Introduction: The biologic safety profile of oral care products is often determined based on simple test models such as monolayer cell culture systems. Such systems can be overly sensitive and are not clinically relevant[1], although progress has been made in tissue-engineered human oral tissue models. It has been suggested that the 3D tissue model is a potential alternative for biologic assessment of oral care products as it provides more information and is more relevant to the clinical models. The aim of this study was to compare the biologic effects of formulations containing lipophilic vehicles and fatty acids on both monolayer human gingival fibroblasts and human gingival 3D tissue.
Materials and Methods: The study includes viability tests of lipophilic vehicles on both monolayer human gingival fibroblasts (HGF) and human gingival tissue (Gin-100, Mattek). MTS assay was carried out on monolayer cells while MTT and LDH assays evaluated the tissue viability. In all assays the test articles were in contact with cells for 24 hours. The positive control was 0.1% Triton X-100 and the negative control was PBS. All aspects of the studies, in terms of extract preparation and handling the cells and tissues, were carried out according to the ISO-10993-5.
Results: The MTS assay on monolayer cells showed lipophilic ingredients are toxic to the HGF cells at all concentrations, including that used in the adhesive formulation (15% w/w). In contrary, viability studies (MTT & LDH) on gingival 3D tissue showed lipophilic ingredients are not toxic at the above levels. Further studies showed the adhesive formulation containing 15% w/w of the lipophilic ingredient had a viability of 80% which is similar to the placebo formulation and as a result it does not have a cytotoxic effect on gingival tissue.
Discussion: The outcome of this study suggests monolayer cells are not ideal for biocompatibility studies of oral care products as they lack the differentiated function and barrier properties of cells found in the normal human oral tissue. In contrary, the tissue used in this study has 9-13 layers of viable nucleated cells which is partially cornified at the apical surface and closely resemble the human gum tissue. Hence, the results obtained from 3D oral tissue model are more applicable to the normal human oral tissue compared to the ones from monolayer cell cultures. Moreover, several studies have been suggesting the benefits of using 3D oral tissue models in medical and dental research over monolayer cell culture models1 including biocompatibility assessment of dental biomaterials and oral care products[2]-[4].
Conclusions: Given the ethical and regulatory restrictions for conducting animal studies in the preclinical stage for medical device and cosmetic products, and the fact that monolayer cell cytotoxicity studies are not necessarily clinically relevant, re-constructed oral soft tissues have the potential for being used as a more realistic model for biocompatibility tests of preclinical studies. This approach has been validated for skin products and more studies need to be done on oral healthcare products to validate oral tissue models.
References:
[1] Moharamzadeh K., Franklin K., Brook IM., van Noort R., “Biologic assessment of antiseptic mouthwashes using a three dimensional human oral muncusal model”, May 2009, J periodontal 2009; 80:769- 775.
[2] Breyfogle1 B., Kubilus1 J., Dale2 B., Wertz3 P., “Epigingival™ (GIN-100) tissue models for oral irritation studies.” 1MatTek Corporation, Ashland, MA, USA, 2University of Washington, Seattle, WA, USA, 3University of Iowa, Iowa City, IA, USA. Presented at 5th World Congress, Berlin, Germany, August, (2005).
[3] Moharamzadeh K., Brook IM., Scutt AM, Thornhill MH., van Noort R., “Mucotoxicity of dental composite resins on a tissue engeenired human oral mucosal model”. J Dent 2008;36: 331- 336.
[4] Lida T., Takami Y., Yamaguchi R., Shimazaki S., Harii K., “Development of a tissue engeneered human oral mucosa equivalent based on acellular allogeneic dermal matrix: A preliminary report of clinical application to burn woods.” Scand J Plast Reconstr Surg Hand Surg 2005;39:138-146.