- Email: [email protected]
Effect of Novel Chitosan-fluoroaluminosilicate Glass Ionomer Cement with Added Transforming Growth Factor Beta-1 on Pulp Cells
Basic Research—Technology
Effect of Novel Chitosan-fluoroaluminosilicate Glass Ionomer Cement with Added Transforming Growth Factor Beta-1 on Pulp Cells Nitra Rakkiettiwong, MSc, DDS,* Chanothai Hengtrakool, PhD, DDS,† Kewalin Thammasitboon, PhD, DDS,† and Ureporn Kedjarune-Leggat, PhD, DDS‡ Abstract Introduction: Vital pulp therapy might benefit from the sustained release of transforming growth factor beta-1 (TGF-b1) from dental restorative materials. Chitosan has previously been shown to enable sustained release of bovine serum albumin (BSA) from glass ionomer cement (GIC). Because BSA can prolong release of growth factor, chitosan-fluoroaluminosilicate GIC with albumin (BIO-GIC) should sustain the effect of growth factor. This study investigated the effect of BIO-GIC with added TGF-b1 on pulp cells. Methods: BIO-GIC was prepared from GIC (conventional type) incorporated with 15% of chitosan and 10% of BSA. TGF-b1 (100 ng) was added in BIO-GIC+TGF-b1 and GIC+TGF-b1 groups during each disk specimen (10 mm diameter, 1 mm high) preparation. Two control groups were BIO-GIC and GIC. The effect of each specimen on pulp cells was investigated by using the Transwell plate technique. Cell proliferation was determined by MTT assay at 2 time periods (each period lasting 3 days). Pulp cell differentiation was examined by alkaline phosphatase activity and also by cell mineralization, which was measured by calculating the area of mineralization with von Kossa staining. Results: Percentage of viable cells of GIC+TGF-b1 group was the highest after the first period. This might suggest an initial rapid release of TGF-b1 from GIC. After the second period, BIO-GIC, BIO-GIC+TGF-b1, and GIC+TGF-b1 had more than 90% cell survival. It was significantly greater than GIC (82% 2%). There was no significant difference in alkaline phosphatase activity. BIO-GIC+TGF-b1 had the highest mineralization area during 21 days. Conclusions: BIO-GIC could retain the effect of TGF-b1. (J Endod 2011;37:367–371)
From the *Dental Department, Narathiwat Hospital, Muang, Narathiwat, Thailand; †Department of Conservative Dentistry, Faculty of Dentistry, Prince of Songkla University, Hat Yai, Songkhla, Thailand; and ‡Department of Oral Biology and Occlusion, Faculty of Dentistry, Prince of Songkla University, Hat Yai, Songkhla, Thailand. Address requests for reprints to Assoc Prof Dr Ureporn Kedjarune-Leggat, Department of Oral Biology and Occlusion, Faculty of Dentistry, Prince of Songkla University, Hat Yai, Songkhla, Thailand 90112. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2011 American Association of Endodontists. doi:10.1016/j.joen.2010.11.031
JOE — Volume 37, Number 3, March 2011
Key Words Bioactive releasing material, chitosan, glass-ionomer cement, growth factor, pulp regeneration
T
issue engineering is emerging as a new model in dentistry and medicine to regenerate natural tissues or to create biological substitutes for defective tissues (1,2), which nowadays also includes endodontic management (3). To regenerate or repair pulp-dentin complex, growth factors play a key role in success (4). Many studies suggest that transforming growth factor beta-1 (TGF-b1) is an important factor involved in dental pulp tissue repair (5–7) by stimulating cell proliferation, cell migration, and type I collagen synthesis (8), as well as mineralization (9). However, growth factors or bioactive molecules are sensitive to temperature, pH, and pressure (1). The development of a possible new generation of biomaterials to control release bioactive molecules is therefore challenging. Glass ionomer cements (GICs) are acid-base cements produced from the reaction of fluoroaluminosilicate glass powder with poly(acrylic acid), which are widely used in dental and medical applications because of their biocompatibility, antibacterial properties, sealing ability, and capacity to prolong the release of fluoride (10). There have been some attempts to enhance sustained release of substances from this cement, such as casein phosphopeptide–amorphous calcium phosphate (CPP-ACP) (11), chlorhexidine (12), and also, in particular, some proteins (13). Chitosan is a biocompatible, biodegradable natural biopolymer that is a copolymer of glucosamine and N-acetylglucosamine derived from chitin. There are many studies about attempting the use of chitosan in dentistry. The addition of chitosan to calcium phosphate cement can increase compressive strength of the cement as well as promote odontoblastic differentiation in human dental pulp cells (14). Moreover, chitosan nanoparticle has an antibacterial property, which has been suggested for root canal disinfection (15). These particles still retain antibacterial activity, even after 90 days (16). In addition, calcium hydroxide–containing chitosan can sustain release of calcium ion for more than 30 days (17). Recently, our group discovered a novel chitosan-fluoroaluminosilicate GIC that can prolong the release of BSA without alteration of its molecular weight, and this cement did not increase toxicity to pulp cells (13). This material can prolong the release of protein, possibly as a result of the formation of a polyelectric complex between the cationic group of chitosan and the anionic group of poly(acrylic acid) (18). Because albumin was introduced to prolong release of growth factor (19), we developed a novel material composed of chitosan-fluoroaluminosilicate GIC and albumin to sustain the release of growth factors. The hypothesis of this study is that the addition of growth factor to this material should sustain its biological effect. This novel GIC could be considered as a material for vital pulp therapy, if it can retain the biological effect of the added growth factor in the cement long enough to activate the healing of pulp tissue by increasing cell numbers or promoting pulp cell differentiation. The objective of this study was to investigate the biological effect of the addition of TGF-b1 in chitosan-modified GIC (BIO-GIC) on human dental pulp (HDP) cells.
