Pulpal Response to a Hybrid Composite Resin Inlay Bonded ...

Dental Materials Journal 24(2) : 178-186, 2005

Pulpal Response to a Hybrid Composite Resin Inlay Bonded with a Newly

Developed Resin-modified Glass Ionomer Luting Cement

Md. Akhtar UZZAMAN1, Yasushi SHIMADA1, Yuichi SEKI1 and Junji TAGAMI1,2 1Cariology and Operative Dentistry, Graduate School, Tokyo Medical and Dental University, 5-45, Yushima 1-Chome,

Bunkyo-ku, Tokyo, Japan 2COE Program, FRMDRTB at TMDU; Graduate School, Tokyo Medical and Dental University, 5-45, Yushima 1-Chome, Bunkyo-ku, Tokyo, Japan Corresponding author, E-mail : [email protected]

Received December 10, 2004/Accepted March 9, 2005

This study evaluated the pulpal response of hybrid composite resin inlay luted with a resin-modified glass ionomer cement, and compared it with a glass ionomer cement and an amalgam. Cervical cavities were prepared in monkey teeth. A resin-modified glass ionomer luting cement (Ionotite F, Tokuyama Dental Corp.) was applied to the teeth in one of the experimen-tal groups, and then hybrid composite resin inlays (Estenia, Kuraray Medical Inc.) were bonded to the cavities. The teeth were extracted after 3, 30, and 90 days and stained with Hematoxylin and Eosin staining or Brown and Brenn gram stain for bacterial observation. No serious inflammatory reaction of the pulp, such as necrosis or abscess formation, was observed in any of the experimental groups. No bacterial penetration along the cavity walls was detected in the resin-modified glass ionomer luting cement group. Hence, the resin-modified glass ionomer luting cement showed an acceptable biological com-patibility with monkey pulp.

Key words : Resin-modified glass ionomer luting cement, Pulpal response, Hybrid composite resin

INTRODUCTION

Contemporary restorative dentistry continues to evolve with the development of new materials, tech-niques and concepts. The growing interest in tooth-colored, nonmetallic posterior restorations has stimu-lated fervor in the development of new materials. Esthetic alternatives to cast gold inlays and amalgam fillings include direct composites and ceramic inlays. Recently, new composite resin materials for indirect restorations were developed, and the mechanical prop-erties were improved by resin and filler technology: including heat curing in addition to visible-light cur-ing. Estenia (Kuraray Medical Inc., Okayama, Japan) , a hybrid ceramic, is an advanced composite material with a large quantity of ultra-fine fillers

(particle size: 0.02um) . A high filler content pro-vides high compressive strength and durability re-

quired for posterior restorations1) . Despite all the improvements, there is still a need for a biomaterial which possesses high biocompatibility, antimicrobial effects, and good mechanical properties.

With the development of dentin bonding systems which offer excellent mechanical properties, durable bonding to tooth structure using resin cement has been achieved2-5) . Resin luting cements are now the choice of material for bonding of esthetic indirect restorations6) . However, marginal adaptation still re-mains an inevitable problem for composite luting ma-terials, especially at the gingival wall of cervical or Class II restorations7-10)

In order to overcome or at least ameliorate the disadvantages associated with conventional resinous adhesives, various efforts have been made to combine

glass ionomer chemistry with methacrylate resin technology11).Glass ionomer materials (which in-clude both conventional and resin-modified glass ionomer cements) and the so-called compomers have been reported to provide anticariogenic effect and promote ion-exchange reaction with tooth tissue12)

Ionotite F (Tokuyama Dental Corp., Tokyo, Japan) , a new resin-modified glass ionomer luting ce-ment, contains MTU-6 (thiouracil functional adhesive monomer) which produces a tenacious chemical bond between various resins and precious metals13) (Table 1) . This resinous cement also contains phosphate monomer and HEMA for bonding to tooth structure. Furthermore, with this luting cement, no priming or bonding steps are necessary.

