- Email: [email protected]
Preferential Attachment of Human Gingival Fibroblasts to the Resin Ionomer Geristore
Basic Research—Technology
Preferential Attachment of Human Gingival Fibroblasts to the Resin Ionomer Geristore Fuwad Al-Sabek, DDS, MS, Sandra Shostad, DDS, MS, and Keith L. Kirkwood, DDS, PhD Abstract The resin ionomer Geristore has been used extensively for root perforation repairs. The purpose of this study was to evaluate oral in vitro biocompatibility of the resin ionomer Geristore compared to two other dental perforation repair materials, Ketac-Fil and Immediate Restorative Material (IRM). Growth and morphology of human gingival fibroblasts (HGFs) was determined using scanning electron microscopy (SEM) of HGFs cells grown on test materials as well as cytotoxicity assays using eluates from test materials. SEM analysis showed that HGFs attached and spread well over Geristore with relatively normal morphology. SEM showed that fibroblasts did not attach and spread well over Ketac-Fil or IRM as cells appeared much fewer with rounded and different morphology than fibroblasts grown on Geristore. Cytotoxicity assays indicated that HGFs proliferated in the presence of Geristore eluates and not in the presence of Ketac-Fil or IRM eluates. In vitro interpretation indicates that Geristore is less cytotoxic to gingival fibroblasts.
From the Departments of Endodontics and Periodontics, Oral Biology, and Pharmacology & Toxicology, State University of New York at Buffalo, Buffalo, NY. Address request for reprints to Keith L. Kirkwood, DDS, PhD, Assistant Professor, Department of Periodontics/Prevention/Geriatrics, University of Michigan 1011 N. University Ave., Ann Arbor, MI 48109-1078; E-mail address: [email protected]. Copyright © 2005 by the American Association of Endodontists
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adicular perforations can occur during Endodonontic treatment, postspace preparation, postremoval, and operative procedures. Perforations that occur below the level of the epithelial attachment can cause infection, which may result in attachment loss. These defects have been treated in the past with a variety of different materials including gutta-percha, calcium hydroxide products, amalgam, Immediate Restorative Material (IRM), cavit, and super Epoxy Benzoic Acid (EBA). Major problems relative to these materials include biocompatibility and inadequate sealing. The use of composite resins, glass ionomer cement and Mineral Trioxide Aggregate (MTA) has been suggested to circumvent these problems. Studies have shown that MTA is the most suitable material for repairing sub-osseous perforations especially furcation perforations, because of its superior sealing and biocompatibility characteristics (1, 2), as well as permitting mineralized matrix formation (3). Subgingival root perforations that are supraosseous have been repaired using glass ionomers and composite resins. However, all studies have demonstrated some degree of cytotoxicity through in vitro and in vivo studies (4 –14). Recently, resin ionomers such as Geristore, originally designed for restorative procedures, have been used in treating subgingival defects such as root resorption and perforation. Geristore is a dual cure (both self and light-curing), hydrophilic, nonaqueous polyacid modified composite resin composed of fluoride releasing glass, mainly barium fluorosilicate, and a polymerizable organic matrix (Modified Bis-GMA, including 2-HEMA) combined with a photoinitiator. Advantages of these materials include insolubility in oral fluids, increased adhesion to tooth structure, dual cure capabilities, low cure shrinkage, low coefficient of thermal expansion, radiopacity, fluoride release, and biocompatibility (15). Relatively few clinical studies have addressed biocompatibility (15), although several clinical studies have demonstrated Geristore could repair subgingival and subosseous defects and could be used as a barrier for guided tissue regeneration (16 –22). Cytotoxicity of resin ionomers (Geristore) towards oral tissues has only started to be investigated (23). The aim of the present study is to evaluate in vitro biocompatibility of human gingival fibroblasts with Geristore using scanning electron microscopy and nucleic acid fluorescent staining to measure cell attachment and morphology as well as cell cytotoxicity assays to eluates from root perforation repair materials namely Geristore, Ketac-Fil, and IRM.
