanism, as illustrated in Fig. 1. Rose Bengal photosensitizes the oxygen molecules to generate singlet oxygen, and the resulting singlet oxygen reacts with furan derivatives to afford crosslinking through the formation of furan endoperoxide. 2. Materials and methods 2.1. Synthesis of furan-conjugated gelatin
* Corresponding author. Tel.: +81 48 467 9302; fax: +81 48 467 9300. E-mail address: [email protected] (Y. Ito).
Porcine skin gelatin (G2500) was purchased from Sigma (St Louis, MO), and 0.25 g was dissolved in 25 ml of water at
1742-7061/$ - see front matter Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2010.05.018
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Fig. 1. Schematic drawing of the photo-oxidation crosslinking (POC) mechanism.
40 °C. An aqueous solution of NaOH was used to adjust the solution pH to 9.0. Furfuryl isocyanate (100 ll) in dimethylsulfoxide (2.5 ml) was added dropwise to the solution. After addition the solution was allowed to stand for 20 h at room temperature, after which it was neutralized with dilute HCl. Finally, the solution was dialyzed using a dialysis membrane for 2 days at 40 °C. The furfurylated gelatin is referred to as F-gelatin. Gel permeation chromatography measurements were performed on samples dissolved in pure water using a TSK-GEL a-M column (Tosoh, Tokyo, Japan) at 0.6 ml min 1. The eluted peaks were detected with a RI-2031 Plus detector (JASCO, Hachioji, Japan). The calibration was performed using a polyethylene glycol kit purchased from Polymer Laboratories (Varian Inc., Palo Alto, CA). For the NMR measurements the sample was dissolved in D2O. Measurements were performed using a JNM-AL400 spectrometer (JASCO). 2.2. Gelation by photo-irradiation An aqueous solution of F-gelatin was prepared and mixed with Rose Bengal. The mixture was cast on a substrate and irradiated after drying with a halogen lamp (higher than 400 nm) or fluorescent lamp without or with a photomask, as shown in Fig. 2. Polyester disks (Thermanox™, NalgenNunc, NY) and 15 mm diameter glass disks (Matsunami, Osaka, Japan) coated with polyethylene glycol as previously reported [25] were used as substrates for micropatterning and cell culture, respectively.
2.3. Cell culture A fusion cell of two mouse embryonic stem cells (EB3 and B6G2) was cultured in Dulbecco’s Modified Eagle Medium (high glucose, Wako Osaka, Japan) with 15% fetal bovine serum (Hyclone, Logan, UT), 1% glutamate (Sigma–Aldrich), penicillin–streptomycin stock (Sigma–Aldrich) at 10 2 dilution, 1% non-essential amino acids (Invitrogen Life Technologies, Carlsbad, CA), 0.1% 2-mercaproethanol (Invitrogen) and 0.1% Leukemia Inhibitory Factor (Wako). The cell suspension was added to a sample plate sterilized with 70% ethanol. The cells were incubated at 37 °C in an atmosphere of 5% CO2 in air for 2 days and then stained with Giemsa stain for observation by microscopy. 2.4. Cytotoxicity COS-7 cells were cultured in Dulbecco’s modified minimum essential medium (high glucose, no phenol red, GIBCO 21068) with 5% fetal bovine serum (Hyclone, Waltham, MA), and penicillin– streptomycin stock (Sigma–Aldrich). The modified gelatin was added to the culture medium. The cells (100 cells ll 1) were incubated in a 96-well plate (50 ll well 1) at 37 °C in an atmosphere of 5% CO2 in air for 2 days. The cells were counted using a cell counting kit (Dojindo Molecular Technologies, Kumamoto, Japan). 2.5. Animal experiments The use of photo-curable gelatin for direct pulp capping was investigated [26]. Cavities with an exposed pulp area were pre-
Fig. 2. Photo-crosslinking procedures: (A) photo-gelation; (B) photo-micropatterning.