Since the development of glass ionomer cement, many investigators have reported on the biological responses to glass ionomer cements and their low ir-ritant effects on pulp tissue14-16) . Dental amalgam has also been used successfully as a restorative mate-rial for more than a century. Despite its unaesthetic appearance, dental amalgam is a remarkably durable and long-lasting restorative material7,18)

This study evaluated the pulpal response of a resin-modified glass ionomer cement, Ionotite F, as a luting for hybrid ceramic inlay. The histological re-sults were compared with those of a conventional

glass ionomer cement and an amalgam. In vivo

UZZAMAN et al. 179

Table 1 Composition of Ionotite F & Chemical structure of MTU-6

MTU - 6

Ionotite F manufactured by Tokuyama Dental Corp., Tokyo, Japan

HEMA indicates 2 hydroxy ethyl methacrylate; UDMA, urethane dimethacrylate; MTU-6: 6 -

methacryloyloxyhexyl 2 - thiouracil - 5 - carboxylate; BPO, benzoyl peroxide

sealing ability of the resin-modified glass ionomer luting cement was also evaluated histologically.

MATERIALS AND METHODS

The animals were used in strict adherence to the pro-tocol and facilities approved by the Committee on Ethical Guidelines for Animal Care of Tokyo Medical and Dental University.

Preparation of composite resin inlay Resin inlays were made using the indirect method. Impressions of approximately 2-mm end of a dia-mond stone (ISO # SB2, GC Dental Industrial Corp., Tokyo, Japan) were obtained with an addition sili-cone material (Exafine, GC Dental Industrial Corp., Tokyo, Japan) using the wash technique; the silicone material was in turn used as a mold for the compos-

ite resin inlays (Fig. 1). A resin composite for indi-rect restoration (Estenia, Shade DA2, Lot # 00209B, Kuraray Medical Inc., Okayama, Japan) was placed into the impressions, irradiated for 60 seconds (Optilux 501, Kerr Corporation, Danbury, CT), and then subsequently exposed to a laboratory visible light-curing unit (a -light II, J. Morita Co., Tokyo, Japan) for five minutes, prior to heat curing at 110 C for 15 minutes in air (KL 100, Kuraray Medical Inc., Okayama, Japan). Very small cylinders of resin inlay, 2 mm high with the same dimensions as the diamond stone, were removed from the impres-sion (Fig. 2) and silanized using a silane agent (Por-celain Liner M, Lot # EK1, Sun Medical Co. Ltd., Moriyama, Japan) for 60 seconds.

Fig. 1 2-mm end of # SB2 diamond stones (arrow 1) were exposed through the paraffin wax sheet, of which impressions were taken using an addition

silicone material (arrow 2) .

Fig. 2 Estenia was placed into the patterns of silicone impression (arrow 1), irradiated for 60 seconds and then subsequently exposed to laboratory visi-

ble photo-irradiation for 5 minutes prior to heat curing at 110°C for 15 minutes. Arrow 2: A cylin-

der-shaped resin inlay of 2 mm high with the same size as the diamond stone.

180 PULPAL RESPONSE TO A RESINOUS LUTING CEMENT

Histological study using monkey teeth Three monkeys (Macaca fuscata) were anaesthetized by intramuscular injection of 2 mg/kg Ketamine (Ketaral, Sankyo Co., Tokyo, Japan) and intrave-nous injection of 2 mg/kg Pentobarbital Sodium (Nembutal Sodium Solution, Abbott Laboratories, Abbott Park, IL) .

Cervical Class V cavities, approximately 2 mm in depth, were prepared in the teeth using diamond stones (ISO # SB2, GC Dental Industrial Corp., Tokyo, Japan) at high speed under water spray cool-ant. The cavosurface margin of the cavities was al-ways surrounded by enamel.