Materials and Methods Cell Culture Conditions Human gingival fibroblasts (HGF-1; American Type Culture Collection, Manassas, VA; #ATCC CRL-2014) were obtained through commercial sources for these studies. Cells utilized for these studies were between passages 14 and 21. Cells were cultured in DMEM (Invitrogen Life Technologies, Grand Island, NY), supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO), penicillin (100 U/ml; Sigma), and streptomycin (100 g/ml; Sigma) at 37°C in a humidified atmosphere of 5% CO2 in air. The culture medium was changed every 3 to 4 days. Sample Preparation and Sterilization Geristore (DEN-MAT Corporation, Santa Maria, CA.), Ketac Fil (ESPE, Seefeld/ Oberbay, Germany), and IRM (Caulk/Dentsply, Milford, DE) were obtained from commercial sources. Discs (6 ⫻ 2 mm) from the three materials were fabricated under aseptic conditions by packing the material after mixing in a Teflon washer (internal diameter of 6 ⫻ 2 mm), and compressed between two glass slides to generate even
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Figure 1. Scanning Electron Microscopic analysis of human gingival fibroblasts attachment and growth following 72-h incubation on (A) glass coverslip controls, (B) Geristore, (C) Ketac Fil, or (D) IRM. SEM views are shown at 100⫻ with reference bar length indicated.
thickness of material. Glass cover slips were used as positive controls in all experimental conditions throughout this study. Extracts from all three materials were prepared by preincubating sterilized discs in 50 cm3 conical tubes with 5 ml of DMEM (Life Technologies) supplemented with 100 U/ml penicillin and 100 g/ml streptomycin at 37°C in a shaker (Labline, Barnstead Int., Dubuque, IA) at 150 rpm.
Scanning Electron Microscopy Growth Assay Attachment, growth and morphology of human gingival fibroblasts over Geristore were evaluated by scanning electron microscopy. Geristore, Ketac Fil and IRM discs and control glass cover slips were sterilized as described above and placed in the bottom of a 12-well culture plate. HGF-1 cells were seeded into the wells at density (3 ⫻ 104 cells per well) in DMEM medium containing 10% fetal bovine serum. After incubation, cells were washed three times with phosphate buffered saline and fixed in 2.5% glutaraldehyde in 1 mmol cacodylate buffer (pH 7.4). Samples were then observed using a JEOL JSM 6400 scanning electron microscope (Jeol USA, Peabody, MA). Cell attachment and viability in each experimental condition were assessed by qualitatively comparing morphology of cells over tested materials with that of cells over glass coverslips, which were considered cells with normal morphology. Cytotoxicity Assays Human gingival fibroblasts cytotoxicity towards Geristore, Ketacfil, and IRM material extracts was determined by means of using the CellTiter Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). HGF-1 cells were seeded into 96-well culture plates at 1 ⫻ 104 cells per well in DMEM supplemented with 10% fetal bovine serum. After 24h cells were rinsed three times with PBS and treated with 100 l of the following material eluates in serum-free media: Geristore (24 and 72 hr extracts), IRM (24 and 72 hr extracts), and Ketac-fil (24 and 72 hr extracts). Controls included media only (no material ex206
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tracts) and media without cells present. Soluble formazan absorbance was recorded using an ELISA plate reader at 490 nm. These experiment were repeated four times (n ⫽ 4). Cytotoxicity was calculated by expressing absorbance values as a percentage of control values (100% represented zero cytotoxicity). ANOVA statistical analysis was used to assess if differences exist between controls and treatment groups and the Tukey-Kramer test was used for multiple comparisons of means to determine differences between treatment groups.
Results Cellular Attachment and Growth Assays For direct visual comparisons of gingival fibroblasts grown on Geristore, Ketac-Fil, and IRM, we employed SEM analysis to view cell morphology and spreading over 72 h in culture under serum-free conditions. As a control, HGF-1 cells attached and spread well over glass coverslips in these studies (Fig. 1A). Compared to glass coverslips, HGF-1 cells grew and spread qualitatively equally well over the surface of Geristore exhibiting characteristic elongated fibroblastic morphology (Fig. 1B). However, HGF-1 cells grown on coverslips appeared to have a smoother cell membrane compared to cells on Geristore. However, human gingival fibroblasts attached poorly to Ketac-Fil (Fig. 1C) and IRM (Fig. 1D) under the same experimental concentrations. In addition, these experiments were repeated in the presence of serum (2 and 10%) to rule out any deleterious effects on HGF-1 cells because of the absence of serum. As shown in Fig. 2, HGF-1 cells could attach to Geristore in the presence of serum (Fig. 2A, B; 10 and 2% FCS, respectively) as well as in the absence of FCS (Fig. 2C). Cells grown on Geristore also had similar cellular morphology as glass coverslip controls (Fig. 2D). In parallel experiments, HGF-1 cells failed to attach to either Ketac-Fil or IRM in the presence of serum (data not shown). JOE — Volume 31, Number 3, March 2005
Basic Research—Technology
Figure 2. Scanning Electron Microscopic analysis of human gingival fibroblasts attachment and growth following 72-h incubation on Geristore under different serum conditions. HGF-1 cells grown on Geristore in media containing (A) 10%, (B) 2%, or (C) 0% fetal calf serum (FCS) in tissue culture media. Panel D indicates HGF-1 cells grown on glass coverslip controls in 10% FCS containing media. SEM views are shown at 500⫻ with reference bar length indicated.
