Attachment of Human Gingival Fibroblasts to Periodontally Involved Root Surface Following Scaling...

7/30/2019 Attachment of Human Gingival Fibroblasts to Periodontally Involved Root Surface Following Scaling and - Or Etchin

1/9

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, Departmentof Preventive Dental Sciences, King Saud University College of Dentistry, P.O. Box 60169,

Riyadh 11545, Kingdom of Saudi Arabia. e-mail: [email protected]

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 into3 groups of 12 specimens each. The root surfaces were root planed with a Gracey 7/8 curette, anEMS or an Amdent piezo-electric scaler and treatedwith 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 significantlygreater 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 andBurdick (3) reported enhancing the new attachment by acid. One of the most widely used


2/9

adjuncts is citric acid, which primarily removes the smear layer (4). Other agents such as

tetracycline (5) and EDTA (6) have been reported to favor new periodontal attachment. This

process renders the root biologically compatible to the adjacent tissues.

Prior to any periodontal therapy, cementum should be removed from the root surface (7).

However, Nyman et al. (8) reported that cementum removal is not a pre-requisite for newattachment. This inconsistency may be due to the inadequate removal of endotoxins (9).

Cogen et al. (10) observed that fibroblasts attached normally to instrumented periodontallydiseased root surfaces, whereas little or no attachment was seen on uninstrumented root surfaces

in vitro. In contrast, Adelson et al. (11) could not confirm that hypothesis. According toLowenberg et al. (12), demineralization of the root surface may provide a more favorable

substrate for enhanced and stronger cell attachment.

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.

MATERIAL AND METHODS

Eighteen periodontally-diseased teeth, with the following criteria, were selected for use in this

study: 1) no history of scaling or root planing in the previous 6 months, 2) proximal attachment

loss of 5 mm or more, 3) no history of acute pain or swelling, 4) absence of caries. The teethwere individually placed in sterile, capped tubes containing saline and were processed

immediately after extraction.

The teeth were cleaned of blood, saliva and irrigating solutions with a soft bristle toothbrush anddeionized water. Only the diseased part of the root surface was used in this study. The

anatomical crown was removed with a water cooled high speed bur. Each root surface was

divided into two equal parts, one half of the specimen (experimental) was scaled with either hand

instruments or one of the piezo-electric scalers. The other half was not instrumented and servedas control. The teeth were randomly divided into three groups: Group 1: the experimental root

surfaces were treated with a Gracey 7/8 curette (Hu-Friedy, Chicago, IL, USA) until a smooth

root surface was obtained; Group 2: the root surfaces were treated with an EMS piezo-electricscaler (Nyon, Switzerland) using back-and-forth strokes until a smooth root surface was

obtained. The power setting was medium; Group 3: the experimentalsurfaces were treated as in

group 2, except an Amdent piezo-electric scaler (Nynshamn, Sweden) was used. Test andcontrol areas were marked with a notch to ascertain the coronal direction.

Following scaling, two blocks, each measuring 3 x 4 mm, were obtained from each tooth. Each

specimen was placed in a 96-well microliter Honeycombplate, (Labsystem, Helsinki, Finland). A

total of 36 specimens representing 36 test/control surfaces were registered according to the roottreatment performed. Each group had a total of 12 specimens. Three specimens from each group

(I, II and III) were treated withsaline, saturated citric acid (pH 1.0), saturated tetracycline


3/9

hydrochloride (pH 1.8) or 8% EDTA dissolved in PBS solution (pH 7.3). After 3 min, the

solutions were removed by suction and the specimens rinsed with distilled water.

All specimens were placed at the bottom of a single, sterile 3.5 x 8 mm Petri dish with the

external surface of the root facing upward. A suspension of human gingival fibroblasts

containing 1 x 10

5

cells/ml propagated prior to use according to the method of Aleo

(9) wasadded to each Petri dish so that the fluid level of the suspension totally immersed the rootsections. 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, 100 IU

penicillin/ml, 0.1 mg streptomycin/ml, and 2 mM glutamine. Primary cell cultures used for thetest were from the third to the eighth passage. The root sections were incubated with cells for 72

h at 37oC. After incubation, the specimens were washed in Hank's balanced salt solution.

Specimens for SEM were rinsed two times in PBS at 37oC and fixed in 0.1 M cacodylate buffer

containing 2.5% glutaraldehyde at room temperature for 45 min. This was followed by post-

fixation in 1% OSO 4 in 0.1 M cacodylate buffer for 15 min, rinsing in water and staining in 2%

aqueous uranyl acetate for 30 min. The specimens were dehydrated through a graded series ofethanol (35 to 100%) and then critical point dried. Each specimen was coated with goldpalladium and examined by scanning electron microscopy (JEOL, Tokyo, Japan), at 15 Kv with

a tilt angle of 0 to 40o.

