In Vitro Effects of Citric Acid Application Techniques
on
Dentin Surfaces C. Rosa Wen, * Raul G. Udayan K. Parikhf
Caffesse, * Edith
C. Morrison,
*
Carlos E.
Nasjleti, * and
The present study evaluated the in vitro effects of different application techniques of citric acid on dentin root surfaces. Ten freshly extracted, periodontally involved teeth were obtained and 4 dentin slabs, approximately 4x6x2 mm, were obtained from the roots of each tooth, for a total of 40 slabs. These slabs were identified by tooth and preserved in 1:1 anhydrous glycerol/absolute alcohol solution. Citric acid pH 1 was applied to 32 of the slabs for 5 minutes with one of 4 different techniques: 1) immersion; 2) placed with a saturated cotton pellet with no rubbing; 3) placed and burnished with a saturated cotton pellet; or 4) applied with a camel hair brush. The remaining 8 dentin slabs were used as negative control specimens, root-planed and non-acid treated. Following the various treatments, the slabs were fixed, dehydrated, critical point dried, and coated for scanning electron microscopic (SEM) evaluation. Scanning photomicrographs were obtained at 2,000, 6,000, and 40,000 magnifications. The surface characteristics of the treated dentin slabs were evaluated descriptively regarding the degree of fiber exposure; the number of exposed tubules and the surface area occupied by tubule orifices were also measured. Friedman's 2-way analysis for block designs was employed. Results demonstrated that root-planed, non-acid treated specimens had an amorphous, irregular surface which corresponded to a smear layer. Corresponding views of instrumented surfaces which had been treated with citric acid by immersion, placed with a cotton pellet, or applied with a camel hair brush exhibited funnel-shaped orifices of dentinal tubules and intertubular zones with exposed connective tissue fibers, mat-like morphology. In contrast, slabs treated with the burnishing technique showed wide surface variations. Surface tufting with widened tubular opening was observed on 2 of the 8 treated slabs. Surface smearing with complete obturation of tubules was present in the remaining 6 slabs. According to the results of the present study, if maximum exposure of intertubular fibrils and dentinal openings is beneficial in periodontal regenerative procedure, it may be undesirable to apply citric acid using excessive pressure. JPeriodontol 1992; 63:883—
889.
Key Words: Tooth root; citric acid; dentin; acid etching, dental.
The use of demineralizing agents in periodontal therapy can be dated back to the turn of the century.1 The interest has again been rekindled by the results of recent reports.2 6 Register2 and Register and Burdick3,4 have shown cementogenesis and new connective tissue attachment following citric acid conditioning. Regeneration of cementum and periodontal ligament was further proven to be biologically possible even in furcation defects in dogs after acid
demineralization.5,6
'Department of Periodontics, Dental Branch, The Health Science Center, Houston, TX. department of Anatomical Science.
University
of Texas
Topical application of citric acid to dentin surfaces prozone of demineralization, exposing the dentin fibrils7 and opening dentinal tubules. These are collagen believed to induce cementogenesis and enhance attachment either by connective tissue ingrowth and remineralization4 or by splicing of newly formed collagen to the exposed duced a 4 µ
dentinal fibrils.7,8 After testing different demineralization agents,3 citric acid (pH 1) application for 2 to 3 minutes was recommended as the optimal choice. Since then, various techniques of citric acid application have been used in animal and human studies. Some used cotton pellets,5,912 while others used a camel hair brush13,14 as an applicator for citric acid; Register and
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J Periodontol November 1992
EFFECT OF CITRIC ACID ON DENTIN
Burdick4
suggested rubbing techniques, Magnusson et al.15
used a continuous drip from a syringe, others5,10,12 had used placement of cotton pellets and Miller1618 has advocated the burnishing (with root planing pressure) technique. However there has been no background information comparing the effects of different acid application techniques on native dentin to warrant the different application modalities. Using scanning electron microscopy (SEM), Sterrett and Bain19 and Sterrett and Murphy20 have demonstrated that citric acid burnishing of formalin fixed dentinal root surfaces produced a distinguishable root topography of tufted collagen fibrils, and that the degree of collagen exposed was greater with citric acid "burnished" than with citric acid "placed." They suggested that the acid demineralization combined with the physical burnishing action results in a chemical/mechanical removal of more hydroxyapatite crystals. However, other studies have shown that citric acid burnishing of native unfixed dentin resulted in a relatively flat yet highly cracked surface, with no evidence of tufted or matted fibrillar structures, while controls (formalin fixed) exhibited a tufted fibrillar surface.21 The purpose of this SEM study was to evaluate and compare the in vitro effects of different citric acid application techniques on naturally preserved dentin slabs. The surface characteristics of the treated dentin slabs were evaluated descriptively; the number as well as the surface area of exposed tubules were measured and analyzed. MATERIALS AND METHODS Ten recently extracted periodontally involved human central incisors and molars with no endodontic therapy or root decay were stored in a mixture of anhydrous glycerol/absolute alcohol, 1:1 by volume, until ready to be used. Using a high-speed handpiece with copious water coolant, the crowns were resected at the cemento-enamel junction (CEJ). Following this, each root surface was thoroughly planed and flattened with a fine diamond tapered bur to remove cementum and expose the underlying dentin. The root was then sectioned longitudinally as 4 equal parts, and the pulpal side was flattened with a straight bur followed by a small inverted cone to make a horizontal groove on the pulpal surface for identification purposes. Lastly, these specimens were harvested by a horizontal cut to yield 4 dentin slabs about 4x6x2 mm in size. These dentin slabs were further identified by tooth and preserved in glycerol/alcohol mixture as mentioned above until the time of treatment. This yielded a total of 40 dentin slabs, 32 experimental and 8 negative control slabs. These control slabs were obtained from planed only, non-acid treated roots. The experimental dentin slabs were rinsed with copious distilled water, and each of the 4 slabs from the same root was randomly assigned to a different treatment group. Freshlymade citric acid solution was used. The citric acid solution was prepared by slowly adding 65 g of anhydrous citric acid crystals into 100 ml of distilled water, at 37°C, with constant stirring. This gave a pH 1.0 solution when checked
with a pH meter. It was then filtered using Whatman filter paper #1. The 4 acid treatment groups were as follows: group A: immersion in citric acid solution; group B: placement of saturated cotton pellets on slabs; group C: burnishing with saturated cotton pellets, vigorously rubbed using root planing pressure; and group D: camel hair brush application. The application time of the citric acid solution was 5 minutes and the pellets were changed 2 to 3 times per minute. Control and experimental dentin slabs were then rinsed with distilled water for 30 seconds, and fixed in 2.5% glutaraldehyde (3% glutaraldehyde diluted 5:1 with 0.2M sodium cacodylate, pH 7.4) overnight (18 to 19 hours) at 4°C. The slabs were then rinsed for 2 minutes in 0.2M cacodylate buffer. In preparation for the SEM study, the specimens were dehydrated in graded ethanol, critical point dried in C02 (impregnation for 10 minutes with liquid C02), then sputter coated 60 seconds with 60/40 gold/palladium. The mounted slabs were evaluated using a Jeol 100CX SEM. Scanning photomicrographs were obtained at 2,000, 6,000, and 40,000 magnification. The surface characteristics of intertubular dentin matrix, the number as well as the surface area of exposed dentinal tubules, were evaluated for each treatment group. An effort was made to take photomicrographs only from the center of the slabs. The surface characteristics of the dentin slabs were evaluated descriptively, concerning the degree of fiber exposure on the intertubular surface and the openness of the exposed tubules. The number of exposed tubules were quantitated from the 4" x 5" frames (under x 2,000 magnification) of black-and-white instant camera prints (area 57.5 x 37.5 µ ). The quantitation was carried out independently by 2 examiners and repeated on 3 different occasions. There were no significant differences within or between examiners for each of the treatments. The average of the 6 readings was used as the actual number of exposed tubules for the slab. The mean and standard deviation for each treatment group were calculated. Friedman's 2-way analysis for block designs was employed.22 Treatments were ranked within tooth and then a main-effects analysis of variance on the ranks was performed. The area occupied by the dentinal tubules orifice was also measured on the photomicrographs taken at magnification x 2000. Measurements were made by tracing the outline of the orifices of the dentinal tubules on the projected image of the photomicrograph using a digitizing tablet.* The digitizing tablet was connected to an Apple II computer with the Bloquant Image Analysis System,* which included programs for measurements of surface areas and perimeters. The individual areas occupied by tubule openings were used to calculate the total surface area of the specimen field occupied by tubule orifices. Data obtained from area measurements and tubule counts were used to calculate the mean area occupied per individual tubule. =
=
*R&M Biometrics, Inc., Nashville, TN.