Cytotoxicity Assay To determine if Geristore was less cytotoxic to human gingival fibroblasts, in vitro cytotoxicity (MTS) assays were performed using material extracts with HGF-1 cells. Results obtained with the MTS assay indicated that Geristore was less toxic to HGF-1 cells after 24 h incubation period than toxicity observed with Ketac Fil or IRM extracts (Fig. 3). In these studies, we used serum free media as the control since 24 and 72 h material extracts were also prepared in serum free media (ascribed 100% viability). Normalized percent viability values from multiple experimental samples (n ⫽ 4) were subjected to statistical analysis. ANOVA statistical analysis indicated that material extracts had a significant effect on cell proliferation (cell cytotoxicity) with a p ⬍ 0.0001 (F ⫽ 13.105). Posthoc pair wise comparison using Tukey-Kramer multiple comparison test indicated that significant differences exist between Geristore and both IRM and Ketac Fil. Results from Tukey-Kramer statistical analysis indicated that Geristore was significantly different than 0% FCS at 72 h (p ⬍ 0.05) but not at 24 h. However, IRM effects on cell viability was not significantly different from 0% FCS control at either 24 or 72 h time points (p ⬎ 0.05). The effects of Ketac Fil extracts on HGF-1 cell viability was not significantly different from controls at 24 h (p ⬎ 0.05) but was significantly different at 72 h (p ⬍ 0.001). Significant differences between Geristore and the other materials existed as well. See Fig. 3 to compare Geristore with Ketac Fil and IRM at 24 and 72 h time points. Significant differences between the same materials using 24 and 72 h extracts were compared using Student t test. Geristore (p ⫽ 0.04) and Ketac Fil (p ⫽ 0.0008) indicated significant differences between material extracts but not for IRM (p ⫽ 0.057).
Discussion Materials used adjacent to oral tissues should have minimal cytotoxicity towards oral cells. Geristore is currently being used to repair
JOE — Volume 31, Number 3, March 2005
subgingival defects and the interaction between Geristore in the defect with adjacent periodontal tissues must be critical in the wound healing process. Evaluation of cellular growth and attachment has been used to test cytotoxicity of dental materials (4, 6, 10, 24, 25). It has been suggested that cell growth on the surface of a material is a more sensitive indicator of cytotoxicity than surrounding cell growth (5, 26). Scanning electron microscopy has been used to evaluate adhesion of cells on materials used in a proximity to periodontal tissues as a part of evaluating the cytotoxicity of these materials (27). Geristore did not inhibit growth of gingival fibroblasts as evaluated by scanning electron microscopy. Fibroblasts in this study grew and spread well over Geristore with a morphology close to that of the controls. Interestingly, we found that fibroblasts attached well to Geristore even in the absence of serum in the tissue culture media. When evaluated by the scanning electron microscopy, cells did not grow or spread well over Ketac Fil or IRM. These cells appeared rounded and balled up when compared with cells over Geristore and the controls in the presence and absence of serum (Figs. 1 and 2). Adhesion and spreading of cells on a material surface are the initial phase for cellular function. The persistence of rounded cells with little or no spreading suggests the surface material may be toxic (28). A recent study has also evaluated gingival fibroblast and periodontal ligament fibroblast attachment to Geristore (23). These investigators determined that cellular attachment occurred significantly greater than other endodontic root-end filling materials tested with approximately 90% attachment after 72 h. These studies are consistent with our present study where we observed that gingival fibroblasts attached more readily to Geristore than even glass cover slip controls. The same investigators also determined that gingival fibroblasts attached to Geristore in an integrin-independent manner (23) indicating that integrins do not directly mediate attachment to this material.