RESULTS

Controls (non-scaled specimens)

The untreated root surfaces were relatively smooth and free of collagen fibers, with spots ofattached calculus. The root surfaces were not affected by saline, citric acid or EDTA (Figure 1).

Gracey 7/8 scaled surfaces

The smooth planed surfaces showed fibroblast clusters mainly in the notched areas (Figure 2).

The etched or chelated specimens had a rougher surface with scattered fibroblasts. The surfacewas covered with a material suggestive of organic debris with occasional exposure of dentinal

tubules (Figure 3).

Amdent treated root surfaces

The ultrasonic treated root surfaces were smooth, well planed but with no migration of

fibroblasts. One specimen of the same group displayed a fibrous network resembling collagenfibers (Figure 4). When the roots were etched or chelated there was a significant increase in the

number of attached fibroblasts. Most of the adherent cells were flat with prominent microvilli

and filopodia forming a multilayer on the specimen surface (Figure 5). The root surface was not

visible between the multiple layers of fibroblasts (Figure 6).


4/9

Piezon treated root surfaces

The appearance of the root surfaces were similar to the Amdent treated and etched or chelatedroot surfaces.

Figure 1. SEM photomicrograph of untreated root surface. Spots of attached calculus are visible

(arrow). (Original magnification, 500X).

Figure 2. SEM photomicrograph of root surface scaled with a Gracey 7/8 curette. Presence of

cell inhabiting crack areas (arrow). (Original magnification, 350X).


5/9

Figure 3. SEM photomicrograph of root surface scaled with a Gracey 7/8 curette and etched with

EDTA. Fibroblast attached to the etched surface (arrow).(Original magnification, 1500X).

Figure 4. SEM photomicrograph of root surface ultrasonically treated with Amdent. Note the

presence of a fibrous network (arrow) and the absence of cells. (Original magnification, 500X).

Figure 5. SEM photomicrograph ultrasonically scaled with Amdent and etched with tetracyclinehydrochloride for 3 min. Note the well-spread presence of cells (arrow). (Original magnification,

200X).

Figure 6. SEM photomicrograph ultrasonically scaled with Amdent and etched with citric acid

for 3 minutes. Note the spreading of cells (arrow) completely masking the root surfaces.


6/9

(Original magnification, 350X).

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. Fibroblast

attachment is a common method used to evaluate the biocompatibility of root surfaces (15).

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. Acid treatment of

periodontally diseased human teeth has given both successful and unsuccessful results. The

observations in this study confirm and extend previous work.

More cells were attached to demineralized surfaces with no difference between the variousdemineralizing agents in enhancing cell attachment. This suggests that cell attachment to the

demineralized root can be enhanced by the detoxification of the root and also indicates that rootplaning removed substances that inhibited or prevented fibroblasts. Lucas et al. (16) suggested

that endotoxins from periodontal pathogens penetrate the root surface and inhibit cellular

attachment. In contrast, Fardal et al. (17) reported that periodontally diseased roots did not

inhibit the initial attachment in vitro.

In this study, fibroblasts did not attach to periodontally involved roots; nor did they attach toultrasonically treated root surfaces. This study suggests that the biochemical modifications of the

dentinal tubules induced by conditioning with citric acid, tetracycline hydrochloride or EDTA

are responsible for an increase in fibroblast attachment. These modifications could be either adirect 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 recent study by Vanheusden et al. (18) also confirmed that dentin conditioning by citric acid orby mynocycline enhanced the reattachment of human gingival epithelial cells to the root, thus

favoring faster periodontal healing; therefore influencing the behavior of periodontal fibroblasts,

improving their attachment and spreading.

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. (17) observed thatperiodontally diseased teeth incubated with human gingival fibroblasts showed equal attachmentregardless of whether they were instrumented, uninstrumented or non-diseased. They also

observed attachment of fibroblasts to calculus. It appears that fibroblast attachment to

periodontally diseased root surfaces is not uncommon. However, this study did not confirm this

nor did the study by Gamel et al. (19) which demonstrated that cell adherence was significantlyhigher in the healthy control group when compared to periodontally diseased treated control,a

finding that confirms that cultured human PDL fibroblasts prefer not to adhere to periodontally


7/9

diseased treated root surfaces. In this study, fibroblasts did not adhere to periodontally involved

teeth, were low in number in root planed root surfaces and more abundant when the root was

etched or chelated.

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. However, Gamal et al.(20) found that the application of tetracycline hydrochloride on the tooth surface had little effect

on cell adherence except to change their morphology.

Based on the results of this study, periodontal disease alters the biocompatibility of the root

surface and thus, potentially renders it less likely to achieve tissue attachment. It has not beendetermined if the conditioned root can be rendered as biocompatible

as a healthy root surface.

