**3.1.** *In vivo* **testing**

#### *3.1.1. Patch test*

Patch testing involves applying patches dipped in supposedly antigenic substance on a freerash segment and off the rash periods and maintaining them in contact with the skin for 48 h. The area for testing is the mid-upper back, anterior sides of forearms and upper external region of the arms, as shown in **Figure 14** [13].

IQ Ultra Chambers Box (Chemotechnique Diagnostics) tests were used, with methyl methacrylate as an allergen. All 10 subjects showed tenderness (sensibility) to methyl methacrylate [14].

#### *3.1.2. TNF alpha test*

TNF alpha (tumour necrosis alpha-factor) test which is eloquent for cytotoxicity detection was used in three cases of mucosal reactions in patients wearing acrylic dentures. The enzymelinked immunosorbent assay (ELIZA) method was applied to the patient's serum [15]. Five hundred microliters were harvested from each patient (having a duplicate sample) in order to determine the TNF alpha concentration. Quantikine Human TNF alpha immunoassay which

**Figure 14.** Patch test.

The measurements of the fracture toughness (*K*IC) were found to be 2.31 MPa√m for Meliodent

Meliodent 29.54 2.26 2.31 (*v* = 0.05 mm/min) Eclipse 25.35 3.18 3.26 (*v* = 0.05 mm/min)

**[MPa√m] SENB**

**Fracture toughness (***K***IC)** 

**[MPa√m] IS**

Results obtained by the strength indentation (IS) method are comparable to those obtained by

Acrylates are well known for their potential allergies. To evaluate the allergy potential of acrylic resins, we used *in vivo* and *in vitro* tests. For *in vivo* testing, we have used the patch test and the tumour necrosis alpha-factor (TNF) test, which is eloquent for cytotoxic detection and can detect early signs of allergy and inflammation by measuring endotoxins in patient serum. Acrylate toxicity is mainly due to the residual monomer. Therefore, we have also carried out tests to determine the amount of residual monomer present in the resin by the volatile-

Patch testing involves applying patches dipped in supposedly antigenic substance on a freerash segment and off the rash periods and maintaining them in contact with the skin for 48 h. The area for testing is the mid-upper back, anterior sides of forearms and upper external

IQ Ultra Chambers Box (Chemotechnique Diagnostics) tests were used, with methyl methacrylate as an allergen. All 10 subjects showed tenderness (sensibility) to methyl methacrylate

TNF alpha (tumour necrosis alpha-factor) test which is eloquent for cytotoxicity detection was used in three cases of mucosal reactions in patients wearing acrylic dentures. The enzymelinked immunosorbent assay (ELIZA) method was applied to the patient's serum [15]. Five hundred microliters were harvested from each patient (having a duplicate sample) in order to determine the TNF alpha concentration. Quantikine Human TNF alpha immunoassay which

the SENB method at low loading rates (~0.05 mm/min), as shown in **Table 3**.

**3. Studies concerning biocompatibility of acrylic resins**

**Material Hardness (H) [MPa] Fracture toughness (***K***IC)** 

component-content method and bromine index methods [11, 12].

region of the arms, as shown in **Figure 14** [13].

and 3.26 MPa√m for Eclipse resins.

12 Acrylic Polymers in Healthcare

**Table 3.** Measurement results for Meliodent and Eclipse.

**3.1.** *In vivo* **testing**

*3.1.2. TNF alpha test*

*3.1.1. Patch test*

[14].

allows the quantitatively determination of cytokines on patient serum was used. The substrate solution was obtained by mixing equal volumes of A and B reagents from the kit, maximum 15 min before usage. The supply solution (the undiluted standard) was prepared by reconstructing the cytokine standard using calibrator thinner RD6-21. For standard preparation, 500 μL of calibrator thinner RD6-21 was used. After pipetting 500 μL of solution in the first tube, the mixing concentration was calculated. The solution was well stirred and 500 μL was transferred into the next tube. The concentration is once again calculated. Standards for different concentrations are prepared in the same way (500, 125, 62.5, 31.2, 15.6 pg/mL). The supply solution was used as high standard (1000 pg/mL) and the calibrating thinner RD6-21 was used as zero standard (0 pg/mL). Note that 100 μL of thinner RD1-51 was pipetted in each hole of the plate and 100 μL of each standard was pipetted in the first strip in the following order: 0, 15.6, 31.2, 62.5, 125, 250, 500 and 1000 pg/mL. Starting with the second strip, 100 μL was pipetted. The plate was covered with an adhesive film and incubated for 2 h at room temperature. After that, the plate was washed automatically four times with 400 μL of washing solution and placed on an absorbent paper to clean the samples without damage. Note that 200 μL of cytokine conjugate was added in each hole and the plate was covered with an adhesive film and incubated for 2 h at room temperature. After the incubation period, the adhesive film was removed and the plate washed in the same way. Note that 200 μL of substrate solution was then added to each hole of the plate, the plate was covered again with an adhesive film and incubated for 30 min at room temperature, in darkness. After incubation time was over, 50 μL of stopping solution was added in each hole of the plate. When the reaction stops, the colour turns from blue to yellow. If the colour is green or does not modify uniformly, the plate will be slightly stirred for complete homogenization. The optical densities are determined 30 min after the reaction was stopped using an automatic reader at a wavelength of 450 nm, with a reference filter of 540 or 570 nm (for correction) (**Figure 15**). In the case of samples that are read only at 450 nm without correction, the accuracy may be influenced. Results are obtained depending on the logarithmic calibration curve of each cytokine, build on absorption and standard concentration (0–1000 pg/mL). The results are shown as optical density units auto-

**Figure 15.** Results reading for TNF alpha test.

matically converted into pg/mL for each of the sample tested. Determination of TNF-alpha concentration in the first case revealed slightly increased values of this cytokine (50.48–50.7 pg/mL). In the second case, the values were higher (90.3–90.9 pg/mL) and in the third case the values were the highest (100.4–107 pg/mL), which indicate certain inflammation. The results obtained with TNF-alpha test are eloquent for cytotoxic detection of early signs of allergy and inflammation by measuring the endotoxins from the patient serum. It is always recommended to associate multiple types of clinical tests and these should always be histologically confirmed [16].

#### **3.2.** *In vitro* **testing**

### *3.2.1. Volatile-component content method*

Three acrylic resin samples were used, each one being harvested from a different full denture base. The samples were preconditioned at 80°C for 2 h for removing moist. The weight of the samples is S1: 0.7863, S2: 0.05638 and S3: 0.8421 g. A Petri box is sterilized at 150°C for 1 h and then weighed in the analytical scale with high precision. The samples are positioned in the Petri box and weighed again very accurately. They are kept in the oven for 10 h at 150°C, and weighed again, very precisely. The weight difference is given by the amount of existing residual monomer: S1: 0.7864 g, S2: 0.5640 g, S3: 0.8422 g. In all these three cases, no significant weight loss was noticed. These results conclude that almost no residual monomer was found in the three full dentures [14].

#### *3.2.2. Bromine index method*

In order to verify the above results, we have used the bromine index method which determines the percentage of monomer in the sample, based on the amount of bromine added to the double bond (C=C) links. All the three samples were found to be free from the residual monomer. We have concluded that in the three PMMA samples, there is no residual monomer in the acrylic resin samples. But, there is less than a 0.0000 g order which is not detectable. This shows that a very accurate manufacturing method was adopted in the case of the three considered full acrylic denture bases [17].
