J. Periodontal Res. 13: 155-163, 1978
Effects of citric acid on diseased root surfaces JOHN S. GARRt-Tr, MAX CRIGGER AND JAN EGELBERO
Loma Linda University, School of Dentistry, Loma Linda, California, U.S.A. The effect of topically applied citric acid on periodontally diseased root surfaces was evaluated using the scanning (SEM) and transmission (TEM) electron microscopes. Results with the SEM indicate that acid application had no effect on specimens that had not been root planed. After application to root planed surfaces, however, the acid produced a fiber-like surface with frequent depressions. TEM observations showed that the acid application produced a four micron wide demineralized zone, which was characterized by exposed collagen fibrils. These fibrils seemed to be continuous between the mineralized and demineralized zones of the root. It appears that the relative success of the citric acid application in periodontal reattachment procedures is related to the fact that the acid causes exposure of collagen fibrils in the dentin matrix, thus providing a suitable nidus for splicing with new fibrils during the healing process. (Accepted for publication May JJ, 1977)
Introduction
One of the earliest attempts in periodontal therapy to facilitate new connective tissue attachment dealt with application of a weak acid to the root surfaces (Younger 18971898, Stewart 1899). Recently Register (1973) and Register and Burdick (1975, 1976), have applied saturated solutions of citric acid (pH 1) to surgically created root surface defects in experimental animals, and reported accelerated reattachment atid cementogenesis during healing. While the effects of acid application on dental enamel and dentin have been studied extensively, the effects of citric acid on periodontally diseased root surfaces do not seem to have been investigated. The purpose of this study was to examine the ef-
fects of citric acid applied to periodontally diseased root surfaces utilizing scanning and transmission electron microscopy. A methodology was used which allowed for both observational results and objeetive analysis of the data.
iVIateriais and iVIetiiods
Sixteen human teeth extracted because of chronic periodontal disease were used. They were obtained from 11 patients, six females and five males, aged 35-81 years. The teeth fulfilled the following criteria: 1) No history of root planing, scaling or prophylaxis for at least five years. 2) No history of acute pain or swelling (abscess). 3) Periodontal pocket depth five millimeters or
156
G A R R E T T ,
C R I G G E R
A N D
E G E L B E R G
16 Periodontally Diseased Teetta
I
Scrubbed and Rinsed
8 Non Root Planed Teetb
8 Root Planed Teeth
I
Fractured 4 Teeth with Calculus
4 Teetb without Calculus
I
Fractured
Calculus Removed
8 Half Teeth Control
I Fractured
4 Half Teeth Control
4 Half Teeth Control 4 Half Teetb Citric Acid 3 mln.
4 Half Teetb Citric Acid 3 min.
8 Half Teetb Citric Add
3 min.
Rinsed Tap Water
Rinsed Tap Water
Rinsed Tap Water Fig.
1. Diagrammatic representation of the experimental procedures.
more. 4) Radiographic evidence of at least 30 % alveolar bone loss. Care was taken during extraction not to instrument the root surfaees to be studied. Following extraction the teeth were scrubbed in distilled water with a toothbrush. They were stored in double deionized water until further preparation. The teeth were divided into two groups of eight which varied with regard to experimental preparation of the root surface: 1) Periodontally diseased without root planing and 2) Periodontally diseased root planed (Fig. 1). Non-root planed teeth were further divided into groups of four teeth; those with and those without clinically visible calculus. Calculus deposits were removed by light scaling. The surfaces were not root planed. In the root planed group all eight teeth were root planed 10 strokes using a Gracey curette No. 13-14. Next the crowns of all sixteen teeth were notched
from the incisal occlusal surface to the cemento-enamel junction with a 558 carbide bur under water irrigation, and the pocket area was delineated by circumscribing it with a No. '/2 round bur. The teeth were then sectioned longitudinally through the notch with a chisel and mallet, bisecting the toot surface area to be studied. Thus, the root area was sectioned into two surfaces which, along the fracture line, were complementary to each other (Fig. 2). One of the bisected surfaces was designated experimental and etched by applying a cotton pellet saturated with citric acid for three minutes followed by a rinse in tap water (Fig. 1). Both control and experimental specimens were fixed in 2.5 % glutaraldehyde buffered at pH 7.4 by 0.2 M cacodylate buffer, for 24 hours at 4°C. They were then rinsed and stored at 4°C in the cacodylate buffer until processed for electt"on microscopy.
