0099-2399/89/1604-0162/$02.00/0 JOURNAL OF ENDODONTICS Copyright 9 1990 by The American Association of Endodontists
Printed in U_S.A. VOL. 16, NO. 4, APRIL 1990
In Vitro Evaluation of the Sealing Ability of a Calcium Phosphate Cement When Used as a Root Canal Sealer-Filler Akiyoshi Sugawara, DDS, PhD, Laurence C. Chow, PhD, Shozo Takagi, PhD, and Hanan Chohayeb, DDS
A calcium phosphate cement (CPC) was examined for its ability to seal the root canal when used as a sealer-filler. Extracted human teeth were divided into three groups. Root canals were filled with either CPC paste containing dicalcium phosphate anhydrous (group 1), CPC containing dicalcium phosphate dihydrate (group 2), or gutta-percha points sealed with Grossman's cement (group 3). After filling, all specimens were kept in 100% humidity for 1 day, immersed in a CaPO4 solution or distilled water at 37~ for 1 wk, and then immersed in 1% Poly-R dye solution at 37~ for 1 wk, after which they were rinsed and sectioned longitudinally for microscopic examination. Specimens in groups 1 and 2, especially those aged in the CaPO4 solution showed considerably less dye penetration than those in group 3. The good sealing ability of the CPC against dye penetration in vitro suggests that it may provide an adequate seal of the canal without a separate sealer.
filling was significantly improved by using glycerine, which tends to ease the extrudability of CPC, as the liquid phase. In a recent in vitro study, Krell and Madison (5) compared the extent of apical leakage in teeth obturated with laterally condensed GP with those sealed with CPC or GC. They found that CPC permitted greater dye penetration than did GC. We hypothesized that the relatively poor performance of CPC might be due in part to the following experimental conditions used in the above study: 1. Teeth were placed in methylene blue dye solutions to test the sealing ability of the cement "immediately" after the treatments. Thus, as Krell and Madison. (5) indicated, the CPC may not have been allowed to set fully. 2. Fully cured CPC is a semiporous solid containing fine pores with diameters estimated to be in the order of 0.001 to 0.01 um (6). Although such a material is most likely to be impervious to bacterial infiltration, it is penetrable by small molecules such as methylene blue used in the Krell and Madison (5) study. Polymeric dyes that have dimensions closer to those of many bacteria and do not readily penetrate into the root dentin might be a more suitable penetrant for studies on the sealing ability of CPC. In light of these two factors, this study was conducted to determine the apical sealing ability of CPC when tested with a higher molecular dye than previously reported.
A setting cement consisting solely of calcium phosphate compounds was recently reported (1). The major components of this calcium phosphate cement (CPC) are finely ground particles of tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) or dicalcium phosphate dihydrate (DCPD). The cement has been shown to be compatible with hard and soft tissues (2) and capable of setting into a hard mass in the presence of body fluids. In an in vitro study of CPC as a root canal sealer, Krell and Wefel (3) reported that under scanning electron microscopic analysis CPC appeared to be similar to Grossman's cement (GC) sealer in apical and dentinal tubule occlusions. CPC also seemed to have an affinity for the canal walls and preferentially adhered to the dentinal surfaces. Results from an animal study conducted by Chohayeb et al. (4) showed that the cement filler had better adaptation to the canal walls than GC and gutta-percha (GP). When voids did appear, however, they seemed to be due to problems associated with the process of delivering the material into the canal. The
M A T E R I A L S AND M E T H O D S The compositions of the two CPC mixtures used in this study are shown in Table 1. TTCP was prepared by heating an equimolar mixture of CaHPO4 and CaCO3 at 1500~ for 24 h (7). All other ingredients of the mixtures were commercially available reagent grade chemicals. The TTCP, DCPA, DCPD, and hydroxyapatite were first ground separately in ethanol in a centrifugal ball mill (model P6; International Equipment, Needham Heights, MA) to obtain particle sizes in the range of 1 to 2 urn. These components were then combined in a blender (Micro Mill 1-7882; Bel-Art Product, Pequannock, N J) according to the desired composition for the cement. The compositions of the two CPCs corresponded to equimolar mixtures of the TTCP and DCPA for CPC-A and TTCP and DCPD for CPC-D. In each mixture 2.8 wt% hydroxyapatite was present as a seed to facilitate the cement 162
Vol. 16, No. 4, April 1990
CaPO4 Root Canal Sealer-Filler