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Basic Research—Technology Materials and Methods Materials and Specimen Preparation GIC used in this study was a conventional type III material (GC lining; GC Corporation, Tokyo, Japan). The powder was composed of calcium fluoroaluminosilicate glass (batch no. 0701151), and the liquid part contained 40% poly(acrylic acid), tartaric acid, and water (batch no. 070191). The novel GIC here was called BIO-GIC. This was prepared from fluoroaluminosilicate glass (conventional type III) incorporated thoroughly (by weight) with 15% of chitosan and 10% of BSA (Sigma Chemical, St Louis, MO). Chitosan used in this study has molecular weight about 545 kd and 80% degree of deacetylation (Fluka, Buchs, Switzerland; batch no. 1138071). BIO-GIC powder was mixed with the original liquid of conventional GIC. Disk specimens (10 mm in diameter, 1 mm in height) were prepared by using a stainless steel spatula and mixing pad at the powder:liquid ratio of 1.17:1 by weight. The cement was then packed into split ring Teflon molds by using the mixing spatula. A polythene sheet and glass slide were then placed over the filled mold, after which firm hand pressure was applied. Four groups of specimens (GIC, BIO-GIC, GIC+TGF-b1, and BIOGIC+TGF-b1) were prepared at room temperature of 23 C 2 C. GIC and BIO-GIC were 2 negative control groups. Both experimental groups, GIC+TGF-b1 and BIO-GIC+TGF-b1, were GIC and BIO-GIC, respectively, with added 2 mL of TGF-b1 (solution concentration, 50 ng/mL = 100 ng). This TGF-b1 (PeproTech Inc, Rocky Hill, NJ) was added during the preparation of each specimen. Specimens were retained in their mold during storage in an incubator at 37 C for 1 hour before biological investigation. Cell Culture HDP cells were cultured from normal human third molars without dental decay from 2 adolescent patients aged about 18 years at the Dental Hospital, with approval of the Research Ethics Committee, Faculty of Dentistry, Prince of Songkla University (approval number 0521.1.03). Patients gave informed consent for donation of these cells. The culture media and supplements were the products of Gibco (Invitrogen Corporation, Grand Island, NY), unless indicated elsewhere. Primary culture of pulp cells was performed by using an enzymatic method. Briefly, pulp tissue was minced into pieces and digested in a solution of 3 mg/mL of collagenase type I and 4 mg/mL of dispase for 1 hour at 37 C. After centrifugation, cells were cultured in alpha modified Eagle medium (aMEM), supplemented with 10% fetal calf serum, 100 mmol/L L-ascorbic acid 2-phosphate (Sigma-Aldrich), 2 mmol/L L-glutamate, 100 units/mL penicillin, and 100 mg/mL streptomycin and incubated at 37 C with 5% CO2. Pulp cells used in this study were from the third to eighth passages. Cell Proliferation Methol-thiazol-diphenyltetrazolium (MTT) assay was used to determine cell proliferation after the release of substances from the specimens exposed to cells at 2 periods of time, each period lasting 3 days. There were 6 groups in this part of the experiment. Four groups were cells exposed to specimens. Two other groups were cells cultured in the normal medium with added 1 ng/mL of TGF-b1 (positive comparator) and cells fed with normal medium only (culture control). The experiment was repeated 4 times. HDP cells were seeded at 5.5 104 cells/cm2 on a 12-well Transwell cluster plate (Costar, NY). Cells were fed with 1.5 mL of culture medium and incubated under 5% CO2 at 37 C. After incubation for 24 hours, the medium was refreshed, and each specimen was placed on the upper compartment (Transwell insert, with 12-mm membrane 368
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diameter and 3-mm pore size), and 0.5 mL of the medium was added. After 3 days, MTT was performed, and each specimen was moved to another new Transwell insert that had already been seeded with 5.5 104 cells/cm2 for 24 hours. After 3 days, cell viability was determined by MTT assay (20). Reduced MTT was measured by optical density (OD) in spectrophotometer at a wavelength of 570 nm. The OD values, corrected for a blank (medium only), of the experimental groups were divided by the OD of the control and expressed as a percentage of the control, which represented the percent of cell viability. The results were compared by using two-way analysis of variance (ANOVA) and the Tukey honestly significant difference post hoc test, with statistical significance set at p <.05.
Alkaline Phosphatase Activity Alkaline phosphatase (ALP) is a marker for odontoblast-like differentiation. We used a 12-well Transwell cluster plate, and the experimental groups were allocated in the same way as the MTT assay, but HDP cells were seeded at 4.5 104 cells/cm2. The medium was changed every 2 days. After 14 days, ALP activity was determined according to the principle of Bessey et al (21) by using p-nitrophenol phosphate as a substrate. Cells were lysed with 1 RLB (Promega Corporation, Madison, WI). After centrifugation, total cell protein of the supernatant was determined by using bicinchoninic acid method of BCA kit (Pierce Biotechnology, Holmdel, NJ), and ALP activity was measured by ALP kit (Human Gesellschaft F€ur Biochemica und Diagnostica GmbH, Wiesbaden, Germany). The procedures followed the manufacturers’ protocols. The experiment was repeated 4 times in each group and reported as ALP activity in U/L per mg of total protein. Results were analyzed with one-way ANOVA. Von Kossa Staining Cells were seeded at 6.4 103 cells/cm2 (6 wells, Transwell) in odontogenic inductive medium (22) consisting of 10 mmol/L b-glycerolphosphate, 0.2 mmol/L ascorbic acid in a-MEM with 15% fetal calf serum. After 24 hours, each specimen was placed on the upper compartment, as described previously. To determine the mineralizing capability of HDP cells, after 21 days of culture, the cells were fixed with solution mixture of 50% ethanol and 18% formaldehyde for 30 minutes, and von Kossa staining was performed (23). The mineralized or calcium nodule area (black nodule), defined as [(Stained area/Total dish area) 100] %, was determined by a digitalized image analysis system (Image Pro Plus; Media Cybernetic Inc, Bethesda, MD). The experiment was repeated 3 times, and the results were compared by TABLE 1. Mean Standard Deviation Percentages of Cell Viability from MTT Assay Percentages of surviving cells Group
First period (day 3)
Second period (day 6)
GIC BIO-GIC GIC+TGF-b1 BIO-GIC+TGF-b1 Medium + TGF-b1
22.8 6.9aA 13.8 4.6aA 47.0 9.6bB 11.3 4.2aA 104.8 3.9cE
81.5 2.4aC 91.0 3.8bC 94.0 9.2bC 93.3 2.5bC 114.8 19.7cE
BIO-GIC, chitosan-fluoroaluminosilicate GIC with albumin; GIC, glass ionomer cement; TGF-b1, transforming growth factor beta-1. Mean values with dissimilar small letters within each column and capital letters within each row are statistically significantly different at p <.05.
