in vivo Evaluation of Marginal Microgaps of Sheep Incisors filled with Two Composite Materials

The aim of the study was to evaluate the marginal microgaps of two light-induced polymerization composite materials: Filtek P60 (3M ESPE) and Opticor New (Spofa Dental) in ovine teeth in vivo. The restorative materials were placed to type A-cavity to buccal surfaces of permanent teeth (Triadan system 301, 302, 303, 401, 402, 403). The variables of composite materials were evaluated in 3 groups of animals, 2 animals in each, at different time intervals (1, 6 and 9 months after beginning of the experiment). In various time intervals, 12 teeth per group were extracted under general injection anaesthesia, 6 teeth from each animal). Altogether 36 teeth were extracted throughout the experiment. During the experiment we observed neither cracks nor marginal discoloration in both Filtek P60 and Opticor New restorations. Retention of all restorations was 100%. Significant (P = 0.029 ANOVA) differences were observed in the dentin with Filtek P60 packable restorations which exhibited smaller marginal microgaps (OPTICOR NEW and dentin 11.09 , FILTEK P60 and dentin 5.64 μ). The ean size of icrogaps between dentin and the packable coposite aterial Filtek P60 was significantly lower (P = 0.029 ANOVA) in comparison with the microhybrid Opticor New composite restorations. These materials are suitable as permanent restoration of dental cervical caries in sheep and other herbivores, such as those kept in zoological gardens and companion animals. Light-cure filling, dentition, histology, restoration, sheep, experiment At present, veterinary dentistry often deals with the issue of caries. Due to wrong feeding this disease is diagnosed especially in companion animals kept in apartments (Hale 1998; Capík 2007). Caries (caries dentinum) is a disease of hard dental tissues. It is a multifactorial disease (Vaško et al. 1994; Capík 2008). Advanced dental caries may result in indication for dental crown reconstruction using metal materials (Capík 2005). In stomatology practice, different types of restorations are used. The size of arginal leakages between a filling and cavity wall represents an iportant evaluation criterion of filling aterials in clinical stoatology. These icrogaps arise ainly due to a polyerization shock as well as the coefficient of linear theral expansion of the filling and hard dental tissues. In vitro study by Loguercio at al. (2004) evaluated these microleakages. Specimens were thermocycled (500 cycles between 5 to 55 °C, analogous to changes of cold and hot feed and ingestion of liquids) and immersed in 0.5% methylene blue. The packable composites (e.g. Filtek P60) showed lower polymerization shrinkage compared to the hybrid resin. Food as a negative factor of polymerization shrinkage of composites is described by Att in et al. (1995) at the neck area of a tooth by post-operative sensitivity. When comparing the thermal expansion, Pucklet t et al. (1995) concluded that classical glass-ionomeric cements have the lowest thermal expansion. The composites, on the other hand, exhibited the highest thermal expansion. Microgaps arising between a coposite filling and the cavity wall for an entry for percolation of both micro-organisms and liquids, possibly resulting in hypersensitivity, ACTA VET. BRNO 2009, 78: 649-655; doi:10.2754/avb200978040649 Address for correspondence: MVDr. Mária Figurová Department of Small Animals University of Veterinary Medicine Komenského 73,041 81 Košice, Slovak Republic Phone: +421 904 584 102 E-ail: figurova@konex.sk coposite@orangeail.sk http://www.vfu.cz/acta-vet/actavet.htm pulp irritation, marginal discolorations and caries (Gojdišová 1997). At present, there is deand for coposite fillings that would ensure a quality aesthetic filling (Attin et al. 1995). In general, coposite aterials are hydrophobic and do not ake a fir connection with a wet surface of both dentine and cement. This connection is possible to be made only with the help of primers and dentine adhesives. With enamel, a connection is made with the help of micro-retention achieved by enamel etching with phosphoric acid (35% or 37% in accordance with dentistry manufacturer SpofaDental, 3M ESPE). After restoration of enamel hypoplasia in canine teeth by a composite, Capík (1996) described better results in the neck and central part of a tooth than in the area of crown top where the fillings were detached frequently (occlusive pressure in dogs is 10 × higher than that in huans). The defects in the first third of the crown were treated by reoving unevenness and polishing the surface (Capík 1996). Materials and Methods The experiment was carried out between June 2002 and May 2003 at the Clinic of Surgery, Orthopaedics and Roentgenology, UVM Košice, accredited for experients on anials conducted for scientific purposes (Act No. 115/95 of the Civil Code on Animal Protection), accreditation No. 12 766/02-220. The study was carried out on 6 adult sheep with permanent teeth. Two composite materials were used to reconstruct class A cavities: Opticor New (microhybrid) and Filtek P60 (packable composite), both of lightinduced polymerisation exhibits. We evaluated the following variables: marginal microgaps, marginal colour stability, quality and retention of restorations of class A cavities in vivo. The experiment was conducted on animals with healthy dental tissues. The characteristics of composites were evaluated in 3 groups of animals, (2 animals per group) at various intervals (at 1, 6 and 9 months in sheep after the beginning of the experiment). At the above-mentioned intervals, we extracted and evaluated 12 incisors from sheep (a total 36 teeth during the experiment), under general anaesthesia. Histological and statistical data are on 36 teeth. We were not concerned with occlusive forces. Sheep were selected as experimental animals suitable for this purpose. Preparation of class A cavity and their restoration was on animals under general anaesthesia. Preparation procedure Calculus was cleaned off the teeth and isolated from the buccal mucosa by means of paper cylinders. The prepared class A cavities were on the buccal surface of teeth. Those located on the left side of dentitions were restored with Filtek P60 (packable composite material) and those on the right side were restored with Opticor New (microhybrid composite material). Opticor New: Box-shaped cavities were cut in buccal surfaces. The cavities were made with a diamond round bur and cone bur in a water-cooled high-speed handpiece. After that, the enamel and dentin were etched with 37% ortophosphoric acid (Etching gel) for 30 s for enamel and 15 s for dentin. Excess water was removed with air, avoiding dentin dehydration. Then we applied the adhesive system Retensin MT to the dentin and enamel surface for 10 s. After that a thin layer (2 mm) of microphil composite material Opticor New was applied to the cavity and was light-cured for 30-40 s, each layer (lightcured polymerization with 630 mW·cm-2). Filtek P60 Box-shaped cavities were cut in buccal surfaces. The cavities were made with diamond round bur and cone bur in a water-cooled high-speed handpiece. After that the enamel and dentin were etched with 35% phosphoric acid (Scotchbond-etchant) for 15 s for enamel and 15 s for dentin. Excess water was removed with air, avoiding dentin dehydration. We then applied adhesive syste Adper Single Bond 2 × to the dentin and enael surface for 2-5 s. After that a thin layer (2.5 mm) of packable composite material Filtek P60 was applied to the cavity and was light-cured for 20 s each layer (light-cured polymerization with 630 mW·cm-2). Checking on the status of fillings: 3 times per week Premedication of all animals was achieved by administration of atropin a. u. v. at a dose of 0.05 mg·kg-1 i.m. General anaesthesia in sheep was induced by i.m. administration of combination of xylazin a. u. v. and ketamin a. u. v. at a dose of 0.4 mg·kg-1 and 5-10 mg·kg-1. The extracted teeth were conserved with 10% formalin. Specimens were prepared using water-hardened cynoacrylates. Sections were cut with a diamond saw and after hardening were embedded in solacryl. They were cut longitudinally and their thickness ranged between 100 and 150 μ. The width of icrogaps was easured in μ, separately for the dentin and enael. Statistical evaluation of the size of icrogaps was carried out by ANOVA and MANOVA. 650