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WEN, CAFFESSE, MORRISON, NASJLETI, PARIKH
885
Figure 1. Negative control specimen. Instrumented, non-acid-treated dentin slab. The surface of the slab has the morphological characteristics of a surface smear layer, and orifices of dentina! tubules are not visible (original magnification x 2,000). Dentin slabs from 5 roots (a total of 20) were selected the interface between the acid-treated root surface and the underlying dentin. The samples were fractured perpendicularly to their long axes and again sputter-coated and the interface examined. Three measurements were taken from the 4" 5" print (under 1,500 magnification) to obtain the width of the demineralized zone. A KruskalWallis test was performed on these measurements. to reveal
RESULTS
Morphologic Description
SEM examination of control, root planed, non-acid-treated specimens showed an irregular, amorphous surface which corresponded to a smear layer (Fig. 1). Corresponding views of instrumented surfaces which had been treated with citric acid were substantially different in appearance compared to the non-acid-treated surfaces. The results of different treatments are shown in Figures 2A, 2B, 2C, and 2D. The dentin slabs treated by acid immersion (group A; Fig. 2A) consistently demonstrated tufting of intertubular dentin fibrils and wide open dentinal orifices. Those treated by pellet placement (group B; Fig. 2B) revealed a more "matted" surface character and some debris was often present inside the tubular orifices. On those slabs treated with the burnishing technique (group C; Figs. 2C, 3A, and 3B), a wide variation was often observed. Surface tufting with widened tubular opening was observed on 2 of the 8 slabs (Fig. 2C), however surface smearing with complete obturation of tubules (Fig. 3A) was present on 6 of the 8 slabs with peeling of the fibrous layer (Fig. 3B) on 2 slabs. On those slabs treated with a camel hair brush (group D; Fig. 2D), the surface character was close to that observed in group A
Figure 2. Dentin slabs with 4 different citric acid application techniques. A. immersion, B. placement with saturated cotton pellet, C. burnishing with saturated cotton pellet, D. brushing with saturated camel hair brush. To varying degrees, all techniques exposed collagen fibers (original mag-
nification
x
6,000).
(immersion) where tufting of dentinal fibrils were predominant features.
of tubules
and
widening
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EFFECT OF CITRIC ACID ON DENTIN
Table 2. Frequency Distribution of Citric Acid-Treated Samples According to Number of Exposed Dentinal Tubules*
_Treatment_
Number of Dentinal Tubules 0-10 11-20 21-30 30 + *Area evaluated
Immersion
=
57.5
x
37.5
Table 3. Total Surface Area*
(
=
8)
Treatment Immersion Placement
Burnishing Brushing
Placement
Burnishing
Brushing
|im2.
Occupied by Tubule Orifices (µ 2)
Mean 56.00 39.75 34.70 40.43
Range
SD 27.28 25.40 39.75 26.90
Two-factor ANOVA: Treatment F 1.0000. 0.00, 'Total area measured 31 31 µ
=
1.82;
21.23- 96.65 2.11- 76.55 0.0 -100.23 12.21- 86.49 =
0.1746; Tooth F
=
=
=
2.
Table 4. Calculated Mean Area per Individual Tubule
(µ 2) (
=
8)
Treatment
Mean
SD
Range
Immersion Placement
4.78 4.03 2.46 3.77
2.04 2.30 2.53 2.93
2.74- 8.02 1.06- 7.92 0.0 7.47 9.61-31.53
Burnishing Brushing
Two-factor ANOVA: Treatment F 1.0000. 0.000,
=
1.42,
-
=
0.2647; Tooth F
=
=
Figure 3. Some other variations often observed with the burnishing technique. A. "Smear layer" which obliterated the dentinal tubules. This "smearing" effect was found in 6 out of the 8 slabs. B. "Peeling" of the demineralized fibrous layer was evident in 2 of the 8 slabs (original magnification x 2,000).
Total Surface Area
Occupied by Tubule Openings The morphometric analysis of the surface area occupied by tubule openings (Table 3) and the calculated mean area of individual tubule (Table 4) showed the lowest value in the burnishing group, but these differences were not statisti-
cally significant. Table 1. Mean Number of Exposed Dentinal Tubules Acid-Treated Roots* (n = 8) Treatment Immersion Placement
Mean 24.00 17.69 12.04 17.26
Burnishing Brushing
Citric
SD
Range
8.77 11.64 13.08 9.07
14.95-38.45 0.0 -40.45 0.0 -31.00 2.50-32.00
Two-factor ANOVA: Treatment F 1.87, 1.0000. 0.00, *Area evaluated 57.5 x 37.5 |im2. =
on
=
Depth of Demineralization A fibrillar zone was present on the surface of the acidtreated root surfaces. It represented the collagenous fibrils of the dentin exposed by the acid treatment (Fig. 4). The depth of the fibrillar zone was measured. The mean and the range for each treatment group are presented in Table 5.
0.1664; Tooth F
=
=
Number of Dentinal Tubular Openings The mean and standard deviation of dentinal openings for each treatment group are shown in Table 1. The mean number of openings was highest in the acid immersion group followed by the cotton pellet placement groups and camel hair brushing. The least number of openings was found in the burnishing group. Frequency distributions of tubular openings are presented in Table 2.