JOE — Volume 37, Number 3, March 2011
Basic Research—Technology TABLE 2. Mean Standard Deviation ALP Activity of HDP Cells Group
ALP activity (U/L/mg of protein)*
GIC BIO-GIC GIC+TGF-b1 BIO-GIC+TGF-b1 Medium Medium + TGF-b1
2.8 0.5 3.7 0.7 4.1 1.8 3.4 1.1 4.9 1.1 3.8 0.9
ALP, alkaline phosphatase; BIO-GIC, chitosan-fluoroaluminosilicate GIC with albumin; GIC, glass ionomer cement; TGF-b1, transforming growth factor beta-1. *1 unit = 16.67 nmol p-nitrophenol/min.
using one-way ANOVA and Tukey honestly significant difference post hoc test.
Results Cell Proliferation MTT assay evaluated cell viability at 2 periods of time. The first period of cell viability was tested after complete setting of the material and then placed on the insert part of Transwell for 3 days. The result (Table 1) showed that GIC with added TGF-b1 (GIC+TGF-b1) had significantly higher percentages of viable cells (p < .05) compared with GIC, BIO-GIC, and BIO-GIC+TGF-b1. In the second period, percentages of surviving cells were significantly higher than during the first period, whereas GIC showed the lowest percentage of viable cells (p < .05) when compared with all groups. BIO-GIC, BIOGIC+TGF-b1, and GIC+TGF-b1 had no significant difference in cell
viability, and their percentages of surviving cells were all greater than 90%. The positive comparator group, which was cells cultured in medium with added TGF-b1, demonstrated more than 100% of average percentage of viable cells when compared with culture control group (medium only).
ALP Activity There were no statistically significant differences (p > .05) in ALP activity between all groups (Table 2). Von Kossa Staining Figure. 1 shows the calcification nodules after 21 days of cells exposed to each type of specimen. The mineralized area in BIOGIC+TGF-b1 and odontogenic medium +TGF-b1 groups had significantly higher percentages of calcium deposit area compared with other groups (Table 3).
Discussion The ideal material for vital pulp treatment should have antibacterial effect, stimulate the remaining pulp tissue to return to a normal condition, and promote the formation of dentin. In recent years, a variety of materials have been introduced into dentistry to achieve a restorable functional pulp-dentin complex. These materials might include calcium hydroxide, mineral trioxide aggregate, and GIC. Recently, there have been some attempts to modify these materials to release various bioactive molecules. Of particular interest, TGF-b1
Figure 1. Deposition of mineralized nodules of HDP cells exposed to each specimen for 21 days, with von Kossa staining. Black area was the calcium deposit area. (A) GIC, (B) GI+TGF-b1, (C) BIO-GIC, (D) BIO-GIC+TGF-b1.
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Basic Research—Technology TABLE 3. Mean Standard Deviation Percentages of Calcium Deposit Area of HDP Cells Group
Percentage of calcium deposit area
GIC BIO-GIC GIC+TGF-b1 BIO-GIC+TGF-b1 Odontogenic medium Odontogenic medium + TGF-b1
28.6 0.1a 32.8 2.1a 31.9 2.1a 51.9 5.7b 33.8 2.5a 44.9 3.7b
BIO-GIC, chitosan-fluoroaluminosilicate GIC with albumin; GIC, glass ionomer cement; TGF-b1, transforming growth factor beta-1. Mean values with different letters are statistically significantly different at p < .05.