At present, veterinary dentistry often deals with the issue of caries.Due to wrong feeding this disease is diagnosed especially in companion animals kept in apartments (Hale 1998;Capík 2007).Caries (caries dentinum) is a disease of hard dental tissues.It is a multifactorial disease (Vaško et al. 1994;Capík 2008).Advanced dental caries may result in indication for dental crown reconstruction using metal materials (Capík 2005).In stomatology practice, different types of restorations are used.
The size of arginal leakages between a filling and cavity wall represents an iportant evaluation criterion of filling aterials in clinical stoatology.These icrogaps arise ainly due to a polyerization shock as well as the coefficient of linear theral expansion of the filling and hard dental tissues.In vitro study by Loguercio at al. (2004) evaluated these microleakages.Specimens were thermocycled (500 cycles between 5 to 55 °C, analogous to changes of cold and hot feed and ingestion of liquids) and immersed in 0.5% methylene blue.The packable composites (e.g.Filtek P60) showed lower polymerization shrinkage compared to the hybrid resin.Food as a negative factor of polymerization shrinkage of composites is described by Attin et al. (1995) at the neck area of a tooth by post-operative sensitivity.When comparing the thermal expansion, Pucklett et al. (1995) concluded that classical glass-ionomeric cements have the lowest thermal expansion.The composites, on the other hand, exhibited the highest thermal expansion.
Microgaps arising between a coposite filling and the cavity wall for an entry for percolation of both micro-organisms and liquids, possibly resulting in hypersensitivity, pulp irritation, marginal discolorations and caries (Gojdišová 1997).At present, there is deand for coposite fillings that would ensure a quality aesthetic filling (Attin et al. 1995).
In general, coposite aterials are hydrophobic and do not ake a fir connection with a wet surface of both dentine and cement.This connection is possible to be made only with the help of primers and dentine adhesives.With enamel, a connection is made with the help of micro-retention achieved by enamel etching with phosphoric acid (35% or 37% in accordance with dentistry manufacturer SpofaDental, 3M ESPE).
After restoration of enamel hypoplasia in canine teeth by a composite, Capík (1996) described better results in the neck and central part of a tooth than in the area of crown top where the fillings were detached frequently (occlusive pressure in dogs is 10 × higher than that in huans).The defects in the first third of the crown were treated by reoving unevenness and polishing the surface (Capík 1996).