setting reaction. A small amount of NaF also was present in these mixtures (Table 1). Forty-two single-rooted teeth were chemomechanically prepared according to standard endodontic procedure. Stepback preparation was completed to the size of#80 K-type file. After the preparation, root canals were irrigated with 1.0% H202 and 2.5% NaOC1. The teeth were then randomly divided into three groups. Fourteen teeth in group 1 were filled with CPC-A paste. Fourteen teeth in group 2 were filled with CPC-D paste. In both groups 1 and 2, CPC powder was mixed with glycerine (powder to liquid ratio, 1.0 g:0.4 ml) for 90 s. Glycerin was used as a vehicle for the CPC paste because it does not react with the calcium phosphate components of the cement. Thus, the CPC-glycerin pastes would remain soft for an indefinite period, and provide the operator with as much working time as needed. The paste was injected into the canals using a blunt 19-gauge needle attached to a syringe until a slight amount of CPC extruded from the foramen. The cement paste was then allowed to harden in the canals where it absorbed the water needed for the setting reaction through the dentinal tubules and the apical foramen when the teeth were placed in 100% humidity as described below. Fourteen teeth in group 3 were filled with #80 master G P cones, accessory cones, and GC as the sealer using lateral and vertical condensation with spreaders and pluggers. All master cones were fitted to within 1 m m from the root apex before cementation. After filling, the coronal side of the root was coated with sticky wax, but all other surfaces of the root were left uncoated. To obtain large and relatively constant apical openings for all specimens, the apical end of the root in each specimen was gently ground by hand on sandpaper (#400 grit) wetted slightly with water until about 2 m m was removed and the filling materials in the stepback area of the canal were exposed. All specimens were then kept in 100% humidity at 37~ for 1 day. Krell and Wefel (3) incubated the apex of the filled specimens in calf serum to simulate the effects of a physiological solution on solubility of the sealer. In this study a CaPO4 solution, having a composition of 1 ppm F, 1.5 mM/1 CaCI2, 0.9 mM/l KH2PO4, 50 mM/l HEPES, 130 mM/l KCI (pH 7.0), which is similar to the inorganic composition of ultrafiltered serum (8), was used. Half the specimens in each group were stored in 4 ml of this p H 7.0 CaPO4 solution for 1 wk, and the other half were stored in 4 ml of distilled water at 37~ for 1 wk. The CaPO4 solution or water was changed every 2 days. In preliminary studies the infrared spectra of the CPC fillers recovered from the samples indicated the absence of glycerin (unpublished data). It therefore appears
that, during incubation in water or the CaPO4 solution, the glycerin in CPC-filled specimens leaches out of the filler completely. Powder X-ray diffraction analysis confirmed that the final product of the CPC material was hydroxyapatite whether or not glycerin was present initially. After removal from the incubation solutions, all specimens were rinsed with distilled water for 30 s and then immersed in 4 ml of 1% Poly-R (Sigma, St. Louis, MO) dye solution buffered (with KOH) to pH 7.0 at 37~ for 1 wk. The molecular weight of Poly-R was 8000 daltons, and the estimated effective diameter was below 20 A because the dye solution easily passed an ultrafiltration membrane with a nominal pore size of 20 A. All specimens were then rinsed with distilled water for 1 min and sectioned longitudinally using a water-cooled diamond blade (Isomet; Buehler Ltd, Lake Bluff, IL) for visual and microscopic (x6 to x50) examinations. The distance o f dye penetration into both the filler-canal wall interface and the filler material itself were measured using a microscope eyepiece reticle. Duncan's method of multiple comparison of means (9) was used to determine the significance of the differences between the groups. The data were also examined by a two-way factorial model with the filler materials and incubation solutions as main effects. To observe the filler adaptation to the canal walls, three specimens from each group were taken coated with gold for scanning electron microscopic examination. RESULTS The specimens obturated with GP and sealed with GC, and immersed in either water or CaPO4 solution, appeared to have good adaptation to the canal walls under visual inspection. However, microscopic observations showed dye penetration along the entire filler-canal wall interface (>10 mm). Conversely, no dye penetration into the G P material was observed. Because of the nature of the results, the data from these two groups were not included in the statistical analysis of the results from the CPC-filled groups. The apical leakage observed in the CPC-filled groups is given in Table 2. Most specimens filled with CPC-A followed by immersion in the CaPO4 solution showed no dye penetration into the filler-canal wall interface. The mean depth (+ SD) of penetration was 0.15 + 0.18 m m (Table 2). The specimens in this group showed no discernible dye penetration into the filling material itself. The specimens filled with CPCA followed by immersion in distilled water showed a greater TABLE 2. Apical leakage observed in various experimental groups
TABLE 1. Composition of CPC mixtures
Cement Mixture
Compound
Weight (%)
CPC containing DCPA
Ca4(PO4)20 CaHPO4 Ca~(PO4)3OH NaF Ca4(PO4)20 CaHPO4.2H20 Cas(PO4)3OH NaF
70.72 26.28 2.80 0.19 66,24 30.77 2.81 0.i6
CPC containing DCPD
163
Sealer-Filler
Incubation Solution
Distance of Dye Penetration (mm)
Interface CPC-A CPC-D CPC-A CPC-D
CaPO4 CaPO4 H20 H20
Filler Material
0.15+0.18"J 0.01 _+0.01"[ 1.75___1.26 J 0.60 + 0.69 1 2.81 _+ 0.85 J 1.60_+ 1.09 4.00___ 0.51 J 1.86 + 1.44
* Mean_+SD; n = 7 in all cases. 1 Differencesin meansconnectedby a verticallineare not statisticallysignificant(p > 0.05).