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pared from the first molars in rats. The cavity was formed using a 1/4 round bar and the pulp was eroded. The prepared cavity was washed with 10% sodium hypochlorite and 3% hydrogen peroxide, consecutively. All cavities were divided into two groups: in one group the exposed pulp was covered with the photo-crosslinkable gelatin preparation; in the other it was covered with conventional calcium hydroxide preparation. The cavities covered with the photo-crosslinkable gelatin preparation were photo-irradiated. A protective liner sealed the margins of the treated areas of the molars. The animals were killed 1 week after the operation. All experiments were approved and were performed following the guidelines of the Okayama University Intramural Animal Use and Care Committee. 3. Results and discussion 3.1. Preparation of photosensitive gelatin Gel permeation chromatography (Fig. 3) was used to measure the molecular weight of F-gelatin. After dialysis no reactant furfuryl isocyanate was detected. The results indicated that dialysis of the product could remove unreacted furfuryl isocyanate. Using polyethylene glycol as a molecular weight standards, the molecular weight of F-gelatin was estimated to be 80,000 (Fig. 3). The NMR spectrum of F-gelatin had singlet peaks at 6.1, 6.2 and 7.3 p.p.m., attributed to the furan group (Fig. 4). The splitting of
Fig. 4. The NMR spectra of gelatin and F-gelatin.
peaks was considered to indicate heterogenicity of the gelatin. This confirmed that the polymer contained furan groups, as designed. From the peak area it was demonstrated that the F-gelatin contained 2.66% furan groups. It is known that the content of lysine residues in gelatin is 2.69%. Considering this content, almost all the gelatin amino groups (2.66/2.69) were calculated to be coupled with furan groups. 3.2. Photo-crosslinking Aqueous solutions of F-gelatin with Rose Bengal were cast on a glass plate and the solution irradiated by visible light. Rose Bengal has an absorbance range of 500–590 nm, with a maximum at 549 nm. Therefore, light at wavelengths close to 549 nm was used to irradiate the cast sample. After a prescribed time of irradiation on each cast sample the plate was raised vertically. The solidified sample did not flow downwards but unsolidified sample did. The distance between the point at which the sample was spotted and the end point of flow was measured. In the case of no Rose Bengal or visible light irradiation no solidification occurred. This flow distance was referred to as 100%. In the presence of Rose Bengal solidification was observed after irradiation by visible light and the flow distance was dependent on irradiation time. When the
Fig. 3. Gel permeation chromatography to analyse the molecular weight distribution. The measurements were performed using a TSK-GEL a-M column, model No. 018344 (Tosoh, Tokyo, Japan) with MilliQ water used as the eluent at 0.6 ml min 1. The calibration line was obtained using a polyethylene glycol kit (No. 2080–0200) purchased from Polymer Laboratories (Varian Inc., Palo Alto, CA).
Fig. 5. The time course of solidification. The number of experiments was three and typical data are shown.
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Fig. 6. (A) Photomask for micropatterning and (B) micropatterned gelatin on a polyester disk. Scale bar 100 lm.
concentrations of F-gelatin and Rose Bengal were 3% and 1%, respectively, the solidification time was less than 3 min, as shown in Fig. 5. After drying the solution the sample was photo-irradiated in the presence of a photomask. Micropattern immobilization, as shown in Fig. 6, was observed, matching the micropattern of the photomask. Photo-irradiation through the transparent section led to a crosslinking reaction, resulting in the formation of micropatterns. Microscale patterning was useful for convenient confirmation by optical microscopy. This result indicates that solidification occurred only in the irradiated areas. Tajima et al. [23,24] reported that furan resin was produced by polycondensation of methyl 2-furoate (MFA) via oxygenation by singlet oxygen generated with fullerene as a photosensitizer. They surmised that the endoperoxides of MFA underwent polycondensation to form the conjugated polymer, the furan resin. Similarly, they reported photo-excited, fullerene-generated singlet oxygen, which migrated to the furan units of poly(2-fuoic acid [2-(methacryloyl-oxy)-ethyl]ester) (PFMA) which were oxygenated. The oxygenated furan units combined with the endoperoxides to construct a network as a result of intermolecular bonding between the furan units on different PFMA chains. In this study crosslinking occurred according to the same mechanism, but using Rose Bengal instead of fullerene, as shown in Fig. 1. 3.3. Cell adhesion When mammalian cells were seeded on the micropatterned surface they only adhered to the surface immobilized with F-gelatin, as shown in Fig. 7. This result indicates that the modified gelatin had cell adhesive activity.
