Long-term Biocompatibility Test of Composite Resins and ...

100 Dental Materials Journal 2 (1): 100-112, 1983

Long-term Biocompatibility Test of Composite Resins and Glass

Ionomer Cement (in vitro)

Masaaki NAKAMURA, Haruyuki KAWAHARA, Koichi IMAI, Susumu TOMODA,

Yoshinori KAWATA and Shiro HIKARI

Department of Biomaterials, Osaka Dental University (1-47, Kyobashi, Higashiku, Osaka 540, Japan)

(Head: Prof. H. Kawahara)

Received on May 31, 1983

In order to examine long-term biocompatibility of restorative dental materials, three composite resins

and one glass ionomer cement were tested. Hardened specimen was immersed in extraction medium (MEM)

for a unit period of two weeks, after which extraction was continued in the same manner for twenty weeks.

Extraction was carried out by gyration at 200rpm at 37•Ž that allowed the specimens to move freely, thus

making the extracting environment much closer to a clinical situation. Each extract obtained was used for

cell colony formation of HeLa S3 cells. Cell colony forming rates were utilized as biocompatibility measure

in vitro. As a result, it was found that initial cytotoxicity of the materials tested ranged from moderate to

marked levels at the 2nd week in the dynamic experimental environment. This was followed by none to

weak cytotoxicity toward the end of the experiments. Importance of in vitro simulation experiments of a

clinical situation and the establishment of an in vitro long-term biocompatibility testing were discussed .

Key Words: Composite Resins, Glass Ionomer Cement, Long-term Biocompatibility Test

INTRODUCTION

Restorative materials such as dental amalgams, composite resins and restorative cements, remain in the mouth for a long period once inserted. It is, therefore, considered that a bio-

compatibility check up should include initial, short- and long-term testing. The present authors have shown a dramatic difference in the results of the above two testings by utilizing dental amalgams and have emphasized the necessity for a long-term testing method in

vitro1-5). It is apparent that data of short-term testing does not necessarily reflect material behaviour in the long run6-12).

The present study deals with the long-term in vitro testing of hardened restorative materials, three composite resins and one glass ionomer cement.

MATERIALS AND METHODS

Cell culture-The cells used in the experiments were HeLa S3 cells (Puck et al., 1955)13). A synthetic medium, MEM* (Eagle, 1959)14), supplemented with 10% calf serum* was

employed in a weekly subculture, the replicate culture of which was utilized in the experiments.

Aided, in part, by Grant-in-Aid for Developmental Scientific Research from the Ministry of Education, Science and Culture in Japan (No.56870103).*GIBCO, USA.

LONG-TERM BIOCOMPATIBILITY OF RESTORATIVE MATERIALS 101

Preparation of extracts-

1) Specimens: The experimental method was generally the same as that described in our

previous report4); the points of difference will be described. Three composite resins and

one glass ionomer cement for restorative use were utilized. Two of the three composite

resins were newly developed materials15,16). One of them, TMM (tetramethylolmethane

acrylate derivatives)-SiO2 resin** was designated as an anterior restorative material. The

second composite resin, TMM-Si3N4 resin*** was developed as a posterior restorative

material. The third composite resin used was Adaptic+, and has a relatively long service

record in dental practice. The glass ionomer cement used was a Fuji ionomer++. Mixing

of the materials was in accordance to the respective manufacturers' recommendations.

Mixed materials were placed into a glass tube (inner diameter, 6.0mm; length, 5.0mm) to

give the specimens a cylindrical form. The specimens were taken out of the glass tube after

setting and then placed in an ultra-violet light irradiation chamber for 17 hours for sterili-

zation before use. This period also served as a time for further setting of the resin materials.

No polishing was done in order to exclude any possible effects of the polishing agents.

2) Method of extraction: Extraction was made by immersing the specimen in MEM in a

tightly sealed Erlenmeyer flask for 2 weeks, at the end of which period each extract was

examined for the effects on cell colony formation. The relationship between the surface

area of a specimen and the volume of medium for extraction was maintained at 1cm2 versus

10ml, as previously reported4) and was maintained in whole form throughout the in vitro

biocompatibility check up. The total experimental period lasted for 20 weeks. Extraction

was carried out in a gyrotory shaker+++ at 200rpm at 37•Ž (Figures 1 and 2). The dynamic

extraction allowed the specimens to move freely in the flask, thus making the extraction

environment much closer to a clinical situation. This was exactly same experimental set

Figure 1 Dynamic extraction with a freely moving specimen. The specimen was moved freely

in a 20ml Erlenmeyer flask. Dynamic load upon the specimen during gyration made

experimental condition closer to a real clinical environment.