E F F E C T S
O F C I T R I C
A C I D
C N D I S E A S E D
R O C T
S U R F A C E S
157
duce any maeroscopic surface changes after three weeks. During the experiment the acid was mixed at the beginning of each week and was not used after five days. Specitnens for scanning electron micChisel roscopy (SEM) were post fixed for one hour in ostnium tetroxide buffered to pH 7.4 with 0.2 M cacodylate buffer. They were then washed in cacodylate buffer and dehydrated in graded ethanol. The specimens were dried using the critical point drying method with carbon dioxide as the dtying medium (Boyde & Wood 1969), Outline coated with a thin layer of gold, and Groove tiiounted for SEM study with the AMR 1000 microscope. Photography of SEM Specimens: Scanning electron micrographs were obtained by first examining the control surface at 50X until the delineated pocket area was located. Sites that had an identifying indentation or groove were then selected along the fracture line and electron micrographs taken at 750X, 1500X, 3000X and 9000X. The exFig. 2. Diagram illustrating the tocation of the cirperitnental specimen was then examined cumscribed experimental area of a pericdontally exand the indentation or groove that corresposed root surface. As the tooth was bisected longitudinaiiy, the experimental surface would be divided ponded to the previously photographed in two halves, one half serving as a control specimen control .specimen located. Electron microwhile fhe other half was exposed to citric aoid. graphs were taken at the satne magnificaIrregularities along the fracture line served to locate closely approximated areas on the control and extions. This allowed control and experimenperimental halves of each specimen for comparison. tal electron micrographs frotn an area apPDL, periodontal ligament. proximately IJ^ mm square on the root surface. Scanning electron micrographs were The citric acid solution was prepared by adding citric acid in anhydrous form analyzed for differences between control (Pfizer Co., New York) into distilled water and experimental areas in the following at room temperature under eontinuous mix- tnanner: First, after examination of all the ing until the solution became saturated. It photographie prints by six independent obwas then filtered using Whatman filter servers with no prior knowledge of the paper # 1 . The solution (pH «3 1) was experimentation, four consistently identifistored at room temperature in a dark glass able surface characteristics were agreed container. The shelf life of the acid solution upon. These were 1) surface projections was tested in the following manner: Freshly (pebbles), 2) surf aee depressions (depresextracted teeth were root planed and treat- sions), 3) smooth or flat areas (SF), and ed with the same solution of citric acid at 4) areas showing a mat of fibrouslike five day intervals. The acid failed to pro- material with numerous small projections
158
G A R R E T T ,
C R I G G E R
(fibers). The six observes also agreed that the 3000X micrographs most readily showed the above changes. Further analysis was then done by the principal investigator in two ways. First, the number of pebbles and depressions was counted on all 3000X micrographs. Next, a grid to fit the size of the photographic prints was manufactured and superimposed on each photo. The grid was made up of 285 six mm squares. The number of grid Squares with any of the four above surface characteristics were counted. A square was counted when any part of the square included the characteristic. Differences between control and experimental pairs within each group were evaluated statistically using the Student's t-test for paired observations. Ten randomly selected tnicrographs were re-analyzed two weeks later to determine the accutacy of the analyses, and the error of the method was found to be within 5 %. Specimens for transmission electron microscopy (TEM) were postfixed for one hour in osmium tetroxide. Small pieces were then dissected from the larger root surface specimens. These pieces were dehydrated in graded solutions of alcohol and embedded in Epon (Luft 1961) in gelatin capsules. The specitnens were oriented so that subsequent sectioning would be at right angles to the surface of interest. Ultra thin sections (800 A) were cut with a diatnond knife. These sections were collected on copper grids (200 mesh) and stained with uranyl acetate and lead citrate. They were studied with a Siemens IA microscope. Another series of ultra thin sections were demineralized by floating them on 2 % phosphotungstic acid for 30 minutes.
A N D
E G E L B E R G
Fig. 3. SEM of tooth without macroscopically visible calculus. Nonetched specimen. This figure shows a periodontally diseased root surface with, what is apparently subclinical calculus formation (C), some residual bacteria (B), and pebbles (P). 3000X
root surfaces which were not root planed failed to produce any significant tnorphologic changes regardless of the presence or absence of initial calculus deposits. The non-etched specimens showed tremendous variability ranging from flat to completely pebble covered specimens. Acid application had no effect other than to decrease the prominence of the surface characteristics. The various eharacteristics were still easily distinguishable however.