was selected as a bioactive molecule for release from BIO-GIC for this study. The results of MTT assay in positive comparator group (cells cultivated in medium with added TGF-b1) revealed the proliferation promotion of TGF-b1 to HDP cells. However, there was no significant difference in ALP activity in all groups from this study. This result might differ from the study of Nie et al (9), which revealed significantly increased ALP activity in human pulp cells after incubation in culture medium with 5 ng/mL of TGF-b1 for both 3 and 6 days. But our results correspond with the study of Shirakawa et al (24). They evaluated ALP activity at 0.1 and 5 ng/mL of TGF-b1 in 4 lines of pulp cells and found increased enzyme activity in only 1 cell line at 0.1 ng/mL of TGF-b. In all cultures, TGF-b1 at 5 ng/mL decreased ALP activity. They concluded that the ALP activity of human pulp cells depended on the odontogenic phenotype of the cell line and the concentration of the growth factor. It might require further evaluation of ALP activity in pulp cells exposed to these materials by using cells from more subjects and for a longer period of time. However, TGF-b1 increased mineralization of pulp cells when cultured in odontogenic medium, which confirmed its ability to promote differentiation (9). From our previous study, we found that chitosan added to GIC was able to prolong the release of BSA for 2 weeks, and also BSA by itself was able to reduce the toxicity of the material (13). In this study, we reduced the percentages of chitosan from 20% by weight from that previous study to 15% to reduce the viscosity of the cement. From another study (unpublished data), we investigated the released pattern of epidermal growth factor from chitosan fluoroaluminosilicate cement with added epidermal growth factor, and the result suggested that 10% of BSA gave better release of the growth factor than 1% of BSA (data not shown). In this study, the results showed the persistent biological effect of TGF-b1 from the BIO-GIC+TGF-b1 group. It was apparent that this can promote mineralization in HDP cells during a period of 21 days when compared with GIC+TGF-b1 group (Table 3). The results from MTT assay suggested that most toxic substances might be released within the first 3-day period from BIO-GIC and BIOGIC +TGF-b1 groups, because more than 90% of cells survived at the second period of the assay. The reason that GIC gave the least percentage of viable cells in the second period might come from the lack of TGF-b1, when compared with GIC+TGF-b1 and BIOGIC+TGF-b1 specimens. BIO-GIC specimens also had significantly higher percentages of viable cells than GIC group at the second period. This result might come from the effect of chitosan and albumin, because in their study Limapornvanich et al (13) found that chitosan did not increase the cytotoxicity of GIC, and albumin might even reduce the cytotoxicity of GIC. The real mechanism of chitosan to reduce cytoxicity is not known, whereas BSA itself has the property to bind toxic chemical (25). However, a recent study showed that chitosan can reduce the 370
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percentages of DNA damage caused by 2-hydroxyethyl methacrylate (26). Chitosan monomer (D-glucosamine hydrochloride) also can promote pulp cell regeneration in both in vivo and in vitro experiments (27). However, it was noted that GIC+TGF-b1 gave the highest percentages of surviving cells compared with other specimens at the first period. This point suggested that GIC+TGF-b1 might have an initial release of TGF-b1 in early period of time. But BIO-GIC+TGF-b1 might have a better prolonged release effect, because it can promote more mineralization after 21 days of cultivation. However, the released pattern of TGF-b1 from this material should be further evaluated. In all experiments we used Transwell plates because they allowed the substances continually released from the specimen to pass through the 3-mm pore size membrane and react with HDP cells. This methodology presented better validity result than using the extracts from the specimens to react with cells, especially the released growth factor that might degrade easily. This preliminary study demonstrated that it was possible to develop a novel material that could provide sustained release of growth factor. Further research is required, in particular to examine the released pattern of the growth factor from this material and whether there are any toxic substances released during the early setting of cement.
Conclusion This novel material showed potential for the retained effect of added TGF-b1, which was longer than the conventional GIC. There might be applications in regenerative endodontics or for use as vital pulp therapy material, which could promote repair of the dentinpulp complex.
Acknowledgments This work was supported by a grant from Postgraduate Educational Affairs, Faculty of Dentistry, Graduate School, Prince of Songkla University. The authors deny any conflicts of interest related to this study.
References 1. Dard M, Sewing A, Meyer J, Verrier S, Roessler S, Scharnweber D. Tools for tissue engineering of mineralized oral structures. Clin Oral Invest 2000;4:126–9. 2. Slavkin HC, Bartold PM. Challenges and poteintial in tissue engineering. Periodontol 2000 2006;41:9–15. 3. Huang GT. A paradigm shift in endodontic management of immature teeth: conservation of stem cells for regeneration. J Dent 2008;36:379–86. 4. Smith AJ. Vitality of the dentin-pulp complex in health and disease: growth factors as key mediators. J Dent Educ 2003;67:678–89. 5. Tziafas D, Belibasakis G, Veis A, Papadimitriou S. Dentin regeneration in vital pulp therapy: design principles. Adv Dent Res 2001;15:96–100. 6. Magloire H, Romeas A, Melin M, Couble ML, Bleicher F, Farges JC. Molecular regulation of odontoblast activity under dentin injury. Adv Dent Res 2001;15:46–50. 7. Yongchaitrakul T, Pavasant P. Transforming growth factor-beta1 up-regulates the expression of nerve growth factor through mitogen-activated protein kinase signaling pathways in dental pulp cells. Eur J Oral Sci 2007;115:57–63. 8. Shiba H, Fujita T, Doi N, et al. Differential effects of various growth factors and cytokines on the syntheses of DNA, type I collagen, laminin, fibronectin, osteonectin/ secreted protein, acidic and rich in cysteine (SPARC), and alkaline phosphatase by human pulp cells in culture. J Cell Physiol 1998;174:194–205. 9. Nie X, Tian W, Zhang Y, et al. Induction of transforming growth factor-beta 1 on dentine pulp cells in different culture patterns. Cell Biol Int 2006;30:295–300. 10. Nicholson JW. Chemistry of glass-ionomer cements: a review. Biomaterials 1998;19: 485–94. 11. Mazzaoui SA, Burrow MF, Tyas MJ, Dashper SG, Eakins D, Reynolds EC. Incorporation of casein phosphopeptide-amorphous calcium phosphate into a glass-ionomer cement. J Dent Res 2003;82:914–8. 12. Palmer G, Jones FH, Billington RW, Pearson GJ. Chlorhexidine release from an experimental glass ionomer cement. Biomaterials 2004;25:5423–31.
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Basic Research—Technology 13. Limapornvanich A, Jitpukdeebodintra S, Hengtrakool C, Kedjarune-Leggat U. Bovine serum albumin release from novel chitosan-fluoro-aluminosilicate glass ionomer cement: stability and cytotoxicity studies. J Dent 2009;37:686–90. 14. Lee SK, Lee SK, Lee SI, et al. Effect of calcium phosphate cements on growth and odontoblastic differentiation in human dental pulp cells. J Endod 2010;36: 1537–42. 15. Kishen A, Shi Z, Shrestha A, Neoh KG. An investigation on the antibacterial and antibiofilm efficacy of cationic nanoparticulates for root canal disinfection. J Endod 2008;34:1515–20. 16. Shrestha A, Shi Z, Neoh KG, Kishen A. Nanoparticulates for antibiofilm treatment and effect of aging on its antibacterial activity. J Endod 2010;36:1030–5. 17. Ballal NV, Shavi GV, Kumar R, Kundabala M, Bhat KS. In vitro sustained release of calcium ions and pH maintenance from different vehicles containing calcium hydroxide. J Endod 2010;36:862–6. 18. de Oliveira HC, Fonseca JL, Pereira MR. Chitosan-poly(acrylic acid) polyelectrolyte complex membranes: preparation, characterization and permeability studies. J Biomater Sci Polym Ed 2008;19:143–60. 19. Murray JB, Brown L, Langer R, Klagsburn M. A micro sustained release system for epidermal growth factor. In vitro 1983;19:743–8.