**79-2, Kanebo, Tokyo, Japan.

***79 -1 , Kanebo, Tokyo, Japan.+081G

, Johnson & Johnson, USA.++Type IIF

, FS10, G-C, Tokyo, Japan.+++G -24, New Brunswick, USA.

102 M. NAKAMURA, H. KAWAHARA, K. IMAI, S. TOMODA, Y. KAWATA and S. HIKARI

Figure 2 Gyrotory shaker (G-24, New Brunswick). The Erlenmeyer flask was clamped on a

platform of the shaker that was moved at a speed of 200rpm. A chamber was maintained

at 37•Ž after the lid was closed.

up as previously described4) in an attempt to provide an experimental condition toward a

simulation of clinical situation.

3) Experiments, fixing and staining of the cell colonies: The experimental method was the

same as that of the previous report, as well as the fixing and staining methods1-5). Four

thousand cells per dish were cultured with the diluted extract for one week, then fixed and

stained. Effects of the extracts on cell growth were determined by examining colony forming

activity of the cells.

4) Criteria of cell survival: Cell survival was assessed by colony forming activity determined

by the area of the cell colonies formed. Measurements were made with an automated

colony counting system. The apparatus used in the present experimentsƒ¢ was different

from that of the previous report4) (Figure 3). Both systems operated exactly on the same

principle. Pictures taken by a television camera were fed to an analyzing personal computerƒ¢ƒ¢,

and finally displayed on video and data screens following calculation. The present system

provided not only the same quality data as the previous one, but also calculated and displayed

much more quickly. The area of cell colonies within a unit circular area (17mm) were

calculated. Five measurements were made on each dish. The data measured were pro-

cessed with 95% statistically significant limits under the respective experimental conditions

ƒ¢ Biotran III system, New Brunswick, USA.

MB-6890, Hitachi, Tokyo, Japan.


Figure 3 Automated cell colony counting system (Biotran III system, New Brunswick) & personal computer (MB-6890, Hitachi). Image of cell colonies in a petri dish on an illuminating equipment (b) was first obtained by a television camera (a), fed to a equipped micro-computer of the Biotran III system (d & e), and finally calculated data were obtained on a monitor screen (d) and digital display (e). The accompanying desk-top personal computer (f) directly connected with the system was utilized for statistical treatment of the data. An adjacent dissecting microscope (c) was used for examining the cell colonies in the petri dish prior to each counting.

Table 1 Definition and classification of cytotoxic scores based on Relative growth rate

and compared with the control values. Two kinds of evaluation were adopted as in the

previous study: Relative growth rate (%) and expression in 6 step cytotoxic scores (Table 1).

RESULTS

1. Extract observation

Following the first extraction period, the extracts of the three composite resins remained

clear with no clouding or precipitation. The pH value of the extracts were normal. How-

ever, the glass ionomer cement yielded white float and lowered the pH value significantly.

The specimens appeared normal.

The extracts of the three composite resins were normal up to the sixth week of extraction.




The colour of the glass ionomer cement extract still appeared orange to yellow during the corresponding period of extraction, meaning a lower pH value. However, no clouding or

precipitation occurred.By the eighth week of extraction, a fine, gray, powdery precipitate appeared in the

extract of the TMM-Si3N4 resin. The pH of the extracts remained normal. The other two composite resins yielded no precipitate nor showed change in the pH values of the extracts. At the tenth week of extraction and afterward, a fine powdery precipitate of ivory colour appeared also in the extracts of Adaptic and TMM-SiO2 resin. This precipitate was found after each extraction period throughout the experiments, i.e. at the twentieth week in the case of TMM-Si3N4 resin and at the sixteenth week in the case of Adaptic and TMM-SiO2 resin. At the eighteenth and twentieth weeks, the Adaptic and TMM-SiO2 resins did not

yield any clouds or precipitates. The extracts appeared normal. The glass ionomer cement, on the other hand, yielded no cloud or precipitates toward the end of the experiments. A lower pH value was always found in the extract. At the end of the experiments, the shape of each specimen was found to have remained unchanged.2. Effects of the materials on cell colony formation

Relative growth rates under the respective experimental conditions during the twenty week period are shown in Tables 2 and 3. Cytotoxic scores were obtained from the mean value for each experimental condition (Table 4).1) Cytotoxicity of the materials tested

