The investigation of biocompatibility and apical microleakage of tricalcium phosphate based root canal sealers

The investigation of biocompatibility and apical microleakage of tricalcium phosphate based root canal sealers

0099-2399/97/2302-0105503.00/0 JOURNALOF ENDODONTfCS Copyright©1997 by The American Association of Endodontists Printed in U.S.A. VOL. 23, No. 2, FE...

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0099-2399/97/2302-0105503.00/0 JOURNALOF ENDODONTfCS Copyright©1997 by The American Association of Endodontists

Printed in U.S.A.

VOL. 23, No. 2, FEBRUARY1997

The Investigation of Biocompatibility and Apical Microleakage of Tricalcium Phosphate Based Root Canal Sealers Suzan Bilginer, DDS, PhD, i. Timur Esener, DDS, PhD, Figen S6ylemezo01u, MD, and All Muhtar Tiftik, DVM, PhD

The biocompatibility and apical microleakage of tricalcium phosphate based Sankin Apatite (SA) Type I, II, and III root canal sealers were investigated. Teflon tubes containing freshly mixed test materials were implanted in the subcutaneous tissue of mice. The observation periods were 24 h, 7, and 30 days, after which the areas of tissue reaction to the implanted materials were histopathologically analyzed. A dye-recovery, spectrophotometric method was used to evaluate apical microleakage. Results showed that the severity of tissue reaction among the tested materials decreased with time and at the end of the observation period both SA Type II and Type III were found more biocompatible than either Type I or Grossman's cement (GC). On the other hand, a fibrous tissue capsule was seen around the implants. There was no significant difference in spectrophotometrically measured leakage among teeth obturated with the test materials.

have been reported to be effective in healing mechanical perforations of the pulp chamber floor (6), pulp capping (7), and enhancement of bone fill after periapical surgical procedures (8). A great many researchers investigated these materials as a root canal sealer. Krell and Wefel (9) reported that under scanning electron microscopic analysis calcium phosphate cement (CPC) appears to be similar to GC in apical and dentinal tubule occlusions, adaptation, cohesion, and morphological appearance. Chohayeb et al. (10) in animal studies showed that CPC has a uniform and tight adaptation to the dentinal surfaces of the pulp chamber and root canal walls. On the other hand Krell and Madison (11) reported that CPC sealer did not provide as good an apical seal as GC. Sugawara et al (12) also evaluated the root canal sealing ability of two different formulations of CPC. They were compared with laterally condensed gutta-percha and GC. Both CPC preparations showed considerably less dye penetration than the gutta-percha and GC combination. In another study (2) a new hydroxyapatite root canal system filling was investigated and two formulas, designated 716 and 718, were tested by comparing their sealing abilities to those of a GC used with vertical condensation of warm gutta-percha. Bacterial challenges of the sealing abilities of the formulations and of a gutta-percha/GC combination showed that the formulations were not superior to the gutta-percha/sealer combination in their seal. In light of this information, this study was conducted to determine the biocompatibility and apical sealing ability of c~-tricalcium phosphate based SA Type I, Type II, and Type III.

Complete obturation of the root canal with an inert filling material and creation of a hermetic apical seal have been proposed as goals for successful endodoutic treatment. Grossman (l) has enumerated the requirements for the ideal root canal system filling materials, both sealer and solid core. These requirements, advanced many years ago, remain valid today. They include ease of introduction, adequate sealing both laterally and apically, impermeability to moisture, lack of shrinkage, radiopacity, and bacteriostasis. Materials must be nonirritating to apical tissue, not stain the teeth, and be easily sterilized and removed from the root canal system. Many different obturation techniques and materials have been introduced to increase the quality of the apical seat. Recently several root canal sealers composed of hydroxyapatite (HA) and related tricalcium phosphate (TCP) have been promoted (2, 3). Both HA and TCP appear to be biocompatible, because they do not produce an inflammatory response when placed in contact with bone or soft tissues (4, 5). Calcium phosphate biomaterials

MATERIALS AND METHODS The filling cements used in this study have the following chemical compositions: 1. Sankin Apatite Type I Powder: 80% a-TCP and 20% hydroxyapatite. Liquid: 25% polyacrylic acid, 75% water. 2. Sankin Apatite Type H Powder: 56% a-TCP, 14% hydroxyapatite, 30% iodoform. Liquid: 25% polyacrylic acid, 75% water. 3. Sankin Apatite Type III Powder: 80% a-TCP, 14% hydroxyapatite, 5% iodoform, 1% bismuth subcarbonate.

