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ORIGINAL ARTICLE
Year : 2019  |  Volume : 11  |  Issue : 1  |  Page : 32-36

In Vitro Analysis of the Proliferation of HaCaT Cells Stimulated by Pigments Used for Maxillofacial Prostheses


1 Department of Dental Materials and Prosthodontics, Araçatuba Dental School, University Estadual Paulista – UNESP, Ara-çatuba, São Paul, Brazil
2 Department of Surgery and Integrated Clinic, Araçatuba Dental School, University Estadual Paulista – UNESP, Ara-çatuba, São Paul, Brazil
3 Department of Basic Science, Araçatuba Dental School, University Estadual Paulista – UNESP, Ara-çatuba, São Paul, Brazil

Date of Web Publication9-Aug-2019

Correspondence Address:
Prof. Marcelo Coelho Goiato
Department of Dental Materials and Prosthodontics, Aracatuba Dental School, University Estadual Paulista – UNESP, José Bonifácio street, #1193, Araçatuba, São Paulo, 16015-050
Brazil
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jofs.jofs_1_19

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  Abstract 


Introduction: Although physical and mechanical properties are extremely important for the effectiveness of prosthetic treatment, materials used for facial rehabilitation should be biocompatible, as the maxillofacial prosthesis is in close contact with the patient’s skin. The objective of this study was to evaluate the influence of different eluates formation periods obtained from the methyl tetrazolium salt (MTT) and Alamar Blue assays, analyzed in different silicones and pigments used in the manufacture of maxillofacial prostheses in the proliferation of human keratinocytes (HaCaT cells). Materials and Methods: For the preparation of samples, A2 A-2186 and Silastic MDX4-4210 silicones and three types of pigments (bronze, black, and medium pink) were selected. Two of the pigments were specific for the characterization of the prosthesis and already available in dental market in bronze and black colors, and a new pigment was tested in the medium pink color. Five samples were prepared for each group and distributed according to the type of silicone and pigment added, aiming to prepare the eluates after 24 and 72 h of sample’s immersion in culture medium. In vitro assays of MTT and Alamar Blue were performed for cytotoxic analysis. Data obtained were submitted to analysis of variance and the Tukey test, with a 5% significance level. Results: All groups had cell proliferation percentages higher than 75%, indicating absence of cytotoxicity. Conclusions: It can be concluded that all the materials used are not cytotoxic, including the proposed new pigment, for the preparation of maxillofacial prostheses.

Keywords: Coloring agents, cytotoxicity, immunologic, keratinocytes, materials testing, maxillofacial prosthesis


How to cite this article:
Goiato MC, Nobrega AS, Malavazi EM, Takamiya AS, Penha de Oliveira SH. In Vitro Analysis of the Proliferation of HaCaT Cells Stimulated by Pigments Used for Maxillofacial Prostheses. J Orofac Sci 2019;11:32-6

How to cite this URL:
Goiato MC, Nobrega AS, Malavazi EM, Takamiya AS, Penha de Oliveira SH. In Vitro Analysis of the Proliferation of HaCaT Cells Stimulated by Pigments Used for Maxillofacial Prostheses. J Orofac Sci [serial online] 2019 [cited 2019 Aug 25];11:32-6. Available from: http://www.jofs.in/text.asp?2019/11/1/32/264179




  Introduction Top


The perception of beautiful is an important factor in a person’s life, as it directly interferes in their social and interpersonal relationships.[1] Mutilations, mainly in the face, caused by genetic alterations, traumas, and/or surgical resections[2],[3],[4],[5],[6] can cause severe psychological consequences.[7]

Frequently, maxillofacial prostheses are used to disguise facial defects, aiming to provide a more pleasing appearance, and improvement in function.[3],[8] This allows the mutilated patient to recover their social routine, by improving their self-esteem and their quality of life.[4],[5],[6]

