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Attachment of Human Gingival Fibroblasts to Periodontally Involved Root Surface Following Scaling and/or Etching Procedures: A Scanning Electron

Microscopy Study
Nadir BABAY Division of Periodontics, Department of Preventive Dental Sciences, King Saud University College of Dentistry, Riyadh, Kingdom of Saudi Arabia

Correspondence: Dr. N Babay, Associate Professor, Head Division of Periodontics, Department of Preventive Dental Sciences, King Saud University College of Dentistry, P.O. Box 60169, Riyadh 11545, Kingdom of Saudi Arabia. e-mail: nbabay@ksu.edu.sa

Braz Dent J (2001) 12(1): 17-21 ISSN 0103-6440
INTRODUCTION | MATERIAL AND METHODS | RESULTS | DISCUSSION | ACKNOWLEDGEMENTS | RESUMO | REFERENCES

This study evaluated, in vitro, fibroblast attachment to periodontally involved root surfaces which were either root planed or acid/chelated by different agents. Specimens were divided into 3 groups of 12 specimens each. The root surfaces were root planed with a Gracey 7/8 curette, an EMS or an Amdent piezo-electric scaler and treated with saline, citric acid, tetracycline hydrochloride or EDTA to produce different surface textures. They were then cultured with fibroblasts for 72 h and examined by scanning electron microscopy. There was a significantly greater number of fibroblasts attached to specimens treated with citric acid, tetracycline and EDTA than to those root planed only. Furthermore, fibroblasts were more likely to attach to rough-surfaced than to smooth-surfaced specimens. Key Words: fibroblasts, scaling, etching, SEM.

INTRODUCTION The ultimate goal of periodontal therapy is the regeneration of periodontal support lost due to periodontitis. On the basis of the work by Urist (1) who described the potential of intramuscular demineralized dentin implants to induce new bone formation, Register (2) and Register and Burdick (3) reported enhancing the new attachment by acid. One of the most widely used

(10) observed that fibroblasts attached normally to instrumented periodontally diseased root surfaces. MATERIAL AND METHODS Eighteen periodontally-diseased teeth. Adelson et al. (Labsystem. each measuring 3 x 4 mm. The other half was not instrumented and served as control. The purpose of this investigation was to investigate the correlation between the roughness of periodontally involved root surfaces and the ability of cultured fibroblasts to attach to these root surfaces treated with different scaling and etching procedures. Each root surface was divided into two equal parts. However. were selected for use in this study: 1) no history of scaling or root planing in the previous 6 months. saturated citric acid (pH 1. whereas little or no attachment was seen on uninstrumented root surfaces in vitro. Switzerland) using back-and-forth strokes until a smooth root surface was obtained. which primarily removes the smear layer (4). saliva and irrigating solutions with a soft bristle toothbrush and deionized water.0). Only the diseased part of the root surface was used in this study. Nyman et al. In contrast. II and III) were treated with saline. Finland). Helsinki. This inconsistency may be due to the inadequate removal of endotoxins (9). The teeth were individually placed in sterile. The teeth were cleaned of blood. were obtained from each tooth. According to Lowenberg et al. Sweden) was used. IL. Following scaling. Group 2: the root surfaces were treated with an EMS piezo-electric scaler (Nyon. 4) absence of caries. except an Amdent piezo-electric scaler (Nynäshamn. Each specimen was placed in a 96-well microliter Honeycombplate. USA) until a smooth root surface was obtained. Other agents such as tetracycline (5) and EDTA (6) have been reported to favor new periodontal attachment. two blocks. (12). Each group had a total of 12 specimens. 2) proximal attachment loss of 5 mm or more. This process renders the root biologically compatible to the adjacent tissues. Prior to any periodontal therapy. Test and control areas were marked with a notch to ascertain the coronal direction. Cogen et al. A total of 36 specimens representing 36 test/control surfaces were registered according to the root treatment performed. 3) no history of acute pain or swelling. saturated tetracycline . demineralization of the root surface may provide a more favorable substrate for enhanced and stronger cell attachment. (11) could not confirm that hypothesis. capped tubes containing saline and were processed immediately after extraction. one half of the specimen (experimental) was scaled with either hand instruments or one of the piezo-electric scalers. The anatomical crown was removed with a water cooled high speed bur. The teeth were randomly divided into three groups: Group 1: the experimental root surfaces were treated with a Gracey 7/8 curette (Hu-Friedy. Three specimens from each group (I. The power setting was medium. with the following criteria. (8) reported that cementum removal is not a pre-requisite for new attachment. Chicago.adjuncts is citric acid. cementum should be removed from the root surface (7). Group 3: the experimental surfaces were treated as in group 2.

