In vivo study on the biocompatibility of new resin-based root canal sealers

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I. Introduction

Endodontic materials are used for the permanent obturation of root canals and consist of gutta percha core and sealer combinations. Grossman has adressed the requirements for the ideal root canal system filling materials1). They include ease of introduction, adequate sealing to canal wall, 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.

Endodontic filling materials are peculiar in that they are placed directly onto vital tissue. Therefore, the tissue response to these materials becomes of importance and may influence the outcome of the endodontic treatment2). In clinical practice, it is the sealer cement that comes into contact with the periapical tissue for a long time. So, one of the requirements of an ideal root canal sealer is that it should be nonirritating to the periapical tissues and should be compatible with living connective tissue3).

The biological properties of the sealers are therefore considered to be especially important, and several in vitro and in vivo tests have been applied to assess these properties.

Several methods have been used to evaluate tissue responses to endodontic materials. One of the most practical and widely used methods is the implantation of the material into the subcutaneous connective tissue of rodents4,5,6). The irritative effect of endodontic materials is evaluated by the histopathological examination of the tissue response around the implants7,8,9,10).

The subcutaneous implantation test possesses several of the features of a secondary test for the biological evaluation of endodontic materials11). In this study we used Teflon tubes because of their inert nature and suitability for bringing a test material in contact with living tissue in a controlled and effective manner12). Torneck firstly used polyethylene tube. The thin, cellular response alongside the tube serves as a negative control13).

The purpose of the present, in vivo, research was to study the biocompatibility of five root canal sealers. Two of them (AH 26, AH Plus) contain in their formula epoxy resin. A third, Pulp Canal Sealer is a typical zinc oxide-eugenol sealer. The forth and fifth materials are newly developed resin-based root canal sealers.

II. Materials and Methods

1. Materials

Sixty-four white male Sprague-Dawley rats were used in this study. They were 3 to 4 months old and 200 to 250 g. There were five groups of three animals each for every experimental period. The used sealers, composition and manufacture were Table 1. For every period there was also a control group.

Table 1

Composition of materials

5. Evaluation of Histological Tissue Response

For state of the surrounding tissue, the below biological parameter were examined14).

① Extent of fibrosis/fibrous capsule and inflammation

② Degeneration as determined by changes in tissue morphology

③ Number and distribution as function of distance from the material/tissue interface of the inflammation cell type, namely polymorphonuclear luecocytes, lymphocyte, plasma cell, eosinophils, macrophages and multinucleated cells

④ Presence of necrosis as determined by nuclear debris and/or capillary wall breakdown

⑤ Other parameter such as material debris, fatty infiltration, granuloma

Tissue reactions were graded as mild, moderate, and severe, according to the criteria suggested by Olsson et al.15) and Orstavik and Mjor16). This criteria is represented in Table 2.

Table 2

Criteria for scoring of inflammatory tissue response

III. Results

All specimens were undergone blinded examination by the single examiner who did not know which sealer or which period was being examined. Histopathological results are summarized in Table 3. Data were statistically analyzed with the Kruskall-Wallis test. There was no statistically significant difference among the test materials (p > 0.05).

Table 3

Histological findings

To know the tendency with experimental period within group, data were statistically analyzed with Kruskall-Wallis test. There was statistically significant difference in all sealer groups (p < 0.05). These results are summarized in Table 4 and Figure 1.

Table 4

The mean and interpretation

Fig. 1

Means of the each period

1. Control

Slight reactions generally were observed in the control group (Fig 2). These reactions were characterized by the presence of macrophages and of few plasma cells next to fibrous connective tissue, especially at 1 week specimens. The inflammation was reduced further with time (2, 4 and 12 weeks). But only two specimens at 1 week showed moderate inflammation.

Fig. 2

Control 2 weeks (×40)

2. AH 26

Severe inflammation was observed in the AH 26 specimens at 1 week (Fig. 3). The intensity of the reaction decreased little at 2 weeks. The tissue was infiltrated with many histiocytes and lymphocytes. And there are foreign body reactions around the spilled material (Fig. 4). At 4 weeks, the inflammatory reaction had diminished, compared with the 1 and 2 weeks. There were very few inflammatory cells (Fig. 5). Fibroblasts and collagen fibers with very few inflammatory cells were observed (Fig. 6).