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20. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55–63. 21. Bessey O, Lowry O, Brock M. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J Biol Chem 1946;164:321–9. 22. Wei X, Ling J, Wu L, Liu L, Xiao Y. Expression of mineralization markers in dental pulp cells. J Endod 2007;33:703–8. 23. Nakamura H, Saruwatari L, Aita H, Takeuchi K, Ogawa T. Molecular and biomechanical characterization of mineralized tissue by dental pulp cells on titanium. J Dent Res 2005;84:515–20. 24. Shirakawa M, Shiba H, Nakanishi K, et al. Transforming growth factor-beta-1 reduces alkaline phosphatase mRNA and activity and stimulates cell proliferation in cultures of human pulp cells. J Dent Res 1994;73:1509–14. 25. Gulden M, Morchel S, Seibert H. Serum albumin binding at cytotoxic concentrations of chemicals as determined with a cell proliferation assay. Toxicol Lett 2003;137:159–68. 26. Pawlowska E, Poplawski T, Ksiazek D, Szczepanska J, Blasiak J. Genotoxicity and cytotoxicity of 2-hydroxyethyl methacrylate. Mutat Res 2010;696:122–9. 27. Matsunaga T, Yanagiguchi K, Yamada S, Ohara N, Ikeda T, Hayashi Y. Chitosan monomer promotes tissue regeneration on dental pulp wounds. J Biomed Mater Res A 2006;76:711–20.
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Effect of Novel Chitosan-fluoroaluminosilicate Glass Ionomer Cement with Added Transforming Growth Factor Beta-1 on Pulp Cells Nitra Rakkiettiwong, MSc, DDS,* Chanothai Hengtrakool, PhD, DDS,† Kewalin Thammasitboon, PhD, DDS,† and Ureporn Kedjarune-Leggat, PhD, DDS‡ Abstract Introduction: Vital pulp therapy might benefit from the sustained release of transforming growth factor beta-1 (TGF-b1) from dental restorative materials. Chitosan has previously been shown to enable sustained release of bovine serum albumin (BSA) from glass ionomer cement (GIC). Because BSA can prolong release of growth factor, chitosan-fluoroaluminosilicate GIC with albumin (BIO-GIC) should sustain the effect of growth factor. This study investigated the effect of BIO-GIC with added TGF-b1 on pulp cells. Methods: BIO-GIC was prepared from GIC (conventional type) incorporated with 15% of chitosan and 10% of BSA. TGF-b1 (100 ng) was added in BIO-GIC+TGF-b1 and GIC+TGF-b1 groups during each disk specimen (10 mm diameter, 1 mm high) preparation. Two control groups were BIO-GIC and GIC. The effect of each specimen on pulp cells was investigated by using the Transwell plate technique. Cell proliferation was determined by MTT assay at 2 time periods (each period lasting 3 days). Pulp cell differentiation was examined by alkaline phosphatase activity and also by cell mineralization, which was measured by calculating the area of mineralization with von Kossa staining. Results: Percentage of viable cells of GIC+TGF-b1 group was the highest after the first period. This might suggest an initial rapid release of TGF-b1 from GIC. After the second period, BIO-GIC, BIO-GIC+TGF-b1, and GIC+TGF-b1 had more than 90% cell survival. It was significantly greater than GIC (82% 2%). There was no significant difference in alkaline phosphatase activity. BIO-GIC+TGF-b1 had the highest mineralization area during 21 days. Conclusions: BIO-GIC could retain the effect of TGF-b1. (J Endod 2011;37:367–371)
From the *Dental Department, Narathiwat Hospital, Muang, Narathiwat, Thailand; †Department of Conservative Dentistry, Faculty of Dentistry, Prince of Songkla University, Hat Yai, Songkhla, Thailand; and ‡Department of Oral Biology and Occlusion, Faculty of Dentistry, Prince of Songkla University, Hat Yai, Songkhla, Thailand. Address requests for reprints to Assoc Prof Dr Ureporn Kedjarune-Leggat, Department of Oral Biology and Occlusion, Faculty of Dentistry, Prince of Songkla University, Hat Yai, Songkhla, Thailand 90112. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2011 American Association of Endodontists. doi:10.1016/j.joen.2010.11.031
JOE — Volume 37, Number 3, March 2011
Key Words Bioactive releasing material, chitosan, glass-ionomer cement, growth factor, pulp regeneration
T
issue engineering is emerging as a new model in dentistry and medicine to regenerate natural tissues or to create biological substitutes for defective tissues (1,2), which nowadays also includes endodontic management (3). To regenerate or repair pulp-dentin complex, growth factors play a key role in success (4). Many studies suggest that transforming growth factor beta-1 (TGF-b1) is an important factor involved in dental pulp tissue repair (5–7) by stimulating cell proliferation, cell migration, and type I collagen synthesis (8), as well as mineralization (9). However, growth factors or bioactive molecules are sensitive to temperature, pH, and pressure (1). The development of a possible new generation of biomaterials to control release bioactive molecules is therefore challenging. Glass ionomer cements (GICs) are acid-base cements produced from the reaction of fluoroaluminosilicate glass powder with poly(acrylic acid), which are widely used in dental and medical applications because of their biocompatibility, antibacterial properties, sealing ability, and capacity to prolong the release of fluoride (10). There have been some attempts to enhance sustained release of substances from this cement, such as casein phosphopeptide–amorphous calcium phosphate (CPP-ACP) (11), chlorhexidine (12), and also, in particular, some proteins (13). Chitosan is a biocompatible, biodegradable natural biopolymer that is a copolymer of glucosamine and N-acetylglucosamine derived from chitin. There are many studies about attempting the use of chitosan in dentistry. The addition of chitosan to calcium phosphate cement can increase compressive strength of the cement as well as promote odontoblastic differentiation in human dental pulp cells (14). Moreover, chitosan nanoparticle has an antibacterial property, which has