The degrees of cytotoxicity remained minimum throughout the 20-week experimental

Table 4 Mean scores of cytotoxicity as expressed by cell colony area*

*Based on Relative growth rate .


period, except for the second week. All materials tested yielded a somewhat higher degree

of cytotoxicity after the initial extraction. TMM-Si3N4 resin and Fuji ionomer recorded

weak to moderate cytotoxic scores at 25% and 50% extract concentration levels and moderate

to marked cytotoxic scores at extract concentration levels of 75% and 91%. From the

fourth week to the end of the experiments, the cytotoxic degree recorded for the materials

was from none to weak cytotoxicity, except moderate cytotoxicity in TMM-SiO2 resin at

the sixth week period at higher extract concentration levels.

During the twenty weeks, change in cytotoxicity was minimum. Cytotoxic scores were

mostly 0 to 2, except for the second week (Table 4).

2) Change in cytotoxicity at four extract concentration levels

Despite the increase in the original extract concentration, cytotoxic scores did not greatly

change at higher extract levels, i.e. 75% or 91%. The only exception was that at the

second week. These data show that the materials tested did not impart much cytotoxicity

to the cells except at the very early stage of the post-setting period.

DISCUSSION

The present results indicated that the degrees of cytotoxicity of all materials tested

ranged from weak to moderate. However, moderate to marked cytotoxicity was found at

the very early stage of the post-setting period, i.e. the second week. As for biocompatibility

of the composite resins, quite a few reports based on the results of either animal experiments

and in vitro tests or clinical usage have been accumulated17-25). These reports showed that

composite resins yielded strong to extreme cytotoxicity, particularly during the period of

polymerizing process, and recommended that proper treatment to protect the pulp tissue

underneath was essential for successful clinical results. The present findings give parallel

data in regard to the presence of cytotoxicity, but differ with respect to the degree

of cytotoxicity. Much less cytotoxicity rather than that anticipated was recorded. This

difference seems to stem from differences in experimental objective. As described earlier,

our chief objective was to find how hardened materials behave toward cells over a long

period of time from the viewpoint of biocompatibility. Since the information is scant,

compared with that for short-term experiments dealing mainly with the polymerizing process

period. It may be said that the 17-hour period following mixing, when extraction was

started, comes well after the so called, •gthe clinical•h setting period. This seems to mean

that the presence of even less cytotoxicity indicates the possible effects of residual monomer.

Nearly all the monomers might possibly be extracted during the first extraction. Thus ,

subsequent extraction may not have exerted any further cytotoxicity on the cells.

Different degrees of cytotoxicity among the materials tested were apparent. A difference

in the new composite resins was noticed. Two of these resins consisted of tetramethylolme-

thane acrylate derivatives. The TMM-SiO2 resin contained ƒ¿-quartz crystal approximately

80% by volume, while TMM-Si3N4 resin contained silicone nitride and ƒ¿-quartz crystal15,16).

Polymerization was based on the benzoil peroxide-tertiary amine (paratridiethanol amine)

system. The resins were provided with two pastes. On mixing, one part of paste A con-

taining tertiary amine was necessary for 1 part of paste B containing benzoil peroxide in

TMM-SiO2 resin. The TMM-Si3N4 resin, on the other hand, required 3 parts of paste A


to 1 part of paste B. There are five possible causes for the different results in these two materials: i) base resin itself and various additives, difference of ii) the fillers or iii) mixing

ratio of the two pastes, iv) difference of adhesion of fillers to base resin, and v) combined effects of more than two of the causes described above. As for the base resin and various

additives, these should not be ignored as possible causes. There can not, however, be one factor causing the different results, since both composite resins contain equal amounts of base resin. The fillers were confirmed to be non-cytotoxic26). Therefore, the difference in the fillers can not be a causative factor. It still remains unclear whether there is any

difference in the adhesion of two kinds of fillers to the base resin. If there is any difference, there could be a percolation phenomena at the interface between filler and base resin, causing inner residual monomer to leak, thus creating a dangerous situation to the cells. The most

probable cause is considered to be the difference in the mixing ratio. Three parts of the paste containing tertiary amine are recommended to 1 part of the other paste for the TMM-Si3N4 resin, compared with 1 to 1 ratio for the TMM-SiO2 resin. This difference in the mixing ratio may naturally lead to a greater volume of tertiary amine within hardened materials, if the total amounts were equal. The amount of residual monomer, or more specifically, the tertiary amine in the original pastes, widely regarded as one of main cytotoxic components17-25), might naturally remain in the hardened state of both materials. It should be taken into account that a very small amount of tertiary amine usually found in common restorative resins, is enough to cause deleterious effects in the local tissues. Based on these facts, the difference in the amount of tertiary amine in the two materials may explain the diverse cytotoxicity.