Materials and Methods
The experiment was carried out between June 2002 and May 2003 at the Clinic of Surgery, Orthopaedics and Roentgenology, UVM Košice, accredited for experients on anials conducted for scientific purposes (Act No. 115/95 of the Civil Code on Animal Protection), accreditation No. 12 766/02-220.
The study was carried out on 6 adult sheep with permanent teeth.Two composite materials were used to reconstruct class A cavities: Opticor New (microhybrid) and Filtek P60 (packable composite), both of lightinduced polymerisation exhibits.We evaluated the following variables: marginal microgaps, marginal colour stability, quality and retention of restorations of class A cavities in vivo.The experiment was conducted on animals with healthy dental tissues.The characteristics of composites were evaluated in 3 groups of animals, (2 animals per group) at various intervals (at 1, 6 and 9 months in sheep after the beginning of the experiment).At the above-mentioned intervals, we extracted and evaluated 12 incisors from sheep (a total 36 teeth during the experiment), under general anaesthesia.Histological and statistical data are on 36 teeth.We were not concerned with occlusive forces.Sheep were selected as experimental animals suitable for this purpose.
Preparation of class A cavity and their restoration was on animals under general anaesthesia.
Preparation procedure Calculus was cleaned off the teeth and isolated from the buccal mucosa by means of paper cylinders.The prepared class A cavities were on the buccal surface of teeth.Those located on the left side of dentitions were restored with Filtek P60 (packable composite material) and those on the right side were restored with Opticor New (microhybrid composite material).
Opticor New: Box-shaped cavities were cut in buccal surfaces.The cavities were made with a diamond round bur and cone bur in a water-cooled high-speed handpiece.
After that, the enamel and dentin were etched with 37% ortophosphoric acid (Etching gel) for 30 s for enamel and 15 s for dentin.Excess water was removed with air, avoiding dentin dehydration.Then we applied the adhesive system Retensin MT to the dentin and enamel surface for 10 s.After that a thin layer (2 mm) of microphil composite material Opticor New was applied to the cavity and was light-cured for 30-40 s, each layer (lightcured polymerization with 630 mW•cm -2 ).