ACKNOWLEDGEMENTS

The author wishes to express his appreciation to Prof. Axel Bergenholtz for his help in thefibroblast culture technique, to Arturo Palustre for his help with the scanning electron

microscope and to Pressy dR Oba for typing the manuscript.

REFERENCES

1. Urist MR. Bone histogenesis and morphogenesis in implants of demineralized enamel anddentin. Oral Surg 1971;29:88-94.

2. Register AA. Bone and cementum induction by dentin demineralized in situ. J Periodontol

1973;44:49-59.

3. Register AA, Burdick FA. Accelerated reattachment with cementogenesis to dentin

demineralized in situ. I. Optimum range. J Periodontol 1975;46:646-655.

4. Polson AM, Frederick GT, Ladenheim S, Hanes P. The production of a root surface smear

layer by instrumentation and its removal by citric acid. J Periodontol 1984;55:443-446.

5. Wikesj UME, Baker PJ, Christersson LA, Genco LA, Genco RJ, Lyall RM, Hic S, DiFlorio

RM, Terranova VP. A biochemical approach to periodontal regeneration: Tetracycline treatment

conditions dentin surfaces. J Periodont Res 1986;21:322-329.

6. Blomlf J, Lindskog S. Root surface texture and early cells and

tissue colonization after different etching modalities. Eur J Oral Sc 1995;103:17-24.


8/9

7. Garett JS, Crigger M, Egelberg J. Effects of citric acid on diseased root surfaces. J Periodontol

Res 1978;13:155-163.

8. Nyman S, Lindhe J, Karring T. Healing following surgical treatment and root demineralization

in monkeys with periodontal disease. J Clin Periodontol 1981;8:249-258.

9. Aleo JJ, DeRenzis FA, Farber PA, Varboncoeur AP. The presence of biologic activity of

cementum-bound endotoxin. J Periodontol 1974;45:672-677.

10. Cogen RB, Garrison DC, Weatherford TW. Effect of various root surface treatments on the

viability and attachment of human gingival fibroblasts. J Periodontol 1983;54:700-704.

11. Adelson LJ, Hanks CT, Ramfjord SJ, Caffesse RG. Cytotoxicity of periodontally diseased

root surfaces. J Periodontol 1980;54:700-704.

12. Lowenberg BF, Aubin JE, Pitaru S, Melcher AH. A new chromium assay for accurate

measurement of cell attachment to demineralized and nondemineralized dentin in vitro. JPeriodont Res 1984;19:1106-1110.

13. Terranova VP, Franzetti LC, Hic S, Di Florio RM, Lyall RM, Wikesj UM, Baker PJ,

Christersson LA, Genco RJ. A biochemical approach to periodontal regeneration: Tetracycline

treatment of dentin promotes fibroblast adhesion and growth. J Periodont Res 1986;21:330-337.

14. Lowenberg BF, Aubin JE, Deporter DA, Sodek S, Melcher AH. Attachment, migration and

orientation of human gingival fibroblasts to collagen-coated, surface demineralized, and

untreated root slices. J Dent Res 1985;64:1106-1110.

15. Aleo JJ, DeRenzis FA, Farber PA. In vitro attachment of human gingival fibroblasts to rootsurfaces. J Periodontol 1975;46:639-645.

16. Lucas R, Cohen S, Aleo J. Histochemical study of strain I. Fibroblasts exposed to endotoxin.

The effect on cellular organelles. J Periodontol 1979;50:20-25.

17. Fardal O, Aubin JE, Lowenberg BF, Freeman E. Initial attachment of fibroblast like cells to

periodontally diseased root surfaces in vitro. J Clin Periodontol 1986;13:735-739.

18. Vanheusden AJ, Goffinet G, Zahedi S, Nusgeus B, Lapiere CM, Rompen EH. In vitro

stimulation of human gingival epithelial cell attachment to dentin by surface conditioning. J

Periodontol 1999;70:594-603.

19. Gamal AY, Mailhot JM. The effect of local delivery of PDGF-BB on attachment of human

periodontal ligament fibroblasts to periodontitis affected root surfaces _ in vitro. J Clin

Periodontol 2000;27:347-353.


9/9

20. Gamal AY, Mailhot JM, Garnick JJ, Newhouse R, Sharawy MM. Human periodontal

ligament fibroblast response to PDGF-BB and IGF-1 application on tetracycline HCl conditioned

root surfaces. J Clin Periodontol 1998;25:404-412.

Attachment of Human Gingival Fibroblasts to Periodontally Involved Root Surface Following Scaling...

Documents

Transcript of Attachment of Human Gingival Fibroblasts to Periodontally Involved Root Surface Following Scaling...