been suggested for root canal disinfection (15). These particles still retain antibacterial activity, even after 90 days (16). In addition, calcium hydroxide–containing chitosan can sustain release of calcium ion for more than 30 days (17). Recently, our group discovered a novel chitosan-fluoroaluminosilicate GIC that can prolong the release of BSA without alteration of its molecular weight, and this cement did not increase toxicity to pulp cells (13). This material can prolong the release of protein, possibly as a result of the formation of a polyelectric complex between the cationic group of chitosan and the anionic group of poly(acrylic acid) (18). Because albumin was introduced to prolong release of growth factor (19), we developed a novel material composed of chitosan-fluoroaluminosilicate GIC and albumin to sustain the release of growth factors. The hypothesis of this study is that the addition of growth factor to this material should sustain its biological effect. This novel GIC could be considered as a material for vital pulp therapy, if it can retain the biological effect of the added growth factor in the cement long enough to activate the healing of pulp tissue by increasing cell numbers or promoting pulp cell differentiation. The objective of this study was to investigate the biological effect of the addition of TGF-b1 in chitosan-modified GIC (BIO-GIC) on human dental pulp (HDP) cells.
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Basic Research—Technology Materials and Methods Materials and Specimen Preparation GIC used in this study was a conventional type III material (GC lining; GC Corporation, Tokyo, Japan). The powder was composed of calcium fluoroaluminosilicate glass (batch no. 0701151), and the liquid part contained 40% poly(acrylic acid), tartaric acid, and water (batch no. 070191). The novel GIC here was called BIO-GIC. This was prepared from fluoroaluminosilicate glass (conventional type III) incorporated thoroughly (by weight) with 15% of chitosan and 10% of BSA (Sigma Chemical, St Louis, MO). Chitosan used in this study has molecular weight about 545 kd and 80% degree of deacetylation (Fluka, Buchs, Switzerland; batch no. 1138071). BIO-GIC powder was mixed with the original liquid of conventional GIC. Disk specimens (10 mm in diameter, 1 mm in height) were prepared by using a stainless steel spatula and mixing pad at the powder:liquid ratio of 1.17:1 by weight. The cement was then packed into split ring Teflon molds by using the mixing spatula. A polythene sheet and glass slide were then placed over the filled mold, after which firm hand pressure was applied. Four groups of specimens (GIC, BIO-GIC, GIC+TGF-b1, and BIOGIC+TGF-b1) were prepared at room temperature of 23 C 2 C. GIC and BIO-GIC were 2 negative control groups. Both experimental groups, GIC+TGF-b1 and BIO-GIC+TGF-b1, were GIC and BIO-GIC, respectively, with added 2 mL of TGF-b1 (solution concentration, 50 ng/mL = 100 ng). This TGF-b1 (PeproTech Inc, Rocky Hill, NJ) was added during the preparation of each specimen. Specimens were retained in their mold during storage in an incubator at 37 C for 1 hour before biological investigation. Cell Culture HDP cells were cultured from normal human third molars without dental decay from 2 adolescent patients aged about 18 years at the Dental Hospital, with approval of the Research Ethics Committee, Faculty of Dentistry, Prince of Songkla University (approval number 0521.1.03). Patients gave informed consent for donation of these cells. The culture media and supplements were the products of Gibco (Invitrogen Corporation, Grand Island, NY), unless indicated elsewhere. Primary culture of pulp cells was performed by using an enzymatic method. Briefly, pulp tissue was minced into pieces and digested in a solution of 3 mg/mL of collagenase type I and 4 mg/mL of dispase for 1 hour at 37 C. After centrifugation, cells were cultured in alpha modified Eagle medium (aMEM), supplemented with 10% fetal calf serum, 100 mmol/L L-ascorbic acid 2-phosphate (Sigma-Aldrich), 2 mmol/L L-glutamate, 100 units/mL penicillin, and 100 mg/mL streptomycin and incubated at 37 C with 5% CO2. Pulp cells used in this study were from the third to eighth passages. Cell Proliferation Methol-thiazol-diphenyltetrazolium (MTT) assay was used to determine cell proliferation after the release of substances from the specimens exposed to cells at 2 periods of time, each period lasting 3 days. There were 6 groups in this part of the experiment. Four groups were cells exposed to specimens. Two other groups were cells cultured in the normal medium with added 1 ng/mL of TGF-b1 (positive comparator) and cells fed with normal medium only (culture control). The experiment was repeated 4 times. HDP cells were seeded at 5.5 104 cells/cm2 on a 12-well Transwell cluster plate (Costar, NY). Cells were fed with 1.5 mL of culture medium and incubated under 5% CO2 at 37 C. After incubation for 24 hours, the medium was refreshed, and each specimen was placed on the upper compartment (Transwell insert, with 12-mm membrane 368
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diameter and 3-mm pore size), and 0.5 mL of the medium was added. After 3 days, MTT was performed, and each specimen was moved to another new Transwell insert that had already been seeded with 5.5 104 cells/cm2 for 24 hours. After 3 days, cell viability was determined by MTT assay (20). Reduced MTT was measured by optical density (OD) in spectrophotometer at a wavelength of 570 nm. The OD values, corrected for a blank (medium only), of the experimental groups were divided by the OD of the control and expressed as a percentage of the control, which represented the percent of cell viability. The results were compared by using two-way analysis of variance (ANOVA) and the Tukey honestly significant difference post hoc test, with statistical significance set at p <.05.