DISCUSSION After mechanical instrumentation of a root surface, a smear layer is usually present.23 It has been characterized as an amorphous structure obscuring the underlying dentin surface.24 After acid treatment, this smear layer is usually removed, leaving patent dentina! tubules and varying degrees of dentin matrix exposed.24,25 The proposed mechanisms for the success of citric acid treatment include the hypothesis that demineralized dentin substrate increases fibroblastic attachment by creating a more "hospitable" environment26 29 or it promotes formation of a fibrin net-
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WEN, CAFFESSE, MORRISON, NASJLETI,
PARIKH
887
Acid demineralization has been shown to increase clinical attachment level34-35 as well as to promote histologie evidence of cementogenesis or fibrous attachment.11-36-37 On the other hand, however, several studies failed to demonstrate the benefits of citric acid treatment, either clinically9-14-38-42 or histologically.12-38 In this study, the intertubular surface character, the number of exposed tubules, and the surface area occupied by tubule orifices were evaluated. The ingrowth of collagen fibrils from the soft tissue flap into the opened tubules and subsequent formation of cementum pins during healing has been suggested by Register and Burdick4 and Poison and Frederick43 have also shown the attachment of cell processes down in the exposed collagen substrate within the dentinal tubules. In short, the exposed dentinal fibrils in the open tubules44 and on the root surface may have greatly increased the surface area for future attachment in regeneration procedures. On the average, the number of dentinal openings per µ 2 found after acid treatment is lower than what has been reported by Hanes et al.45 One reasonable explanation could be that periodontally involved roots are known to have hypermineralized surfaces,46 and fewer dentinal tubules with smaller tubular diameter. Also in this study, several dentin slabs from each treatment group were split perpendicularly to their long axes and evaluated under SEM. The width of the demineralized fibrillar zone varied from 3 to 7.5 µ , which is in agreement with the 3 to 8 µ reported by Hanes et al.47 Using transmission electron microscopy (TEM), the demineralization zone has been reported to average 4 µ 7 or 3 to 5
unpredictable.
Figure 4. Twenty (20) dentin slabs were fracturedperpendicularly to their long axes, and the interface between the dentin root surface and the fractured surface was examined under SEM. The fibrillar zone is evident on the acid-treated dentin surface (original magnification x 1,500). Table 5. Mean Width of Demineralized Zone Treated Roots
(µ ) for Citric Acid-
Mean
Treatment
Immersion Placement
Burnishing Brushing
5 3 4 5
Kruskal-Wallis test
X2
SD 1.8 0.4 1.9 0.8
5.5 3.6 4.8 4.6 =
2.7871;
=
Range 4.0-7.5 3.0-5.0 4.0-7.0 4.0-5.7
0.4256.
work with the exposed dentinal fibrils in the initial healing stage and interferes with epithelial downgrowth,30 allowing more time for the formation and maturation of a new connective tissue attachment. The exposed dentinal fibrils are believed to serve as nidus for splicing with the fibrils of the healing flap.7-8 Miller17-18 has performed free gingival grafts in conjunction with citric acid. Excellent clinical results are reported in an attempt to cover denuded root surfaces. In the technique described "burnishing" (vigorous rubbing using root planing pressure) of the root surface with citric acid pH 1 for 5 minutes was employed. It was claimed that the added mechanical effect is important in exposing dentinal fibrils for periodontal regeneration.1618 The role of citric acid treatment on regeneration of periodontal tissues is controversial. In animal studies, acid treatment provided promising results. Register et al.,4 and Ririe et al.8 have demonstrated accelerated reattachment, mediated by cementogenesis and Osteogenesis in dogs treated with citric acid. In experimental or natural occurring furcation defects in dogs, acid demineralization has also succeeded in gaining new attachment.5-6-31 However, Nyman et al.10 and Gottlow and co-workers32 have failed to induce cementogenesis in monkeys and dogs, respectively, with the same treatment. Sometimes, the result was not clinically =
significant.33 In human
studies, the results
are more
conflicting if not
µ .8
After evaluating the effects of the 4 different acid application techniques it became clear that the variability is especially high in the "burnishing" group. For instance, burnishing could either make the dentin surface more fibrous with more open tubules (in 2 of 8 samples), create peeling (2 out of 8 samples), or smearing the surface (6 out of 8 samples), obturating the dentinal openings. In quantitating the number of exposed tubules, the criteria for "openness" of a tubule are difficult to set. One specimen in group C (burnishing) has as many as 36 tubules counted; however, the vast majority of these tubules were actually partially obliterated. This ambiguity may have accounted for the high standard deviation reported. In general, both brushing the acid with a camel hair brush (group D) and placement of saturated cotton pellets (group B) have exposed the intertubular fibrils and a high number of patent tubules comparable to what was observed in group A (immersion). However, with excessive pressure, as with burnishing (group C), the exposed dentin matrix may often be masked or flattened creating a "smear" layer with obliterated tubules or peeling the exposed fibrillar structure. These observations are further supported by the morphometric analyses of the surface area occupied by tubule orifices. Pashley et al.48 studied the effects of burnishing
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EFFECT OF CITRIC ACID ON DENTIN
different pastes on dentin permeability and concluded that burnishing alone created a partial smear layer occluding the orifices of dentinal tubules and produced a significant decrease in dentin permeability. Formalin fixation has been shown to denature or change collagen structure making it relatively insoluble to acid.49 Combined with the mechanical burnishing of the acid, this may have contributed to the deeply tufted dentin fibrils seen in the formalin-fixed dentin slabs reported by Sterret and colleagues.19'20 Glycerol has been used to preserve the tissue at the cellular organelle level, and it is felt that glycerol may preserve the dentin matrix without denaturing, thus this in vitro study using glycerol as a preservative may better reflect the in vivo situation. In this study, a smear layer with total or partial obliteration of dentinal tubules was often observed on slabs treated with the burnishing technique. This may coincide with the "amorphous" appearance observed by Sterrett et al.21 when citric acid was burnished on native (unfixed) dentin, and the relatively flat yet highly cracked surface with no evidence of tufted or matted fibrillar structures found. According to the results of the present study, if maximum exposure of intertubular fibrils and dentinal openings is beneficial in periodontal regenerative procedure, it may be undesirable to apply citric acid using excessive pressure. REFERENCES 1. Stewart H. Partial removal of cementum and décalcification of tooth in treatment of pyorrhea alveolaris. Dent Cosmos 1895; 41:617626. 2. Register AA. Bone and cementum induction by dentin, demineralized in situ. J Periodontol 1973; 44:49-54. 3. Register AA, Burdick FA. Accelerated reattachment with cementogenesis to dentin, demineralized in situ. I. Optimum range. / Periodontol 1975; 46:646-655. 4. Register AA, Burdick FA. Accelerated reattachment with cementogenesis to dentin, demineralized in situ. . Defect repair. / Periodontol 1976; 47:497-505. 5. Crigger M, Bogle G, Nilveus R, et al. The effect of topical citric acid application on the healing of experimental furcation defects in dogs. J Periodont Res 1978; 13:538-549. 6. Nilveus R, Bogle G, Crigger M, et al. The effect of topical citric acid application on the healing of experimental furcation defects in dogs. . Healing after repeated surgery. / Periodont Res 1980; 15:544550. 7. Garrett JS, Crigger M, Egelberg J. The effects of citric acid on diseased root surfaces. J Periodont Res 1978; 13:155-163. 8. Ririe CM, Crigger M, Selvig KA. Healing of periodontal connective tissues following surgical wounding and application of citric acid in dogs. J Periodont Res 1980; 15:314-327. 9. Cole R, Nilveus R, Ainamo J, et al. Pilot clinical studies on the effect of topical citric acid application on healing after replaced periodontal flap surgery. J Periodont Res 1981; 16:117-122. 10. 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. 11. Frank RM, Fiore-Donno G, Cimasoni G. Cementogenesis and soft tissue attachment after citric acid treatment in a human. An electron microscopic study. J Periodontol 1983; 54:389-401.