Fig. 7. Cell adhesion on the F-gelatin micropatterned glass disk observed by phase contrast microscopy. Scale bar 100 lm.
We previously prepared ultraviolet-curable gelatin, which was derivatized with phenylazido groups, and micropatterned the surface by lithography [27,28]. This study reveals that the micropatterning can be produced by visible light irradiation. Photoimmobilization of biological molecules is very important for the development of various types of biomaterials [29,30]. It is known that ultraviolet irradiation causes damage, including DNA damage, such as thymine dimer formation, resulting in mutagenesis, carcinogenesis and ageing [31]. Considering that less damage is caused by visible light than by ultraviolet, this methodology is considered of importance for future progress.
3.4. Cytotoxicity The cytotoxicity of Rose Bengal was compared with that of other compounds, such as 2,2-bis[(p-2-hydroxy-3-methacryloxy-propoxy)phenyl]propane (Bis-GMA) and 2-hydroxy methacrylate (HEMA), which are used as standards in the field of dental materials (Fig. 8). As Ratanasathie et al. [32] reported, the TC50 (50% toxic concentration) value of HEMA was about 1000 times that of Bis-GMA. The cytotoxicity of Rose Bengal is less than that of Bis-GMA and similar to that of HEMA. This result indicates that Rose Bengal could be applicable in the medical field. F-gelatin showed no cytotoxicity, even with more than 20% Rose Bengal. Cell culture was performed in the presence of samples irradiated for different times. When the concentration of Rose Bengal in the sample was lower than the cytotoxic concentration the irradiation time did not affect the growth of cells.
Fig. 8. Cytotoxicities of F-gelatin, Rose Bengal, HEMA and G-BIS. The number of experiments was four for each concentration.
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Fig. 9. (A) Histological changes after pulp capping with the photo-crosslinkable gelatin preparation. Hematoxylin–eosin (H&E) staining. Slight inflammation (star) was seen. (B) Histological changes after pulp capping with calcium hydroxide preparation. Severe inflammation (star) was seen. The number of experiments was 30 for each sample and typical photos are shown.
3.5. Animal experiments In both groups the capped, treated teeth were observed 1 week after the operation. Although at 1 week after cavity preparation coalescence of inflammatory cells was observed subjacent to the site of the exposed dental pulp for teeth treated with both the calcium hydroxide preparation and the photo-crosslinkable gelatin preparation, the inflammatory response was significantly weaker when the cavities had been treated with the photo-crosslinkable gelatin preparation (Fig. 9). In this animal test we did not observe any remaining gelatin in the cross-section.
[5]
[6] [7]
[8]
[9] [10]
4. Conclusion [11]
A new type of visible light-curable gelatin derivative was prepared by conjugation with furfuryl groups and mixing with Rose Bengal, which is a visible light sensitizer. When the solution was cast on a plate, dried and photo-irradiated in the presence of a photomask a micropattern was formed that matched the micropattern on the photomask. The gelatin-immobilized regions enhanced cell adhesion. The photo-crosslinkable gelatin was useful as a direct pulp capping material in the dental field. Considering that all the materials are non-toxic, the system could be applicable for bioadhesives or biosealants in the future.
[12] [13]
[14]
[15]
[16]
Acknowledgement The authors thank Mr. M. Kawashima at Kuraray Medical Co. for his helpful suggestions.
[17]
[18]
Appendix A. Figures with essential color discrimination [19]
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