Biological Evaluation on Glass lonomer Cement HARUYUKI KAWAHARA, YOSHITSUGU IMANISHI, and HIROSHI OSHIMA Department of Biomaterials and Tissue Culture Center, Osaka Dental University, 1-4 7 Kyobashi, Osaka 540, Japan

Biological properties of a new dental cement of glass ionomer cement were compared with other types of conventional cement. The biological test was carried out by tissue culture method and animal examination. The culture cells showed weaker reaction to the glass ionomer cement than zinc oxide-eugenol or polycarboxylate cement. Pulp tissue reaction showed no significant difference between glass ionomer and zinc oxide-eugenol cement from in vivo experiments using monkeys. J Dent Res 58(3):1080-1086, March 1979

Introduction.

cement of Carbolit 100** and one zinc

oxide-eugenol cement of G-C's eugenol cement** were used in this study. The cyto-

toxic effect was evaluated on the basis of cell growth and morphological change.3A4 Test pieces were prepared in un-set cement, which is freshly-mixed, and set cement. The powder/liquid ratio and mixing were treated by the method according to the direction of the manufacturer. A test piece for the purpose of deciding cell growth was made with glass tubes (inside: 01.59 x 10 mm)*. Test cement of equal quantity was filled in one side of the tube, and the other side was sealed by white paraffin wax

Glass ionomer cement was developed for new dental cement by A. D. Wilson and B. E. Kent in 1969. The new cement is a powder- to make a constant condition of active area liquid material, the powder being mainly a of interface between the cement and cell culglass material of SiO2 and A12 03, the liquid ture medium. The white paraffin wax was not an aqueous solution of polyacrylic acid. cytotoxic in the preliminary experiments. When the powder and liquid are mixed, the The un-set test piece was used immeliquid erodes the glass powder surface, diately after the preparation. Test piece of and the metal ions of A13+, Ca2+, Na+, etc., set cement was stored for 24 hours at 370C are drawn from the powder by hydrogen and 100% humidity, after mixing. ions of carboxyl radical in the liquid. Each Test piece of morphological observation polyacrylic acid molecule reacts with metal was prepared on a coverglass. One drop of ions and, linked together into a network mixed cement from a dental explorer was structure, form a cement of insoluble gel put on the coverglass to make constant size and volume. The un-set and set test structure in water and set. According to the report of Wilson and piece were made by the method similar to Kent,1 and Kawahara, et al.,2 the new ce- that of the cell growth experiment. ment is apparently sufficient for practical The cells used were human pulp cells use in cementation, filling and fissure seal- (Hp strain cells) established from healthy ant. Glass ionomer cement in this experi- human pulp by H. Kawahara, Y. Imanishi ment was tested from the viewpoint of bio- and T. Nishida in 1976.5 Hp cells were logical requirements by means of cell cul- grown in Eagle's Basal medium containing ture, in vitro. Polycarboxylate cement was 10% calf serum. For deciding the cell growth activity, tested to compare with data of glass ionomer 4 x 104 cells suspended in 1 ml medium cement and discussed. were cultured in each small test tube (01 5 x 105 mm) with the test piece. The test tube was tightly closed by a silicone cup and inMaterials and methods. at 370C. Cell growth of parallel Two glass ionomer cements of Aspa* cubated with each test piece at days 2, 4 and and Fuji Ionomer**, one polycarboxylate cultures 7 was measured by cell nuclei counting, Received for publication March 6, 1978. using Burker Turk hemocytometer. Control

Accepted for publication June 5, 1978. *Amalgamated Dental Company, London. **GC Dental Industrial Corp., Japan. *Jintan Terumo Co., Ltd., Japan. 1080 Downloaded from jdr.sagepub.com at UCSF LIBRARY & CKM on April 25, 2015 For personal use only. No other uses without permission.

IONOMER CEMENT 58 NVo. 3 Vol.VGLASS

cells without any test piece were cultured by the same way as in the experimental culture. For the morphological observation, S X 105 cells suspended in 3 ml medium were inoculated to each plastic multidish (35 x 10 mm)* and cultured in the CO2 incubator at 370C. The cells made a monolayer of continuous cell sheet on the multidish after 24 hours' cultivation in normal medium (Fig. 1). A test piece of cement was attached to the center of the cell sheet on the bottom wall of the multidish. After 2 and 24 hours' incubation, cellular responses were observed with an inverted phase-contrast microscope (x 100)** and taken with a 16 mm movie camera ** In addition to tissue culture method, animal examination was used to evaluate the pulpal responses induced by glass ionomer and zinc oxide-eugenol cement as a control. Sixteen cavities with exposed pulp were made on the lavial surface of anterior teeth of four monkeys by using No. 201 diamond point**** with air turbine hand piece under adequate water coolant. The cements were directly filled in the cavities with exposed pulp. Monkeys were killed at 2 months after the filling, and histological specimens were made.

