DENTURE BASE RESINS BIOCOMPATIBILITY TESTING IN VIVO

Background. The wearing of acrylic dentures is the cause of the inflammatory reaction of the oral mucosa. The aim of this study was to investigate the response of rat tissues to subcutaneous and intramuscular implantation of acrylic samples. The aim of the study was to perform pathohistological analysis of tissues after subcutaneous and intramuscular implantation of samples of different acrylic materials. Methods and materials. The study included two samples of hard and three samples of soft acrylic resins (heat and cold polymerized), that were subcutaneously and intramuscularly implanted in rats tissues. Implantation tests were designed to test the biological response of the surrounding tissue of the tested materials after their application for a period of two weeks and four months. Results. After two weeks, regardless of the type of implantation, histopathologic analysis shows an acute inflammatory response. There was an intense hyperplasia of inflammatory cells, multiplication of connective tissue, as well as formation of many new blood vessels. The highest level of inflammatory changes was observed after the apliaction of cold-polymerized resins. A lower intensity of inflammation in the case of heat polymerised resin was the result of its more complete polymerization. After the second observation period, fibrotic capsules were formed around the implanted samples indicating a chronic course of the inflammatory process. Less visible signs of inflammation and chronicity of the processes indicate that with time, respectively, the length of observation period, reduces inflammation. Conclusion. Subcutaneous and intramuscular implantation of acrylic resin material samples led to inflammatory response whose intensity was decreased over time. Heat polymerized resin was a biologically more acceptable in comparison to the cold polymerized acrylates.


INTRODUCTION
Wearing of denture plates is very frequently the cause of inflammatory reactions of submucose of the oral cavity 1,2 .Hypersensitivity to acrylates was observed in almost 17% of patients wearing dentures 3 .
Adverse effects related to acrylates are, in most cases, of local character and may present in form cheilitis and stomatitis, stinging and burning in the mouth, painful sensations of different intensity and candidiasis [4][5][6][7][8] .Allergic reaction to acrylic denture may occur in more severe form such as erythema multiforme 9 .Potential toxicity of temporary acrylic dentures has been well documented.Contact stomatitis in children caused by wearing orthodontic appliances has been described in clinical practice as well 10 .
The above mentioned changes are more frequent in patients with already infected, inflammed submucose of the oral cavity damaged with different drugs or vomitting 11,12 .Some regions of the oral cavity are particularly sensitive to irritating effects of acrylic dentures 13 .Apart from being places that can endure additional strain, zones with keratinized epithelium represent places less sensitive to effect of harmful components of acrylates as well 14 .
The greatest number of irritations in the oral cavity disappear very quickly if their cause is eliminated 15 .Chronic prosthodontic stomatites is significantly less common and mainly occur in elder patients in the form of fibrous hyperplasia 16 .
Intense gingival inflammation under acrylic veneers of bridges, which may, among other things, be explained by porousness and superficial roughness as the result of great abrasion of acrylic material.Regarding the complexity of the potential clinical investigation biocompatibility of acrylic matherials used to manufacture dentures, it is easier to analyse tissue reaction to acrylic materials after implantation of the samples in tissues of experimental animals.Such a study comparing effects of different types of commercially available acrylic materials in vivo conditions has not been performed so far.
The aim.Study performed pathohistological analysis of tissues after subcutaneous and intramuscular implantation of samples of different acrylic materials.

Tested material
The tested material included two hard and three soft acrylates used in prosthodontic dentistry for construction and readaptation of mobile dental restorations.Cold and hot polymerized acrylates were used in the study (Table 1).
Upon polymerization the samples were polished using standard procedure in order to avoid mechanical irritation during the implantation in the tissue of experimental animal.
According to the type of the tested materials the samples were divided into five experimental groups (G1-G5), each of which were further subdivded into two groups depending on the place of implantation (subcutaneous and intramuscular).Each experimental group consisted of twelve samples (n=12) (six samples for subcutaneous and six samples fore intramuscular implantation).
A pink wax (Cavex, Holland BV) sample with identical shape and dimensions was made as a negative control for each implanted sample of the tested material (n=60).
Samples were combination of paraffin, microwax and bee's wax and their neutral effect on tissues previously experimentally was proven 17 .
All of the tested samples were desinfected with 70% ethanol and rinsed with saline (0.9% NaCl).The samples were stored in sterile Petri dishes at room temperature untill implantation.Immediately before implantation they were removed to Petri dish with sterile saline (no more than 60 minutes).