Filtek P60
Box-shaped cavities were cut in buccal surfaces.The cavities were made with diamond round bur and cone bur in a water-cooled high-speed handpiece.After that the enamel and dentin were etched with 35% phosphoric acid (Scotchbond-etchant) for 15 s for enamel and 15 s for dentin.Excess water was removed with air, avoiding dentin dehydration.We then applied adhesive syste Adper Single Bond 2 × to the dentin and enael surface for 2-5 s.After that a thin layer (2.5 mm) of packable composite material Filtek P60 was applied to the cavity and was light-cured for 20 s each layer (light-cured polymerization with 630 mW•cm -2 ).
Checking on the status of fillings: 3 times per week Premedication of all animals was achieved by administration of atropin a. u. v. at a dose of 0.05 mg•kg -1 i.m.General anaesthesia in sheep was induced by i.m. administration of combination of xylazin a. u. v. and ketamin a. u. v. at a dose of 0.4 mg•kg -1 and 5-10 mg•kg -1 .
The extracted teeth were conserved with 10% formalin.Specimens were prepared using water-hardened cynoacrylates.Sections were cut with a diamond saw and after hardening were embedded in solacryl.They were cut longitudinally and their thickness ranged between 100 and 150 μ.The width of icrogaps was easured in μ, separately for the dentin and enael.Statistical evaluation of the size of icrogaps was carried out by ANOVA and MANOVA.

Clinical evaluation of the sheep
All restorations in the 3 groups (n = 2 each) were intact and showed no marginal discolouration.
In sheep of the 1 st (1 month following the restoration), 2 nd (6 months following the restoration) and 3 rd group (9 onths following the restoration), all the fillings were intact and without any marginal colour changes.The retention of restorations was 100% (Table 1).
No health complications occurred in the sheep throughout the study and their food intake and digestion corresponded to their physiology.

Histology
Evaluating methods of MANOVA and ANOVA were employed in the study.The intra-group factors were as follows: method / 2 levels: Opticor New and Filtek P60 / tooth (3 teeth were treated in each sheep using both methods).
The ethods provided significantly different results (P = 0.033).
The results of MANOVA were used for ANOVA to detect which conversion (enamel / dentine) causes the overall significance of MANOVA (Tables 1-3).
The measurements in sheep showed that the mean span of leakages between dentine and coposite OPTICOR NEW was 11.09 μ, and coposite FILTEK P60 restorations was 5.64 μ and the difference between the sizes was significant (P = 0.029 ANOVA).The icrogaps between sheep enael and OPTICOR New was 3.08 μ and FILTEK P60 was 1.69 μ were non-significant (P = 0.338 ANOVA) (Table 4), see Plate XIII, Figs 2, 3, Plate XIV, Figs 3, 4.