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Fig. I.-Normal Hp cell sheet in the control culture. eration caused by the un-set cement were observed in the morphological observation. Aspa, Fuji lonomer, Carbolit 100 and G-C's eugenol cement showed cytotoxic effects (Fig. 3). It appeared, however, that the cements of both Aspa and Fuji Jonomer have less cytotoxic action than Carbolit 100 or G-C's eugenol cement. Only the cells within about 200-300p from the test piece of Aspa and Fuji lonomer degenerated after 24 hours' administration, but no degeneration was observed in the long distance area from the test piece (Fig. 3a, 3b). Almost all cells around the test piece of Carbolit 100

Result. Cytotoxicity of un-set cement was recognized in all cements from the findings of the cell growth curve (Fig. 2). Glass ionomer cement of Aspa and Fuji lonomer slightly inhibited cell growth compared with the control, but the cells were growing as the culture day proceeded, as shown in the increasing cell growth curve after 4 days' culture. It appeared that two glass ionomer cements had a very mild cytotoxic effect under the un-set conditions. On the contrary, polycarboxylate cement of Carbolit 100 and zinc oxide-eugenol cement of G-C's eugenol cement strongly inhibited cell growth and had a strong cytotoxic effect, such that the inoculated cells were invariably damaged in the early stage of the experiment. Various types of cell degen-

*Linbro Scientific

c:

la] z

Co., Inc., Model FB-6TC,

USA.

**Nippon Kogaku K. K., Model M, Japan. **Bolex, H1I6RX-Matic, Switzerland. ***Shofu Dental Mfg. Co., Ltd., Japan. *

Fig. 2.-Effect of un-set cements upon the multiplication of Hp cells.

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IFig. 3.-Cell degeneration around the un-set cement of glass ionomer, polycarboxylate and zinc oxide-eugenol cement at 24 hours' cultivation. Mild reaction with a test piece of glass ionomer cements of Aspa (a) and F'uji lonomer (b) from which many small particles dispersed into the medium. Severe response in Carbolit 100 (c); severe change with normal cell form in eugenol cement (d).

and G-C's eugenol cement were apparently damaged and changed to round form with cytoplasmic shrinkage (Fig. 3c, 3d). The damaged cells were observed in the wider zone than that of Aspa and Fuji lonomer. It was indicated that the cytotoxicity of Carbolit 100 and G-C's eugenol cement was stronger than that of Aspa and Fuji lonomer under the un-set condition. In the case of set cement, there was no significant difference among the cell growth activities of Aspa, Fuji Ionomer cements and the control culture. But the cell growth curve of Carbolit 100 and G-C's eugenol cement did not show normal cell growth as shown in the control culture, and it decreased with the number of culture days (Fig. 4). The morphological changes of Aspa and Fuji lonomer cement showed no significant difference in the cytotoxicity compared with that of the control culture. The cells around both test pieces were still in a normal state after 2 hours' administration of the set cements. Oin the contrary, almost all of the cells around the set test pieces of Carbo-

lit 100 and G-C's eugenol cement degenerated. After 24 hours' cultivation, only the cells in the region of 1 0-20, from set cement of Aspa (Fig. 5a) and Fuji Jonomer (Fig. 5b) were slightly damaged. It was not recognized whether these damaged cells were still living or not. Other cells around the test piece were cultured in normal. On the contrary, in the culture with Carbolit 100 (Fig. Sc) or G-C's eugenol cement (Fig. 5d), the cells degenerated in the distance of 4.5-8.0 mnm and 8.5-14.0 mm from the set test piece. There is no significant difference between both glass ionomer cements of Aspa and Fuji Ionomer in the present experiment, although these cements showed milder cytotoxicity than carboxylate cemient of Carbolit 100 and G-C's eugenol cement. Pulp tissue response of the monkey teeth for zinc oxide-eugenol cemeint filling (Fig. 6a) showed bleeding, degeneration, round cell infiltration and hyperemia. Degeneration and reduced odontoblasts showed near the cement, but a normal odontoblastic layer