Experimental animals
Laboratory Wistar male rats, 10 to 12 weeks of age and 180-200gr of average weight were used in the experiment.Twelve animals were used for each of the tested materials (n=60).
The animals were healthy and acclimatized to laboratory environment and standard laboratory nutrition.They were followed for the behaviour change, disease onset and weight loss to eliminate potential irregularity that will affect plausibility of the obtained data.
Experimental investigations on animals were performed in accordance with Helsinki declaration (Approval of the Ethics Committee of the Faculty of Medicine in Niš, number 01-2066-1)

Experimental design
Implantation tests were designed for examining biological response of the surrounding tissue to the tested materials upon their application (ISO 10994-6: 2007) 18 .
Anesthetised animals were placed in prone position on a special wooden framework.
The operating field was prepared by removing hairs on interscapulary portion of the back and both thighs.
Implantation region was rinsed with Povidone iodine.The implantation procedure was performed using a sterile needle 4/18.The samples were subcutaneously implanted in the interscapulary portion of the back.The sample of the tested material was implanted on the left side of medial dorsal line and the sample of sterile pink wax was implanted on the right side.
The sample of the tested material was implanted in m. gastrocnemius of the left leg of the experimental animal, while the sample of the pink wax was implanted in the muscle on the opposite side.The wounds were healed with Povidone iodine and left to heal spontaneously.
No antibiotic protection of experimental animals was performed.Postoperative recovery was monitored every day, and there were no signs of infection.
The two-week observation period and four-month observation period were designed.
After each of the observation periods three animals from each experimental subgroup were sacrificed.

Euthanasia of experimental animals was performed by exanguination of left ventricle
and extirpation of complete blood.Changes in the subcutaneous tissue of the expereimental animal after a four-week implantation period were both macroscopically and microscopically observed.

Preparation of samples for microscopic analysis
Tube-like portions of subcutaneous and muscular tissue in the lumen of which there was an inplanted sample were taken as samples for analysis.The samples of the implanted materials were carefully separated from tissue by twizzers and fixated in 100% formalin.
The material was further dehydrated in growing concentrations of ethanol (from 50% to absolute).Upon xylol illumination the material was put in paraffin molds.Tissue blocks molded in paraplast were cut on microtom (LKB Broma, Sweden) (1.5 µm) and stained using classical method -Hematoksilin & Eozin (HE) and special method for staining of collagen fibres-Trihrom staining according to Masson.
The stained preparations were analysed pathohistologically on the image analysis system Lucia 3,2 G (Laboratory Imaging, the Czech Republic) on microscope NU-2 (Carl Zeiss, Germany).
The histomorphological analysis of tissues was designed for all samples of subcutaneous and muscular tissue that were in contact with the tested material and negative control for both observation periods.
The evaluation of results by microscopic analysis was performed on the basis of the presence of inflammatory reaction and tissue fibrosis, number and distribution of inflammatory cells and the existence of degenerative changes, as well as potential necrosis and destructive changes in capillary walls (Table 2).Table 2. Evaluation of degree of inflammatory reaction 19 .

RESULTS
Upon a two-week implantantion period it was noticed that implantation of Lang Immediate sample material caused macroscopically apparent changes on subcutaneous tissue in the form of mild erythema and local hemorrhage (Fig. 1).There were no local macroscopic changes on the implantation site of control samples and other tested materials.The highest degree of inflammatory changes were observed in cold polymerized acrylates (Fig. 2).The presence of Triplex Hot sample caused low intensity inflammatory changes in relation to other tested materials, which may be attributed to its more complete polymerization (Fig. 3).
There was no inflammatory reaction on the site of subcutaneous implantation of control sample, which eliminates mechanical trauma during application as the cause of occurrence of the above mentioned inflammatory reaction.Upon a four-week observation period there were no macroscopic changes on the implantation site of acrylic materials in all tested experimental groups.
Fibrous capsules were formed around the implanted samples as the result of the presence of material in the subcutaneous tissue.The hyperplasia degree of inflammatory cells was lower in comparison to the first observation period.Tissue fibrosis with lower number of connective tissue cells indicated chronic course of inflammatory process (Fig. 4).Less prominent inflammatory signs and chronic course showed reduction of inflammation over the observed period of time.Implantation of acrylic samples in m. gastrocnemius of experimental animals for a twoweek observation period caused no macroscopic changes on tissue.Pathohistological analysis of the surrounding muscle tissue after removal of material samples showed mild to strong inflammatory reaction, hyperplasia of connective inflammatory cells, intramuscular proliferation of connective tissue and a great number of newly formed blood vessels in the tested materials of all experimental groups (Fig. 5 and 6).Inflammation caused by the presence of implantes acrylic samples reduced over time, that is, along with the duration of observation period.Implantation of control samples of pink wax caused no significant changes in subcutaneous tissue and muscle tissue in the whole observation period (Table 3) .Table 3.Average values of the prominence degree of inflammatory reaction of tissue after subcutaneous and intramuscular implantation of acrylic samples.