Although physical and mechanical properties are extremely important for the effectiveness of prosthetic treatment,[9] it is even more important that the materials used for facial rehabilitation are biocompatible.[10],[11] As the maxillofacial prosthesis is in close contact with the patient’s skin, it should not generate any type of local or systemic inflammatory response.[12],[13] Biocompatibility tests can be performed in vivo or in vitro. In vivo tests to assess toxicity have always been criticized for scientific, economic, and ethical reasons.[14] On the other hand, in vitro tests are able to safely and quickly demonstrate the cellular response to a studied material with a low-cost and a well-defined standardization.[12],[15]

The literature shows different in vitro tests to determine the material’s cytotoxicity, such as the methyl tetrazolium salt (MTT)[11],[15] and the Alamar Blue assays,[16] which reflect cellular metabolism through mitochondrial activity. Cell proliferation or inhibition can be observed through the cell culture method, by using the mentioned assays, due to the contact with cytotoxic substances.

The aim of this study was to evaluate the influence of different periods of formation of eluates obtained from different pigments used for maxillofacial prosthesis fabrication on the cytotoxicity of human keratinocytes (HaCaT cells), through MTT and Alamar Blue assays. The null hypothesis was that the different periods of formation of eluates obtained from different pigments used for maxillofacial prosthesis fabrication do not produce cytotoxic effects on the cell culture studied.


  Materials and Methods Top


For the preparation of samples, A2 A-2186 (Factor II, Lakeside, AZ, USA) and Silastic MDX4-4210 (Dow Corning Corporation Medical Products, Midland, MI, USA) silicones and three types of pigments were selected. Two of the pigments were specific for the characterization of the prosthesis and already available in dental market in bronze (Tan FI-215) and black (Black FI-205) colors (Functional Intrinsic II, Factor II, Lakeside, AZ, USA), and a new pigment was tested in the medium pink color (Pigmento Rosa Médio Orbital, Orbital Colors, Birigui, SP, Brazil). The pink pigment was obtained from primary colors − red and yellow − mixed with the shading and shadowing colors − white and black, respectively.

The present study was approved by the Research Ethics Committee of the Aracatuba Dental School, São Paulo, Brazil. procotol no. 594 on 12th July 2017.

Five samples were prepared for each group and distributed according to the type of silicone and pigment added as follows: Group MDX − MDX4-4210 Silicone; Group Pink MDX − MDX4-4210 Silicone + Pink pigment; Group Bronze MDX − MDX4-4210 Silicone + Bronze pigment; Group Black MDX − MDX4-4210 Silicone + Black pigment; Group A Factor − A2 A-2186 Silicone; Group Pink A Factor − A2 A-2186 Silicone + Pink pigment; Group Bronze A Factor − A2 A-2186 Silicone + Bronze pigment; and Group Black A Factor − A2 A-2186 Silicone + Black pigment.

Silicones and pigments were weighed in a digital analytical balance (Adventurer, Ohaus Corporation, NJ, USA) for sample confection.[9] The bronze and black pigments were added to 0.2% of the weight of the silicone. The medium pink pigment was added according to the manufacturer’s specification (White: 0.6%; Yellow: 0.122%; Red: 0.03%; Black: 0.006%).

The pigments were added to the silicone through an industrial manipulation using a grinding machine in the Orbital Colors Industry (Orbital Ind. E Com. Ltda., Birigui, SP, Brazil). The process consisted of three stages. Initially, the pigment was added to the silicone through a propeller disperser, then passed through a tricylinder mill, and subsequently by a quality control test, according to DIN standard. The catalyst was added to the material at laboratory level, according to manufacturer’s specification, and poured into a metallic matrix (10-mm diameter × 1-mm thickness).[17] The matrix was placed into a equipment for polymerizing at 20 pounds for 20 min. After this period, the silicone, contained in the matrix, was exposed to room environment for 72 h until complete polymerization of the material and release of the by-product (formaldehyde), according to manufacturer’s recommendation. After this period, each sample was carefully separated from the matrix to avoid distortions.