164
Sugawara et al.
Journal of Endodontics
dye penetration into the interface (2.81 +-- 0.85 mm) as well as into the filling material (1.60 4- 1.09 ram). The specimens filled with CPC-D and immersed in the CaPO4 solution showed a greater dye penetration into both the interface and the filler than the corresponding specimens in the CPC-Afilled group. Analysis of the data using Duncan's multiple comparison test indicated that the mean dye penetration distance into the interface in each of the four CPC filled groups was significantly different (Table 2) with the CPC-A-filIed and the CaPO4 solution incubated group having the least leakage. A 2 • 2 factorial analysis of variance of the dye penetration data with the filling material and the incubation solution as the main effects revealed that leakage into the interface was dependent (p < 0.01) on both factors. The interaction of the effects from the material and the incubation solution was insignificant (p > 0.05). Similar analyses applied to the data on dye penetration into the filling material itself showed that only the CPC-Afilled and the CaPO, solution incubated group had significantly (p < 0.05) lower leakage; differences between all others were insignificant (p > 0.05). Two-way analysis of variance results suggested that dye penetration into the material was affected by incubation solution (p < 0.05) but not by type of CPC material (p > 0.05). There were no significant interactions between the two factors. The scanning electron microscopic observations showed that both CPC-A and CPC-D fillers appeared to adapt to the canal wall closely (Fig. 1A and B, respectively). Some gaps seen in the interface might be a result of loss of the CPC materials during sectioning of specimens. Under this magnification (xl,000) both CPC materials appeared to be an amorphous-looking paste containing microscopic voids and crevices. Higher magnification (x 10,000 to • 100,000) observations revealed that the CPC fillers are microporous solids comprised primarily of small rod-like crystals and some platelike crystals with diameter of the rods or thickness of the plates in the range of 0.03 to 0.1 ~zm. The GC sealer in the GC-sealed and GP-filled specimens also appeared to have excellent adaptation to the canal wall (Fig. IC). Although in some areas the GC had a smooth appearance, most GC sealer was composed of irregularly shaped particles with significant spaces between the particles.
DISCUSSION Results from a recent study (5) indicated that root canals filled with GP and GC showed significantly better resistance against methylene blue dye penetration than canals sealed with CPC. However, results from this study showed essentially the opposite trend: Canals filled with GP sealed with GC showed significantly greater leakage to the Poly-R dye than those filled with CPC. This is especially true for specimens immersed in the CaPO4 solution. In the present study sandpapering the apical region may have altered the interface between cement and canal wall, thereby increasing or reducing microleakage. However, since the samples in all treatment groups were similarly sandpapered, this should not have been the cause of the better sealing ability of the CPC-filled samples. Possible reasons for the improved sealing ability of the CPC
FIG 1. Scanning electron micrographs of longitudinally sectioned root canal specimens filled with CPC-A (A), CPC-D (B), and GP and GC (C). All specimens were incubated in the CAP04 solution for 1 wk. D, dentin. Bar represents 10 #m.