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Figure 3. HGF cell viability assay. Geristore, IRM, and Ketac Fil material extracts were prepared following 24 (A) and 72 (B) hour incubation periods in serumfree tissue culture media. HGF-1 cells were incubated for 24 h in the presence of material extracts and MTS cell proliferation assay was used to determine cell viability in the presence of material extracts. Control cells were incubated in serum free media in the absence of any material. Statistically significant differences (ANOVA, p ⬍ 0.001) using posthoc Tukey multiple comparison are indicated between control and test materials. Refer to text for details.
Regardless of the mechanism of cellular attachment to Geristore, gingival fibroblasts appeared to have less cellular cytotoxicity in the presence of extracts prepared from Geristore. We observed that gingival fibroblasts proliferated significantly better (p ⬍ 0.05) in the presence of Geristore extracts compared to controls after 72 h in culture. Gersitore extracts were significantly less toxic to gingival fibroblasts than IRM (p ⬍ 0.01) or Ketac-Fil (p ⬍ 0.001) with 72-h extracts over the same period of time. It is unclear why Geristore has more favorable cellular response. It might be a result of certain surface characteristics that Geristore, especially because cellular attachment is not dependent upon integrins (23). Another reason could be that Geristore elutes less toxic materials into the medium. Our current results support this conclusion, however, the exact component differences are not known at the present time. Several components of dental resin composite monomers or additives are cytotoxic to fibroblasts, including gingival fibroblasts (10). Therefore, future studies will be conducted to evaluate the organic constituents present in Gersitore and compare it to other resin or perforation repair materials and test their effects on different oral cells.
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1. Osorio RM, Hefti A, Vertucci FJ, Shawley AL. Cytotoxicity of Endodonontic materials. J Endod 1998;24:91– 6. 2. Torabinejad M, Hong CU, McDonald F, Pitt Ford TR. Physical and chemical properties of a new root-end filling material. J Endod 1995;21:349 –53. 3. Thomson TS, Berry JE, Somerman MJ, Kirkwood KL. Cementoblasts maintain expression of osteocalcin in the presence of mineral trioxide aggregate. J Endod 2003;29: 407–12. 4. Willershausen B, Schafer D, Pistorius A, Schulze R, Mann W. Influence of resin-based restoration materials on cytotoxicity in gingival fibroblasts. Eur J Med Res 1999;4: 149 –55. 5. Hensten-Pettersen A, Helgeland K. Evaluation of biologic effects of dental materials using four different cell culture techniques. Scand J Dent Res 1977;85:291– 6. 6. Caughman WF, Caughman GB, Dominy WT, Schuster GS. Glass ionomer and composite resin cements: effects on oral cells. J Prosthet Dent 1990;63:513–21. 7. Caughman WF, Caughman GB, Shiflett RA, Rueggeberg F, Schuster GS. Correlation of cytotoxicity, filler loading and curing time of dental composites. Biomaterials 1991; 12:737– 40. 8. Makkawy HA, Koka S, Lavin MT, Ewoldsen NO. Cytotoxicity of root perforation repair materials. J Endod 1998;24:477–9. 9. Franz A, Konig F, Anglmayer M, et al. Cytotoxic effects of packable and nonpackable dental composites. Dent Mater 2003;19:382–92. 10. Geurtsen W, Lehmann F, Spahl W, Leyhausen G. Cytotoxicity of 35 dental resin composite monomers/additives in permanent 3T3 and three human primary fibroblast cultures. J Biomed Mater Res 1998;41:474 – 80. 11. Schwarze T, Fiedler I, Leyhausen G, Geurtsen W. The cellular compatibility of five Endodonontic sealers during the setting period. J Endod 2002;28:784 – 6. 12. Kan KC, Messer LB, Messer HH. Variability in cytotoxicity and fluoride release of resin-modified glass-ionomer cements. J Dent Res 1997;76:1502–7. 