The initial cytotoxicity of the glass ionomer cement was also recorded. It is reported that aluminium phosphate and calcium fluoride in the cement powder caused severe to moderate cytotoxicity in vitro, while acidity and irritability of the cement liquid showed cytotoxicity27,28). It is also reported that in vitro cytotoxicity rapidly decreases as the setting process continues. The reason for this may be that cytotoxic components form stable reaction products and thus become increasingly less cytotoxic. The present results are apparently consistent with these reports. The first extraction seemed to decrease the number of residual cytotoxic elements. Thus, subsequent extraction could not exert much, if any cytotoxicity.

It is noteworthy that the present results have been presented from the aspects of clinical usage, though only in vitro data have been dealt with. As already mentioned above, the objective of this study has been to elucidate the in vitro biocompatibility check up of hardened materials over a long period of time. Moreover, material behaviour at a very early stage

between the beginning of mixing and setting, has not been included. Naturally, not very excessive reactions were obtained following the initial period of the experiments. Thus, it is apparent that our results can hardly be applied to an oral environment. The presence of cytotoxicity at the very early stage seems to emphasize the necessity for the common practice

of cavity lining that protects pulp tissue effectively from possible stimuli of restorative materials. The same precaution will be needed in the case of glass ionomer cement restoration. Either some or many restorations in a mouth or direct contact of local tissues with this material, will not necessarily be safe, despite reports of near biocompatible27).


Also, considerable differences have been found in data on restorative materials1-12), i.e. composite resins and glass ionomer cement vs. dental amalgam. Mostly none to slight

cytotoxicity in the present study contrasts well with the data of dental amalgams in the

previous study, in which the cytotoxic degree ranged from severe to extreme. The results of the present paper certainly provide a good basis for judging the applicability of these

materials for dental restorative work. It is a well known fact that dental amalgams have been widely used in dentistry with fairly good clinical records over a long period of time29)

. This long service record could not have been made by a restorative material, other than

dental amalgam. On the other hand, it is apparent that there are many shortcomings involved, the most serious of which is the chief component, mercury. There are quite a few reports which question the use of this material as a restorative material30-39), despite excellent handling and no clinical records of mercury poisoning. Dentists will surely be

in a dilemma, when contrary data appear. It seems necessary that the appropriateness of this biomaterial should be seriously discussed so as to scrutinize and judge the applicability of all these materials including dental amalgams. This is particularly important, since hazardous substances are considered to destroy biological homeostasis. People around the world have become more and more aware of the efforts of governments and industry to take the necessary precaution against use of hazardous substances. The enactment of various regulations can be viewed as a direct result of this action. The dental profession can not be exempted from these regulations. The materials tested in the present experiments showed a minimum cytotoxicity and can therefore be considered to partially fulfil the re-

quirements for biomaterials, compared with dental amalgams used at the present. However, it is apparent that our results do not require adherence to all requirements for using bio-materials. Tests for further elucidation of biocompatibility are required.

The Commission on Dental Materials, Instruments, Equipment and Therapeutics, Federation Dentaire Internationale has currently issued recommendations for making a

standard biological evaluation of dental materials40). The recommended tests involve three levels of testing, initial, secondary and usage tests. The total cost for these tests is quite considerable and the Organization has not requested that every test be performed, but so only appropriate tests. It is said the initial and secondary tests should be followed by extensive usage tests. Two aspects of the Recommendation should be revised: First, a

general method is lacking for long-term tests at the initaial test level. Only a maximum period of 7 days for observation for in vivo tests and mere 24 hours for in vitro tests are required. Even the maximum duration of 12 weeks for two of the secondary tests is con-sidered insufficient. It needs hardly be said that long-term tests are as important as short-term tests. One can easily understand the importance of long-term tests on considering the duration of restorative, prosthetic, endodontic or orthodontic materials once inserted. Second, there is no general concept for simulation of an oral environment in the three in

vitro tests. It has been a long time since the poor correlation between two data of in vitro and of in vivo was pointed out41,42). Since then, more people have come to support in vivo tests rather than oppose them. One reason for this unfortunate result is lack of effort to simulate an oral environment effectively. As a result, most of the advantages of the in vitro approach have not fully been shown yet. Appropriate simulation might enable all testing