105

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Bilginer, et al.

Liquid: 25% polyacrylic acid, 75% water.

Biocompatibility Study One hundred twenty-four sterile teflon tubes, 10 mm long and 1.3 mm in internal diameter, were filled flush with freshly mixed SA Type I, Type II, Type III (Sankin Trading, Tokyo, Japan), or GC (Endoco Incorporated, Memphis, TN). They were immediately implanted into the subcutaneous connective tissue of 31 white female mice that weighed 35 g each. Each mouse received four implants. The test materials were handled aseptically and prepared according to the manufacturer's instruction. The excess material was carefully removed from the ends of the tube with sterile gauze. In addition 12 empty teflon tubes, equal in size, were also implanted into subcutaneous connective tissue of 3 white female mice and used as control group. Mice were anesthetized with intramuscular administration of Ketalar (50 mgr/ml, Parke-Davis), 0.001 g per 1.000 g body weight. After the mice were shaved and disinfected with 5% iodine in alcohol the dorsal skin of left and right shoulders and hips of animals was superficially perforated with a needle of the same diameter as the tubes. This allowed insertion of tubes beneath the superficial muscle layer at least 2 cm from the perforation zone. Care was taken to prevent smearing of the test materials on the outside areas of the tubes. At the end of each experimental period (24 h, 7, and 30 days) animals were killed by ether suffocation. The implants along with the surrounding tissues were removed and then fixed in 10% formalin solution. After fixation the tissues were processed for paraffin embedding and then longitudinally sectioned through the implants. Serial sections approximately 5/~m thick were obtained from each specimen and stained with hematoxylin and eosin. Seven of the 136 implants were discarded from the evaluation because of surgical and histological procedure problems. The histopathological appearance was assessed as to degree of inflammation, type of inflammation, and thickness of the fibrous capsule surrounding the implants. Degree of inflammation was classified as mild, moderate, and severe. Mild described small number of inflammatory cells (polymorphonuclear leukocytes, lymphocytes, macrophages, and plasma cells) and moderate, greater accumulation of inflammatory cells. An intense concentration of inflammatory cells indicated a severe reaction to the implant. The type of inflammation was defined as acute or chronic. Acute inflammation represented a predominance of polymorphonuclear leukocytes with few Chronic inflammatory cells (lymphocytes, macrophages or plasma cells) and chronic consisted of predominance of chronic inflammatory cells with few polymorphonuclear neutrophils. The results of the fibrous capsule were statistically analyzed using the one way of variance (ANOVA) and Least Significant Difference (LSD) test.

Apical Leakage Study Fifty-four human permanent teeth with single straight canals, stored in a 10% formalin solution following extraction, were used in the study. They were immersed in 5.25% sodium hypochlorite (NaOCI) for 24 h to remove soft tissue from root surfaces. After cleansing they were rinsed and stored in saline. Anatomical crowns of the teeth were removed with a high-speed fissure bur under water spray, and pulp tissues were extirpated with barbed broaches. To determine the working length of the root canal, a 4: 15K file

Journal of Endodontics

(Kerr, Romulus, MI) was passed into the root canal until it was visible at the apical foramen. One milimeter was subtracted from this length to establish the working length. Each root canal was serially prepared to a minimum size of a 4: 45K file at the apical seat and the rest of the canal was flared to a 4: 70K file using a conventional step-back technique. Hedstrom files were used to blend the apical and coronal canal preparations. During instrumentation 5.25% NaOCI was used for irrigation. The roots were kept in moist gauze during instrumentation and subsequent filling. The teeth were randomly divided into four experimental groups of 11 teeth each, plus a negative col,trol of 5 teeth and a positive control of 5 teeth. The root canals were dried with paper points and the apices were coated with sticky Wax. A 4: 25K type file was passed through the apices to stapEardize the area of root exposed to dye. The wax was removed circumferentially from 1 mm of the apical area. Five unfilled teeth completely coated with wax were used as negative controls to show that sticky wax was capable of preventing dye penetration. In group 1, 2, 3, and 4 entire root canals were obturated with lateral condensation of gutta-percha and experimental sealer of groups SA Type I, II, III, and GC. For the positive control group, 5 teeth were obturated with lateral condensation of gutta-percha without sealer. After the teeth were obturated, a temporary filling cement was placed over the coronal portion of every tooth. The cervical portion of the root was again coated with multiple coats of sticky wax. Following storage in 100% humidity at 37°C for 48 h, specimens were immersed in 2% methylene blue dye for 48 h. Teeth were then rinsed twice with water, and the wax coatings were removed. The root surfaces were inspected for surface dye. Dye leakage on the exterior surface of the roots was removed with a rubber wheel. The apical root end of the each tooth was cut off with a tapered carpide bur perpendicular to the canal until the tip of the gutta-percha was visible to eliminate the dye that remained in 1 mm of the delta area of the apex. For quantitative measurement of the amount of dye leakage, the roots were dissolved in 2 ml of 50% nitric acid for 48 h. The resulting solution was filtered and then centrifuged at 15,000 rpm for 15 min. The solution was analyzed using a spectrophotometer (Shimadzu - UV 2100, Japan). The concentration of methylene blue dye in the solution was determined by using a lineer regression curve formulated from the optical density readings of known dilutions of methylene blue dye. Data were statistically analyzed using the analysis of variance and Kruskall-Wallis test.