13. Theilig C, Tegtmeier Y, Leyhausen G, Geurtsen W. Effects of BisGMA and TEGDMA on proliferation, migration, and tenascin expression of human fibroblasts and keratinocytes. J Biomed Mater Res 2000;53:632–9. 14. Hanks CT, Wataha JC, Sun Z. In vitro models of biocompatibility: a review. Dent Mater 1996;12:186 –93. 15. Dragoo MR. Resin-ionomer and hybrid-ionomer cements: part I. Comparison of three materials for the treatment of subgingival root lesions. Int J Periodontics Restorative Dent 1996;16:594 – 601. 16. Dragoo MR. Resin-ionomer and hybrid-ionomer cements: part II. Human clinical and histologic wound healing responses in specific periodontal lesions. Int J Periodontics Restorative Dent 1997;17:75– 87. 17. Resillez-Urioste F, Sanandajt K, Davidson RM. Use of a resin-ionomer in the treatment of mechanical root perforation: report of a case. Quintessence Int 1998;29:115– 8. 18. Shuman IE. Repair of a root perforation with a resin-ionomer using an intentional replantation technique. Gen Dent 1999;47:392–5. 19. Scherer W, Dragoo MR. New subgingival restorative procedures with Geristore resin ionomer. Pract Periodontics Aesthet Dent 1995;7(Suppl):1– 4. 20. Abitbol T, Santi E, Scherer W. Use of a resin-ionomer in guided tissue regeneration: case reports. Am J Dent 1995;8:267–9. 21. Abitbol T, Santi E, Scherer W, Palat M. Using a resin-ionomer in guided tissue regenerative procedures: technique and application— case reports. Periodontal Clin Investig 1996;18:17–21. 22. Behnia A, Strassler HE, Campbell R. Repairing iatrogenic root perforations. J Am Dent Assoc 2000;131:196 –201. 23. Camp MA, Jeansonne BG, Lallier T. Adhesion of human fibroblasts to root-end-filling materials. J Endod 2003;29:602–7. 24. Willershausen B, Marroquin BB, Schafer D, Schulze R. Cytotoxicity of root canal filling materials to three different human cell lines. J Endod 2000;26:703–7. 25. Geurtsen W, Leinenbach F, Krage T, Leyhausen G. Cytotoxicity of four root canal sealers in permanent 3T3 cells and primary human periodontal ligament fibroblast cultures. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:592–7. 26. Hensten-Pettersen A. Comparison of the methods available for assessing cytotoxicity. Int Endod J 1988;21:89 –99. 27. Zhu Q, Haglund R, Safavi KE, Spangberg LS. Adhesion of human osteoblasts on root-end filling materials. J Endod 2000;26:404 – 6. 28. Zmener O, Cabrini RL. Adhesion of human blood monocytes and lymphocytes to different Endodonontic cements. A methodological in vitro study. J Endod 1986;12: 150 –5.
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Preferential Attachment of Human Gingival Fibroblasts to the Resin Ionomer Geristore Fuwad Al-Sabek, DDS, MS, Sandra Shostad, DDS, MS, and Keith L. Kirkwood, DDS, PhD Abstract The resin ionomer Geristore has been used extensively for root perforation repairs. The purpose of this study was to evaluate oral in vitro biocompatibility of the resin ionomer Geristore compared to two other dental perforation repair materials, Ketac-Fil and Immediate Restorative Material (IRM). Growth and morphology of human gingival fibroblasts (HGFs) was determined using scanning electron microscopy (SEM) of HGFs cells grown on test materials as well as cytotoxicity assays using eluates from test materials. SEM analysis showed that HGFs attached and spread well over Geristore with relatively normal morphology. SEM showed that fibroblasts did not attach and spread well over Ketac-Fil or IRM as cells appeared much fewer with rounded and different morphology than fibroblasts grown on Geristore. Cytotoxicity assays indicated that HGFs proliferated in the presence of Geristore eluates and not in the presence of Ketac-Fil or IRM eluates. In vitro interpretation indicates that Geristore is less cytotoxic to gingival fibroblasts.