SJ, Kushner L, Stahl SS. Healing responses of human intraoslesions following the use of debridement, grafting and citric acid root treatment. I. Clinical and histologie observations six months postsurgery. J Periodontol 1983; 54:67-76. 13. Poison AM, Proye MP. Effect of root surface alterations on periodontal healing. II. Citric acid treatment of the denuded root. J Clin Periodontol 1982; 9:441-454. 14. Mark SC Jr, Mehta NR. Lack of effect of citric acid treatment of root surface on the formation of new connective tissue attachment. / Clin Periodontol 1986; 13:109-116. 15. Magnusson I, Claffey N, Bogle G, et al. Root résorption following periodontal flap procedures in monkeys. J Periodont Res 1985; 20:79— 85. 16. Miller PD. Root coverage using a free soft tissue autograft following citric acid application. I. Technique. Int J Periodontics Restorative Dent 1982; 2(l):65-70. 17. Miller PD. Root coverage using a free soft tissue autograft following citric acid application. II. Treatment of the carious root. Int J Periodontics Restorative Dent 1983; 3(5):38-51. 18. Miller PD. Root coverage using a free soft tissue autograft following citric acid application. III. A successful and predictable procedure in areas of deep-wide recession. Int J Periodontics Restorative Dent 1985; 5(2): 14-37. 19. Sterrett JD, Bain CA. Citric acid burnishing of dentinal root surfaces. A preliminary scanning electron microscopy report. / Can DentAssoc 1987; 53:395-397. 20. Sterrett JD, Murphy HJ. Citric acid burnishing of dentinal root surfaces. A scanning electron microscopy report. / Clin Periodontol 1989; 16:98-104. 21. Sterrett JD, Hawkins CH, Thomas J. Citric acid burnishing of native radicular dentin: A scanning electron microscopic report. Quintessence Int 1989; 20:423-426. 22. Conover WJ. Practical Nonparametric Statistics, 2nd ed. New York: John Wiley and Sons; 1980:299-305. 23. Jones SJ, Lozdan J, Boyde A. Tooth surface treated in situ with periodontal instruments. SEM studies. Br Dent J 1972; 132:57-64. 24. Poison 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; 55:443^146. 25. Brännström M, Johnson G. Effects of various conditioners and cleaning agents on prepared dentin surfaces. A SEM investigation. J Prosthet Dent 1974; 31:422-430. 26. Boyko GA, Brunette DM, Melcher AH. Cell attachment to demineralized root surfaces in vitro. J Periodont Res 1980; 15:297-303. 27. Cogen RB, Garrison DC, Weatherford TW. Effect of various root surface treatments on the viability and attachment of human gingival fibroblasts. / Periodontol 1983; 54:277-282. 28. Fernyhough W, Page RC. Attachment, growth, and synthesis by human gingival fibroblasts on demineralized or fibronectin-treated normal and diseased tooth roots. J Periodontol 1983; 54:133-140. 29. Lopez NJ. Connective tissue regeneration to periodontally diseased roots, planed and conditioned with citric acid and implanted into oral mucosa. / Periodontol 1984; 55:381-390. 30. Poison AM, Proye MP. Fibrin linkage: A precursor for new attachment. J Periodontol 1983; 54:141-147. 31. Bogle G, Adams D, Crigger M, et al. New attachment after surgical treatment and acid conditioning of roots in naturally occurring periodontal disease in dogs. J Periodont Res 1981; 16:130-133. 32. Gottlow J, Nyman S, Karring T. Healing following citric acid conditioning of roots implanted into bone and gingival connective tissue. / Periodont Res 1984; 19:214-220. 33. Woodyard SG, Snyder AJ, Henley G, O'Neal RB. A histometric evaluation of the effect of citric acid preparation upon healing of coronally positioned flaps in nonhuman primates. J Periodontol 1984; 55:203-212. 34. Liu W, Solt CW. A surgical procedure for the treatment of localized 12. Froum seous
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gingival recession in conjunction with root tioning. / Periodontol 1980; 51:505-509.
WEN, CAFFESSE, MORRISON, NASJLETI, surface citric acid condi-
35. Shiloah J. The clinical effects of citric acid and laterally positioned pedicle grafts in the treatment of denuded root surfaces A pilot study. J Periodontol 1980; 51:652-654. 36. Cole R, Crigger M, Bogle G, et al. Connective tissue regeneration to periodontally diseased teeth. A histological study. J Periodont Res 1980; 15:1-9. 37. Albair WB, Cobb CM, Killoy WJ. Connective tissue attachment to periodontally diseased roots after citric acid demineralization. / Periodontol 1982; 53:515-526. 38. Stahl SS, Froum SJ. Human clinical and histologie repair responses following the use of citric acid in periodontal therapy. J Periodontol 1977; 48:261-266. 39. Renvert S, Egelberg J. Healing after treatment of periodontal intraosseous defects. II. Effect of citric acid conditioning of the root surface. J Clin Periodontol 1981; 8:459-473. 40. Smith BA, Mason WE, Morrison EC, Caffesse RG. The effectiveness of citric acid as an adjunct to surgical reattachment procedures in humans. / Clin Periodontol 1986; 13:701-708. 41. Caffesse RG, Alspach SR, Morrison EC, Burgett FG. Lateral sliding flaps with and without citric acid. Int J Periodontics Restorative Dent
43.
-
1987; 7(6):43-58. JA, Ashley FP, Waterman CA. The effect
42. Moore
on
healing of the
44.
45. 46. 47.
48.
49.
PARIKH
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application of citric acid during replaced flap surgery. / Clin Periodontol 1987; 14:130-135. Poison AM, Frederick GT. Cell processes in dentinal tubules during early phases of attachment to demineralized, periodontitis-affected surfaces. J Clin Periodontol 1985; 12:162-169. Selvig KA. Ultrastructural changes in human dentin exposed to a weak acid. Arch Oral Biol 1968; 13:719-734. Hanes PJ, Poison AM, Frederick GT. Root and pulpal dentin after surface demineralization. Endod Dent Traumatol 1986; 2:190-195. Selvig KA. Biological changes at the tooth-saliva interface in periodontal disease. J Dent Res 1969; 48:846-855. Hanes PJ, Poison AM, Frederick GT. Initial wound healing attachments to demineralized dentin. J Periodontol 1988; 59:176-183. Pashley DH, Leibach JG, Horner JA. The effects of burnishing NaF/ kaolin/glycerin paste on dentin permeability. J Periodontol 1987; 58:1923. Cook SF, Ezra-Cohn HE. A comparison of methods for decalcifying bone. / Histochem Cytochem 1962; 10:560-563.