fillers found in the present study compared with that obtained previously (5) include: 1. Smaller CPC particles and a higher powder to liquid ratio were used. Because CPC was delivered into the canal by
Voh 16, No. 4, April 1990
injection through a 19-gauge needle, a relatively thick paste could be used. This should help lower the porosity of the filler and increase its resistance to dye penetration. 2. Incubation of the specimens first in 100% humidity and then in CaPO4 solution allowed adequate setting of CPC before the dye penetration test. 3. Poly-R dye used in the dye penetration test is several times larger than methylene blue used in the previous study. As a result, a dye penetration test using Poly-R is not as severe a test of sealing ability as the test using methylene blue. Since Poly-R is still about two orders of magnitude smaller than most bacteria, Poly-R dye penetration should still provide a realistic test for the sealing ability against the invasion of bacteria into the canals. Conversely, the performance of the GC sealer may have been worse in the present study due to a greater exposure of the sealer to the aqueous environment compared with the condition of the previous study (5). This is because in the present study the root surfaces of the specimens were not covered with wax so that liquid may penetrate through the root cementum and dentin as well as from the apical opening during incubation and immersion of specimens in the dye solution. Since GC is slightly soluble in water, this may have allowed some GC to be solubilized throughout the length of the canal. The enlargement of the apical opening may also cause a greater exposure of the GC to an aqueous environment. Three specimens from each treatment group were examined by scanning electron microscopic analysis. Because of the large variations in the appearance of the filler-sealer within a given sample, it was not possible to make quantitative assessment of the adaptation of the filler or sealer to the canal walls. Consequently, only qualitative observations were made, and these appear to be consistent with the dye penetration test results. The results of scanning electron microscopic analysis are also in general agreement with those reported by Krell and Wefel (3) in that both CPC and GC adapted well to the canal wall. The CPC materials used in the present study seemed to have a higher density and lower porosity than those used previously probably because a higher powder to liquid ratio was used. However, the GC sealer appeared to be more porous than previously observed, probably because of greater exposure of the GC sealer to the aqueous environment in the present study as described earlier. Despite the fact that the GC sealer was completely penetrated by the Poly-R dye in this study, it is a totally adequate sealer clinically. Therefore, the dye penetration test procedure used in the present study may still be too stringent a test even though it is less so than methylene blue. The good sealing ability observed in the CPC samples suggests that CPC should be as effective a sealer as GC, but it may not necessarily be better than GC clinically. CPC consists solely of CaPO4 compounds and forms hydroxyapatite as the final product. Thus, it should be highly compatible with the periradicular tissue, and extrusion of CPC beyond the apical foramen produced no discernible chronic inflammation in dogs (4) or monkeys (Y. C. Hong, unpublished data). This along with the injection method of
CaPO, Root Canal Sealer-Filler
165
delivering CPC as the filler-sealer may simplify the root canal filling procedure. CPC may also be a more suitable material for roots with open apex, split canals, and so forth, where the traditional canal filling materials do not produce satisfactory results. Further studies would be warranted to determine if CPC can be a useful sealer or sealer-filler combination in terms of other requirements of an acceptable endodontic filling material. CONCLUSION The ability of two CaPO4 cements used as root canal sealerfiller to prevent Poly-R dye penetration procedure was compared with that of GC and GP. The CaPOn cements were superior under the experimental conditions tested. Certain commercial materials and equipment are identified in this article to specify the experimental procedure. In no instance does such identification imply recommendation or endorsement by the National Institute of Standards and Technology or the American Dental Association Health Foundation or that the material or equipment identified is necessarily the best available for the purpose. This investigation was supported, in part, by USPHS Research Grant DE05030 to the American Dental Association Health Foundation from the National Institutes of Health-National Institute of Dental Research and is part of the dental research program conducted by the National Institute of Standards and Technology in cooperation with the American Dental Association Health Foundation. The authors are grateful to Mr. D. Blackbum and Mr. D. Kauffrnan of the National Institute of Standards and Technology for preparation of tetracalcium phosphate and to Hitachi Scientific Instruments (Gaithersburg, MD) for the use of a scanning electron microscope. Drs. Sugawara, Chow, Takagi, and Chohayeb are affiliated with Paffenbarget Research Center, American Dental Association Health Foundation, National Institute of Standards and Technology, Galthersburg, MD. Address requests for reprints to Dr. Shozo Takagi, ADAHF Paffenbarger Research Center, National Institute of Standards and Technology, Building 224, Room A153, Galthersburg, MD 20899. Dr. Sugawara's current address is Nihon University, School of Dentistry, Tokyo, Japan. Dr. Chohayeb's current address is York Hospital, York, PA 17403.
References 1. Brown WE, Chow LC. A new calcium phosphate setting cement. J Dent Res 1983;62:672. 2. Gruninger SE, Slew C, Chow LC, O'Young A, Ts'ao NK, Brown WE. Evaluation of the biocompatibility of a new calcium phosphate setting cement. J Dent Res 1984;63:200. 3. Krell KV, Wefel JS. A calcium phosphate cement root canal sealerscanning electron microscopic analysis. J Endodon 1984; 10:571-6. 4. Chohayeb AA, Chow LC, Tsaknis P. Evaluation of calcium phosphate as a root canal sealer-filler material. J Endodon 1987;13:384-7. 5. Krell KV, Madison S. Comparison of apical leakage in teeth obturated with a calcium phosphate cement and Grossman's cement using lateral condensation. J Endodon 1985;11:336-9. 6. Chow LC, Takagi S, Sugawara A, Eanes ED, Heywood BR. X-ray diffraction and electron microscopic characterization of calcium phosphate cement setting reactions. J Dent Res 1987;66:190. 7. Brown WE, Epstein EF. Crystallography of tetracalcium phosphate. J Res Nat Bur Stand 1965;69A:547-51. 8. Eidelman N, Chow LC, Brown WE. Calcium phosphate saturation level in ultrafiltered serum. Calcif Tissue Int 1987;40: 71-8. 9. Wall FJ. Statistical data analysis handbook. New York: McGraw-HUl, 1986:4.1-4.25.