A total of 90 cavities were divided into three groups of 30 cavities each for different restorative materials given below. All materials were placed into the prepared cavities as per their manufacturers' instructions. Group 1: Resin-modified glass ionomer luting ce-ment (Ionotite F, Powder Lot # 208M, Liquid Lot # 109M, Tokuyama Dental Corp., Tokyo, Japan) was hand-mixed according to manufacturer's instructions and placed into the cavity using a composite injection syringe. The composite resin inlay was then inserted and bonded to the cavity.

Resin cement flash and small projections from inlays were removed with a high-speed, super-fine diamond bur (ISO # V16ff, GC Dental Industrial Corp., Tokyo, Japan) .

Group 2: Glass ionomer cement (Fuji II, Lot # 101211, GC Dental Industrial Corp., Tokyo, Japan) was hand-mixed for 30 seconds and placed into the cavity using a composite injection syringe. After the initial setting phase, a thin coat of varnish was ap-

plied on the surface to prevent dehydration and cracking of the restoration.

Group 3: High-copper amalgam alloy and mer-cury (Dispersalloy, Lot # 30GH, Johnson & John-son, East Windsor, NJ) were mixed in an amalgama-tor for 15 seconds, and condensed into the cavity.

The experimental periods for the pulp test were 3, 30, and 90 days. All the experimental materials and experimental periods were randomly distributed among each monkey's teeth. After the prescribed pe-riods, the monkeys were sacrificed by an intravenous injection of 250 mg/kg of thiopental sodium

(Ravonal, Tanabe Pharmaceutical Co., Osaka, Japan) , and the teeth were removed from the jaws. Following fixation in 10% neutral buffered formalin solution for seven days, the teeth were demineralized with Plank-Rychlo's decalcifying solution at 4 °C for four days, neutralized with 5% sodium sulfate for six hours, and then washed under running water for six hours. After removing the restorations, the teeth were dehydrated and finally embedded in paraffin.

Histological serial sections of 5-um thickness were prepared and stained with Hematoxylin and Eosin staining (H&E staining) or Brown & Brenn

gram staining to assess bacterial penetration. For each section, four histological features were evalu-ated: disarrangement of the odontoblastic layers, in-flammatory cell infiltration or response, reparative dentin formation, and bacterial staining. The fea-tures were graded under a light microscope as none

(0) , slight (1) , moderate (2) , or severe (3)16,19).

Evaluation criteria Characterization criteria of the evaluated histopathol-ogical gradings are shown in Table 2.

Statistical analysis Statistical analysis was done using KyPlot (Kyence Incorporated, Tokyo, Japan) statistical package for Windows version 3.0.2 software. Results of the dis-arrangement of odontoblastic layers, inflammatory cell infiltration, reparative dentin formation, and bac-terial staining were statistically analyzed using the non-parametric version of two-way analysis of vari-ance (Friedman Test) , as well as using non-

parametric multiple comparisons (Steel-Dwass Test) . Table 3 shows the statistical analysis for comparison within the materials and experimental time groups. The confidence level for all analyses was set at 95% level (p< 0.05) . The remaining dentin thickness be-tween the cavity floor and pulp was also recorded, and variance of the thickness was analyzed using one-way ANOVA (p< 0.05) .

RESULTS

Table 4 shows a summary of the findings on histological sections and thickness of remaining dentin. No statistically significant differences in re-maining dentin thickness (RDT) were found among the groups (one-way ANOVA, p>0.05) .

Disarrangement of odontoblastic layers The disappearance of primary odontoblasts of re-maining dentin underlying the cavity preparation and the aspiration of cell nuclei into these same dentinal tubules were observed. All the groups re-vealed changes to the odontoblastic layer to some ex-tent. Resin-modified glass ionomer luting cement showed one severe change at three days, followed by one moderate and two slight changes at 30 days. Glass ionomer cement showed one severe, two moder-ate, and three slight changes at three days, and also one moderate and one slight change at 30 days. In amalgam, three severe, three moderate, and two slight changes were detected at three days, one mod-erate and three slight changes at 30 days, and also three slight changes at 90 days.