Fig. 3

AH 26 1 week (×40)

Fig. 4

AH 26 2 weeks (×400)

Fig. 5

AH 26 4 weeks (×400)

Fig. 6

AH 26 12 weeks (×400)

3. AH Plus

Moderate to severe inflammatory reaction was observed with AH Plus at 1 week. Inflammatory cell infiltration was around spilled sealer (Fig. 7). The inflammatory response was similar at 2 week. It was characterized by the presence of macrophages, plasma cell, lymphocyte and fibroblasts (Fig. 8). Reduction of inflammation continued progressively to 4 and 12 weeks. The tissue was almost mild to moderate. The connective tissue became almost normal architectures (Fig. 9). Very thick fibrous capsule was observed (Fig. 10).

Fig. 7

AH Plus 1 week (×100)

Fig. 8

AH Plus 2 weeks (×400)

Fig. 9

AH Plus 4 weeks (×400)

Fig. 10

AH Plus 12 weeks (×400)

4. Pulp Canal Sealer

Severe inflammatory reaction was observed at 1 week after the implantation of sealer. Accumulation of macrophages, lymphocytes, plasma cells, and foreign body giant cells were seen in the specimens (Fig. 11). The inflammatory response was similar at 2 weeks (Fig. 12), whereas at 4 weeks it was graded as mild to moderate. It was characterized by the presence of a few lymphocytes, fibroblasts and fibrous capsule (Fig. 13). But at 12 weeks the severity of inflammation increased to moderate. There were plasma cells and macrophages as much as at 2 weeks (Fig. 14).

Fig. 11

Plup Canal Sealer 1 week (×400)

Fig. 12

Plup Canal Sealer 2 weeks (×400)

Fig. 13

Plup Canal Sealer 4 weeks (×400)

Fig. 14

Plup Canal Sealer 12 weeks (×400)

5. Adseal-1

Moderate to severe inflammation with concentration of macrophages and lymphocytes was observed at 1 week after implantation (Fig. 15). Some foreign body giant cells with engulfed particles of the material were seen in the areas surrounding the ends of the tubes. The intensity of the reaction was diminished at 2 weeks (Fig. 16). Inflammation consisted of lymphocytes. Fibrous capsule and normal architecture were also observed at 4 weeks (Fig. 17). In the tissue adjacent to the material, fibroblast and collagen fiber were detected. Inflammation almost subsided and thin fibrous capsule was observed (Fig. 18).

Fig. 15

Adseal-1 1 week (×400)

Fig. 16

Adseal-1 2 weeks (×400)

Fig. 17

Adseal-1 4 weeks (×400)

Fig. 18

Adseal-1 12 weeks (×400)

6. Adseal-2

Moderate to severe inflammation was observed in the implant area at 1 week. The connective tissue was infiltrated by lymphocytes, plasma cells and macrophages (Fig. 19). The intensity of inflammatory reaction at 2 week was similar with 1 week. Macrophages, plasma cells, and lymphocytes were also present (Fig. 20). In the specimens of 4 weeks, the intensity of the inflammtion was diminished very much (Fig. 21). At 12 weeks fibrous connective tissue infiltrated with few plasma cells and lymphocytes was observed (Fig. 22).

Fig. 19

Adseal-2 1 week (×400)

Fig. 20

Adseal-2 2 weeks (×400)

Fig. 21

Adseal-2 4 weeks (×400)

Fig. 22

Adseal-2 12 weeks (×400)

IV. Discussion

Many methods have been used to evaluate the biocompatibility of endodontic sealers. But, in vivo methods correlate better than the in vitro tests17). Because the direct contact between root canal sealer and periapical tissue is important, the subcutaneous implantation was used in this study.

And this study used an ordinal scoring system for tissue response evaluation15,16). The results of the ordinal scale and mean of them provided numerical illustration of the impressions and histopathological findings.

A common feature of the tissue response to implantation was the development of a well-defined reaction zone. Tissue response to the materials showed the increase in the width of the reaction zone and localized infiltration by inflammatory cells. In addition, inflammatory responses may occur around spilled material fragments. The filling tubes with freshly-mixed sealers happened spillage of them. It is could be prevented if the tubes were implanted after the sealers had set, but high toxicity that most endodontic material have observed before setting 15).