From the Departments of Endodontics and Periodontics, Oral Biology, and Pharmacology & Toxicology, State University of New York at Buffalo, Buffalo, NY. Address request for reprints to Keith L. Kirkwood, DDS, PhD, Assistant Professor, Department of Periodontics/Prevention/Geriatrics, University of Michigan 1011 N. University Ave., Ann Arbor, MI 48109-1078; E-mail address: [email protected]. Copyright © 2005 by the American Association of Endodontists
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adicular perforations can occur during Endodonontic treatment, postspace preparation, postremoval, and operative procedures. Perforations that occur below the level of the epithelial attachment can cause infection, which may result in attachment loss. These defects have been treated in the past with a variety of different materials including gutta-percha, calcium hydroxide products, amalgam, Immediate Restorative Material (IRM), cavit, and super Epoxy Benzoic Acid (EBA). Major problems relative to these materials include biocompatibility and inadequate sealing. The use of composite resins, glass ionomer cement and Mineral Trioxide Aggregate (MTA) has been suggested to circumvent these problems. Studies have shown that MTA is the most suitable material for repairing sub-osseous perforations especially furcation perforations, because of its superior sealing and biocompatibility characteristics (1, 2), as well as permitting mineralized matrix formation (3). Subgingival root perforations that are supraosseous have been repaired using glass ionomers and composite resins. However, all studies have demonstrated some degree of cytotoxicity through in vitro and in vivo studies (4 –14). Recently, resin ionomers such as Geristore, originally designed for restorative procedures, have been used in treating subgingival defects such as root resorption and perforation. Geristore is a dual cure (both self and light-curing), hydrophilic, nonaqueous polyacid modified composite resin composed of fluoride releasing glass, mainly barium fluorosilicate, and a polymerizable organic matrix (Modified Bis-GMA, including 2-HEMA) combined with a photoinitiator. Advantages of these materials include insolubility in oral fluids, increased adhesion to tooth structure, dual cure capabilities, low cure shrinkage, low coefficient of thermal expansion, radiopacity, fluoride release, and biocompatibility (15). Relatively few clinical studies have addressed biocompatibility (15), although several clinical studies have demonstrated Geristore could repair subgingival and subosseous defects and could be used as a barrier for guided tissue regeneration (16 –22). Cytotoxicity of resin ionomers (Geristore) towards oral tissues has only started to be investigated (23). The aim of the present study is to evaluate in vitro biocompatibility of human gingival fibroblasts with Geristore using scanning electron microscopy and nucleic acid fluorescent staining to measure cell attachment and morphology as well as cell cytotoxicity assays to eluates from root perforation repair materials namely Geristore, Ketac-Fil, and IRM.
Materials and Methods Cell Culture Conditions Human gingival fibroblasts (HGF-1; American Type Culture Collection, Manassas, VA; #ATCC CRL-2014) were obtained through commercial sources for these studies. Cells utilized for these studies were between passages 14 and 21. Cells were cultured in DMEM (Invitrogen Life Technologies, Grand Island, NY), supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO), penicillin (100 U/ml; Sigma), and streptomycin (100 g/ml; Sigma) at 37°C in a humidified atmosphere of 5% CO2 in air. The culture medium was changed every 3 to 4 days. Sample Preparation and Sterilization Geristore (DEN-MAT Corporation, Santa Maria, CA.), Ketac Fil (ESPE, Seefeld/ Oberbay, Germany), and IRM (Caulk/Dentsply, Milford, DE) were obtained from commercial sources. Discs (6 ⫻ 2 mm) from the three materials were fabricated under aseptic conditions by packing the material after mixing in a Teflon washer (internal diameter of 6 ⫻ 2 mm), and compressed between two glass slides to generate even
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Figure 1. Scanning Electron Microscopic analysis of human gingival fibroblasts attachment and growth following 72-h incubation on (A) glass coverslip controls, (B) Geristore, (C) Ketac Fil, or (D) IRM. SEM views are shown at 100⫻ with reference bar length indicated.
thickness of material. Glass cover slips were used as positive controls in all experimental conditions throughout this study. Extracts from all three materials were prepared by preincubating sterilized discs in 50 cm3 conical tubes with 5 ml of DMEM (Life Technologies) supplemented with 100 U/ml penicillin and 100 g/ml streptomycin at 37°C in a shaker (Labline, Barnstead Int., Dubuque, IA) at 150 rpm.