Resuits
SEM observatiotis Acid application to periodontally diseased
Fig. 4. SEM of tooth after root planing. Non-etched specimen. A flat, smooth (FS) surface. 3000X
E F F E C T S
C F C I T R I C
A C I D
C N D I S E A S E D
Fig. 5. SEM of tooth after root planing. Non-etched specimen. By rotating the specimen the fractured surface, created when the tooth was split, can be studied. Dentinal tubules (DT) extending to the surface (RS) indicate that the cementum was completely removed during root planing. 1000X
Three of the four periodontally diseased specimens which appeared free of calculus visually showed calculus-like deposits wheti viewed with the SEM. There were also a few bacteria remaining in these areas. The calculus-like areas were honeycotnbed in shape (Fig. 3). Teeth After Root Platutig. Periodontally diseased root surfaces that were root planed revealed the satne results regardless of the
R O C T
S U R F A C E S
159
Fig. 7. SEM of tooth after root planing. Acid-etched specimen. Higher magnification of an area in Fig. 6 illustrating fibrillar structures of the surface. 14,000X
presence or absence of calculus initially. The control noji-etched specimens had a stnooth, flat, pebble free surface (Fig. 4). Examination of the fractured surface created when the specimens were split indicated that the cementum had been completely retnoved in all teeth (Fig. 5). Detitinal tubules could be seen extending to the surface although openings of those tubules were not readily apparent on the root surface (compare with Fig. 4). Acid etched speeimens showed a flat surface with frequent depressions and numerous fiber-like structures (Fig. 6). Higher magnification revealed a fibrous appearing surface with fiberlike projections extending above the surface (Fig. 7). Statistical Analysis. Results of the statistical analyses ate summarized m Table 1. With acid application there was a statistically significant increase in the number of depressions and libers in the root planed specimens. No other significant differences were observed.
Fig. 6. SEM of tooth after root planing. Aoid-etched specimen. Frequent depressions (D) and fiber-like (F) structures are evident. Compare with Fig. 4. 3000X
TEM observations Root planed specimens studied with the TEM showed that acid application produced a demineralized zone approximately
160
G A R R E T T ,
C R I G G E R
A N D
E G E L B E R G
Table I Statistical comparison of SEM observations between experimental (etched) and control (non-etched) root surfaces. Results of Student's t-test for paired observations. Group 1 Non Root (8 spec imens) Surface characteristics Pebbies, count Pebbles, grid Depressions, count Depressions, grid Smooth/flat, grid Fibers, grid
t
P
1.4
N.S. N.S. N.S. N.S. N.S. N.S.
1.3 1.7 2.0 1.0 1.1
Group II Root Planed (8 specimens)
t 1.2 1.1 3.1 4.0 1.6
41.2
P
N.S. N.S.
Effects of citric acid on diseased root surfaces JOHN S. GARRt-Tr, MAX CRIGGER AND JAN EGELBERO
Loma Linda University, School of Dentistry, Loma Linda, California, U.S.A. The effect of topically applied citric acid on periodontally diseased root surfaces was evaluated using the scanning (SEM) and transmission (TEM) electron microscopes. Results with the SEM indicate that acid application had no effect on specimens that had not been root planed. After application to root planed surfaces, however, the acid produced a fiber-like surface with frequent depressions. TEM observations showed that the acid application produced a four micron wide demineralized zone, which was characterized by exposed collagen fibrils. These fibrils seemed to be continuous between the mineralized and demineralized zones of the root. It appears that the relative success of the citric acid application in periodontal reattachment procedures is related to the fact that the acid causes exposure of collagen fibrils in the dentin matrix, thus providing a suitable nidus for splicing with new fibrils during the healing process. (Accepted for publication May JJ, 1977)
Introduction
One of the earliest attempts in periodontal therapy to facilitate new connective tissue attachment dealt with application of a weak acid to the root surfaces (Younger 18971898, Stewart 1899). Recently Register (1973) and Register and Burdick (1975, 1976), have applied saturated solutions of citric acid (pH 1) to surgically created root surface defects in experimental animals, and reported accelerated reattachment atid cementogenesis during healing. While the effects of acid application on dental enamel and dentin have been studied extensively, the effects of citric acid on periodontally diseased root surfaces do not seem to have been investigated. The purpose of this study was to examine the ef-
fects of citric acid applied to periodontally diseased root surfaces utilizing scanning and transmission electron microscopy. A methodology was used which allowed for both observational results and objeetive analysis of the data.
iVIateriais and iVIetiiods
Sixteen human teeth extracted because of chronic periodontal disease were used. They were obtained from 11 patients, six females and five males, aged 35-81 years. The teeth fulfilled the following criteria: 1) No history of root planing, scaling or prophylaxis for at least five years. 2) No history of acute pain or swelling (abscess). 3) Periodontal pocket depth five millimeters or
156
G A R R E T T ,
C R I G G E R
A N D
E G E L B E R G
16 Periodontally Diseased Teetta
I
Scrubbed and Rinsed
8 Non Root Planed Teetb
8 Root Planed Teeth
I
Fractured 4 Teeth with Calculus
4 Teetb without Calculus
I
Fractured
Calculus Removed
8 Half Teeth Control
I Fractured
4 Half Teeth Control
4 Half Teeth Control 4 Half Teetb Citric Acid 3 mln.
4 Half Teetb Citric Acid 3 min.
8 Half Teetb Citric Add
3 min.
