Comparison of primary human gingival fibroblasts from an older and a young donor on the evaluation of cytotoxicity of denture adhesives

Abstract Denture adhesives (DA) improve the retention and stability of ill-fitting dentures, especially for older adults. These materials should be biocompatible, i.e., they cannot cause undesired biological responses and be non-cytotoxic to oral tissues. However, in vitro testing of DA biocompatibility employing primary cell culture may possibly be affected by other factors, such as the donor age. Objective To compare the cytotoxicity of three different denture adhesives when assessed in primary gingival fibroblasts from a young donor or from an older donor, as well as the release of the basic fibroblast growth factor (bFGF), and the inflammatory response marker interleukin-6 (IL-6). Material and Methods Gingival fibroblasts isolated from a 30- and a 62-year-old donor were assayed for proliferation (1-7 days) and sensitivity to latex (positive control). Fibroblasts were indirectly exposed to Corega Ultra (cream), Corega powder and Fixodent Original for a 24 h period and assayed by XTT and Crystal Violet tests. The release of IL-6 and bFGF by exposed cells was determined by ELISA. Results While cells from the young donor presented higher cell growth after 7 days, the sensitivity to increasing concentrations of latex extracts was very similar between young and older cells. Both XTT and CVDE detected no difference between the DA and the control group. All materials induced higher levels of IL-6 and bFGF compared to control. Cells from the older donor exposed to Corega Ultra released lower levels of cytokine and growth factor. Conclusions All materials were considered non-cytotoxic, but affected cytokine and growth factor release. The biological differences found between fibroblasts from both donors could be due to individual or age-related factors. The authors suggest the use of cells from older donors on studies of dental products aimed at older patients, to better simulate their physiological response.


Introduction
Dentists often recommend the use of denture adhesives (DA) to correct prosthetic failures and to improve the comfort and function of dentures worn by patients presenting ridge resorption, decreased salivary flow, poor neuromuscular coordination, a low denture adaptability, and cognitive impairment 17 7,8 . In this regard, other studies have already employed primary human cell models, such as human gingival fibroblasts, to assess the cytotoxicity of several dental materials 12 , including the widely used DA Fixodent Fresh 10 . Thus, we demonstrated that primary human oral mucosal cells may provide more valuable information in toxicity screening of denture adhesives when compared to animal-related cell lines 5 .
Human gingival fibroblasts are among the most abundant resident cells from the oral mucosa, they feature a higher ability for scarless wound healing when compared to skin fibroblasts 20 , mostly due to a differential release of growth factors, such as TGF-Beta, connective tissue growth factor (CTGF) and basic fibroblast growth factor (bFGF). Besides producing the extracellular matrix (ECM) in oral connective tissues, human gingival fibroblasts express the cell surface proteins CD14, TLR4, and MD-2, and produce proinflammatory cytokines such as interleukin 6 (IL-6) 3 , indicating an important immunomodulatory role in response to stress and diseases such as periodontitis.
Therefore, these cells may represent interesting models for in vitro assessment of dental materials by also allowing the investigation of the release of growth factors and cytokines that affect biocompatibility.
A cytokine that has long been suggested as a parameter for the evaluation of the biological activity under nontoxic experimental conditions of dental materials in vitro 26 is IL-6, being a representative of stress and immune response. Similarly, other in vitro dental materials biocompatibility studies have also investigated the release of bFGF 6 , mostly due to its effects on proliferation, differentiation and extracellular matrix production of cells involved in wound healing and response to stress 4   Cell sensitivity assessment A dose-response assay was performed with both cell systems to assess the cell sensitivity to cytotoxic substances, they were exposed to increasing concentrations of latex extract, a well-known positive control for cytotoxicity of dental materials 19  The cell viability of the gingival fibroblasts from the young and older donors exposed to the different extracts was assayed by a test involving sequential analysis (i.e., using the same culture) for metabolic activity (XTT test) and cell density (CVDE test). The assays were performed using a commercial kit (In   analysis an alpha error of 5% was considered, therefore, a p<0.05 was considered significant. Figure 2 shows the assessment of cell growth during the first week of culture for fibroblasts from the both donors. While no difference was found between both cultures within 24 hours, a significant increase of 46% in proliferation was observed for fibroblasts of the young donor when compared to cells from the older donor (t-test, p<0.05) by the 7 th day after cell seeding.