For the cell viability analysis, a pilot study was conducted to determine the concentration of bovine fetal serum (BFS) that would be used for eluate’s preparations. Then, five samples of each group were placed in tubes containing 9 mL of DMEM culture medium (Dulbecco’s modified Eagle’s) supplemented with 10% BFS and incubated at 37°C for 24 and 72 h. These periods allowed leaching of hydrosoluble substances from the samples into the culture medium, for the analysis of their cytotoxic effect.[18],[19],[20] A control group consisted in the culture medium without sample. After the leaching, the equates were filtered with 0.22-μm Millex filters (Millipore) for sterilization, due to the great ease contamination of the medium due to its richness in nutrients.

The possible cytotoxic effect of the substances released by the tested materials was evaluated by the cell culture method. Thus, keratinocytes (HaCaT) were expanded in DMEM culture medium with a 10% supplementation with BFS in an environment with 5% of CO2, controlled humidity, and at 37°C until reaching confluence for the assays. Cell suspensions of 1.0 × 105 cells/mL were prepared,[10],[18],[21],[22] with 1 mL being pipetted into each well of a 24-well plate. After 24 h, the culture medium was discarded and 500 µL of the different eluates were added to each well. The negative control consisted of 1 mL of DMEM culture medium with a 10% supplementation with BFS. The positive control consisted of 3 µL of Tween diluted in 1 mL of DMEM with a 10% supplementation with BFS. Both were submitted to the same incubation and temperature conditions used for obtaining eluates.[23]

After each 24 and 72-h treatment period, the MTT and Alamar Blue assays were performed. For the MTT assay, culture media with the eluates were removed and replaced by 0.5 mL of DMEM medium without the addition of BFS containing 0.5 mg/mL of MTT (3- [4,5-dimethyl-2-thiazyl]-2,5-diphenyl-2H-tetrazolium bromide), and incubated for 2 h at 37°C. After incubation, the wells were checked for the presence of intracellular formazan crystals. The medium was replaced by 500 µL of isopropyl alcohol for the reading in triplicate at 570 nm in 96-well plate in an ultraviolet-visible spectrophotometer.[23]

For the Alamar Blue assay, mitochondrial activity was evaluated by the reduction of resazurin to resofurin in the presence of Nicotinamide adenine dinucleotide an oxidized and reduced form (NADH). After the incubation periods of the cells in contact with the eluates, 20 µL of Alamar Blue extract were diluted in 100 µL of culture medium and added to each well of the plate and incubated for 4 h at 37°C. Then, the contents were transferred to a 96-well plate and the reading was performed in triplicate at 570 and 600 nm a spectrophotometer.[24]

Results were evaluated using GraphPad Prism 5.0 software, in which a two-way analysis of variance was applied, followed by the Tukey test with a 5% significance level.


  Results Top


The interaction between period and group did not significantly interfere in cell proliferation by the MTT assay (df = 9; F = 0.880; P = 0.551), but only the group factor (df = 9; F = 99.361; P < 0.001). In the period of 24 h of eluate’s formation, the MDX (86.5%), Pink MDX (85.3%), A Factor (79.2%), and Pink A Factor (85.5%) groups presented significantly lower percentages of cell proliferation than the control group. In the period of 72 h of eluate’s formation, lower percentages were observed for the Bronze MDX (84.3%), A Factor (86.2%), and Bronze A Factor (85.1%) groups. When the groups were evaluated separately, there was no difference between the time periods [Figure 1].
Figure 1 Percentage ± standard deviation of cell proliferation obtained in the MTT assay for the different groups tested in the periods of eluate formation for 24 and 72 h. Different upper and lowercase letters indicate statistical difference in relation to the respective nonstimulated group in the periods of 24 and 72 h, respectively. MTT, methyl tetrazolium salt.