Rinsed Tap Water
Rinsed Tap Water
Rinsed Tap Water Fig.
1. Diagrammatic representation of the experimental procedures.
more. 4) Radiographic evidence of at least 30 % alveolar bone loss. Care was taken during extraction not to instrument the root surfaees to be studied. Following extraction the teeth were scrubbed in distilled water with a toothbrush. They were stored in double deionized water until further preparation. The teeth were divided into two groups of eight which varied with regard to experimental preparation of the root surface: 1) Periodontally diseased without root planing and 2) Periodontally diseased root planed (Fig. 1). Non-root planed teeth were further divided into groups of four teeth; those with and those without clinically visible calculus. Calculus deposits were removed by light scaling. The surfaces were not root planed. In the root planed group all eight teeth were root planed 10 strokes using a Gracey curette No. 13-14. Next the crowns of all sixteen teeth were notched
from the incisal occlusal surface to the cemento-enamel junction with a 558 carbide bur under water irrigation, and the pocket area was delineated by circumscribing it with a No. '/2 round bur. The teeth were then sectioned longitudinally through the notch with a chisel and mallet, bisecting the toot surface area to be studied. Thus, the root area was sectioned into two surfaces which, along the fracture line, were complementary to each other (Fig. 2). One of the bisected surfaces was designated experimental and etched by applying a cotton pellet saturated with citric acid for three minutes followed by a rinse in tap water (Fig. 1). Both control and experimental specimens were fixed in 2.5 % glutaraldehyde buffered at pH 7.4 by 0.2 M cacodylate buffer, for 24 hours at 4°C. They were then rinsed and stored at 4°C in the cacodylate buffer until processed for electt"on microscopy.
E F F E C T S
O F C I T R I C
A C I D
C N D I S E A S E D
R O C T
S U R F A C E S
157
duce any maeroscopic surface changes after three weeks. During the experiment the acid was mixed at the beginning of each week and was not used after five days. Specitnens for scanning electron micChisel roscopy (SEM) were post fixed for one hour in ostnium tetroxide buffered to pH 7.4 with 0.2 M cacodylate buffer. They were then washed in cacodylate buffer and dehydrated in graded ethanol. The specimens were dried using the critical point drying method with carbon dioxide as the dtying medium (Boyde & Wood 1969), Outline coated with a thin layer of gold, and Groove tiiounted for SEM study with the AMR 1000 microscope. Photography of SEM Specimens: Scanning electron micrographs were obtained by first examining the control surface at 50X until the delineated pocket area was located. Sites that had an identifying indentation or groove were then selected along the fracture line and electron micrographs taken at 750X, 1500X, 3000X and 9000X. The exFig. 2. Diagram illustrating the tocation of the cirperitnental specimen was then examined cumscribed experimental area of a pericdontally exand the indentation or groove that corresposed root surface. As the tooth was bisected longitudinaiiy, the experimental surface would be divided ponded to the previously photographed in two halves, one half serving as a control specimen control .specimen located. Electron microwhile fhe other half was exposed to citric aoid. graphs were taken at the satne magnificaIrregularities along the fracture line served to locate closely approximated areas on the control and extions. This allowed control and experimenperimental halves of each specimen for comparison. tal electron micrographs frotn an area apPDL, periodontal ligament. proximately IJ^ mm square on the root surface. Scanning electron micrographs were The citric acid solution was prepared by adding citric acid in anhydrous form analyzed for differences between control (Pfizer Co., New York) into distilled water and experimental areas in the following at room temperature under eontinuous mix- tnanner: First, after examination of all the ing until the solution became saturated. It photographie prints by six independent obwas then filtered using Whatman filter servers with no prior knowledge of the paper # 1 . The solution (pH «3 1) was experimentation, four consistently identifistored at room temperature in a dark glass able surface characteristics were agreed container. The shelf life of the acid solution upon. These were 1) surface projections was tested in the following manner: Freshly (pebbles), 2) surf aee depressions (depresextracted teeth were root planed and treat- sions), 3) smooth or flat areas (SF), and ed with the same solution of citric acid at 4) areas showing a mat of fibrouslike five day intervals. The acid failed to pro- material with numerous small projections
158
G A R R E T T ,
C R I G G E R
(fibers). The six observes also agreed that the 3000X micrographs most readily showed the above changes. Further analysis was then done by the principal investigator in two ways. First, the number of pebbles and depressions was counted on all 3000X micrographs. Next, a grid to fit the size of the photographic prints was manufactured and superimposed on each photo. The grid was made up of 285 six mm squares. The number of grid Squares with any of the four above surface characteristics were counted. A square was counted when any part of the square included the characteristic. Differences between control and experimental pairs within each group were evaluated statistically using the Student's t-test for paired observations. Ten randomly selected tnicrographs were re-analyzed two weeks later to determine the accutacy of the analyses, and the error of the method was found to be within 5 %. Specimens for transmission electron microscopy (TEM) were postfixed for one hour in osmium tetroxide. Small pieces were then dissected from the larger root surface specimens. These pieces were dehydrated in graded solutions of alcohol and embedded in Epon (Luft 1961) in gelatin capsules. The specitnens were oriented so that subsequent sectioning would be at right angles to the surface of interest. Ultra thin sections (800 A) were cut with a diatnond knife. These sections were collected on copper grids (200 mesh) and stained with uranyl acetate and lead citrate. They were studied with a Siemens IA microscope. Another series of ultra thin sections were demineralized by floating them on 2 % phosphotungstic acid for 30 minutes.