Printed in U_S.A. VOL. 16, NO. 4, APRIL 1990
In Vitro Evaluation of the Sealing Ability of a Calcium Phosphate Cement When Used as a Root Canal Sealer-Filler Akiyoshi Sugawara, DDS, PhD, Laurence C. Chow, PhD, Shozo Takagi, PhD, and Hanan Chohayeb, DDS
A calcium phosphate cement (CPC) was examined for its ability to seal the root canal when used as a sealer-filler. Extracted human teeth were divided into three groups. Root canals were filled with either CPC paste containing dicalcium phosphate anhydrous (group 1), CPC containing dicalcium phosphate dihydrate (group 2), or gutta-percha points sealed with Grossman's cement (group 3). After filling, all specimens were kept in 100% humidity for 1 day, immersed in a CaPO4 solution or distilled water at 37~ for 1 wk, and then immersed in 1% Poly-R dye solution at 37~ for 1 wk, after which they were rinsed and sectioned longitudinally for microscopic examination. Specimens in groups 1 and 2, especially those aged in the CaPO4 solution showed considerably less dye penetration than those in group 3. The good sealing ability of the CPC against dye penetration in vitro suggests that it may provide an adequate seal of the canal without a separate sealer.
filling was significantly improved by using glycerine, which tends to ease the extrudability of CPC, as the liquid phase. In a recent in vitro study, Krell and Madison (5) compared the extent of apical leakage in teeth obturated with laterally condensed GP with those sealed with CPC or GC. They found that CPC permitted greater dye penetration than did GC. We hypothesized that the relatively poor performance of CPC might be due in part to the following experimental conditions used in the above study: 1. Teeth were placed in methylene blue dye solutions to test the sealing ability of the cement "immediately" after the treatments. Thus, as Krell and Madison. (5) indicated, the CPC may not have been allowed to set fully. 2. Fully cured CPC is a semiporous solid containing fine pores with diameters estimated to be in the order of 0.001 to 0.01 um (6). Although such a material is most likely to be impervious to bacterial infiltration, it is penetrable by small molecules such as methylene blue used in the Krell and Madison (5) study. Polymeric dyes that have dimensions closer to those of many bacteria and do not readily penetrate into the root dentin might be a more suitable penetrant for studies on the sealing ability of CPC. In light of these two factors, this study was conducted to determine the apical sealing ability of CPC when tested with a higher molecular dye than previously reported.
A setting cement consisting solely of calcium phosphate compounds was recently reported (1). The major components of this calcium phosphate cement (CPC) are finely ground particles of tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) or dicalcium phosphate dihydrate (DCPD). The cement has been shown to be compatible with hard and soft tissues (2) and capable of setting into a hard mass in the presence of body fluids. In an in vitro study of CPC as a root canal sealer, Krell and Wefel (3) reported that under scanning electron microscopic analysis CPC appeared to be similar to Grossman's cement (GC) sealer in apical and dentinal tubule occlusions. CPC also seemed to have an affinity for the canal walls and preferentially adhered to the dentinal surfaces. Results from an animal study conducted by Chohayeb et al. (4) showed that the cement filler had better adaptation to the canal walls than GC and gutta-percha (GP). When voids did appear, however, they seemed to be due to problems associated with the process of delivering the material into the canal. The
M A T E R I A L S AND M E T H O D S The compositions of the two CPC mixtures used in this study are shown in Table 1. TTCP was prepared by heating an equimolar mixture of CaHPO4 and CaCO3 at 1500~ for 24 h (7). All other ingredients of the mixtures were commercially available reagent grade chemicals. The TTCP, DCPA, DCPD, and hydroxyapatite were first ground separately in ethanol in a centrifugal ball mill (model P6; International Equipment, Needham Heights, MA) to obtain particle sizes in the range of 1 to 2 urn. These components were then combined in a blender (Micro Mill 1-7882; Bel-Art Product, Pequannock, N J) according to the desired composition for the cement. The compositions of the two CPCs corresponded to equimolar mixtures of the TTCP and DCPA for CPC-A and TTCP and DCPD for CPC-D. In each mixture 2.8 wt% hydroxyapatite was present as a seed to facilitate the cement 162
Vol. 16, No. 4, April 1990
CaPO4 Root Canal Sealer-Filler