Send reprint requests to: Dr. Raul G. Caffesse, Department of Periodontics, Dental Branch, The University of Texas Health Science Center, 6516 John Freeman Avenue, Room 305, Houston, TX 77030. Accepted for publication May 8, 1992.
on
Dentin Surfaces C. Rosa Wen, * Raul G. Udayan K. Parikhf
Caffesse, * Edith
C. Morrison,
*
Carlos E.
Nasjleti, * and
The present study evaluated the in vitro effects of different application techniques of citric acid on dentin root surfaces. Ten freshly extracted, periodontally involved teeth were obtained and 4 dentin slabs, approximately 4x6x2 mm, were obtained from the roots of each tooth, for a total of 40 slabs. These slabs were identified by tooth and preserved in 1:1 anhydrous glycerol/absolute alcohol solution. Citric acid pH 1 was applied to 32 of the slabs for 5 minutes with one of 4 different techniques: 1) immersion; 2) placed with a saturated cotton pellet with no rubbing; 3) placed and burnished with a saturated cotton pellet; or 4) applied with a camel hair brush. The remaining 8 dentin slabs were used as negative control specimens, root-planed and non-acid treated. Following the various treatments, the slabs were fixed, dehydrated, critical point dried, and coated for scanning electron microscopic (SEM) evaluation. Scanning photomicrographs were obtained at 2,000, 6,000, and 40,000 magnifications. The surface characteristics of the treated dentin slabs were evaluated descriptively regarding the degree of fiber exposure; the number of exposed tubules and the surface area occupied by tubule orifices were also measured. Friedman's 2-way analysis for block designs was employed. Results demonstrated that root-planed, non-acid treated specimens had an amorphous, irregular surface which corresponded to a smear layer. Corresponding views of instrumented surfaces which had been treated with citric acid by immersion, placed with a cotton pellet, or applied with a camel hair brush exhibited funnel-shaped orifices of dentinal tubules and intertubular zones with exposed connective tissue fibers, mat-like morphology. In contrast, slabs treated with the burnishing technique showed wide surface variations. Surface tufting with widened tubular opening was observed on 2 of the 8 treated slabs. Surface smearing with complete obturation of tubules was present in the remaining 6 slabs. According to the results of the present study, if maximum exposure of intertubular fibrils and dentinal openings is beneficial in periodontal regenerative procedure, it may be undesirable to apply citric acid using excessive pressure. JPeriodontol 1992; 63:883—
889.
Key Words: Tooth root; citric acid; dentin; acid etching, dental.
The use of demineralizing agents in periodontal therapy can be dated back to the turn of the century.1 The interest has again been rekindled by the results of recent reports.2 6 Register2 and Register and Burdick3,4 have shown cementogenesis and new connective tissue attachment following citric acid conditioning. Regeneration of cementum and periodontal ligament was further proven to be biologically possible even in furcation defects in dogs after acid
demineralization.5,6
'Department of Periodontics, Dental Branch, The Health Science Center, Houston, TX. department of Anatomical Science.
University
of Texas
Topical application of citric acid to dentin surfaces prozone of demineralization, exposing the dentin fibrils7 and opening dentinal tubules. These are collagen believed to induce cementogenesis and enhance attachment either by connective tissue ingrowth and remineralization4 or by splicing of newly formed collagen to the exposed duced a 4 µ
dentinal fibrils.7,8 After testing different demineralization agents,3 citric acid (pH 1) application for 2 to 3 minutes was recommended as the optimal choice. Since then, various techniques of citric acid application have been used in animal and human studies. Some used cotton pellets,5,912 while others used a camel hair brush13,14 as an applicator for citric acid; Register and
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J Periodontol November 1992
EFFECT OF CITRIC ACID ON DENTIN
Burdick4
suggested rubbing techniques, Magnusson et al.15
used a continuous drip from a syringe, others5,10,12 had used placement of cotton pellets and Miller1618 has advocated the burnishing (with root planing pressure) technique. However there has been no background information comparing the effects of different acid application techniques on native dentin to warrant the different application modalities. Using scanning electron microscopy (SEM), Sterrett and Bain19 and Sterrett and Murphy20 have demonstrated that citric acid burnishing of formalin fixed dentinal root surfaces produced a distinguishable root topography of tufted collagen fibrils, and that the degree of collagen exposed was greater with citric acid "burnished" than with citric acid "placed." They suggested that the acid demineralization combined with the physical burnishing action results in a chemical/mechanical removal of more hydroxyapatite crystals. However, other studies have shown that citric acid burnishing of native unfixed dentin resulted in a relatively flat yet highly cracked surface, with no evidence of tufted or matted fibrillar structures, while controls (formalin fixed) exhibited a tufted fibrillar surface.21 The purpose of this SEM study was to evaluate and compare the in vitro effects of different citric acid application techniques on naturally preserved dentin slabs. The surface characteristics of the treated dentin slabs were evaluated descriptively; the number as well as the surface area of exposed tubules were measured and analyzed. MATERIALS AND METHODS Ten recently extracted periodontally involved human central incisors and molars with no endodontic therapy or root decay were stored in a mixture of anhydrous glycerol/absolute alcohol, 1:1 by volume, until ready to be used. Using a high-speed handpiece with copious water coolant, the crowns were resected at the cemento-enamel junction (CEJ). Following this, each root surface was thoroughly planed and flattened with a fine diamond tapered bur to remove cementum and expose the underlying dentin. The root was then sectioned longitudinally as 4 equal parts, and the pulpal side was flattened with a straight bur followed by a small inverted cone to make a horizontal groove on the pulpal surface for identification purposes. Lastly, these specimens were harvested by a horizontal cut to yield 4 dentin slabs about 4x6x2 mm in size. These dentin slabs were further identified by tooth and preserved in glycerol/alcohol mixture as mentioned above until the time of treatment. This yielded a total of 40 dentin slabs, 32 experimental and 8 negative control slabs. These control slabs were obtained from planed only, non-acid treated roots. The experimental dentin slabs were rinsed with copious distilled water, and each of the 4 slabs from the same root was randomly assigned to a different treatment group. Freshlymade citric acid solution was used. The citric acid solution was prepared by slowly adding 65 g of anhydrous citric acid crystals into 100 ml of distilled water, at 37°C, with constant stirring. This gave a pH 1.0 solution when checked
with a pH meter. It was then filtered using Whatman filter paper #1. The 4 acid treatment groups were as follows: group A: immersion in citric acid solution; group B: placement of saturated cotton pellets on slabs; group C: burnishing with saturated cotton pellets, vigorously rubbed using root planing pressure; and group D: camel hair brush application. The application time of the citric acid solution was 5 minutes and the pellets were changed 2 to 3 times per minute. Control and experimental dentin slabs were then rinsed with distilled water for 30 seconds, and fixed in 2.5% glutaraldehyde (3% glutaraldehyde diluted 5:1 with 0.2M sodium cacodylate, pH 7.4) overnight (18 to 19 hours) at 4°C. The slabs were then rinsed for 2 minutes in 0.2M cacodylate buffer. In preparation for the SEM study, the specimens were dehydrated in graded ethanol, critical point dried in C02 (impregnation for 10 minutes with liquid C02), then sputter coated 60 seconds with 60/40 gold/palladium. The mounted slabs were evaluated using a Jeol 100CX SEM. Scanning photomicrographs were obtained at 2,000, 6,000, and 40,000 magnification. The surface characteristics of intertubular dentin matrix, the number as well as the surface area of exposed dentinal tubules, were evaluated for each treatment group. An effort was made to take photomicrographs only from the center of the slabs. The surface characteristics of the dentin slabs were evaluated descriptively, concerning the degree of fiber exposure on the intertubular surface and the openness of the exposed tubules. The number of exposed tubules were quantitated from the 4" x 5" frames (under x 2,000 magnification) of black-and-white instant camera prints (area 57.5 x 37.5 µ ). The quantitation was carried out independently by 2 examiners and repeated on 3 different occasions. There were no significant differences within or between examiners for each of the treatments. The average of the 6 readings was used as the actual number of exposed tubules for the slab. The mean and standard deviation for each treatment group were calculated. Friedman's 2-way analysis for block designs was employed.22 Treatments were ranked within tooth and then a main-effects analysis of variance on the ranks was performed. The area occupied by the dentinal tubules orifice was also measured on the photomicrographs taken at magnification x 2000. Measurements were made by tracing the outline of the orifices of the dentinal tubules on the projected image of the photomicrograph using a digitizing tablet.* The digitizing tablet was connected to an Apple II computer with the Bloquant Image Analysis System,* which included programs for measurements of surface areas and perimeters. The individual areas occupied by tubule openings were used to calculate the total surface area of the specimen field occupied by tubule orifices. Data obtained from area measurements and tubule counts were used to calculate the mean area occupied per individual tubule. =
=
*R&M Biometrics, Inc., Nashville, TN.