Alkaline Phosphatase Activity Alkaline phosphatase (ALP) is a marker for odontoblast-like differentiation. We used a 12-well Transwell cluster plate, and the experimental groups were allocated in the same way as the MTT assay, but HDP cells were seeded at 4.5 104 cells/cm2. The medium was changed every 2 days. After 14 days, ALP activity was determined according to the principle of Bessey et al (21) by using p-nitrophenol phosphate as a substrate. Cells were lysed with 1 RLB (Promega Corporation, Madison, WI). After centrifugation, total cell protein of the supernatant was determined by using bicinchoninic acid method of BCA kit (Pierce Biotechnology, Holmdel, NJ), and ALP activity was measured by ALP kit (Human Gesellschaft F€ur Biochemica und Diagnostica GmbH, Wiesbaden, Germany). The procedures followed the manufacturers’ protocols. The experiment was repeated 4 times in each group and reported as ALP activity in U/L per mg of total protein. Results were analyzed with one-way ANOVA. Von Kossa Staining Cells were seeded at 6.4 103 cells/cm2 (6 wells, Transwell) in odontogenic inductive medium (22) consisting of 10 mmol/L b-glycerolphosphate, 0.2 mmol/L ascorbic acid in a-MEM with 15% fetal calf serum. After 24 hours, each specimen was placed on the upper compartment, as described previously. To determine the mineralizing capability of HDP cells, after 21 days of culture, the cells were fixed with solution mixture of 50% ethanol and 18% formaldehyde for 30 minutes, and von Kossa staining was performed (23). The mineralized or calcium nodule area (black nodule), defined as [(Stained area/Total dish area) 100] %, was determined by a digitalized image analysis system (Image Pro Plus; Media Cybernetic Inc, Bethesda, MD). The experiment was repeated 3 times, and the results were compared by TABLE 1. Mean Standard Deviation Percentages of Cell Viability from MTT Assay Percentages of surviving cells Group
First period (day 3)
Second period (day 6)
GIC BIO-GIC GIC+TGF-b1 BIO-GIC+TGF-b1 Medium + TGF-b1
22.8 6.9aA 13.8 4.6aA 47.0 9.6bB 11.3 4.2aA 104.8 3.9cE
81.5 2.4aC 91.0 3.8bC 94.0 9.2bC 93.3 2.5bC 114.8 19.7cE
BIO-GIC, chitosan-fluoroaluminosilicate GIC with albumin; GIC, glass ionomer cement; TGF-b1, transforming growth factor beta-1. Mean values with dissimilar small letters within each column and capital letters within each row are statistically significantly different at p <.05.
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Basic Research—Technology TABLE 2. Mean Standard Deviation ALP Activity of HDP Cells Group
ALP activity (U/L/mg of protein)*
GIC BIO-GIC GIC+TGF-b1 BIO-GIC+TGF-b1 Medium Medium + TGF-b1
2.8 0.5 3.7 0.7 4.1 1.8 3.4 1.1 4.9 1.1 3.8 0.9
ALP, alkaline phosphatase; BIO-GIC, chitosan-fluoroaluminosilicate GIC with albumin; GIC, glass ionomer cement; TGF-b1, transforming growth factor beta-1. *1 unit = 16.67 nmol p-nitrophenol/min.
using one-way ANOVA and Tukey honestly significant difference post hoc test.
Results Cell Proliferation MTT assay evaluated cell viability at 2 periods of time. The first period of cell viability was tested after complete setting of the material and then placed on the insert part of Transwell for 3 days. The result (Table 1) showed that GIC with added TGF-b1 (GIC+TGF-b1) had significantly higher percentages of viable cells (p < .05) compared with GIC, BIO-GIC, and BIO-GIC+TGF-b1. In the second period, percentages of surviving cells were significantly higher than during the first period, whereas GIC showed the lowest percentage of viable cells (p < .05) when compared with all groups. BIO-GIC, BIOGIC+TGF-b1, and GIC+TGF-b1 had no significant difference in cell
viability, and their percentages of surviving cells were all greater than 90%. The positive comparator group, which was cells cultured in medium with added TGF-b1, demonstrated more than 100% of average percentage of viable cells when compared with culture control group (medium only).
ALP Activity There were no statistically significant differences (p > .05) in ALP activity between all groups (Table 2). Von Kossa Staining Figure. 1 shows the calcification nodules after 21 days of cells exposed to each type of specimen. The mineralized area in BIOGIC+TGF-b1 and odontogenic medium +TGF-b1 groups had significantly higher percentages of calcium deposit area compared with other groups (Table 3).
Discussion The ideal material for vital pulp treatment should have antibacterial effect, stimulate the remaining pulp tissue to return to a normal condition, and promote the formation of dentin. In recent years, a variety of materials have been introduced into dentistry to achieve a restorable functional pulp-dentin complex. These materials might include calcium hydroxide, mineral trioxide aggregate, and GIC. Recently, there have been some attempts to modify these materials to release various bioactive molecules. Of particular interest, TGF-b1
Figure 1. Deposition of mineralized nodules of HDP cells exposed to each specimen for 21 days, with von Kossa staining. Black area was the calcium deposit area. (A) GIC, (B) GI+TGF-b1, (C) BIO-GIC, (D) BIO-GIC+TGF-b1.