Non-parametric version of two-way analysis of variance revealed that the disarrangement of odontoblastic layers was influenced by the material used and the experimental time period [ x^2= 19.951;

UZZAMAN et al. 181

Table 2 Histopathologic Evaluation Criteria & Characterization

Table 3 Grading of Histopathologic Features of Sections


Table 4 Steel-Dwass Analysis for Comparison within the Materials and the Experimental Time Groups

p<0.001 (Friedman Test, p<0.05) ] . Also, non-parametric multiple comparison analysis showed that a significant difference existed between the resin-modified glass ionomer luting cement and amalgam

groups only at three days [p = 0.01859; (Steel-Dwass test,p<0.05) ] .

Inflammatory cell infiltration No inflammatory cell infiltration was detected in resin-modified glass ionomer luting cement (Ionotite F) group, except one moderate reaction at 30 days

(Figs. 3 and 4) . Glass ionomer cement group showed only one moderate reaction at three days and one slight reaction at 90 days. In amalgam, one severe reaction and two moderate reactions were detected at

UZZAMAN et al. 183

Fig. 3 Resin-modified glass ionomer luting cement

(Ionotite F) at 3 days. Stained with H&E. Re- maining dentin thickness is approximately 0.65 mm. No disarrangement of odontoblastic layers or

inflammatory cell infiltration is seen (magnification X40).

Fig. 4 Resin-modified glass ionomer luting cement

(Ionotite F) at 90 days. Stained with H&E. Re- maining dentin thickness is approximately 0.45 mm. No inflammatory cell infiltration or repara-

tive dentin formation is seen (magnification X40).

Fig. 5 Glass ionomer cement (Fuji II) at 90 days. Stained with H&E. Remaining dentin thickness is approxi-

mately 0.5 mm. Moderate reparative dentin formation can be seen (magnification X40).

three days, and two cases of moderate reaction at 90 days were also seen.

Non-parametric version of two-way analysis of variance revealed that inflammatory cell infiltration was influenced by the material used and the experi-mental time period [ x^2=6.1; p=<0.05 (Friedman Test, p<0.05)]. However, no significant differences in inflammatory cell infiltration were found among the restorative materials or time periods [p>0.05; (Steel-Dwass test, p<0.05) ].

Reparative dentin formation At three days in all three groups, there were no indi-cations of any reparative dentin below the cut tu-bules of the remaining dentin.

In resin-modified glass ionomer luting group,

only two cases of slight reparative dentin formation were seen at 30 days. In glass ionomer group, three slight reparative dentin formations were observed at 30 days, whereas two moderate and two slight for-mations were observed at 90 days (Fig. 5) . In the case of high-copper amalgam, two slight and two moderate formations of reparative dentin were seen at 30 days, whereas one severe, two moderate, and four slight formations were observed at 90 days.

Non-parametric version of two-way analysis of variance revealed that reparative dentin formation was influenced by the material used and the experi-mental time period [ x^2=17.885; p<0.001 (Friedman Test, p<0.05)]. Also, non-parametric multiple com-

parison analysis of reparative dentin formation showed that a significant difference existed between the resin-modified glass ionomer luting cement and amalgam groups only at 90 days [p=0.005103; (Steel-Dwass test, p<0.05)].

Bacterial penetration Bacterial penetration along the cavity walls could not be detected in any of the resin-modified glass ionomer luting cement or glass ionomer cement speci-mens, except for one slight case at 30 days in glass ionomer cement group. The leakage ratings of resin-modified glass ionomer luting cement and glass ionomer cement groups showed no significant differ-ences. Amalgam showed five cases of bacterial pene-tration at three days, one case at 30 days, and three cases at 90 days.

Non-parametric version of two-way analysis of variance revealed that bacterial penetration was in-fluenced by the material used and the experimental time period [ x^2=16.222; p<0.001 (Friedman Test,

p<0.05)]. Bacterial penetration of amalgam at three days was of a significantly higher level compared


with resin-modified glass ionomer luting cement and

glass ionomer cement groups [p = 0.033242; (Steel-Dwass test, p<0.05) ] .