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WEN, CAFFESSE, MORRISON, NASJLETI, PARIKH
885
Figure 1. Negative control specimen. Instrumented, non-acid-treated dentin slab. The surface of the slab has the morphological characteristics of a surface smear layer, and orifices of dentina! tubules are not visible (original magnification x 2,000). Dentin slabs from 5 roots (a total of 20) were selected the interface between the acid-treated root surface and the underlying dentin. The samples were fractured perpendicularly to their long axes and again sputter-coated and the interface examined. Three measurements were taken from the 4" 5" print (under 1,500 magnification) to obtain the width of the demineralized zone. A KruskalWallis test was performed on these measurements. to reveal
RESULTS
Morphologic Description
SEM examination of control, root planed, non-acid-treated specimens showed an irregular, amorphous surface which corresponded to a smear layer (Fig. 1). Corresponding views of instrumented surfaces which had been treated with citric acid were substantially different in appearance compared to the non-acid-treated surfaces. The results of different treatments are shown in Figures 2A, 2B, 2C, and 2D. The dentin slabs treated by acid immersion (group A; Fig. 2A) consistently demonstrated tufting of intertubular dentin fibrils and wide open dentinal orifices. Those treated by pellet placement (group B; Fig. 2B) revealed a more "matted" surface character and some debris was often present inside the tubular orifices. On those slabs treated with the burnishing technique (group C; Figs. 2C, 3A, and 3B), a wide variation was often observed. Surface tufting with widened tubular opening was observed on 2 of the 8 slabs (Fig. 2C), however surface smearing with complete obturation of tubules (Fig. 3A) was present on 6 of the 8 slabs with peeling of the fibrous layer (Fig. 3B) on 2 slabs. On those slabs treated with a camel hair brush (group D; Fig. 2D), the surface character was close to that observed in group A
Figure 2. Dentin slabs with 4 different citric acid application techniques. A. immersion, B. placement with saturated cotton pellet, C. burnishing with saturated cotton pellet, D. brushing with saturated camel hair brush. To varying degrees, all techniques exposed collagen fibers (original mag-
nification
x
6,000).
(immersion) where tufting of dentinal fibrils were predominant features.
of tubules
and
widening
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EFFECT OF CITRIC ACID ON DENTIN
Table 2. Frequency Distribution of Citric Acid-Treated Samples According to Number of Exposed Dentinal Tubules*
_Treatment_
Number of Dentinal Tubules 0-10 11-20 21-30 30 + *Area evaluated
Immersion
=
57.5
x
37.5
Table 3. Total Surface Area*
(
=
8)
Treatment Immersion Placement
Burnishing Brushing
Placement
Burnishing
Brushing
|im2.
Occupied by Tubule Orifices (µ 2)
Mean 56.00 39.75 34.70 40.43
Range
SD 27.28 25.40 39.75 26.90
Two-factor ANOVA: Treatment F 1.0000. 0.00, 'Total area measured 31 31 µ
=
1.82;
21.23- 96.65 2.11- 76.55 0.0 -100.23 12.21- 86.49 =
0.1746; Tooth F
=
=
=
2.
Table 4. Calculated Mean Area per Individual Tubule
(µ 2) (
=
8)
Treatment
Mean
SD
Range
Immersion Placement
4.78 4.03 2.46 3.77
2.04 2.30 2.53 2.93
2.74- 8.02 1.06- 7.92 0.0 7.47 9.61-31.53
Burnishing Brushing
Two-factor ANOVA: Treatment F 1.0000. 0.000,
=
1.42,
-
=
0.2647; Tooth F
=
=
Figure 3. Some other variations often observed with the burnishing technique. A. "Smear layer" which obliterated the dentinal tubules. This "smearing" effect was found in 6 out of the 8 slabs. B. "Peeling" of the demineralized fibrous layer was evident in 2 of the 8 slabs (original magnification x 2,000).
Total Surface Area
Occupied by Tubule Openings The morphometric analysis of the surface area occupied by tubule openings (Table 3) and the calculated mean area of individual tubule (Table 4) showed the lowest value in the burnishing group, but these differences were not statisti-
cally significant. Table 1. Mean Number of Exposed Dentinal Tubules Acid-Treated Roots* (n = 8) Treatment Immersion Placement
Mean 24.00 17.69 12.04 17.26
Burnishing Brushing
Citric
SD
Range
8.77 11.64 13.08 9.07
14.95-38.45 0.0 -40.45 0.0 -31.00 2.50-32.00
Two-factor ANOVA: Treatment F 1.87, 1.0000. 0.00, *Area evaluated 57.5 x 37.5 |im2. =
on
=
Depth of Demineralization A fibrillar zone was present on the surface of the acidtreated root surfaces. It represented the collagenous fibrils of the dentin exposed by the acid treatment (Fig. 4). The depth of the fibrillar zone was measured. The mean and the range for each treatment group are presented in Table 5.
0.1664; Tooth F
=
=
Number of Dentinal Tubular Openings The mean and standard deviation of dentinal openings for each treatment group are shown in Table 1. The mean number of openings was highest in the acid immersion group followed by the cotton pellet placement groups and camel hair brushing. The least number of openings was found in the burnishing group. Frequency distributions of tubular openings are presented in Table 2.