The medium used for propagating and culturing the cells with the root sections was Dulbecco's Modified Eagle medium supplemented with 10% fetal calf serum. Primary cell cultures used for the test were from the third to the eighth passage. sterile 3. After incubation. When the roots were etched or chelated there was a significant increase in the number of attached fibroblasts. the specimens were washed in Hank's balanced salt solution. rinsing in water and staining in 2% aqueous uranyl acetate for 30 min.8) or 8% EDTA dissolved in PBS solution (pH 7. This was followed by postfixation in 1% OSO 4 in 0. The root surfaces were not affected by saline. 0. Japan). RESULTS Controls (non-scaled specimens) The untreated root surfaces were relatively smooth and free of collagen fibers. Specimens for SEM were rinsed two times in PBS at 37oC and fixed in 0. Each specimen was coated with gold palladium and examined by scanning electron microscopy (JEOL. The root surface was not visible between the multiple layers of fibroblasts (Figure 6).5% glutaraldehyde at room temperature for 45 min. Tokyo. Gracey 7/8 scaled surfaces The smooth planed surfaces showed fibroblast clusters mainly in the notched areas (Figure 2). 100 IU penicillin/ml. After 3 min.3). with spots of attached calculus. the solutions were removed by suction and the specimens rinsed with distilled water.1 M cacodylate buffer for 15 min.5 x 8 mm Petri dish with the external surface of the root facing upward.hydrochloride (pH 1.1 mg streptomycin/ml. Most of the adherent cells were flat with prominent microvilli and filopodia forming a multilayer on the specimen surface (Figure 5). The root sections were incubated with cells for 72 h at 37oC. The etched or chelated specimens had a rougher surface with scattered fibroblasts. All specimens were placed at the bottom of a single. and 2 mM glutamine. One specimen of the same group displayed a fibrous network resembling collagen fibers (Figure 4). Amdent treated root surfaces The ultrasonic treated root surfaces were smooth. The surface was covered with a material suggestive of organic debris with occasional exposure of dentinal tubules (Figure 3). . citric acid or EDTA (Figure 1). well planed but with no migration of fibroblasts. A suspension of human gingival fibroblasts containing 1 x 105 cells/ml propagated prior to use according to the method of Aleo (9) was added to each Petri dish so that the fluid level of the suspension totally immersed the root sections. The specimens were dehydrated through a graded series of ethanol (35 to 100%) and then critical point dried.1 M cacodylate buffer containing 2. at 15 Kv with a tilt angle of 0 to 40o.

500X). (Original magnification. (Original magnification. Figure 1. Spots of attached calculus are visible (arrow). SEM photomicrograph of root surface scaled with a Gracey 7/8 curette. Presence of cell inhabiting crack areas (arrow). .Piezon treated root surfaces The appearance of the root surfaces were similar to the Amdent treated and etched or chelated root surfaces. SEM photomicrograph of untreated root surface. 350X). Figure 2.