A N D
E G E L B E R G
Fig. 3. SEM of tooth without macroscopically visible calculus. Nonetched specimen. This figure shows a periodontally diseased root surface with, what is apparently subclinical calculus formation (C), some residual bacteria (B), and pebbles (P). 3000X
root surfaces which were not root planed failed to produce any significant tnorphologic changes regardless of the presence or absence of initial calculus deposits. The non-etched specimens showed tremendous variability ranging from flat to completely pebble covered specimens. Acid application had no effect other than to decrease the prominence of the surface characteristics. The various eharacteristics were still easily distinguishable however.
Resuits
SEM observatiotis Acid application to periodontally diseased
Fig. 4. SEM of tooth after root planing. Non-etched specimen. A flat, smooth (FS) surface. 3000X
E F F E C T S
C F C I T R I C
A C I D
C N D I S E A S E D
Fig. 5. SEM of tooth after root planing. Non-etched specimen. By rotating the specimen the fractured surface, created when the tooth was split, can be studied. Dentinal tubules (DT) extending to the surface (RS) indicate that the cementum was completely removed during root planing. 1000X
Three of the four periodontally diseased specimens which appeared free of calculus visually showed calculus-like deposits wheti viewed with the SEM. There were also a few bacteria remaining in these areas. The calculus-like areas were honeycotnbed in shape (Fig. 3). Teeth After Root Platutig. Periodontally diseased root surfaces that were root planed revealed the satne results regardless of the
R O C T
S U R F A C E S
159
Fig. 7. SEM of tooth after root planing. Acid-etched specimen. Higher magnification of an area in Fig. 6 illustrating fibrillar structures of the surface. 14,000X
presence or absence of calculus initially. The control noji-etched specimens had a stnooth, flat, pebble free surface (Fig. 4). Examination of the fractured surface created when the specimens were split indicated that the cementum had been completely retnoved in all teeth (Fig. 5). Detitinal tubules could be seen extending to the surface although openings of those tubules were not readily apparent on the root surface (compare with Fig. 4). Acid etched speeimens showed a flat surface with frequent depressions and numerous fiber-like structures (Fig. 6). Higher magnification revealed a fibrous appearing surface with fiberlike projections extending above the surface (Fig. 7). Statistical Analysis. Results of the statistical analyses ate summarized m Table 1. With acid application there was a statistically significant increase in the number of depressions and libers in the root planed specimens. No other significant differences were observed.
Fig. 6. SEM of tooth after root planing. Aoid-etched specimen. Frequent depressions (D) and fiber-like (F) structures are evident. Compare with Fig. 4. 3000X
TEM observations Root planed specimens studied with the TEM showed that acid application produced a demineralized zone approximately
160
G A R R E T T ,
C R I G G E R
A N D
E G E L B E R G
Table I Statistical comparison of SEM observations between experimental (etched) and control (non-etched) root surfaces. Results of Student's t-test for paired observations. Group 1 Non Root (8 spec imens) Surface characteristics Pebbies, count Pebbles, grid Depressions, count Depressions, grid Smooth/flat, grid Fibers, grid
t
P
1.4
N.S. N.S. N.S. N.S. N.S. N.S.
1.3 1.7 2.0 1.0 1.1
Group II Root Planed (8 specimens)
t 1.2 1.1 3.1 4.0 1.6
41.2
P
N.S. N.S.