DISCUSSION After mechanical instrumentation of a root surface, a smear layer is usually present.23 It has been characterized as an amorphous structure obscuring the underlying dentin surface.24 After acid treatment, this smear layer is usually removed, leaving patent dentina! tubules and varying degrees of dentin matrix exposed.24,25 The proposed mechanisms for the success of citric acid treatment include the hypothesis that demineralized dentin substrate increases fibroblastic attachment by creating a more "hospitable" environment26 29 or it promotes formation of a fibrin net-
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PARIKH
887
Acid demineralization has been shown to increase clinical attachment level34-35 as well as to promote histologie evidence of cementogenesis or fibrous attachment.11-36-37 On the other hand, however, several studies failed to demonstrate the benefits of citric acid treatment, either clinically9-14-38-42 or histologically.12-38 In this study, the intertubular surface character, the number of exposed tubules, and the surface area occupied by tubule orifices were evaluated. The ingrowth of collagen fibrils from the soft tissue flap into the opened tubules and subsequent formation of cementum pins during healing has been suggested by Register and Burdick4 and Poison and Frederick43 have also shown the attachment of cell processes down in the exposed collagen substrate within the dentinal tubules. In short, the exposed dentinal fibrils in the open tubules44 and on the root surface may have greatly increased the surface area for future attachment in regeneration procedures. On the average, the number of dentinal openings per µ 2 found after acid treatment is lower than what has been reported by Hanes et al.45 One reasonable explanation could be that periodontally involved roots are known to have hypermineralized surfaces,46 and fewer dentinal tubules with smaller tubular diameter. Also in this study, several dentin slabs from each treatment group were split perpendicularly to their long axes and evaluated under SEM. The width of the demineralized fibrillar zone varied from 3 to 7.5 µ , which is in agreement with the 3 to 8 µ reported by Hanes et al.47 Using transmission electron microscopy (TEM), the demineralization zone has been reported to average 4 µ 7 or 3 to 5
unpredictable.
Figure 4. Twenty (20) dentin slabs were fracturedperpendicularly to their long axes, and the interface between the dentin root surface and the fractured surface was examined under SEM. The fibrillar zone is evident on the acid-treated dentin surface (original magnification x 1,500). Table 5. Mean Width of Demineralized Zone Treated Roots
(µ ) for Citric Acid-
Mean
Treatment
Immersion Placement
Burnishing Brushing
5 3 4 5
Kruskal-Wallis test
X2
SD 1.8 0.4 1.9 0.8
5.5 3.6 4.8 4.6 =
2.7871;
=
Range 4.0-7.5 3.0-5.0 4.0-7.0 4.0-5.7
0.4256.
work with the exposed dentinal fibrils in the initial healing stage and interferes with epithelial downgrowth,30 allowing more time for the formation and maturation of a new connective tissue attachment. The exposed dentinal fibrils are believed to serve as nidus for splicing with the fibrils of the healing flap.7-8 Miller17-18 has performed free gingival grafts in conjunction with citric acid. Excellent clinical results are reported in an attempt to cover denuded root surfaces. In the technique described "burnishing" (vigorous rubbing using root planing pressure) of the root surface with citric acid pH 1 for 5 minutes was employed. It was claimed that the added mechanical effect is important in exposing dentinal fibrils for periodontal regeneration.1618 The role of citric acid treatment on regeneration of periodontal tissues is controversial. In animal studies, acid treatment provided promising results. Register et al.,4 and Ririe et al.8 have demonstrated accelerated reattachment, mediated by cementogenesis and Osteogenesis in dogs treated with citric acid. In experimental or natural occurring furcation defects in dogs, acid demineralization has also succeeded in gaining new attachment.5-6-31 However, Nyman et al.10 and Gottlow and co-workers32 have failed to induce cementogenesis in monkeys and dogs, respectively, with the same treatment. Sometimes, the result was not clinically =
significant.33 In human
studies, the results
are more
conflicting if not
µ .8
After evaluating the effects of the 4 different acid application techniques it became clear that the variability is especially high in the "burnishing" group. For instance, burnishing could either make the dentin surface more fibrous with more open tubules (in 2 of 8 samples), create peeling (2 out of 8 samples), or smearing the surface (6 out of 8 samples), obturating the dentinal openings. In quantitating the number of exposed tubules, the criteria for "openness" of a tubule are difficult to set. One specimen in group C (burnishing) has as many as 36 tubules counted; however, the vast majority of these tubules were actually partially obliterated. This ambiguity may have accounted for the high standard deviation reported. In general, both brushing the acid with a camel hair brush (group D) and placement of saturated cotton pellets (group B) have exposed the intertubular fibrils and a high number of patent tubules comparable to what was observed in group A (immersion). However, with excessive pressure, as with burnishing (group C), the exposed dentin matrix may often be masked or flattened creating a "smear" layer with obliterated tubules or peeling the exposed fibrillar structure. These observations are further supported by the morphometric analyses of the surface area occupied by tubule orifices. Pashley et al.48 studied the effects of burnishing
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EFFECT OF CITRIC ACID ON DENTIN
different pastes on dentin permeability and concluded that burnishing alone created a partial smear layer occluding the orifices of dentinal tubules and produced a significant decrease in dentin permeability. Formalin fixation has been shown to denature or change collagen structure making it relatively insoluble to acid.49 Combined with the mechanical burnishing of the acid, this may have contributed to the deeply tufted dentin fibrils seen in the formalin-fixed dentin slabs reported by Sterret and colleagues.19'20 Glycerol has been used to preserve the tissue at the cellular organelle level, and it is felt that glycerol may preserve the dentin matrix without denaturing, thus this in vitro study using glycerol as a preservative may better reflect the in vivo situation. In this study, a smear layer with total or partial obliteration of dentinal tubules was often observed on slabs treated with the burnishing technique. This may coincide with the "amorphous" appearance observed by Sterrett et al.21 when citric acid was burnished on native (unfixed) dentin, and the relatively flat yet highly cracked surface with no evidence of tufted or matted fibrillar structures found. According to the results of the present study, if maximum exposure of intertubular fibrils and dentinal openings is beneficial in periodontal regenerative procedure, it may be undesirable to apply citric acid using excessive pressure. REFERENCES 1. Stewart H. Partial removal of cementum and décalcification of tooth in treatment of pyorrhea alveolaris. Dent Cosmos 1895; 41:617626. 2. Register AA. Bone and cementum induction by dentin, demineralized in situ. J Periodontol 1973; 44:49-54. 3. Register AA, Burdick FA. Accelerated reattachment with cementogenesis to dentin, demineralized in situ. I. Optimum range. / Periodontol 1975; 46:646-655. 4. Register AA, Burdick FA. Accelerated reattachment with cementogenesis to dentin, demineralized in situ. . Defect repair. / Periodontol 1976; 47:497-505. 5. Crigger M, Bogle G, Nilveus R, et al. The effect of topical citric acid application on the healing of experimental furcation defects in dogs. J Periodont Res 1978; 13:538-549. 6. Nilveus R, Bogle G, Crigger M, et al. The effect of topical citric acid application on the healing of experimental furcation defects in dogs. . Healing after repeated surgery. / Periodont Res 1980; 15:544550. 7. Garrett JS, Crigger M, Egelberg J. The effects of citric acid on diseased root surfaces. J Periodont Res 1978; 13:155-163. 8. Ririe CM, Crigger M, Selvig KA. Healing of periodontal connective tissues following surgical wounding and application of citric acid in dogs. J Periodont Res 1980; 15:314-327. 9. Cole R, Nilveus R, Ainamo J, et al. Pilot clinical studies on the effect of topical citric acid application on healing after replaced periodontal flap surgery. J Periodont Res 1981; 16:117-122. 10. 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. 11. Frank RM, Fiore-Donno G, Cimasoni G. Cementogenesis and soft tissue attachment after citric acid treatment in a human. An electron microscopic study. J Periodontol 1983; 54:389-401.