RESULTS

Biocompatibility All specimens underwent blinded examination by a single examiner who did not know which sealer or which period was being examined. Macroscopic examination showed that wound healing was satisfactory in all observation periods. Histopathological resuits are summarized in Table 1. At the end of the 24-h observation period the tissue reaction to SA Type II, Type III, and control (Teflon) groups was similar. A mild acute inflammatory reaction was observed in the adjacent tissue of these materials. A mild/moderate inflammatory response was observed in SA Type I, and a moderate acute inflammatory reaction was found in GC. After 7 days a mild/moderate inflammatory response with intensive polymorphonuclear leukocytes and many lymphocytes, macrophages, and plasma cells was observed in the SA Type II and

Vol. 23, No. 2, February

1997

Evaluation of tricalcium TABLE 1. S u m m a r y

phosphate

sealers

107

of the histological findings

TISSUE REACTION TYPE SEALERS

Experimental Period

No. o f

ACUTE

Specimens Mild

Sankin A p a t i t e Type I

24 h 7 days

Sankin Apatite T y p e II

30 d a y s 24 h 7 days

Sankin Apatite T y p e Ill

30 d a y s 24 h 7 days 30 d a y s

Grossman

Control Group (Teflon)

24 h 7 days 30 d a y s 24 h 7 days 30 d a y s

CHRONIC

Moderate

Severe

.

Severe

9

4

5

1

6

2

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--

--

13

--

--

--

9

4

--

9

8

1

.

8 12

--

__

k

9 7

7 3

1 3

11

--

--

--

9

4

5

.

2

.

Moderate

9

4

.

Mild

.

.

.

.

2

--

--

--

6

6

--

1 1

---

---

---

4

3

4 .

.

.

9

2

4

3

--

--

--

12

--

--

--

3

7

2

4

4

.

4 4

1 --

3 --

1

--

Type III specimens. Some necrotic tissue as well as the presence of macrophages was also observed in direct contact with these materials. A moderate inflammatory response was observed in SA Type I and control (Teflon) specimens. While necrosis was evident in half of the SA Type I specimens, the necrosis was present in only one specimen of the control group. Moderate/severe inflammatory response was observed in GC specimens. In addition, a fibrous capsule composed of young fibroblasts was also found around all of these materials. After 30 days all reactions were of a chronic nature with many lymphocytes, macrophages, and plasma cells. The general aspect of the reaction was that the inflammatory responses were much less intense. While mild/moderate inflammatory responses were observed in SA Type I, II, and III specimens, moderate inflammatory response in the GC specimens and mild inflammatory response in control (Teflon) specimens were evident. Some amount of necrosis was found in SA Type 1 and GC specimens. A fibrous capsule composed of young fibroblasts was seen around SA Type I specimens. SA Type II, Type III, GC and control (Teflon) implants were surrounded by a well-defined fibrous capsule with fibroblast, fibrocyte and collagen bundles (Fig. 1, A to E). Concerning the presence of fibrous capsules there was no statistically significant difference amount the test materials (p >

0.05). Apical Microleakage The apical microleakage results are summarized in Table 2. There was no significant difference in spectrophotometrically measured leakage among teeth obturated with the test materials (p > O.O5).

DISCUSSION The study of the biological properties of a root canal sealer is at present one of the major means of evaluating its suitability for clinical application. There have been many methods of testing materials over the years. Subcutaneous implantation of teflon tubes filled with materials is one of major methods. This methodology allows a precise control of the amount of the materials that will be

.

. . --

.

.

.