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Basic Research—Technology TABLE 3. Mean Standard Deviation Percentages of Calcium Deposit Area of HDP Cells Group
Percentage of calcium deposit area
GIC BIO-GIC GIC+TGF-b1 BIO-GIC+TGF-b1 Odontogenic medium Odontogenic medium + TGF-b1
28.6 0.1a 32.8 2.1a 31.9 2.1a 51.9 5.7b 33.8 2.5a 44.9 3.7b
BIO-GIC, chitosan-fluoroaluminosilicate GIC with albumin; GIC, glass ionomer cement; TGF-b1, transforming growth factor beta-1. Mean values with different letters are statistically significantly different at p < .05.
was selected as a bioactive molecule for release from BIO-GIC for this study. The results of MTT assay in positive comparator group (cells cultivated in medium with added TGF-b1) revealed the proliferation promotion of TGF-b1 to HDP cells. However, there was no significant difference in ALP activity in all groups from this study. This result might differ from the study of Nie et al (9), which revealed significantly increased ALP activity in human pulp cells after incubation in culture medium with 5 ng/mL of TGF-b1 for both 3 and 6 days. But our results correspond with the study of Shirakawa et al (24). They evaluated ALP activity at 0.1 and 5 ng/mL of TGF-b1 in 4 lines of pulp cells and found increased enzyme activity in only 1 cell line at 0.1 ng/mL of TGF-b. In all cultures, TGF-b1 at 5 ng/mL decreased ALP activity. They concluded that the ALP activity of human pulp cells depended on the odontogenic phenotype of the cell line and the concentration of the growth factor. It might require further evaluation of ALP activity in pulp cells exposed to these materials by using cells from more subjects and for a longer period of time. However, TGF-b1 increased mineralization of pulp cells when cultured in odontogenic medium, which confirmed its ability to promote differentiation (9). From our previous study, we found that chitosan added to GIC was able to prolong the release of BSA for 2 weeks, and also BSA by itself was able to reduce the toxicity of the material (13). In this study, we reduced the percentages of chitosan from 20% by weight from that previous study to 15% to reduce the viscosity of the cement. From another study (unpublished data), we investigated the released pattern of epidermal growth factor from chitosan fluoroaluminosilicate cement with added epidermal growth factor, and the result suggested that 10% of BSA gave better release of the growth factor than 1% of BSA (data not shown). In this study, the results showed the persistent biological effect of TGF-b1 from the BIO-GIC+TGF-b1 group. It was apparent that this can promote mineralization in HDP cells during a period of 21 days when compared with GIC+TGF-b1 group (Table 3). The results from MTT assay suggested that most toxic substances might be released within the first 3-day period from BIO-GIC and BIOGIC +TGF-b1 groups, because more than 90% of cells survived at the second period of the assay. The reason that GIC gave the least percentage of viable cells in the second period might come from the lack of TGF-b1, when compared with GIC+TGF-b1 and BIOGIC+TGF-b1 specimens. BIO-GIC specimens also had significantly higher percentages of viable cells than GIC group at the second period. This result might come from the effect of chitosan and albumin, because in their study Limapornvanich et al (13) found that chitosan did not increase the cytotoxicity of GIC, and albumin might even reduce the cytotoxicity of GIC. The real mechanism of chitosan to reduce cytoxicity is not known, whereas BSA itself has the property to bind toxic chemical (25). However, a recent study showed that chitosan can reduce the 370
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percentages of DNA damage caused by 2-hydroxyethyl methacrylate (26). Chitosan monomer (D-glucosamine hydrochloride) also can promote pulp cell regeneration in both in vivo and in vitro experiments (27). However, it was noted that GIC+TGF-b1 gave the highest percentages of surviving cells compared with other specimens at the first period. This point suggested that GIC+TGF-b1 might have an initial release of TGF-b1 in early period of time. But BIO-GIC+TGF-b1 might have a better prolonged release effect, because it can promote more mineralization after 21 days of cultivation. However, the released pattern of TGF-b1 from this material should be further evaluated. In all experiments we used Transwell plates because they allowed the substances continually released from the specimen to pass through the 3-mm pore size membrane and react with HDP cells. This methodology presented better validity result than using the extracts from the specimens to react with cells, especially the released growth factor that might degrade easily. This preliminary study demonstrated that it was possible to develop a novel material that could provide sustained release of growth factor. Further research is required, in particular to examine the released pattern of the growth factor from this material and whether there are any toxic substances released during the early setting of cement.
Conclusion This novel material showed potential for the retained effect of added TGF-b1, which was longer than the conventional GIC. There might be applications in regenerative endodontics or for use as vital pulp therapy material, which could promote repair of the dentinpulp complex.
Acknowledgments This work was supported by a grant from Postgraduate Educational Affairs, Faculty of Dentistry, Graduate School, Prince of Songkla University. The authors deny any conflicts of interest related to this study.
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20. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55–63. 21. Bessey O, Lowry O, Brock M. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J Biol Chem 1946;164:321–9. 22. Wei X, Ling J, Wu L, Liu L, Xiao Y. Expression of mineralization markers in dental pulp cells. J Endod 2007;33:703–8. 23. Nakamura H, Saruwatari L, Aita H, Takeuchi K, Ogawa T. Molecular and biomechanical characterization of mineralized tissue by dental pulp cells on titanium. J Dent Res 2005;84:515–20. 24. Shirakawa M, Shiba H, Nakanishi K, et al. Transforming growth factor-beta-1 reduces alkaline phosphatase mRNA and activity and stimulates cell proliferation in cultures of human pulp cells. J Dent Res 1994;73:1509–14. 25. Gulden M, Morchel S, Seibert H. Serum albumin binding at cytotoxic concentrations of chemicals as determined with a cell proliferation assay. Toxicol Lett 2003;137:159–68. 26. Pawlowska E, Poplawski T, Ksiazek D, Szczepanska J, Blasiak J. Genotoxicity and cytotoxicity of 2-hydroxyethyl methacrylate. Mutat Res 2010;696:122–9. 27. Matsunaga T, Yanagiguchi K, Yamada S, Ohara N, Ikeda T, Hayashi Y. Chitosan monomer promotes tissue regeneration on dental pulp wounds. J Biomed Mater Res A 2006;76:711–20.
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