Scanning Electron Microscopy Growth Assay Attachment, growth and morphology of human gingival fibroblasts over Geristore were evaluated by scanning electron microscopy. Geristore, Ketac Fil and IRM discs and control glass cover slips were sterilized as described above and placed in the bottom of a 12-well culture plate. HGF-1 cells were seeded into the wells at density (3 ⫻ 104 cells per well) in DMEM medium containing 10% fetal bovine serum. After incubation, cells were washed three times with phosphate buffered saline and fixed in 2.5% glutaraldehyde in 1 mmol cacodylate buffer (pH 7.4). Samples were then observed using a JEOL JSM 6400 scanning electron microscope (Jeol USA, Peabody, MA). Cell attachment and viability in each experimental condition were assessed by qualitatively comparing morphology of cells over tested materials with that of cells over glass coverslips, which were considered cells with normal morphology. Cytotoxicity Assays Human gingival fibroblasts cytotoxicity towards Geristore, Ketacfil, and IRM material extracts was determined by means of using the CellTiter Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). HGF-1 cells were seeded into 96-well culture plates at 1 ⫻ 104 cells per well in DMEM supplemented with 10% fetal bovine serum. After 24h cells were rinsed three times with PBS and treated with 100 l of the following material eluates in serum-free media: Geristore (24 and 72 hr extracts), IRM (24 and 72 hr extracts), and Ketac-fil (24 and 72 hr extracts). Controls included media only (no material ex206
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tracts) and media without cells present. Soluble formazan absorbance was recorded using an ELISA plate reader at 490 nm. These experiment were repeated four times (n ⫽ 4). Cytotoxicity was calculated by expressing absorbance values as a percentage of control values (100% represented zero cytotoxicity). ANOVA statistical analysis was used to assess if differences exist between controls and treatment groups and the Tukey-Kramer test was used for multiple comparisons of means to determine differences between treatment groups.
Results Cellular Attachment and Growth Assays For direct visual comparisons of gingival fibroblasts grown on Geristore, Ketac-Fil, and IRM, we employed SEM analysis to view cell morphology and spreading over 72 h in culture under serum-free conditions. As a control, HGF-1 cells attached and spread well over glass coverslips in these studies (Fig. 1A). Compared to glass coverslips, HGF-1 cells grew and spread qualitatively equally well over the surface of Geristore exhibiting characteristic elongated fibroblastic morphology (Fig. 1B). However, HGF-1 cells grown on coverslips appeared to have a smoother cell membrane compared to cells on Geristore. However, human gingival fibroblasts attached poorly to Ketac-Fil (Fig. 1C) and IRM (Fig. 1D) under the same experimental concentrations. In addition, these experiments were repeated in the presence of serum (2 and 10%) to rule out any deleterious effects on HGF-1 cells because of the absence of serum. As shown in Fig. 2, HGF-1 cells could attach to Geristore in the presence of serum (Fig. 2A, B; 10 and 2% FCS, respectively) as well as in the absence of FCS (Fig. 2C). Cells grown on Geristore also had similar cellular morphology as glass coverslip controls (Fig. 2D). In parallel experiments, HGF-1 cells failed to attach to either Ketac-Fil or IRM in the presence of serum (data not shown). JOE — Volume 31, Number 3, March 2005
Basic Research—Technology
Figure 2. Scanning Electron Microscopic analysis of human gingival fibroblasts attachment and growth following 72-h incubation on Geristore under different serum conditions. HGF-1 cells grown on Geristore in media containing (A) 10%, (B) 2%, or (C) 0% fetal calf serum (FCS) in tissue culture media. Panel D indicates HGF-1 cells grown on glass coverslip controls in 10% FCS containing media. SEM views are shown at 500⫻ with reference bar length indicated.
Cytotoxicity Assay To determine if Geristore was less cytotoxic to human gingival fibroblasts, in vitro cytotoxicity (MTS) assays were performed using material extracts with HGF-1 cells. Results obtained with the MTS assay indicated that Geristore was less toxic to HGF-1 cells after 24 h incubation period than toxicity observed with Ketac Fil or IRM extracts (Fig. 3). In these studies, we used serum free media as the control since 24 and 72 h material extracts were also prepared in serum free media (ascribed 100% viability). Normalized percent viability values from multiple experimental samples (n ⫽ 4) were subjected to statistical analysis. ANOVA statistical analysis indicated that material extracts had a significant effect on cell proliferation (cell cytotoxicity) with a p ⬍ 0.0001 (F ⫽ 13.105). Posthoc pair wise comparison using Tukey-Kramer multiple comparison test indicated that significant differences exist between Geristore and both IRM and Ketac Fil. Results from Tukey-Kramer statistical analysis indicated that Geristore was significantly different than 0% FCS at 72 h (p ⬍ 0.05) but not at 24 h. However, IRM effects on cell viability was not significantly different from 0% FCS control at either 24 or 72 h time points (p ⬎ 0.05). The effects of Ketac Fil extracts on HGF-1 cell viability was not significantly different from controls at 24 h (p ⬎ 0.05) but was significantly different at 72 h (p ⬍ 0.001). Significant differences between Geristore and the other materials existed as well. See Fig. 3 to compare Geristore with Ketac Fil and IRM at 24 and 72 h time points. Significant differences between the same materials using 24 and 72 h extracts were compared using Student t test. Geristore (p ⫽ 0.04) and Ketac Fil (p ⫽ 0.0008) indicated significant differences between material extracts but not for IRM (p ⫽ 0.057).