AH 26 seems to be the most irritative material at 1 and 2 weeks. But the intensity of the inflammation was decreased by 4 and 12 weeks. These results are agreement with those of other researches showed high initial and dramatically declining irritation surrounding AH 2617,18).

These toxic effects of AH 26 could be caused mainly by formaldehyde, which is released primarily during the initial setting reaction19). The powder, however, contains hexamethylenetetramine which could be decomposed in acid environment, yielding ammonia and formaldehyde. The concentration of formaldehyde has increased two-fold 12 hours after mixing and increased until 2 days of setting. The concentration of formaldehyde has increased to nearly 200 times over the concentration of freshly mixed AH 26. The approximate setting time for AH 26 is 2 days. The cytotoxicity due to formaldehyde is supported by other researches 20,21)

Additionally, mutagenic substances, epoxy derivatives of bisphenol-A-diglicidylether which may be also cytotoxic, have been found in the set material 20,22). It is known that epoxy-bis-phenol resin (liquid component of AH 26) relate to its severe cytotoxicity 23). So other researcher pointed to the possibility that mixed silver-free AH 26 might contain small amounts of two mutagenic substances : bisphenol A diglycidyl eter and formaldehyde24).

AH Plus represented the moderate to severe inflammation at 1 and 2 weeks and the intensity of the inflammation was decreased by 4 and 12 weeks. But it looked like milder inflammation than AH 26 overall periods. In another research, AH 26 and AH plus exhibited severe reactivity by agar diffusion test 25). The amount of formaldehyde released by AH 26 and AH plus has been measured and found to be 1347 ppm and 3.9 ppm, respectively. The fact that epoxy-bis-phenol resin was related to cytotoxicity could explain why AH Plus, which release minimal formaldehyde, is also as cytotoxic as AH 26.

On the other hand, other study indicated that AH Plus indeed exhibited a lower cytotoxic potential compared to AH 2626). The new formulation, AH Plus, uses new types of amines and a setting reaction based on thermal epoxide-amine addition reaction. It was said that the new sealers may represent progress in the development of more biocompatible endodontic materials.

Pulp Canal Sealer is a zinc oxide-eugenol sealer. The toxic effects of zinc oxide-eugenol sealers have been studied extensively. The cause of cytotoxicity may be free eugenol remaining in the zinc oxide and eugenol mixture. The free eugenol can also be released from mixture subject to hydrolysis. The rate of releasing eugenol is decreased very slowly. So small amounts of eugenol continue to be released from mixture for at least 1 year27). Eugenol may have a beneficial effects at concentration ranging from 10^-8M to 10^-5M, but may be cytotoxic above 10^-3M concentration by cell respiratory inhibition28).

In the present study, severe inflamation was observed at 1 and 2 weeks and the severity was decreased to moderate at 4 weeks. But at 12 weeks increased inflammation was detected. It should be noted that free-eugenol is still present even after the sealer has been set and released over an extended period. But no positive relationship between the release of eugenol and cytotoxicity has been proven. This fact is supported by other study showed that the pattern of eugenol release did not concide with the cytotoxicity expressed by the test solution29). So, other investigator suggested that the cytotoxicity of zinc oxide eugenol sealer may be based on the possible toxic effect of zinc ions30). Adseal-1 and adseal-2 had the least inflammatory reaction at 1 and 2 weeks. But the severity became same with AH Plus at 4 and 12 weeks. Adseal-2 had lower inflammation than Adseal-1 at 1 and 12 weeks. Adseal-1 and Adseal-2 are resin based sealer. Calcium phosphate is added to Adseal-1 to improve the biocompatibility of resin-based root canal seaelr. And 4-aminobenzonate is used for amine as curing agent. This is a kind of polymer which is less irritative than monomer. Ethylglycol monosalicylate used for good flowability remains as liquid which appear to be cytotoxic in mixtures. To remove Ethylglycol monosalicylate, Adseal-2 contains calcium oxide as chelating agent.

Considering the variations between the in vitro and in vivo studies, it is difficult to compare our results with others. But, It is necessary to investigate more extensively.

V. Conclusions

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