. 3

.

in contact with the tissues (13). In this study we used teflon tubes to eliminate some methodological problems. Lack of tissue reactions adjacent to tubes indicated that the reactions at the tube ends were related to the toxicity of the endodontic materials and not to the movement or the surface characteristics of the tubes. SA Type II and III, which contain iodoform, were found more biocompatible than Type I and GC. Iodoform is less toxic than eugenol in spite of its antiseptic and bacterisid properties. Curson (14) reported that the use of iodoform as a periapical bone dressing could inhibit proliferation of epithelium and speeds repair of the abcess cavity. Telli (15) reported that the cytotoxic and hemolytic effects of SA Types were less than the effects of Endomethasone, Dorifill, CRCS, Express, and Spad root canal sealers. However, he found a few damaged cells and granulation tissue in Type II specimens and related this to the iodoform constituent of the material. This contradicts our results that showed better biocompatible responses of SA Type II and III than responses of Type I. Chohayeb et al (10) evaluated CPC as a root canal sealer-filler in beagle dogs. Histological evaluation of the periradicular tissues of CPC materials showed variable degrees of inflammatory response after 62 days. Hong et al (16) also evaluated CPC, GC, and Sargenti N2 in root canals of monkeys' incisors. The reactions to GC were milder than the reactions to N2 but persisted throughout the observation period. The CPC treated animals showed mild tissue irritation after 1 month, but thereafter the adverse tissue reactions were minimal. The presence and thickness of the capsule have been used as a measure of the tissue tolerance to implanted materials (17). The capsules around the implants in this study were poorly organized. They became more organized and more definite with time. A histopathological investigation was made to estimate the effect of a-TCP upon periapical wound healing by Shoji et al (18). A thick hyalinized fibrous tissue was observed around the extruded a-TCP at 8 weeks after the filling. The acute reactions were later reduced and converted into a chronical stage and surrounded by a fibrous capsule. The results of our study confirm previous findings of studies that evaluated calcium phosphate based root canal sealers (10, 16). The success of modem endodontic therapy also relies on establishing a canal that can be adequately obturated without any apical

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Bilginer, et al.

Journal of Endodontics

FIG 1. Tissue reaction to implanted materials at the end of the tubes after 30 days of implantation. A. SA Type I specimen; moderate chronic inflammation (a) and fibrous connective tissue capsule (b) (hematoxylin and eosin; original magnification × 40). B. SA Type II specimen; moderate chronic inflammation (a) and dense fibrous connective tissue capsule (b) (hematoxylin and eosin; original magnification x 200). C. SA Type III specimen; severe chronic inflammation (a) (hematoxylin and eosin; original magnification × 40). D. Grossman sealer; severe chronic inflammation (a) and fibrous connective tissue capsule (b) (hematoxylin and eosin; original magnification × 40). E. Teflon (control) specimen; mild chronic inflammation (a) (hematoxylin and eosin; original magnification × 40).

leakage. Apical leakage studies have been published in large numbers over the years. Materials used to determine the leakage are usually dyes such as methylene blue. Because the actual volume of dye leakage into the root canal system can be calculated by using spectrophotometric analysis (19), we used this method in our work. This method provides for a direct quantitative measurement of the material leaking into the radicular space or spaces (19).

The results of our study showed that SA root canal sealers had minimal dye penetration and no statistically significant difference was found between GC and SA sealers. This result is similar to the results of Barkhordar. Barkhordar et al. (3) reported that Sealapex had the best sealing ability, followed by SA Type II, Type I, IlI, Kerr, and Roth's sealer. But no statistically significant difference was found between SA sealers and Sealapex. Results from our

Evaluation of tricalcium phosphate sealers

Vol. 23, No. 2, February 1997 "FABLE2. Volumetric evaluation of leakage SEALERS Sankin Apatite Type I Sankin Apatite Type II Sankin Apatite Type III Grossman (+) Control G r o u p ( - ) Control G r o u p