Results
Fibroblasts from both donors exposed to increasing concentrations of latex extract, as the positive cytotoxicity control, behaved similarly, with IC 50 values of 8.95 and 9.16 mg/mL, respectively.

Discussion
Evidence suggests that fibroblasts from older individuals are likely to be more sensitive to adverse effects, since age-related frailty can influence cellular senescence, loss of telomeric structures, mitochondrial activity, production of reactive oxygen species and DNA repair capability 29 . However, in this study we showed that the sensitivity to a well-known cytotoxic system (latex extracts) is not affected by the age of the primary gingival fibroblasts donor, having very similar inhibitory curves (Figure 6), and IC 50 values similar to those described for other primary cell models 19 .
Similarly, the age of the donor did not affect the outcome of cytotoxicity tests of the denture adhesives Fixodent, Corega Powder and Corega Ultra, which had already been assessed by other authors 10,11 , using different cell models. Our results for the combined XTT/CVDE tests showed that all tested DAs are non- Figure 4-Release of IL-6 in the culture medium by gingival fibroblasts from the young (black) or the older (white) donor in contact with Fixodent, Corega Ultra or Corega powder. Data were normalized against the density of cells, as measured by the optical density at 540 nm during a CVDE assay (ODCVDE). Tissue culture polystyrene (TCPS) was considered the negative control. The SD values are indicated by error bars. *Significantly different from the control group (Blank) at each age-dependent cell type (P<0.05). Significance bars indicate differences between age-dependent cell types (unpaired t-test) found. While this important growth factor is often related to repair and tissue regeneration by influencing the proliferation of fibroblasts and epithelial cells, the increased release of fibroblasts from otherwise healthy tissues exposed to denture adhesives must be carefully regarded, since this may also imply in a cell response induced by injury or irritation 11 , including those caused by indirect or direct contact with a material.
The model used was unable to detect differences in bFGF release between unexposed cells from young or older donors. However, previous reports indicate that the process of aging decreases the physiological response to injuries and irritation. Therefore, a dual interpretation is raised for the possible benefits or drawbacks of the reduction of bFGF release in older cells exposed to Corega Ultra, as it may imply both a decrease of wound-healing capacity and maintenance of ECM integrity, or a lower response to irritation caused by this material. Therefore, further in vivo or clinical assessments of bFGF release and impact are needed to confirm its role on the safe use of denture adhesives.
One of the main limitations of this preliminary study is that the comparison was performed with a single donor for each age group. Therefore, the possibility that individual, rather than age-related factors, might have affected the outcomes of the cytotoxicity tests is not discarded. Nevertheless, the overall results indicate similarities and relevant differences in the performance of human gingival fibroblasts of a donor with advanced age when compared to a younger donor, during the in vitro biological evaluation of denture adhesives. While simple endpoints such as cytotoxicity were unaffected, this result could be altered with longer experimental times, since the proliferation of fibroblasts from older donors was affected after seven days ( Figure 2). Furthermore, the results indicate that the outcomes of advanced biocompatibility assays, including protein expression, -omics studies (e.g.: genomics, proteomics or metabolomics) or the evaluation of cell-cell interactions, might be affected by the choice of cell system, which must be assessed in future studies, employing larger samples of each experimental age group. This is especially relevant if we consider the need to prioritize more relevant and mechanistic insights of the toxic pathways, instead of evaluating single isolated cytotoxicity endpoints. Thus, even though the clinical relevance of in vitro tests should be considered carefully, considering the results we confirm the proposed hypothesis that cells from a young and an older donor may behave differently in the evaluation of denture adhesives biocompatibility.
In this context, using primary human fibroblasts from older donors for the in vitro testing of materials in which age-dependent factors are relevant, such as denture adhesives, could provide more clinically relevant results.

Conclusions
While all materials were considered non-cytotoxic regardless of the age of the donor, Fixodent and Corega Ultra induced different levels of cytokine and growth factor release in fibroblasts from young and older donors. These differences suggest the relevance of using cells from older donors on studies of dental products aimed at older patients.