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In the Alamar Blue assay, the interaction of all periods and all groups analyzed did not significantly interfere in cell proliferation (df = 9; F = 1.326; P = 0.255). When evaluated separately, the group (df = 9; F = 51.144; P < 0.001) and the period (df =&#9617 F = 4.366; P = 0.043) factors interfered in the cell proliferation. When the periods were evaluated separately, there was no difference among groups. However, when groups were analyzed separately, the cell proliferation was significantly higher in the 24-h period the Black MDX (105.8%) and A Factor (103.4%) groups than in the 72-h period (Black MDX: 86.9%; A Factor: 85.9%) [Figure 2].
Figure 2 Percentage ± standard deviation of cell proliferation obtained in the Alamar Blue assay for the different groups tested in the periods of eluate formation for 24 and 72 h. Different upper and lowercase letters indicate statistical difference in relation to the respective nonstimulated group in the periods of 24 and 72 h, respectively.

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  Discussion Top


Although the periods of eluate’s formation obtained from the different silicones and pigments tested influenced the cell proliferation of keratinocytes, the null hypothesis of this study was accepted, as all groups had percentages of cell proliferation higher than 75%, indicating absence of cytotoxicity. According to ISO standard 10993-523, in vitro methods for cytotoxic analysis, materials can be classified as noncytotoxic, when cell viability is greater than 75%; slightly cytotoxic, when cell viability is between 50% and 75%; moderately cytotoxic, when between 25% and 50%; and highly cytotoxic, when lower than 25%.[25]

This study analyzed two pigments, in the bronze and black colors, already available in the market for the pigmentation of maxillofacial prostheses, as well as a new pigment proposed by the authors, with a high solidity to the light, in an attempt to improve the chromatic stability required for a successful rehabilitation of these prostheses. Thus, in addition of being an unpublished research, this study is interesting because a compatibility between this new pigment and the human cell line HaCaT was verified.

Through the MTT assay, the MDX, Pink MDX, A Factor, and Pink A Factor groups had significantly lower percentages of cell proliferation than the control group at 24-h treatment period. At 72-h treatment period, the control group was different from Bronze MDX, A Factor, and Bronze A Factor groups. However, all percentages were higher than 75%, indicating absence of cytotoxicity of the tested materials.[25]

In general, cell viability was higher at 72 h, when compared to 24 h, possibly due to the higher concentration of soluble substances in the eluates formed for 24 h. It is known that the silicone has continuous polymerization and that the greater release of formaldehyde occurs in the first 72 h.[9],[26] However, after this period, the release of by-products still continues, but in a decreasing way. Although the samples from this study was placed in the medium for eluate’s formation after this critical period of 72 h (manufacturer’s recommendation), due to the continuous release of formaldehyde, a cell inhibition could have occurred, justifying the results found. A complementary biocompatibility analysis was performed using the Alamar Blue assay. Cell proliferation was significantly higher in the 24-h period for Black MDX and A Factor groups than in the 72-h period. It is interesting to note that the increased cell proliferation for these groups in the 24-h period may indicate a stimulus of cellular metabolism for the production of inflammatory mediators and tissue repair.[27]

Future studies analyzing inflammatory mediators and gene expression, such as type IV collagen, should be performed as a complement to this study. In the present study, tests to analyze in vitro cytotoxicity were performed. Although they have limitations, as the results do not fully reflect the toxic properties of the material in their clinical condition, such tests are essential for predicting the biocompatibility in the use of these materials in humans.[15]


  Conclusion Top


It can be concluded that all the tested materials used for maxillofacial prostheses confection, both silicones and pigments, including the proposed new pigment, are not cytotoxic for the human keratinocytes.

Acknowledgements

The authors thank the Sao Paulo Research Foundation for the financial support (No. 2014/11765-0) provided to Adhara Smith Nobrega.

Financial support and sponsorship

Sao Paulo Research Foundation for the financial support (No. 2014/11765-0 AND 2016/03455).

Conflicts of interest

There are no conflicts of interest.



 
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