Base-metal dental casting alloy biocompatibility assessment using a human-derived three-dimensional oral mucosal model
Introduction
Two-dimensional (2-D) oral keratinocyte [1], [2], [3] and gingival fibroblast [4], [5], [6] cell monolayers have been employed to determine dental casting alloy biocompatibility. Oral keratinocytes are the primary tissue target of nickel ions and have been associated with the proinflammatory response elicited by the oral mucosa during nickel hypersensitivity reactions in vivo [7]. However, the ability of cell monolayer structures to faithfully replicate the complexities of full-thickness human oral mucosal tissue have been questioned [7], [8], [9]. Cell monolayer structures possess deficiencies in cell differentiation [10], [11], since anatomically cell monolayers fail to represent complex three-dimensional (3-D) native human oral mucosal tissue. Cell differentiation modifies a cell’s ability to respond to chemical and hormonal signals due to changes in gene expression [12]. Additionally, cell monolayers lack supporting connective tissue, basement membranes and extracellular matrix [13], which can lead to increased toxin susceptibility and inappropriate immune responses.
Recent advances in tissue engineering have led to oral mucosal equivalent development with an in vitro translational advantage for biocompatibility testing [8], [13], [14], [15]. Moharamzadeh et al. [15] incorporated primary gingival fibroblasts and immortalised TR146 oral keratinocytes into a human-derived scaffold structure (Alloderm™) and identified cell differentiation, manifest as a cytokeratin expression profile similar to native human oral mucosal tissue, comprising cytokeratins 5, 10 and 19, indicative of cell type and epithelial differentiation status [15]. However, the Moharamzadeh et al. [15] study could not be described as a primary cell-based structure as immortalised rather than primary oral keratinocytes were employed.
Nickel–chromium (Ni–Cr) dental alloys were developed as a cost-effective alternative to Weinstein’s high-gold patented alloy [16] in response to rising gold prices from 1968 [17]. Co–Cr dental alloys possess similar elastic moduli, strength and hardness as Ni–Cr dental alloys, but are less ductile compared with their Ni–Cr counterparts [18], [19] and are accordingly not as widely employed in clinical dentistry. Today Ni–Cr alloys are used extensively in fixed prosthodontics [20], [21] where appliances can remain in situ, adjacent to the oral mucosa, for substantial periods of time and have been associated with Ni allergy—indicative of a type IV Ni-induced hypersensitivity response [22]. Nickel allergy has been associated with dental appliances with metal–ceramic restorations manufactured using Ni-based dental casting alloys remaining in situ and directly adjacent to the oral mucosa for substantial periods of time [23], [24], [25], [26]. Nickel is a potent allergen and carcinogen that causes hypersensitivity reactions to a greater extent compared with any other metal used in metal–ceramic restorations with approximately one in five female and one in twenty male patients experiencing such a reaction [27]. Consequently, the leaching of the metallic elemental components into the surrounding oral mucosal tissues should therefore be expected.
The oral mucosal equivalent [15] was modified by replacing the immortalised cell line with primary oral keratinocytes, thereby creating an exclusively primary human cell-based tissue, termed the oral mucosal model, following integration into Alloderm™ scaffold structures. We hypothesised that the full-thickness oral mucosal model would provide an improved biocompatibility testing methodology for base-metal dental alloys and provide novel insights into the mechanisms of Ni–Cr alloy-induced toxicity. Assessment involved histological analyses in conjunction with analysis of cell viability, oxidative stress responses, inflammatory cytokine expression and cellular toxicity.
Section snippets
Dental casting alloys
Ni–Cr (d.Sign®10, composition 75.4 Ni, 12.6 Cr and 8.0 Mo (mass%) and d.Sign®15, composition 58.7 Ni, 25.0 Cr and 12.1 Mo; Ivoclar Vivadent, Schaan, Liechtenstein) and Co–Cr (d.Sign®30, composition 30.1 Cr, 1.0 Mo and 60.2 Co; Ivoclar Vivadent) alloy discs (10.0 mm diameter, 1.0 mm thickness) were cast, divested by alumina abrasion, polished to a clinical surface finish using rubber polishing wheels [2], [28] and sterilized at 115 °C for 15 min.
Cell culture
Prior ethical approval for the study was obtained from
Histological analyses
The Ni–Cr (d.Sign®10 and d.Sign®15) alloy-exposed oral mucosal models elicited critical losses of cellular thickness and compactness of epithelial and connective tissue layers (Fig. 1b,c) compared with the untreated control oral mucosal model (Fig. 1a). d.Sign®15 alloy discs induced increased vacuolization of connective tissue (Fig. 1c) compared with d.Sign®10 (Fig. 1b). No adverse morphological alterations were highlighted for the Co–Cr (d.Sign®30) alloy discs (Fig. 1d) compared with untreated
Discussion
The current study produced a primary human cell-based oral mucosal model, integrated into Alloderm™ that permitted multiple-endpoint responses to dental alloy discs. Alloderm™ is a low-antigenic, durable [29], acellular human cadaveric dermis comprising a basal lamina suitable for keratinocyte attachment and growth and a lamina propria facilitating fibroblast infiltration and growth [14]. The oral mucosal model was viable as defined epithelial and connective tissue layers thickened, due to
Conclusions
In summary, Ni–Cr alloy exposure to the oral mucosal models resulted in vacuolization of the connective tissue, loss of tissue integrity and a significant loss of cell viability with the initiation of a proinflammatory cytokine response and a significant reduction in reduced GSH levels, indicative of major oxidative damage. It is proposed that Ni ions permeated the epithelial tissue of the oral mucosal model and interacted with TLR4 proteins, activating the proinflammatory responses observed
Acknowledgements
The authors would like to thank Dr Keyvan Moharamzadeh and Professor Richard van Noort, School of Clinical Dentistry, University of Sheffield, UK for training on their oral mucosal equivalent and Dublin Dental University Hospital for the provision of funding for this project.
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