DISCUSSION

This in vivo study examined the pulpal response and sealing ability of a resin-modified glass ionomer ce-ment when it was used as a luting material for tooth-colored inlay restorations. The results seemed

quite promising. Pulpal irritation caused by resin-modified glass ionomer luting cement was considered to be of acceptable level for clinical applications; moreover, no severe reactions occurred in any of the experimental periods. According to ISO standard20) for in vivo biological testing of dental materials, zinc oxide eugenol (ZOE) cement is suggested as the non-irritating control restorative agent for placement in non-exposed cervical cavities of non-human primates. However, because of the poor physical properties in terms of solubility, setting shrinkage, and tensile strength, ZOE cement is seldom used clinically for

permanent restorations16,18,21) Therefore, it may be necessary to use control materials other than ZOE cement for pulp tissue research as well as for in vivo

leakage test. To date, amalgam and glass ionomer cement have sufficient evidence to be appropriate re-storative materials for clinical usage. In this in vivo

study, an attempt was made to use amalgam and

glass ionomer cement as control materials16) Amongst the materials placed into cavity prepa-

rations, significant differences were observed between disarrangement of odontoblastic layers, inflammatory cell infiltration, reparative dentin formation, and bac-terial penetration. The disarrangement and reduc-tion of odontoblasts, a sign of initial damage caused by restorative procedure and material, was compara-ble in all three groups at three days, and then de-creased with time except for high-copper amalgam. For resin-modified glass ionomer luting cement, its score for disarrangement and reduction of odontoblasts was low at three days (score 0.3; Fig. 3) ; whereas for conventional glass ionomer cement, it showed a slightly higher change (score 1.0) .

Since the effect of low pH (acidity) on pulpal ir-ritation has been suggested22) , materials with higher

pH value may be less irritating to the pulp tissues, resulting in less odontoblastic changes. The setting of conventional glass ionomer cement occurs by a se-ries of neutralization reactions involving the polyacid and calcium and aluminum ions leached from a reac-tive glass, resulting in the formation of a polysalt matrix23). The pH of the initial mix is reported to be low, but rises to a level approaching neutrality dur-ing the course of the setting reaction22,24) Smith and Ruse22) reported that the pH of the liquid component of some conventional glass ionomer cements ranged from 0.25 to 0.6; but immediately after mixing (0

minute) the pH of those cements ranged from 1.3 to 1.6. In the case of Ionotite F, pH value of the liquid component was measured to be 3.3 even though the value might not be stable due to a less-water content. Probably, this higher pH value of Ionotite F caused less irritation to the pulp, resulting in less odontoblastic changes. Our results also supported the results of Attar et al.25) . They reported that a resin-modified glass ionomer (Vitremer, 3M Dental Products) had an initial mean pH of 3.6, which was higher than the initial pH of conventional glass ionomer, thus resulting in low incidence of pulpal sensitivity problems26) .

The amount of reparative dentin formation may reflect the irritation level of initial stage and the re-sults of disarrangement of odontoblastic layers or in-flammatory reaction of pulp tissue at three days. For resin-modified glass ionomer luting cement, its scores for disarrangement of odontoblastic layers and inflammatory cell infiltration at three days were low (score 0.3) and none (0.0) respectively. For conven-tional glass ionomer cement, its corresponding scores at three days were slightly high (score 1.0) and low

(score 0.2) . In terms of reparative dentin formation at 90 days, resin-modified glass ionomer luting ce-ment scored 0.0 while glass ionomer cement scored 0.6 (Figs. 4 and 5) . For high-copper amalgam, its scores were 1.7 in the case of odontoblastic changes, 0.7 in the case of inflammatory reaction at three days, and 1.1 in the case of reparative dentin forma-tion at 90 days. However, it seemed highly likely-that the irritation effect of amalgam was not injuri-ous to pulp tissue since the tissue produced repara-tive dentin without any significant inflammatory re-action at 30 or 90 days, with a score of only 0.4 at 90 days.