Note the presence of a fibrous network (arrow) and the absence of cells. Note the spreading of cells (arrow) completely masking the root surfaces. SEM photomicrograph ultrasonically scaled with Amdent and etched with citric acid for 3 minutes. Figure 6. Figure 4. 1500X). SEM photomicrograph of root surface ultrasonically treated with Amdent. (Original magnification. Fibroblast attached to the etched surface (arrow). (Original magnification. 500X).Figure 3. Figure 5. SEM photomicrograph of root surface scaled with a Gracey 7/8 curette and etched with EDTA. . 200X). Note the well-spread presence of cells (arrow). (Original magnification. SEM photomicrograph ultrasonically scaled with Amdent and etched with tetracycline hydrochloride for 3 min.

It appears that fibroblast attachment to periodontally diseased root surfaces is not uncommon. In this study. DISCUSSION Several in vitro studies have suggested that root surface modifications by tetracycline (13) or citric acid (14) could improve the attachment and spreading of periodontal fibroblasts. Lucas et al. Cells binding to both dentinal and cementum surfaces and root planning alone permit diseased roots to interact with fibroblasts as well as normal roots. Fardal et al. this study did not confirm this nor did the study by Gamel et al. They also observed attachment of fibroblasts to calculus. nor did they attach to ultrasonically treated root surfaces. (16) suggested that endotoxins from periodontal pathogens penetrate the root surface and inhibit cellular attachment. However. This suggests that cell attachment to the demineralized root can be enhanced by the detoxification of the root and also indicates that root planing removed substances that inhibited or prevented fibroblasts. Fardal et al. These modifications could be either a direct consequence of root conditioning by the exposure of some of the extracellular matrix constituents acting on the attachment mechanism of fibroblasts or an indirect effect by the increased fixation on the demineralized root surface of biochemical factors.a finding that confirms that cultured human PDL fibroblasts prefer not to adhere to periodontally . therefore influencing the behavior of periodontal fibroblasts. thus favoring faster periodontal healing. tetracycline hydrochloride or EDTA are responsible for an increase in fibroblast attachment. Fibroblast attachment is a common method used to evaluate the biocompatibility of root surfaces (15). (17) observed that periodontally diseased teeth incubated with human gingival fibroblasts showed equal attachment regardless of whether they were instrumented. (17) reported that periodontally diseased roots did not inhibit the initial attachment in vitro. Acid treatment of periodontally diseased human teeth has given both successful and unsuccessful results. The observations in this study confirm and extend previous work. A recent study by Vanheusden et al. improving their attachment and spreading. (18) also confirmed that dentin conditioning by citric acid or by mynocycline enhanced the reattachment of human gingival epithelial cells to the root. This study was designed to investigate the correlation between the roughness of periodontally involved root surfaces and the ability of cultured fibroblasts to attach to them.(Original magnification. 350X). This study suggests that the biochemical modifications of the dentinal tubules induced by conditioning with citric acid. In contrast. (19) which demonstrated that cell adherence was significantly higher in the healthy control group when compared to periodontally diseased treated control. fibroblasts did not attach to periodontally involved roots. More cells were attached to demineralized surfaces with no difference between the various demineralizing agents in enhancing cell attachment. uninstrumented or non-diseased.

J Periodontol 1984. Christersson LA. Blomlöf J. were low in number in root planed root surfaces and more abundant when the root was etched or chelated. potentially renders it less likely to achieve tissue attachment. periodontal disease alters the biocompatibility of the root surface and thus. Hic S. Register AA. J Periodontol 1975. I. DiFlorio RM. Genco LA. . 2. Frederick GT. 6. Based on the results of this study. Burdick FA. J Periodontol 1973. J Periodont Res 1986. Oral Surg 1971. Bone histogenesis and morphogenesis in implants of demineralized enamel and dentin.103:17-24.diseased treated root surfaces. Bone and cementum induction by dentin demineralized in situ. Register AA.29:88-94. Genco RJ. Urist MR. Baker PJ. (20) found that the application of tetracycline hydrochloride on the tooth surface had little effect on cell adherence except to change their morphology. Hanes P. This study would suggest that the direct cytotoxic nature on nonmicrobial root surface irritants may play an important role in the overall nature of root surface toxicity. Eur J Oral Sc 1995. Polson AM. fibroblasts did not adhere to periodontally involved teeth.44:49-59. 5. Lyall RM. The production of a root surface smear layer by instrumentation and its removal by citric acid. Lindskog S. Accelerated reattachment with cementogenesis to dentin demineralized in situ.55:443-446. Gamal et al. Terranova VP. A biochemical approach to periodontal regeneration: Tetracycline treatment conditions dentin surfaces. to Arturo Palustre for his help with the scanning electron microscope and to Pressy dR Oba for typing the manuscript. Axel Bergenholtz for his help in the fibroblast culture technique. ACKNOWLEDGEMENTS The author wishes to express his appreciation to Prof. In this study.46:646-655. Root surface texture and early cells and tissue colonization after different etching modalities.21:322-329. Wikesjö UME. Ladenheim S. However. REFERENCES 1. It has not been determined if the conditioned root can be rendered as biocompatible as a healthy root surface. Optimum range. 4. 3.