DISCUSSION
The test of tissue reaction to the implanted material (ISO 10994-6: 2007) has no direct implication on clinical application 18 .However, immediate contact of implanted material and tissue offers a more precise picure of the bodies reaction to its presence.Subcutaneous implantation turned out to be an efficient metthod for the examination of biological features of dental materials 19,20 .The reaction of subcutaneous tissue may be considered analogous to that of submucous tissue regarding their unique histological form.
On the other hand, implantation of dental materials in muscle is considered to be a less sensitive method for examination of biocompatibility of dental materials, but the obtained data certainly contributes to the analysis of tissue reaction to acrylates.Samples of acrylic materials and control samples of pink wax were implanted in the rats'subcutaneous tissue and muscle during the two-week period and and four-month period.Changes in the tissues of experimental animals after removal of material samples may be considered analogous to the tissue reaction in direct contact with the denture plates.
The disadvantage of the applied test is reflected in the failure to identify the influence of salivary flow and its buffering capacity on the tissue reaction in contact with acrylic material.
Pathohistological analysis of subcutaneous tissue showed acute inflammatory reaction after removal of material samples.Intensive hyperplasia of inflammatory cells or granulomatous reaction, duplication of connective tissue, as well as formation of numerous new blood vessels were the results of a two-week presence of acrylic material.Fusion of macrophages led to the development of giant cells as the response to the presence of a foreign body.A more intensive reaction of subcutaneous tissue to the implantation of cold polzmerized acrylates represented further evidence of its biological inferiority as compared to heat polymerized acrylate.The obtained data may be explained by greater porousness and superficial adherence of materials, as well as greater amount of non-polymerized potentially toxic substances in samples of soft acrylates and Triplex Cold in relation to Triplex Hot.Findings of Kallus et al. also confirmed greater granulomatous reaction of subcutaneous tissue of rats to the presence of cold as compared to hot polymerized acrylate 1,2 .The results of this study did not indicate significant changes in inflammatory reaction of tissues to the presence of different types of soft acrylic materials.
Upon the second observation period fibrous capsules formed around implanted samples as the result of the presence of material.Less prominent inflammatory signs and chronic course of the process showed inflammation reduction over the period of time, which is in accordance with the findings of Kallus  Cold polymerized acrylates induced the most intensive granulomatous reaction after both implantation procedures, which is in accordance with the results of examination of their cytotoxic effect [25][26][27] .The results of this study are in positive correlation with the findings of in vitro studies that proved stronger cytotoxic effect of soft and hard cold polymerized acrylates as compared to hot polymerixed acrylate [28][29][30][31] .

CONCLUSION
Subcutaneous and intramuscular implantation of the samples of the tested acrylic materials led to acute inflammatory reaction became chronic over the period of time.Heat polymerized acrylate showed the least proinflammatory effect.Therefore authors recommend heat polymerited acrylates as the material of choice for construction and readaptation of dentures.

Figure 1 .
Figure 1.Changes in subcutaneous tissue observed on implantation site of Lang Immediate sample.

Figure 2 .
Figure 2. Application of samples in subcutaneous tissue of experimental animal after two weeks caused the occurrence of the mild degree fibrosis, hyperplasia of inflammatory cells and formation of new blood vessels:(a) Bosforth Trusoft, (b) Lang Flexacryl, (c) Triplex Cold.(Trihrom according to Masson, 100).

Figure 3 .
Figure 3. Upon two weeks, subcutaneous application of Triplex Hot sample caused proliferation of connective tissue cells and fibrosis (HE200).

