OSLOBAĐANJE POTENCIJALNO TOKSIČNIH KOMPONENTI IZ AKRILATA ZA MEKO PODLAGANJE PROTEZA A RELEASE OF POTENTIALLY TOXIC COMPONENTS FROM THE ACRYLIC RESINS FOR SOFT RELINING DENTURES

Uvod: Meki akrilati pripadaju grupi hladno polimerizovanih materijala koji se aplikuju na bazalnu površinu zubne proteze kako bi se eliminisale mehaničke iritacije, omogućilo ozdravljenje oštećene ili inflamirane sluzokože ili se oralno tkivo pripremilo (kondicioniralo) za prihvatanje nove nadoknade. Cilj istraživanja bio je ispitivanje ritma oslobađanja komponenti mekih akrilata u tri različita modela veštačke pljuvačke, u toku tridesetodnevnog opservacionog perioda. Materijal i metode: U ispitivanju su korišćena dva meka akrilata:poli (etil metakrilat)/ n-butil metakrilat i poli (etil metakrilat) / metil metakrilat, koji su odlagani u tri različita modela veštačke pljuvačke, u okviru tri opservaciona perioda: jedan, sedam i trideset dana. Ispitivanje je obuhvatilo detekciju metil metakrilata, etil metakrilata, butil metakrilata, di butil ftalata i benzoil peroksida tečnom hromatografijom pod visokim pritiskom. Rezultati: Količina oslobođenih komponenti srazmerno se povećava sa porastom trajanja opservacionog perioda, bez obzira na model veštačke pljuvačke.Najviše vrednosti svih ispitivanih parametara uočene su nakon tridesetodnevnog opservacionog perioda. Model veštačke pljuvačke nije uticao na ritam oslobađanja komponenti, što znači da se one nesmetano oslobađaju u usnu duplju bez obzira na sastav pljuvačke pacijenta. Zaključak: Sa porastom dužine opservacionog perioda došlo je i do očvršavanja materijala, čime se završava njihova upotrebna vrednost.


Introduction
Soft acrylic resins belong to a group of cold curing materials that are applied to the basal surface of the dental prosthesis in order to eliminate mechanical irritation, and to allow for the recovery of damaged or inflamed mucous membranes, and prepare the oral tissue to accept a new compensation. Soft acrylic resins remain resistant for a certain period of time and, thanks to their viscoelastic properties, provide an equal distribution of the masticatory forces to the supporting tissues, thus removing the pain and distress of the prosthesis carrier with very reduced, sharp and submerged alveolar ridge and hypersensitive or movable mucous membrane 1,2 .
The basic polymer (powder) of a twocomponent soft acrylate system is usually poly (ethyl methacrylate) (PEMA) or poly (methyl methacrylate) (PMMA). The liquid is a mixture of ester plasticizers (dibutyl-phthalate (DBP), butyl glycolate) consisting of 30-60% of ethanol as a solvent, which content is about 4-60% 2,3 . Plasticizers, which are added to acrylic resins, make these materials soft at body temperature 4 . Plasticizers are soluble in oral fluids, and their gradual loss in time leads to the hardening of the material in the mouth of the patient 5 . The cold curing agent is benzoyl peroxide (BP).
Ethanol is absorbed by the polymer particles when mixing the powder with the liquid. The particles of the PEMA with smaller molecular weight allow for faster and more extensive penetration of alcohol within the polymer than the conventional PMMA. On the other hand, by the action of intermolecular Van der Waals forces, the chains of the polymer are separated, so that the large molecules of the plasticizer interpenetrate between them. After homogenization, the whole mass is obtained by gelatinous form due to the chains of polymers trapped inside the matrix composed of ethanol and plasticizers 1 .
The release of ethanol from the polymerized conditioner into the oral environment begins immediately after giving the dentures to the patient, which is compensated by water absorption 2,3 . Since the loss of ethanol and water absorption do not occur simultaneously and at the same speed, the physical properties of the material change over time. After the end of the absorption process, the material is fully hardened, so it should be replaced or the dentures used as a definitive one 6,7 .
According to the literature, the components included in the structure of soft acrylic resins may be toxic to oral tissues if they are not completely neutralized in the polymerization process [8][9][10][11] . Since there is no absolute bonding of material during its preparation and development of dental prostheses, the research started from the assumption that at the time of using soft acrylates as a liner, the components will be released into the surrounding liquid medium.
The aim of the study was to examine the rhythm of the release of soft acrylate components in three different models of artificial saliva, during a thirty-day observation period.