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GLASS IONOMER CEMENT

Vol. 58 No. 3

~~~~~~~~~~~~~~~~Control

S

O

2

4 CuItI,aUon time

7 da^y

Fig. 4.-Effect of set cements upon the multiplication of Hp cells at 24 hours after mixing.

was observed some distance from the cement. Round cell infiltration occurred in the pulp tissue area adjacent to the glass ionomer cement (Fig. 6b), and reduced odontoblasts were seen near the filling cement, with normal odontoblasts observed distant from the cement. Glass ionomer cement was not observed in the cavity after the decalcification treatment for histological specimens, because the cement was easily disintegrated by the Ca-chelation effect of EDTA solution. The pulpal reaction showed no significant difference between glass ionomer and zinc oxide-eugenol cement, which produced a mild response.

Discussion. Two glass ionomer cements of Aspa and Fuji lonomer have been tested to evaluate tissue irritability upon the pulp tissue by means of cell culture using Hp cells. Un-set glass ionomer cement showed a very mild cytotoxicity compared with polycarboxylate and zinc oxide-eugenol cement. It is suggested that irritation to the pulp of un-set glass ionomer cement is little evidence comparing with polycarboxylate and zinc oxideeugenol cement. The cytotoxicity of both cements decreased in the setting process after the mixing. In particular, the cytotox-

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icity of glass ionomer cement disappeared after setting, unlike polycarboxylate and zinc oxide-eugenol cement, which kept the cytotoxicity after the setting. It is revealed that glass ionomer cement has no irritant effect upon the living pulp, but polycarboxylate and zinc oxide-eugenol cement kept the irritating effect after setting. Glass ionomer cement has not only superior physical and chemical properties for dental cement2'6 but also good biological properties. It is revealed that glass ionomer cement is a more biocompatible material than other dental cements of zinc phosphate and epoxylate, as reported by H. Kawahara.7 It is necessary in particular to consider significant differences between the cytotoxic effects of both glass ionomer cement and polycarboxylate cement. The liquids of both glass ionomer and polycarboxylate cement are an aqueous solution of appropriate polyacrylic acids. It is considered that the difference in cytotoxicity between the cements may be caused by some elements in the powder. The powder of glass ionomer cement contains SiO2, A12 03, CaF2 and Na3AlF6. The cement is made by mixing the powder and the liquid. Free metal ions of A13+, Ca2+ and Na+ are dissolved from the powder to the liquid. The A13+, Ca2+ and Na+ ions may disperse into the culture medium from the un-set test piece of glass ionomer cement. However, A13+, Ca2+ and Na+ ions are considerably nonirritant or non-toxic for living cells or tissues, as reported by H. Kawahara. SiO2, which is the base substance of glass powder, does no damage. Therefore, the main factor for the mild cytotoxicity of un-set cement of glass ionomer may be in the liquid of polyacrylic acid. As the initial hardening reaction proceeds rapidly, metallic free ions disappeared due to the cross-linking with polyanionic chains of acrylic acid to form a set cement which showed the non-cytotoxic effects. Powder of polycarboxylate cement contains mainly zinc oxide, magnesium oxide and other materials in small quantities. Zinc oxide powder is decomposed in the liquid of polyacrylic acid solution by mixing, and free zinc ions may be dispersed into the culture medium. Metallic zinc and zinc ion have shown a pronounced cytotoxic effect in culture medium, as reported by H. Kawahara.3 It is revealed that release of Zn2+

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1`ig. 5.-Cellular reaction around the set cement of glass iononmer, polycarboxylate and zinc oxideeugenol cemnent at 24 hours' cultivation. Slight reaction with a test piece of glass ionomer cements of Aspa (a) and Fuji lonomer (b); mild reaction in Carbolit 100 (c); severe reaction in eugenol cement (d). ion and free polyacrylic acid dispersed into the culture medium from the test piece of un-set carboxylate cement are main factors for cytotoxicity. Set polycarboxylate cement showed a cytotoxic effect which may be caused by irradiation of zinc ion from the set cement. The elution of zinc from polycarboxylate cement in water was analyzed by using the chemical techniques of Crisp.8