Discussion Materials used adjacent to oral tissues should have minimal cytotoxicity towards oral cells. Geristore is currently being used to repair
JOE — Volume 31, Number 3, March 2005
subgingival defects and the interaction between Geristore in the defect with adjacent periodontal tissues must be critical in the wound healing process. Evaluation of cellular growth and attachment has been used to test cytotoxicity of dental materials (4, 6, 10, 24, 25). It has been suggested that cell growth on the surface of a material is a more sensitive indicator of cytotoxicity than surrounding cell growth (5, 26). Scanning electron microscopy has been used to evaluate adhesion of cells on materials used in a proximity to periodontal tissues as a part of evaluating the cytotoxicity of these materials (27). Geristore did not inhibit growth of gingival fibroblasts as evaluated by scanning electron microscopy. Fibroblasts in this study grew and spread well over Geristore with a morphology close to that of the controls. Interestingly, we found that fibroblasts attached well to Geristore even in the absence of serum in the tissue culture media. When evaluated by the scanning electron microscopy, cells did not grow or spread well over Ketac Fil or IRM. These cells appeared rounded and balled up when compared with cells over Geristore and the controls in the presence and absence of serum (Figs. 1 and 2). Adhesion and spreading of cells on a material surface are the initial phase for cellular function. The persistence of rounded cells with little or no spreading suggests the surface material may be toxic (28). A recent study has also evaluated gingival fibroblast and periodontal ligament fibroblast attachment to Geristore (23). These investigators determined that cellular attachment occurred significantly greater than other endodontic root-end filling materials tested with approximately 90% attachment after 72 h. These studies are consistent with our present study where we observed that gingival fibroblasts attached more readily to Geristore than even glass cover slip controls. The same investigators also determined that gingival fibroblasts attached to Geristore in an integrin-independent manner (23) indicating that integrins do not directly mediate attachment to this material.
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Figure 3. HGF cell viability assay. Geristore, IRM, and Ketac Fil material extracts were prepared following 24 (A) and 72 (B) hour incubation periods in serumfree tissue culture media. HGF-1 cells were incubated for 24 h in the presence of material extracts and MTS cell proliferation assay was used to determine cell viability in the presence of material extracts. Control cells were incubated in serum free media in the absence of any material. Statistically significant differences (ANOVA, p ⬍ 0.001) using posthoc Tukey multiple comparison are indicated between control and test materials. Refer to text for details.
Regardless of the mechanism of cellular attachment to Geristore, gingival fibroblasts appeared to have less cellular cytotoxicity in the presence of extracts prepared from Geristore. We observed that gingival fibroblasts proliferated significantly better (p ⬍ 0.05) in the presence of Geristore extracts compared to controls after 72 h in culture. Gersitore extracts were significantly less toxic to gingival fibroblasts than IRM (p ⬍ 0.01) or Ketac-Fil (p ⬍ 0.001) with 72-h extracts over the same period of time. It is unclear why Geristore has more favorable cellular response. It might be a result of certain surface characteristics that Geristore, especially because cellular attachment is not dependent upon integrins (23). Another reason could be that Geristore elutes less toxic materials into the medium. Our current results support this conclusion, however, the exact component differences are not known at the present time. Several components of dental resin composite monomers or additives are cytotoxic to fibroblasts, including gingival fibroblasts (10). Therefore, future studies will be conducted to evaluate the organic constituents present in Gersitore and compare it to other resin or perforation repair materials and test their effects on different oral cells.
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JOE — Volume 31, Number 3, March 2005