methods to become more simple, reproducible, analytical and economical. This does not necessarily mean to deny secondary in vivo or usage tests. These methods are good for checking the immunologic and carcinogenic aspects of dental materials. The overall effects of the materials on tissues can also be elucidated by these methods. After all, either method, in vitro or in vivo, has certain points which distinguishing it from the other. Therefore,

arguments for the superiority of either method over the other seems meaningless. Now that the recommended standard has been adopted, it seems a wise practice to revise and improve it. Moreover, the goal for the biological evaluation of dental materials will be the in vitro test under all the simulated conditions of every aspect of the oral environment, as pointed out in the previous paper4).

CONCLUSION

Long-term biocompatibility of composite resins and glass ionomer cement was examined under dynamic extraction with a freely moving specimen condition for 20 weeks in vitro. The cells used were HeLa S3 cells whose growth was evaluated by the cell colony formation method and finally expressed in terms of cytotoxic scores.The following results were obtained: 1. Extracts of the specimens remained unchanged until the 6th week, after which time a

powdery precipitate was found in the extracts of the three composite resins, i.e. newly developed TMM-SiO2 resin, TMM-Si3N4 resin and Adaptic. On the other hand, extracts of Fuji ionomer remained clear and unchanged throughout the entire experimental period.2. TMM-SiO2 resin yielded moderate cytotoxicity at higher levels of extract, i.e. 75% or 91% at the 2nd and 6th week, but otherwise ranged from none to weak cytotoxicity.3. TMM-Si3N4 resin yielded moderate to marked cytotoxicity at the 2nd week, after which time none to weak levels continued.4. Adaptic maintained similar levels of cytotoxicity toward the TMM-SiO2 resin.5. The Fuji ionomer yielded marked cytotoxicity at 75% and 91% levels of extracts at the 2nd week. This was followed by none to weak cytotoxicity after the 4th week.

It has been shown in the present study that initial cytotoxicity ranged from moderate to marked levels at the 2nd week in the dynamic experimental environment. Otherwise,

the degree of cytotoxicity of each material tested ranged from none to weak. The different results obtained for the materials examined are considered due to different components involved. A discussion was made of the existence of considerable differences of biocompati-bility between the composite resins or glass ionomer cement and dental amalgam in the

previous study, in which the cytotoxic degree ranged from strong to extreme, and whether or not the present restorative materials are really biocompatible. In this study, we have discussed the urgency of in vitro simulation experiments of a clinical situation and the establishment of an in vitro long-term biocompatibility testing method.

ACKNOWLEDGEMENT

Thanks are due to Drs. H. Kobayashi, S. Maehara, M. Izutani, Ms. Y. Ohta and Ms. J. Nagaoka for their help in this investigation.


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157

長期生物テスト法によるコンポジットレジンおよび充填用

グラスアイオノマーセメントの細胞毒性(in vitro)

中村正明,川原春幸,今井弘一,友田達,川田義典,光司郎

大阪歯科大学歯科理工学教室

長期間にわたるコンポジットレジンおよび充填用グラ

スアイオノマーセメントの硬化体の細胞毒性をしらべる

ために,HeLa S3細胞を用いて細胞コロニー形成法を

使って生物テスト(in vitro)を行った。規格サイズに作

製された各実験試料は培養液MEMに浸漬され,200

rpmの旋回投入浸漬の動的負荷が与えられた。この実験

環境は臨床の場のin vitroにおけるシミュレーションを

目ざすものである。そして,つぎのような結果を得た。

テストした新コンポジットレジン,TMM-SiO2レジン

および従来のアダプティックは実験初期に弱い細胞毒性

を示したものの,その後は実験終了時の20週後までの間

ほとんど細胞毒性を示さなかった。これに反して,新コ

ンポジットレジン,TMM-Si3N4レジンおよびフジアイ

オノマーは実験初期に中等度の細胞毒性を示した。しか

し,その後は他の2材料と同じくほとんど細胞毒性を示

さなかった。

以上の大部分の実験期間を通じての安定した生物学的

性質から,今回しらべた各材料は一応バイオマテリアル

としての適性を有していると言えよう。また,動的環境

下での長期生物テストの必要性も明確にされた。

本研究の一部は文部省科学研究費補助の試験研究

(56870103)による。

Long-term Biocompatibility Test of Composite Resins and ...

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