The difference in cytotoxic effect between the glass ionomer and polycarboxylate cement may be caused by the difference of powder composition or setting reaction. Zinc oxide-eugenol cement showed stronger cytotoxicity than polycarboxylate cement. It may be that the composition of zinc oxide-eugenol cement is basically different from the other two cements. In the result of cell growth investigation, there is no significant difference between the two glass ionomer cements of Aspa and Fuji Ionomer. However, a very small difference was found from cell growth in the two cements and may be caused by the different composition of liquid and powder which contains more

crystalline substance in Aspa compared with Fuji lonomer, and by the sharper setting process in Fuj i Ionomer.9 According to a report by H. Kawahara3 and Y. Mizunoj0 the conventional dental cements have shown irritant effects upon the pulp. It is necessary that protection of the pulp from irritants be recommended for use in deep cavities. Un-set glass ionomer cement may have very little irritant effect upon the pulp tissue, and it may possiblv be used for direct filling material in the cavity with exposed pulp. Some reports11'12'13 have confirmed that the biological experiments so far carried out suggest polycarboxylate cement having less irritant on the tissue of all the conventional dental cements. But polycarboxylate cement showed cytotoxic effects in this experiment. The cytotoxicity of un-set polycarboxylate cement decreased with the setting process, while the cytotoxic effect remained in the set cement, and the cells showed toxic responses. This cement is considered to be dully irritant to the tissue for a long term

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GLASS IONOMER CEMENT

I"ig. 6.-A typical pulpal response with mild round cell infiltration to a test piece of zinc oxideeugenol (a) and glass ionomer cement (b), two months after filling in the cavity with exposed puilp. Degeneration and displaceInent of odontoblastic layer in the adjacent area to the eugenol cement and F uji lonomer.

after setting, since zinc oxide-eugenol cement showed a cytotoxic effect for a long time after setting. A dull irritant may serve to form a secondary dentin, but if the irritant exceeds the limit of living activity of pulp tissue, it causes pulp death, and an amount of material or irritant is always included in these dangerous results. The animal examination on pulpal reaction was taken to find a certain relation to the result from the tissue culture test. The glass ionomer and zinc oxide-eugenol cement showed the same result of mild responses to the pulp of monkey teeth at two months after the filling, in spite of the results of the tissue culture test, in which the cytotoxicity of zinc oxide-eugenol cement was stronger than that of the glass ionomer cement. Similarity of in vivo experiments on the two cements may be caused by the shortness of

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the post-operative interval. These pulp responses may be induced mainly by the mechanical irritation caused by filling pressure, micro-leakage around the cement filling14, and bacterial contamination during cavity preparation15, considering that glass ionomer cement showed less effect than zinc oxide-eugenol cement in the tissue test. In long term studies on these materials, different results may be obtained. The experimental results are only those of table work. There are considerable problems in the dental clinic. There are three levels of biological tests used to evaluate dental materials: tissue culture, animal examination and clinical observation, as reported by FID COMIET'6.1'7, Dental Materials Group of IADR1819, K. Langeland20, 1. A. Mj&r, A. I-lensten-Pettersen and O. Skogedal21 and H. Kawahara22,23. A. Hensten-Pettersen and K. Hlelgeland24 reported (but from a standardization point of view, it should be noted) that cell culture techniques have a high degree of reproducibility. In the present study of in vitro and in vivo, it was recognized that analyzing the various types of cellular response in tissue culture tests was an effective technique for deciding the complicated reactions of living pulp in animal examination.