Discussion
When preparing the cavity it is very important to maintain the health of the pulp-dentine complex.This complex should exhibit a cellular activity demonstrated by high secretion activity of odontoblasts.necessary to retain a sufficient layer of dentine -RDT -residual dentine thickness.A very thin and insufficient layer of RDT (thinner than 0.5 mm) causes very low secretion reaction of dentine.This results in insufficient reparation (Murray et al. 2000).
Enrichment of composite material resins with the antibacterial compound Chlorhexidine (CHXA) has proven very efficient in slowing down the growth of a biofil on the teeth surface copared to the commercial composite material Z 250 and glass-ionomer cements Fuji II LC and Fuji IX.In the course of several weeks, this bacterial mixture penetrated through the microgaps between the composite material Z 250 and dentine (Leung et al. 2005).Much lower volume of bacterial substance penetrated through coposite aterials enriched with CHXA (Leung et al. 2005).The sizes of microgaps observed in our study in pre-determined time intervals did not result in development of caries caused by penetration of liquids into the space between hard dental tissue and filling.Deliperi et al. (2002) investigated the problems related to microgaps arising at polyerization of coposites on huan teeth using a flow-aterial.This is a flowing micro-hybrid composite applied to dentine and enamel as a basis.By this method, echanical properties of the fillings based on coposites have been iproved.Clinically, a lower occurrence of icrogaps was confired.The nuber of broken fillings, repeated caries, and post-operative sensitivity decreased.Kubo et al. (2004) used flow coposite aterials for filling neck cavities on buccal side of bovine incisors in vivo.The authors copared the variables of flow aterials with those of a hybrid coposite.The results of arginal integrity proved that separate use of flow materials resulted in a higher number and larger-size of microgaps compared to the use of hybrid composite.Lin et al. (1997) observed decreased efficiency of coposites in a 3-year study dealing with light polyerizing coposites in aesthetic fillings of the 1 st class of premolars.Especially after the third year they detected failures in the form of secondary caries, detachment of material from the cavity and appearance of marginal stains.
In vivo study by Koliniotou-Koumpia et al. ( 2004) compared the range of microgaps in the cervical area of canine teeth using two techniques of adhesive system with composites.Much lower number of micro-slots has occurred when using total etching (dentine and enamel) compared to the self-etching system.
In the course of our study the durability of all fillings was 100%.The eployed technique of total etching has ensured 100% intactness of fillings throughout the study.The fillings were free of fractures and marginal stains.
In vitro studies by Abuabara et al. (2004) showed marginal leakages between composite material, glass-ionomer cement (GIC), and hard dental tissues (human, swine, and bovine teeth).Much larger marginal leakage was exhibited by samples with GIC than with a coposite aterial.Saples fro swine and bovine teeth showed significantly higher percentage of marginal leakages than those from human teeth.The samples exposed to the action of swine and bovine substrates showed higher marginal leakage than those in the human substrate.Barros et al. (2003) compared the effect of different ways of polymerization of composite materials on development of microgaps.Better results were obtained at polymerization with a soft start and combined polymerization.Loguercio et al. (2004) carried out an in vitro study to examine microgaps in cementenamel connection between different composite materials.Compared to hybrid composites, condensable composites had a lower number of micro-leakages in a cement margin at polymerization shrinkage.When comparing the size of microgaps between dentine, enamel and different filling aterials (hybrid coposite, condensable coposite, flow coposite, and orocer), Yazici et al. (2004) found no significance.
In our study the measurements in sheep showed that the mean span of microgaps between dentine and coposite OPTICOR NEW was 11.09 μ, and coposite FILTEK P60 restorations was 5.64 μ and the difference between the sizes were significant (P = 0.029 ANOVA).The icrogaps between sheep enael and OPTICOR New was 3.08 μ and FILTEK P60 was 1.69 μ were non-significant (P = 0.338 ANOVA).Attar et al. (2004) investigated the significance of flow aterials in ters of reduction of the number of microgaps at gingival margin combined with micro-hybrid and condensable coposites.When using flow aterials, the nuber of icrogaps was reduced.
The results of in vitro studies conducted by Hasshoff et al. (2004) pointed to the importance of selection of suitable adhesive system primers.An ormocer was used in their study with a specific and universal adhesive syste and a hybrid coposite with a universal adhesive system.Statistically, the least measured micro-leakages were recorded with the cobination of a specific adhesive syste and orocer.In our study, we used original, manufacturer-recommended adhesive systems and the technique of total etching (phosphoric acid 35%, Scotchbond 3M ESPE) and etching with phosphoric acid 37% (etching gel, SPOFA Dental).This approach resulted in minimum-size micro-leakages with enael (0 to 3 μ).
The study of Santini et al. (2004), focused on evaluation of microgaps, showed no significant differences between the use of different types of self-etching agents and the technique of total etching with 36% phosphoric acid.Fabianelli et al. (2003) compared marginal leakages between composite materials and hard dental tissues and observed a worse marginal leakage of composite materials in the cervical area copared to occlusion.The application of a thin layer of flow coposite materials to the cervical area increased the marginal adaptation of condensable composite.The above authors observed lower occurrence of occlusive marginal leakages when using phosphoric acid.However, even the use of various flow coposite aterials in neck cavities failed to prevent fully the development of microgaps in a gingival margin of a filling.No flow aterials were used in our study.The ethod of total etching we have used has ensured a minimum to zero size of microgaps between the enamel and the composite materials used.
The measured sizes of microgaps between composite FILTEK P60 and enamel and between OPTICOR New and enael (1.69 μ and 3.08 μ, respectively) as well as sizes of microgaps between packable composite FILTEK P60 and dentine and between OPTICOR New and dentine (5.64 μ and 11.09 μ, respectively) had no effect on the development of caries in hard dental tissues.Civelek et al. (2003) observed a higher number of microgaps in cement-enamel area when using cobinations of coposite aterial and a flow aterial copared to the use of coposite aterial alone in ceent-enael region with all the variations of fillings as compared to occlusive margins.

Table 1 .
To ensure subsequent secretory reaction of dentine at deep preparation it is Retention and colour stability of composite materials in sheep dentition

Table 3 .
ANOVA -intra-group effectsThe values indicate that the onth of evaluation of icrogap size is significant only with dentine P = 0.029