SJ, Kushner L, Stahl SS. Healing responses of human intraoslesions following the use of debridement, grafting and citric acid root treatment. I. Clinical and histologie observations six months postsurgery. J Periodontol 1983; 54:67-76. 13. Poison AM, Proye MP. Effect of root surface alterations on periodontal healing. II. Citric acid treatment of the denuded root. J Clin Periodontol 1982; 9:441-454. 14. Mark SC Jr, Mehta NR. Lack of effect of citric acid treatment of root surface on the formation of new connective tissue attachment. / Clin Periodontol 1986; 13:109-116. 15. Magnusson I, Claffey N, Bogle G, et al. Root résorption following periodontal flap procedures in monkeys. J Periodont Res 1985; 20:79— 85. 16. Miller PD. Root coverage using a free soft tissue autograft following citric acid application. I. Technique. Int J Periodontics Restorative Dent 1982; 2(l):65-70. 17. Miller PD. Root coverage using a free soft tissue autograft following citric acid application. II. Treatment of the carious root. Int J Periodontics Restorative Dent 1983; 3(5):38-51. 18. Miller PD. Root coverage using a free soft tissue autograft following citric acid application. III. A successful and predictable procedure in areas of deep-wide recession. Int J Periodontics Restorative Dent 1985; 5(2): 14-37. 19. Sterrett JD, Bain CA. Citric acid burnishing of dentinal root surfaces. A preliminary scanning electron microscopy report. / Can DentAssoc 1987; 53:395-397. 20. Sterrett JD, Murphy HJ. Citric acid burnishing of dentinal root surfaces. A scanning electron microscopy report. / Clin Periodontol 1989; 16:98-104. 21. Sterrett JD, Hawkins CH, Thomas J. Citric acid burnishing of native radicular dentin: A scanning electron microscopic report. Quintessence Int 1989; 20:423-426. 22. Conover WJ. Practical Nonparametric Statistics, 2nd ed. New York: John Wiley and Sons; 1980:299-305. 23. Jones SJ, Lozdan J, Boyde A. Tooth surface treated in situ with periodontal instruments. SEM studies. Br Dent J 1972; 132:57-64. 24. Poison 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; 55:443^146. 25. Brännström M, Johnson G. Effects of various conditioners and cleaning agents on prepared dentin surfaces. A SEM investigation. J Prosthet Dent 1974; 31:422-430. 26. Boyko GA, Brunette DM, Melcher AH. Cell attachment to demineralized root surfaces in vitro. J Periodont Res 1980; 15:297-303. 27. Cogen RB, Garrison DC, Weatherford TW. Effect of various root surface treatments on the viability and attachment of human gingival fibroblasts. / Periodontol 1983; 54:277-282. 28. Fernyhough W, Page RC. Attachment, growth, and synthesis by human gingival fibroblasts on demineralized or fibronectin-treated normal and diseased tooth roots. J Periodontol 1983; 54:133-140. 29. Lopez NJ. Connective tissue regeneration to periodontally diseased roots, planed and conditioned with citric acid and implanted into oral mucosa. / Periodontol 1984; 55:381-390. 30. Poison AM, Proye MP. Fibrin linkage: A precursor for new attachment. J Periodontol 1983; 54:141-147. 31. Bogle G, Adams D, Crigger M, et al. New attachment after surgical treatment and acid conditioning of roots in naturally occurring periodontal disease in dogs. J Periodont Res 1981; 16:130-133. 32. Gottlow J, Nyman S, Karring T. Healing following citric acid conditioning of roots implanted into bone and gingival connective tissue. / Periodont Res 1984; 19:214-220. 33. Woodyard SG, Snyder AJ, Henley G, O'Neal RB. A histometric evaluation of the effect of citric acid preparation upon healing of coronally positioned flaps in nonhuman primates. J Periodontol 1984; 55:203-212. 34. Liu W, Solt CW. A surgical procedure for the treatment of localized 12. Froum seous
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gingival recession in conjunction with root tioning. / Periodontol 1980; 51:505-509.
WEN, CAFFESSE, MORRISON, NASJLETI, surface citric acid condi-
35. Shiloah J. The clinical effects of citric acid and laterally positioned pedicle grafts in the treatment of denuded root surfaces A pilot study. J Periodontol 1980; 51:652-654. 36. Cole R, Crigger M, Bogle G, et al. Connective tissue regeneration to periodontally diseased teeth. A histological study. J Periodont Res 1980; 15:1-9. 37. Albair WB, Cobb CM, Killoy WJ. Connective tissue attachment to periodontally diseased roots after citric acid demineralization. / Periodontol 1982; 53:515-526. 38. Stahl SS, Froum SJ. Human clinical and histologie repair responses following the use of citric acid in periodontal therapy. J Periodontol 1977; 48:261-266. 39. Renvert S, Egelberg J. Healing after treatment of periodontal intraosseous defects. II. Effect of citric acid conditioning of the root surface. J Clin Periodontol 1981; 8:459-473. 40. Smith BA, Mason WE, Morrison EC, Caffesse RG. The effectiveness of citric acid as an adjunct to surgical reattachment procedures in humans. / Clin Periodontol 1986; 13:701-708. 41. Caffesse RG, Alspach SR, Morrison EC, Burgett FG. Lateral sliding flaps with and without citric acid. Int J Periodontics Restorative Dent
43.
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1987; 7(6):43-58. JA, Ashley FP, Waterman CA. The effect
42. Moore
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44.
45. 46. 47.
48.
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application of citric acid during replaced flap surgery. / Clin Periodontol 1987; 14:130-135. Poison AM, Frederick GT. Cell processes in dentinal tubules during early phases of attachment to demineralized, periodontitis-affected surfaces. J Clin Periodontol 1985; 12:162-169. Selvig KA. Ultrastructural changes in human dentin exposed to a weak acid. Arch Oral Biol 1968; 13:719-734. Hanes PJ, Poison AM, Frederick GT. Root and pulpal dentin after surface demineralization. Endod Dent Traumatol 1986; 2:190-195. Selvig KA. Biological changes at the tooth-saliva interface in periodontal disease. J Dent Res 1969; 48:846-855. Hanes PJ, Poison AM, Frederick GT. Initial wound healing attachments to demineralized dentin. J Periodontol 1988; 59:176-183. Pashley DH, Leibach JG, Horner JA. The effects of burnishing NaF/ kaolin/glycerin paste on dentin permeability. J Periodontol 1987; 58:1923. Cook SF, Ezra-Cohn HE. A comparison of methods for decalcifying bone. / Histochem Cytochem 1962; 10:560-563.
Send reprint requests to: Dr. Raul G. Caffesse, Department of Periodontics, Dental Branch, The University of Texas Health Science Center, 6516 John Freeman Avenue, Room 305, Houston, TX 77030. Accepted for publication May 8, 1992.