Material and methods
Two soft acrylic resins that were used during the study are Lang Flexacryl (PEMA/nbutyl methacrylate) and Lang Immediate (PEMA/methyl methacrylate) (Lang Dental MFG.Co., USA).
27 samples were made of both materials (dimension: 10x10x2 mm) according to the manufacturer's instructions, in a condensation silicone mold. After making the samples, they showed the expected resilience.
Model 1 and 2 differ in the content of the enzyme, α amylase. Incubation of samples in artificial saliva was performed in closed plastic containers, in a water bath, at a temperature of 37 ± 10°C. The mass ratio of samples and volume of artificial saliva was 0.1 g of material/1 ml artificial saliva (ISO 10993-5:1999) [14][15][16] .
A quantity of released substances was examined in the saliva model after removal of the sample after each of the mentioned periods. The study involved the detection of methyl methacrylate (MMA), ethyl methacrylate (EMA), butyl methacrylate (BMA), dibutyl phthalate (dBuFt) and benzoyl peroxide (BP).
Agilent 1100 Series (USA), with the DAD 1200 detector and the analytical column SUPELCO Discovery HS C18 250× 4.6 µm, 5 mm, Sigma-Aldrich, USA was used as a high-pressure liquid chromatography device (HPLC). The methanol was used as an eluent. The flow rate of the mobile phase was 1 cm 3 /min, and the sample injection volume was 20 ml. The column is thermostated at 25°C. The wavelength of detection was 205 nm.
The samples of the solution of three models of artificial saliva were used for HPLC analysis without prior processing. Then, the samples were filtered on a 0.45 mm pore diameter filter, which was followed by an injection of 20 µl into an HPLC device.
The calibration curves were made from a series of solutions of each of the tested substances in methanol. The initial concentration of the tested compound was about 1 mg / cm 3 , from which, after dilution with the methanol, a series of solutions of lower concentrations were made. The required data from the obtained chromatograms were read out: the retention time of each compound (Rt) and the surface of the peak (A). The device reads the UV / VIS spectrum at each peak point on the chromatogram, so that the λ max value can be determined from the spectrum i e., the wavelength at which the compound has a maximum absorbance.
The concentration of the tested components in artificial saliva models is presented as μg of the substance/cm 3 of the saliva solution. Concentrations were viewed through a mean value with standard deviations. A comparison was made of the obtained concentration values within the same group of materials, depending on the length of the incubation period, as well as between the two tested materials.
U tumačenju rezultata treba naglasiti i činjenicu da su uzorci sa porastom dužine opservacionog perioda menjali svoju konzistenciju, te su nakon tridesetdana postali čvrsti, izgubivši rezilijentnost. Table 2 and table 3 show the obtained values of the released components of the tested materials in three models of artificial saliva using HPLC method after three observation periods.

Results
The amount of released components increases proportionally with the increase in the duration of the observation period, regardless of the artificial saliva model. A higher concentration of released components was detected in Lang Immediate material.
In interpreting the results, it should be emphasized that the samples with the increase in the length of the observation period changed their consistency, and after thirty days they became firm, losing their resilience.

Discussion
This study involved the analysis of three different artificial saliva solutions in terms of detection of the required components by HPLC method.
In proportion to the time of disposal, there has been an increase in the concentration of released components in saliva models, which also indicates their decrease in the samples itself, and also the increase in their biocompatibility. The artificial saliva model did not affect the trend of the obtained results, nor the addition of the α amylase enzyme. In accordance with the obtained results, it can be concluded that the composition of the saliva does not affect the release of unbound components from the soft acrylic resins.
Previous studies have indicated the potentially toxic effect of certain unbound substances from acrylic materials 11,[16][17][18] . Research by Kostic et al. indicated a decrease in the amount of these components in the samples of the material after their immersion in the aqueous environment 19 and the post-polymerization procedures 20 .
BP is used in dermatology, where there are described cases of contact dermatitis in about 1% of cases [23][24][25] . Oyama and Imai pointed to the cytotoxic effect of BP 26 . The total amount of BP is not consumed in the starting process of the polymerization of acrylic resins, although it is added to the powder of acrylic material at very low concentrations of 0.2 and 1.28% 23,25 . BP did not show significant variations in the amount released from the Lang Immediate and Lang Flexacryl samples. A uniform release from acrylate samples was observed, and the highest concentration in saliva was after thirty days of sample incubation.
Cytotoxicity of phthalate, a plasticizer of acrylate materials was also demonstrated [26][27][28][29][30] . The values of the released amount of plasticizer (dBuFt) after the first day of immersion, in soft acrylate samples, are also similar. In Lang Immediate case, the amount of dBuFt increases uniformly with the increase of the incubation period. The values obtained after thirty days were less compared to the Lang Flexacryl model. In Lang Flexacryl case, a sudden increase in the concentration of the plasticizer occurs between the seventh and thirtieth day of immersion. With the release of the plasticizer into artificial saliva, changes in the physical characteristics of soft acrylic materials were recorded.
In order to improve the biocompatibility of soft acrylic resins, the conditioners without phthalate were synthesized, whose deficiency is a rapid loss of viscoelasticity. Oral conditioning without ethanol is also synthesized, obtained by the combination of vinyl esters and PEMA 2 .
It is not possible to accurately determine the standard values of the minimum quantities of analyzed components of acrylic materials that could cause a toxic or allergic reaction of the oral tissue. On the other hand, in a concentrated form, they show a strong toxic effect.
Given the proven toxic effect, the maximum reduction by strict observance of the powder and liquid ratio is very important. It is done through the polymerization procedure prescribed by the manufacturer and the disposal of prosthetics made of cold curing acrylics in water for several days before handing over to the patient 31-33 .

Conclusion
Tested potentially toxic substances were uniformly released into the aquatic environment, in proportion to the incubation length.
The highest values of all tested parameters were detected after a thirty-day observation period. The artificial saliva model did not affect the release of the components, which means they are freely released into the oral cavity regardless of the composition of the patient's saliva. A higher concentration of released components was detected in the Lang Immediate material.
As the length of the observation period increased, the material was solidified, thus ending their use value.
The inflammatory effect of the soft acrylic components on the oral mucosa will be the subject of future research. .