setting reaction. A small amount of NaF also was present in these mixtures (Table 1). Forty-two single-rooted teeth were chemomechanically prepared according to standard endodontic procedure. Stepback preparation was completed to the size of#80 K-type file. After the preparation, root canals were irrigated with 1.0% H202 and 2.5% NaOC1. The teeth were then randomly divided into three groups. Fourteen teeth in group 1 were filled with CPC-A paste. Fourteen teeth in group 2 were filled with CPC-D paste. In both groups 1 and 2, CPC powder was mixed with glycerine (powder to liquid ratio, 1.0 g:0.4 ml) for 90 s. Glycerin was used as a vehicle for the CPC paste because it does not react with the calcium phosphate components of the cement. Thus, the CPC-glycerin pastes would remain soft for an indefinite period, and provide the operator with as much working time as needed. The paste was injected into the canals using a blunt 19-gauge needle attached to a syringe until a slight amount of CPC extruded from the foramen. The cement paste was then allowed to harden in the canals where it absorbed the water needed for the setting reaction through the dentinal tubules and the apical foramen when the teeth were placed in 100% humidity as described below. Fourteen teeth in group 3 were filled with #80 master G P cones, accessory cones, and GC as the sealer using lateral and vertical condensation with spreaders and pluggers. All master cones were fitted to within 1 m m from the root apex before cementation. After filling, the coronal side of the root was coated with sticky wax, but all other surfaces of the root were left uncoated. To obtain large and relatively constant apical openings for all specimens, the apical end of the root in each specimen was gently ground by hand on sandpaper (#400 grit) wetted slightly with water until about 2 m m was removed and the filling materials in the stepback area of the canal were exposed. All specimens were then kept in 100% humidity at 37~ for 1 day. Krell and Wefel (3) incubated the apex of the filled specimens in calf serum to simulate the effects of a physiological solution on solubility of the sealer. In this study a CaPO4 solution, having a composition of 1 ppm F, 1.5 mM/1 CaCI2, 0.9 mM/l KH2PO4, 50 mM/l HEPES, 130 mM/l KCI (pH 7.0), which is similar to the inorganic composition of ultrafiltered serum (8), was used. Half the specimens in each group were stored in 4 ml of this p H 7.0 CaPO4 solution for 1 wk, and the other half were stored in 4 ml of distilled water at 37~ for 1 wk. The CaPO4 solution or water was changed every 2 days. In preliminary studies the infrared spectra of the CPC fillers recovered from the samples indicated the absence of glycerin (unpublished data). It therefore appears
that, during incubation in water or the CaPO4 solution, the glycerin in CPC-filled specimens leaches out of the filler completely. Powder X-ray diffraction analysis confirmed that the final product of the CPC material was hydroxyapatite whether or not glycerin was present initially. After removal from the incubation solutions, all specimens were rinsed with distilled water for 30 s and then immersed in 4 ml of 1% Poly-R (Sigma, St. Louis, MO) dye solution buffered (with KOH) to pH 7.0 at 37~ for 1 wk. The molecular weight of Poly-R was 8000 daltons, and the estimated effective diameter was below 20 A because the dye solution easily passed an ultrafiltration membrane with a nominal pore size of 20 A. All specimens were then rinsed with distilled water for 1 min and sectioned longitudinally using a water-cooled diamond blade (Isomet; Buehler Ltd, Lake Bluff, IL) for visual and microscopic (x6 to x50) examinations. The distance o f dye penetration into both the filler-canal wall interface and the filler material itself were measured using a microscope eyepiece reticle. Duncan's method of multiple comparison of means (9) was used to determine the significance of the differences between the groups. The data were also examined by a two-way factorial model with the filler materials and incubation solutions as main effects. To observe the filler adaptation to the canal walls, three specimens from each group were taken coated with gold for scanning electron microscopic examination. RESULTS The specimens obturated with GP and sealed with GC, and immersed in either water or CaPO4 solution, appeared to have good adaptation to the canal walls under visual inspection. However, microscopic observations showed dye penetration along the entire filler-canal wall interface (>10 mm). Conversely, no dye penetration into the G P material was observed. Because of the nature of the results, the data from these two groups were not included in the statistical analysis of the results from the CPC-filled groups. The apical leakage observed in the CPC-filled groups is given in Table 2. Most specimens filled with CPC-A followed by immersion in the CaPO4 solution showed no dye penetration into the filler-canal wall interface. The mean depth (+ SD) of penetration was 0.15 + 0.18 m m (Table 2). The specimens in this group showed no discernible dye penetration into the filling material itself. The specimens filled with CPCA followed by immersion in distilled water showed a greater TABLE 2. Apical leakage observed in various experimental groups
TABLE 1. Composition of CPC mixtures
Cement Mixture
Compound
Weight (%)
CPC containing DCPA
Ca4(PO4)20 CaHPO4 Ca~(PO4)3OH NaF Ca4(PO4)20 CaHPO4.2H20 Cas(PO4)3OH NaF
70.72 26.28 2.80 0.19 66,24 30.77 2.81 0.i6
CPC containing DCPD
163
Sealer-Filler
Incubation Solution
Distance of Dye Penetration (mm)
Interface CPC-A CPC-D CPC-A CPC-D
CaPO4 CaPO4 H20 H20
Filler Material
0.15+0.18"J 0.01 _+0.01"[ 1.75___1.26 J 0.60 + 0.69 1 2.81 _+ 0.85 J 1.60_+ 1.09 4.00___ 0.51 J 1.86 + 1.44
* Mean_+SD; n = 7 in all cases. 1 Differencesin meansconnectedby a verticallineare not statisticallysignificant(p > 0.05).