Coniclusionis. Biocompatibility of glass ionomer cements of Fuji lonomer and Aspa were tested by the tissue culture method and animal examination, comparing polycarboxylate and zinc oxide-eugenol cements. Biological investigation of the glass ionomer cement was similar to that obtained in control culture, but polycarboxylate and zinc oxide-eugenol cements showed a cytotoxic effect in vitro. The pulpal reaction of the glass ionomer and zinc oxide-eugenol cement was mild in vivo. From the results of this biological investigation, glass ionomer cement may be applied as a pulp capping and canal filling material, and may be widely used as a biomaterial in the medical field. REFERENCES 1. WILSON, A. D.; and KENT, B. E.: A New Translucent Cement for Dentistry, Brit. Dent. J. 132:133-135, 1972.

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2. KAWAHARA, H.; IMANISHI, Y.; OSHIMA, H.; and TOMIOKA, K.: Fuji Glass lonomer for Dental Use, Program and Abstracts of Papers, 24th Annual Meeting of The Japanese Division of IADR, 1976. 3. KAWAHARA, H.; YAMAGAMI, A.; and NAKAMURA, M.: Biological Testing of Dental Materials by Means of Tissue Culture, Int. Dent. J. 18:443467, 1968. 4. KAWAHARA, H.; NAKAMURA, M.; YAMAGAMI, A.; and NAKANISHI, T.: Cellular Responses to Dental Amalgam In Vitro, J. Dent. Res. 54:394401, 1975. 5. KAWAHARA, H.; IMANISHI, Y.; and NISHIDA, T.: A New Cell Strain Derived from Human Dental Pulp, Int. J. Dent. Med. 4:

767-768, 1976. 6. KENT, B. E.; LEWIS, B. G.; and WILSON, A. D.: The Properties of A Glass Ionomer Cement, Brit. Dent. J. 135:322-326, 1973. 7. KAWAHARA, H.; and IMANISHI, Y.: Cytotoxic Effect of Glass lonomer Cement (In Vitro), The 32nd Annual Meeting of The Japan Society for Dental Apparatus and Materials, 1977. 8. CRISP, S.; LEWIS, B. G.; and WILSON, A. D.: Zinc Polycarboxylate Cements: A Chemical Study of Erosion and Its Relationship to Molecular Structure, J. Dent. Res. 55: 299-308, 1976. 9. KAWAHARA, H.; and TOMIOKA, K.: Personal Communication, 1977. 10. MIZUNO, Y.: Analytical Study on The Cytotoxicity of Dental Cements by Means of Tissue Culture, J Osaka Odontol. Soc. 32: 48-57, 1969. 11. TRUELOVE, E. L.; MITCHELL, D. F.; and PHILLIPS, R. W.: Biologic Evaluation of a Carboxylate Cement, J Dent. Res. 50:166, 1971. 12. JENDRESEN, M. D.; and TROWBRIDGE, H. O.: Biologic and Physical Properties of a Zinc Polycarboxylate Cement, J. Prosthet. Dent. 28:264-271, 1972. 13. BEAGRIE, G. S.; MAIN, J. H. P.; and SMITH, D. C.: Inflammatory Reaction Evoked by

14. 15.

16. 17.

Zinc Polyacrylate and Zinc Eugenate Cements, Brit. Dent. J. 132:351-357, 1972. MASSLER, M.: Biologic Considerations in the Selection and Use of Restorative Materials, Dent. Clin. N. Amer. 131-147, 1965. BEAGRIE, G. S.: Dental Pulp Reaction to Restorative Procedure, Report of the First U. Iwadare Memorial Lecture, Osaka, Japan, 1977. COMIET, Working Group on Biological Testing of Dental Materials (Leader, LANGELAND, K.),FDI, 1972. COMIET, FDI/ISO, JWG No. 6, On Biological Testing of Dental Materials (Chairman,

LANGELAND, K.), FDI, 1976. 18. Dent. Mater. Group, IADR: Subcommittee on Toxicity Test for Dental Materials (Chairman, RYGE, G.), 1966, 1967, 1968. 19. Dent. Mater. Group, IADR: ANSI Project on Dental Materials and Devices, 2156 Group No. 3, Subcommittee for Toxicity Test on Dental Materials (Chairman, STANLEY, H. R.), 1970. 20. LANGELAND, K.: Biologic Aspects of Oral Restorations, Int. Dent. J. 20:472474, 1970. 21. MJOR, I. A.; HENSTEN-PETTERSEN, A.; and SKOGEDAL, O.: Biologic Evaluation of Filling Materials. A Comparison of Results Using Cell Culture Techniques, Implantation Tests and Pulp Studies, Int. Dent. J. 27: 124-129, 1977. 22. KAWAHARA, H.: Studies on the Dental Materials and Therapeutics by Means of Tissue Culture of In-Vitro System, Development of Dental Science by Means of Tissue Culture, Program and Abstracts of the 9th Annual Meeting Tissue Culture Society for Dental Research, 1971. 23. KAWAHARA, H.: Biological Test of Dental Materials and Therapeutics (In Vitro), Jap. J. Oral. Biol. 14 Supplement 153-154, 1972. 24. HENSTEN-PETTERSEN, A.; and HELGELAND, K.: Evaluation of Biologic Effects of Dental Materials Using Four Different Cell Culture Techniques, Scand. J. Dent. Res. 85: 291-296, 1977.

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Biological evaluation on glass ionomer cement.

Biological Evaluation on Glass lonomer Cement HARUYUKI KAWAHARA, YOSHITSUGU IMANISHI, and HIROSHI OSHIMA Department of Biomaterials and Tissue Culture...
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