No. o f Specimens

Min-Max

11

0.00-14.43

0.45

2.31 ± 1.32

11

0.00-13.14

0.28

1.48 _ 1.17

11

0.00-13.38

0.11

2.78 ± 1.48

11 5 5

0.00-01.52 3.13 ± 20.86 0.00 ± 0.28

0.24 16.6 0.04

0.41 ± 0.14 12.51 ± 3.54 0.09 ± 0.05

Median

M e a n ± SD

study showed essentially the identical trend; there was not any significant difference between SA root canal sealers and GC. Dr. Bilginer is a Research Assistant, Department of Endodontics, Selguk University, Faculty of Dentistry, Konya; Dr. Esener is Professor, Department of Endodontics, Selguk University, Faculty of Dentistry, Konya; Dr. S6ylemezo~lu is an Instructor, Department of Pathology, Hacettepe University, Faculty of Medicine, Ankara; Dr. Tiftik is an Associate Professor, Department of Biochemistry, Selguk University, Faculty of Veterinary Medicine, Konya. Address requests for reprints to Suzan Bilginer, Selguk Universitesi Di~hekimli~i FakiJltesi, Endodonti Bilim Dalt, Kamp0s, 42079, Konya-Turkey.

References 1. Grossman LI, Olivet S, DeI-Rio CE. Endodontic practice. 1 lth ed. Philadelphia: Lea & Fabiger, 1988. 2. White JM, Goodis HE. In vitro evaluation of an hydroxyapatite root canal system filling material. J Endodon 1991 ;17:561-6. 3. Barkhordar RA, Stark MM, Soelberg K. Evaluation of the apical sealing ability of apatite root canal sealer. Quintessence Int 1992;23:515-8.

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4. Costantino PD, Friedman CD, Jones K, Chow LC, Pelzer HJ, Sisson GA. Hydroxyapatite cements. I. Basic chemistry and histologic properties. Arch Otolaryngol Head Neck Surg 1991 ;117:379-84. 5. Saffar JL, Colombier ML, Detienville R. Bone formation in tricalcium phosphate-filled periodontal intrabony lesions, Histologic Observations in humans. J Periodonto11990;61:209-16. 6. Himel VT, Brady J, Weir J. Evaluation of repair of mechanical perforations of the pulp chamber floor using biodegradable tricalcium phosphate or calcium hydroxide. J Endodon 1985;11:161-5. 7. Jaber L, Mascres C, Donohue WB. Reaction of the dental pulp to hydroxyapatite. Oral Surg Oral Med Oral Pathol 1992;73:92-8. 8. Barkhordar RA, Meyer JR. Histologic evaluation of a human periapical defect after implantation with tricalcium phosphate. Oral Surg Oral Med Oral Pathol 1986;61:201-6. 9. Krell KF, Wefel JS. A calcium phosphate cement root canal sealerscanning electron microscopic analysis. J Endodon 1984;10:571-6. 10. Chohayeb AA, Chow LC, Tsaknis PJ. Evaluation of calcium phosphate as a root canal sealer-filler material. J Endodon 1987;13:384-7. t 1. Krell KV, Madison S, Comparison of apical leakage in teeth obturated with a calcium phosphate cement or Grossman's cement using lateral condensation. J Endodon 1985;11:336-9. 12. Sugawara A, Chow LC, Takagi S, Chohayeb H. In vitro evaluation of the sealing ability of a calcium phosphate cement when used as a root canal sealer-filler. J Endodon 1990;16:162-5. 13. Olsson 13, Sliwkowski A, Langeland K. Subcutaneous implantation for the biological evaluation of endodontic materials. J Endodon 1981 ;7:355-69. 14. Curson I. Root canal filling. 13r Dent J 1966;9:424-8. 15. Telli C. Kalsiyum fosfat esash kanal dolgu maddeleri olan Sankin Apatite Tip t, Tip it ve Tip Ill'tin sitotoksik, hemolitik ve antibaktedyel etkilerin!~n beg deOi~ik kanat dolgu maddesi lie kiyaslamah olarak ara~t=rtlmast, H.U. SaOhk Bilimleri Enstit0s0 Doktora Tezi 1991. 16. Hong VC, Wang IT, Hong CY, 13rown WE, Chow LC. The periapical tissue reactions to a calcium phosphate cement in the teeth of monkeys. J 13iomed Mater Res 1991;25:485-98. 17. Cleary PT, Newton CW, Marrison SW, Kafrawy AH. Histological examination of paraformaldehyde-exposed giJtta-percha implanted in rats. J Endodon 1992;18:63-7. 18. Shoji S, Ishtkawa J, Ebina T, Yamaki K, Horiuchi H. Application of c~-tricalcium phosphate ceramics to endodontics, 1. Application to root canal fillings. Jpn J Conserv Dent 1984;27:253-60. 19. Johnson WT, Zakariasen KL. Spektrophotometric analysis of microleakage in the fine curved canals found in the mesial roots of mandibular molars. Oral Surg 1983;56:305-9.

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