Attachment. Adelson LJ. Freeman E. Fibroblasts exposed to endotoxin. J Periodontol 1999. J Dent Res 1985. In vitro attachment of human gingival fibroblasts to root surfaces. Nusgeus B. J Periodont Res 1984. Aleo J. Aleo JJ. A biochemical approach to periodontal regeneration: Tetracycline treatment of dentin promotes fibroblast adhesion and growth. The effect of local delivery of PDGF-BB on attachment of human periodontal ligament fibroblasts to periodontitis affected root surfaces _ in vitro. and untreated root slices. Gamal AY.54:700-704.45:672-677. Cohen S. Lowenberg BF. Terranova VP. Rompen EH. Farber PA. Lapiere CM. 11. Mailhot JM. A new chromium assay for accurate measurement of cell attachment to demineralized and nondemineralized dentin in vitro. Di Florio RM. J Periodont Res 1986. Aleo JJ. Egelberg J.27:347-353. Lucas R. Garrison DC. Cogen RB. Fardal O. Ramfjord SJ. surface demineralized.70:594-603. J Periodontol 1975. Aubin JE. 18. J Periodontol 1980.7.19:1106-1110. Deporter DA. Weatherford TW.8:249-258. J Periodontol Res 1978. Melcher AH. Franzetti LC. Effects of citric acid on diseased root surfaces. Wikesjö UM. Lowenberg BF. Zahedi S. Garett JS. Healing following surgical treatment and root demineralization in monkeys with periodontal disease. Histochemical study of strain I. Aubin JE. J Periodontol 1983. 8. In vitro stimulation of human gingival epithelial cell attachment to dentin by surface conditioning. 16.13:735-739. 19. Genco RJ.64:1106-1110. Hanks CT. DeRenzis FA. . Effect of various root surface treatments on the viability and attachment of human gingival fibroblasts. Pitaru S. migration and orientation of human gingival fibroblasts to collagen-coated.21:330-337. Vanheusden AJ. Caffesse RG.54:700-704. Cytotoxicity of periodontally diseased root surfaces. Baker PJ. J Clin Periodontol 1986. 10. Farber PA. 13. J Clin Periodontol 1981. Hic S. Varboncoeur AP. Aubin JE. Karring T. Nyman S. J Clin Periodontol 2000. 17. Sodek S. J Periodontol 1979. Lindhe J. J Periodontol 1974. 9. Initial attachment of fibroblast like cells to periodontally diseased root surfaces in vitro.46:639-645. 12. The effect on cellular organelles. Melcher AH. Lyall RM.13:155-163. Christersson LA. 15. Lowenberg BF. Goffinet G. 14. Crigger M. DeRenzis FA. The presence of biologic activity of cementum-bound endotoxin.50:20-25.

J Clin Periodontol 1998. Mailhot JM.   .20. Gamal AY. Garnick JJ. Sharawy MM.25:404-412. Newhouse R. Human periodontal ligament fibroblast response to PDGF-BB and IGF-1 application on tetracycline HCl conditioned root surfaces.