164
Sugawara et al.
Journal of Endodontics
dye penetration into the interface (2.81 +-- 0.85 mm) as well as into the filling material (1.60 4- 1.09 ram). The specimens filled with CPC-D and immersed in the CaPO4 solution showed a greater dye penetration into both the interface and the filler than the corresponding specimens in the CPC-Afilled group. Analysis of the data using Duncan's multiple comparison test indicated that the mean dye penetration distance into the interface in each of the four CPC filled groups was significantly different (Table 2) with the CPC-A-filIed and the CaPO4 solution incubated group having the least leakage. A 2 • 2 factorial analysis of variance of the dye penetration data with the filling material and the incubation solution as the main effects revealed that leakage into the interface was dependent (p < 0.01) on both factors. The interaction of the effects from the material and the incubation solution was insignificant (p > 0.05). Similar analyses applied to the data on dye penetration into the filling material itself showed that only the CPC-Afilled and the CaPO, solution incubated group had significantly (p < 0.05) lower leakage; differences between all others were insignificant (p > 0.05). Two-way analysis of variance results suggested that dye penetration into the material was affected by incubation solution (p < 0.05) but not by type of CPC material (p > 0.05). There were no significant interactions between the two factors. The scanning electron microscopic observations showed that both CPC-A and CPC-D fillers appeared to adapt to the canal wall closely (Fig. 1A and B, respectively). Some gaps seen in the interface might be a result of loss of the CPC materials during sectioning of specimens. Under this magnification (xl,000) both CPC materials appeared to be an amorphous-looking paste containing microscopic voids and crevices. Higher magnification (x 10,000 to • 100,000) observations revealed that the CPC fillers are microporous solids comprised primarily of small rod-like crystals and some platelike crystals with diameter of the rods or thickness of the plates in the range of 0.03 to 0.1 ~zm. The GC sealer in the GC-sealed and GP-filled specimens also appeared to have excellent adaptation to the canal wall (Fig. IC). Although in some areas the GC had a smooth appearance, most GC sealer was composed of irregularly shaped particles with significant spaces between the particles.
DISCUSSION Results from a recent study (5) indicated that root canals filled with GP and GC showed significantly better resistance against methylene blue dye penetration than canals sealed with CPC. However, results from this study showed essentially the opposite trend: Canals filled with GP sealed with GC showed significantly greater leakage to the Poly-R dye than those filled with CPC. This is especially true for specimens immersed in the CaPO4 solution. In the present study sandpapering the apical region may have altered the interface between cement and canal wall, thereby increasing or reducing microleakage. However, since the samples in all treatment groups were similarly sandpapered, this should not have been the cause of the better sealing ability of the CPC-filled samples. Possible reasons for the improved sealing ability of the CPC
FIG 1. Scanning electron micrographs of longitudinally sectioned root canal specimens filled with CPC-A (A), CPC-D (B), and GP and GC (C). All specimens were incubated in the CAP04 solution for 1 wk. D, dentin. Bar represents 10 #m.