Figure 5 .
Figure 5. Upon a two-week implantation period of material samples Lang Flexacryl (a) and Lang Immediate (b) hyperplasia of giant cells of foreign body type and intensive proliferation of connective tissue were observed.Fibrous reaction with numerous newly formed blood vessels occurred around hollow spaces with Triplex Cold sample (c) (Trihrom according to Masson, 200).

Figure 6 .
Figure 6.A two-week implantation period of Triplex Hot sample caused proliferation of connective tissue cells and intensive fibrosis in muscle tissue (Trihrom according to Masson, 100).

Figure 7 .
Figure 7. Mild degree fibrosis was present in muscle tissue after a four-month implantation of Lang Flexacryl sample(a) and Triplex Hot (b) sample, while moderate degree fibrosis was present in Lang Immediate sample (c) (Trihrom according to Masson,  200).
et al. and Zmener 2,19 .The presence of Triplex Hot sample caused low intensity inflammatory changes in relation to other tested materials, which may be attributed to its more complete polymerization.Analysing different materials for denture base Ebadian et al. indicate higher biocompatibility of polymerized PMMA in comparison to Co-Cr alloys after implantation in buccal vestibulum of dogs 22 .Pathohistological analysis of muscle tissue surrounding the tested acrylic materials that were removed after the first observation period showed moderate to strong granulomatous inflammatory reaction, hyperplasia of connective inflammatory cells, intramuscular proliferation of connective tissue and a great number of newly formed blood vessels.The most intensive inflammatory response was noticed in soft acrylic material Lang Flexacryl and solid cold polymerized Triplex Cold.After implantation of the Lang Flexacryl sample hyperplasia of giant cells of a foreign body type in muscle tissue was observed.Implantation of Bosforth Trusoft and Lang Immediate samples led to moderate proliferation of young connective tissue in the muscle and duplication of inflammatory cells.The presence of the hot polymerized Triplex Hot samples in muscle tissue led to the proliferation of connective tissue cells and intensive fibrosis and reaction had chronic course from the very beginning.In accordance with this study, Stinson et al. showed that implantation of poly (methil methacrylate) -PMMA sample in gluteal muscles of rats led to formation of fibrous capsule over the period of time 23 .Dillingham et al. found mild toxicity of PMMA after it had been implanted in paravertebral muscle of a rabbit 24 .Biocompatibility of PMMA based materials was proved after their implantation in the bone as well17 .Formation of fibrous capsule surrounding the application site represents the result of a four-month implantation of the cold polymerized material samples on microscopic level.The tissue fibrosis ranged from mild (Bosforth Trusoft, Lang Flexacryl, Triplex Cold, Triplex Hot ) to moderate degree (Lang Immediate).Pathohistological findings of all tested materials confirmed domination of connective fibres in comparison to inflammatory cells, indicating chronic type of inflammation.Inflammation induced by the presence of implanted acrylic samples reduced over the observation period of time.Pathohistological analysis of the prominence degree of inflammatory reaction of tissue that was in immediate contact with the tested material sample showed more prominent inflammatory reaction in subcutaneous implantation as compared to intramuscular implantation in all tested experimental groups of acrylic materials.Control samples caused no inflammation of the surrounding tissue and the occurrence of mild fibrosis in few of them was probably the result of mechanical tissue damage during the implantation procedure.

Fig 1 .
Fig 1. Changes in subcutaneous tissue observed on implantation site of Lang Immediate

Fig 2 .
Fig 2. Application of samples in subcutaneous tissue of experimental animal after two

Fig 3 .
Fig 3. Upon two weeks, subcutaneous application of Triplex Hot sample caused

Fig 4 .
Fig 4. Upon a four-month subcutaneous implantation of material sample focal tissue

Fig 5 .
Fig 5. Upon a two-week implantation period of material samples Lang Flexacryl (a) and

Fig 6 .
Fig 6.A two-week implantation period of Triplex Hot sample caused proliferation of

Fig 7 .
Fig 7. Mild degree fibrosis was present in muscle tissue after a four-month implantation of

Table 2 .
19aluation of degree of inflammatory reaction19

Table 3 .
Average values of the prominence degree of inflammatory reaction of tissue after subcutaneous and intramuscular implantation of acrylic samples.