In the case of bacterial penetration, glass ionomer cement showed good sealing properties. As for amalgam restorations, slight bacterial penetra-tion occurred - which was also found in previous studies17,27) . The higher frequency of bacterial pene-tration in amalgam restorations can be attributed to the inability to provide a complete seal along cavity walls and to the lack of adhesion15,18,19,21). Compared to conventional materials, resin-modified glass ionomers may offer a better seal to enamel/dentin due to their reduced water content, immediate adhe-sion, and superior wetting ability resulting from the use of HEMA28,29) . However, the sealing ability of resin-modified glass ionomer luting cement appeared comparable to that of glass ionomer cement, as it had sealing properties that prevented bacterial pene-tration. The presence of silica fillers - as ingredi-ents - in this luting material also seemed to have helped prevent bacterial penetration30,31) Murray et al.18) also reported that compared to other restorative materials, resin-modified glass ionomer scored better in both sealing ability and in prevention of bacterial

UZZAMAN et al. 185

microleakage.

In recent years, dual-cure resin cements have been used for luting indirect restorations. However,

for resin cement, although its bonding with dentin adhesives has been improved, microleakage studies') have revealed its insufficient marginal sealing of

bonded restorations. Meiers and Miller32) reported that resin-modified glass ionomer cement had greater

inhibition effect on cariogenic bacteria (S mutans. Ssorbinus, A viscosus, L salivarius) , especially on Streptococcus mutans strains. It was suggested that the antibacterial activity was associated with the low

pH of freshly mixed resin-modified glass ionomer ce-ment combined with the release of fluoride ions above a threshold value33). This antibacterial activity of resin-modified glass ionomer luting cement might have helped in preventing bacterial microleakage — an advantage over resin cement as a luting material for indirect restorations34) . Furthermore, HEMA (2-hydroxyethyl methacrylate) was also thought to have contributed to the antimicrobial action32,35) For all the above reasons, resin-modified glass ionomer cement - when used as a luting agent for indirect restorations - could also minimize some clinical

problems related to initial or secondary caries36,37) In this study, low-level pulpal reaction was observed at all time periods in the resin-modified glass ionomer luting cement group. This low-level reaction might be a result of fluoride release from the re-storative material38) , but further study is needed to confirm this.

Clinically, indirect restorations require several steps before setting of inlays, such as impression taking and temporization. Meanwhile, in our study, resin inlays were bonded soon after cavity prepara-tion. In this respect, discrepancy exists between the clinical procedure and our test method. However, temporization may decrease the irritation level of the

pulp tissue caused by cavity preparation or impres-sion taking. This meant that irritation level in den-tal clinics might be lower than the results of our study. Further study is also needed to confirm this.

The results of this study were limited to biocompatibility and in vivo microleakage in monkey teeth. To be assured of long-term desirable results, further in vivo and in vitro studies must be per-formed - for example, in terms of wear resistance within luting spaces and supporting capability for in-direct restorations.

CONCLUSIONS

Based upon the findings and within the limitations of this in vivo study, it was concluded that the evalu-

ated resin-modified glass ionomer luting cement showed an acceptable biological compatibility with

monkey pulps without bacterial penetration along the cavity walls. The in vivo sealing ability of this

resin-modified glass ionomer luting cement appeared

to be promising. Thus, it can be considered as a

good, alternative luting agent for tooth-colored indi-rect restorations in the future.

ACKNOWLEDGEMENTS

This work was supported by a grant for Center of

Excellence Program for Frontier Research on Molecu-

lar Destruction and Reconstruction of Tooth and

Bone at Tokyo Medical and Dental University. The

authors wish to thank Dr. Masaomi Ikeda for his as-

sistance in statistical analysis and Dr. Alireza Sadr

for his assistance with this manuscript.

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