fillers found in the present study compared with that obtained previously (5) include: 1. Smaller CPC particles and a higher powder to liquid ratio were used. Because CPC was delivered into the canal by
Voh 16, No. 4, April 1990
injection through a 19-gauge needle, a relatively thick paste could be used. This should help lower the porosity of the filler and increase its resistance to dye penetration. 2. Incubation of the specimens first in 100% humidity and then in CaPO4 solution allowed adequate setting of CPC before the dye penetration test. 3. Poly-R dye used in the dye penetration test is several times larger than methylene blue used in the previous study. As a result, a dye penetration test using Poly-R is not as severe a test of sealing ability as the test using methylene blue. Since Poly-R is still about two orders of magnitude smaller than most bacteria, Poly-R dye penetration should still provide a realistic test for the sealing ability against the invasion of bacteria into the canals. Conversely, the performance of the GC sealer may have been worse in the present study due to a greater exposure of the sealer to the aqueous environment compared with the condition of the previous study (5). This is because in the present study the root surfaces of the specimens were not covered with wax so that liquid may penetrate through the root cementum and dentin as well as from the apical opening during incubation and immersion of specimens in the dye solution. Since GC is slightly soluble in water, this may have allowed some GC to be solubilized throughout the length of the canal. The enlargement of the apical opening may also cause a greater exposure of the GC to an aqueous environment. Three specimens from each treatment group were examined by scanning electron microscopic analysis. Because of the large variations in the appearance of the filler-sealer within a given sample, it was not possible to make quantitative assessment of the adaptation of the filler or sealer to the canal walls. Consequently, only qualitative observations were made, and these appear to be consistent with the dye penetration test results. The results of scanning electron microscopic analysis are also in general agreement with those reported by Krell and Wefel (3) in that both CPC and GC adapted well to the canal wall. The CPC materials used in the present study seemed to have a higher density and lower porosity than those used previously probably because a higher powder to liquid ratio was used. However, the GC sealer appeared to be more porous than previously observed, probably because of greater exposure of the GC sealer to the aqueous environment in the present study as described earlier. Despite the fact that the GC sealer was completely penetrated by the Poly-R dye in this study, it is a totally adequate sealer clinically. Therefore, the dye penetration test procedure used in the present study may still be too stringent a test even though it is less so than methylene blue. The good sealing ability observed in the CPC samples suggests that CPC should be as effective a sealer as GC, but it may not necessarily be better than GC clinically. CPC consists solely of CaPO4 compounds and forms hydroxyapatite as the final product. Thus, it should be highly compatible with the periradicular tissue, and extrusion of CPC beyond the apical foramen produced no discernible chronic inflammation in dogs (4) or monkeys (Y. C. Hong, unpublished data). This along with the injection method of
CaPO, Root Canal Sealer-Filler
165
delivering CPC as the filler-sealer may simplify the root canal filling procedure. CPC may also be a more suitable material for roots with open apex, split canals, and so forth, where the traditional canal filling materials do not produce satisfactory results. Further studies would be warranted to determine if CPC can be a useful sealer or sealer-filler combination in terms of other requirements of an acceptable endodontic filling material. CONCLUSION The ability of two CaPO4 cements used as root canal sealerfiller to prevent Poly-R dye penetration procedure was compared with that of GC and GP. The CaPOn cements were superior under the experimental conditions tested. Certain commercial materials and equipment are identified in this article to specify the experimental procedure. In no instance does such identification imply recommendation or endorsement by the National Institute of Standards and Technology or the American Dental Association Health Foundation or that the material or equipment identified is necessarily the best available for the purpose. This investigation was supported, in part, by USPHS Research Grant DE05030 to the American Dental Association Health Foundation from the National Institutes of Health-National Institute of Dental Research and is part of the dental research program conducted by the National Institute of Standards and Technology in cooperation with the American Dental Association Health Foundation. The authors are grateful to Mr. D. Blackbum and Mr. D. Kauffrnan of the National Institute of Standards and Technology for preparation of tetracalcium phosphate and to Hitachi Scientific Instruments (Gaithersburg, MD) for the use of a scanning electron microscope. Drs. Sugawara, Chow, Takagi, and Chohayeb are affiliated with Paffenbarget Research Center, American Dental Association Health Foundation, National Institute of Standards and Technology, Galthersburg, MD. Address requests for reprints to Dr. Shozo Takagi, ADAHF Paffenbarger Research Center, National Institute of Standards and Technology, Building 224, Room A153, Galthersburg, MD 20899. Dr. Sugawara's current address is Nihon University, School of Dentistry, Tokyo, Japan. Dr. Chohayeb's current address is York Hospital, York, PA 17403.
References 1. Brown WE, Chow LC. A new calcium phosphate setting cement. J Dent Res 1983;62:672. 2. Gruninger SE, Slew C, Chow LC, O'Young A, Ts'ao NK, Brown WE. Evaluation of the biocompatibility of a new calcium phosphate setting cement. J Dent Res 1984;63:200. 3. Krell KV, Wefel JS. A calcium phosphate cement root canal sealerscanning electron microscopic analysis. J Endodon 1984; 10:571-6. 4. Chohayeb AA, Chow LC, Tsaknis P. Evaluation of calcium phosphate as a root canal sealer-filler material. J Endodon 1987;13:384-7. 5. Krell KV, Madison S. Comparison of apical leakage in teeth obturated with a calcium phosphate cement and Grossman's cement using lateral condensation. J Endodon 1985;11:336-9. 6. Chow LC, Takagi S, Sugawara A, Eanes ED, Heywood BR. X-ray diffraction and electron microscopic characterization of calcium phosphate cement setting reactions. J Dent Res 1987;66:190. 7. Brown WE, Epstein EF. Crystallography of tetracalcium phosphate. J Res Nat Bur Stand 1965;69A:547-51. 8. Eidelman N, Chow LC, Brown WE. Calcium phosphate saturation level in ultrafiltered serum. Calcif Tissue Int 1987;40: 71-8. 9. Wall FJ. Statistical data analysis handbook. New York: McGraw-HUl, 1986:4.1-4.25.
