The human oral mucosa hosts a diverse microbiome and is exposed to potentially toxic biomaterials from dental restoratives. Mucosal health is partly determined by cell and tissue responses to challenges such as dental materials and pathogenic bacteria. An in vitro model to rapidly determine potential layer-specific responses would lead to a better understanding of mucosal homeostasis and pathology. Therefore, this study aimed to develop a co-cultured microfluidic mucosal model on-a-chip to rapidly assess mucosal remodeling and the responses of epithelial and subepithelial layers to challenges typically found in the oral environment. A gingival fibroblast-laden collagen hydrogel was assembled in the central channel of a three-channel microfluidic chamber with interconnecting pores, followed by a keratinocyte layer attached to the collagen exposed in the pores. This configuration produced apical and subepithelial side channels capable of sustaining flow. Keratinocyte, fibroblast, and collagen densities were optimized to create a co-culture tissue-like construct stable over one week. Cells were stained and imaged with epifluorescence microscopy to confirm layer characteristics. As proof-of-concept, the mucosal construct was exposed separately to a dental monomer, 2-hydroxylethyl methacrylate (HEMA), and the oral bacteria Streptococcus mutans. Exposure to HEMA lowered mucosal cell viability, while exposure to the bacteria lowered transepithelial electrical resistance. These findings suggest that the oral mucosa-on-achip is useful for studying oral mucosal interactions with bacteria and biomaterials with a histology-like view of the tissue layers.

The short average service life of traditional dental composite restorative materials and increasing occurrence of secondary caries adjacent to composite restorations and sealants are necessitating the development of new, longer lasting compositions. Novel monomers and their polymers, reinforcing fillers, and adhesive components are needed. The goal of this research is to develop resin systems for use in restorations, sealants, and other dental services that are superior in properties and endurance to currently used bisphenol A glycidyl dimethacrylate/triethylene glycol dimethacrylate (Bis-GMA/TEGDMA) and urethane–dimethacrylate products. Ether-based monomers and their polymers that were not susceptible to enzymatic or hydrolytic degradation were prepared and characterized. They showed no degradation under hydrolytic and enzymatic challenges, whereas the hydrolysis of ester links weakened contemporary resins within 16 days under these challenges. The success of the ether-based materials is promising in making durable systems that are subjected to long-term biochemical and hydrolytic challenges in oral environments.

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Tissue engineering-based therapies rely on the delivery of monolayered fibroblasts on two-dimensional polystyrene-coated and extracellular matrix (ECM) surfaces to regenerate connective tissues. However, this approach may fail to mimic their three-dimensional (3D) native architecture and function. We hypothesize that ECM fibrous proteins, which direct the migration of cells in vivo, may attach and guide polystyrene- and Matrigel™-ECM (M-ECM)-adherent fibroblasts to rearrangement into large multicellular macrostructures with the ability to proliferate. Gingival monolayered fibroblasts and their derived spheroids were added and adhered to tissue culture polystyrene and M-ECM surfaces. The cells were covered with a layer of collagen1 hydrogel combined with vitronectin, fibronectin or fibrin, or 10% M-ECM. The development of 3D cell constructs was characterized by epifluorescence and confocal scanning microscope image analysis. The ECM turnover and the proliferative capabilities of the fibroblasts were determined via gene expression profiling of collagen1, fibronectin, matrix metalloproteinase/metallopeptidase 2, Nanog, and SRY (sex-determining region Y)-box2 (Sox2). Expression of the Sox2 protein was followed by immunostaining. The collagen1 protein had the strongest effect on monolayered and spheroid cell rearrangements, forming large spherical shapes and fused 3D macroconstructs. The addition of fibrin protein was typically required to achieve a similar effect on M-ECM-adherent monolayered fibroblasts. The spheroid fusion process was followed by an increase in cell density and the formation of tight clusters. The fused spheroids continued to maintain their intracellular ECM turnover and proliferation capacities. Collagen1 is a valuable component in the rearrangement of adherent fibroblast monolayers and spheroids. Fibroblast spheroids should preferably be used as basic building blocks to assemble multicellular connective tissue-like macrostructures.

Deep wounds in the gingiva caused by trauma or surgery require a rapid and robust healing of connective tissues. We propose utilizing gas-brushed nanofibers coated with collagen and fibrin for that purpose. Our hypotheses are that protein-coated nanofibers will: (i) attract and mobilize cells in various spatial orientations, and (ii) regulate the expression levels of specific extracellular matrix (ECM)-associated proteins, determining the initial conformational nature of dense and soft connective tissues. Gingival fibroblast monolayers and 3D spheroids were cultured on ECM substrate and covered with gas-blown poly-(DL-lactide-co-glycolide) (PLGA) nanofibers (uncoated/coated with collagen and fibrin). Cell attraction and rearrangement was followed by F-actin staining and confocal microscopy. Thicknesses of the cell layers, developed within the nanofibers, were quantified by imageJ software. The expression of collagen1α1 chain (Col1α1), fibronectin, and metalloproteinase 2 (MMP2) encoding genes was determined by quantitative reverse transcription analysis. Collagen- and fibrin- coated nanofibers induced cell migration toward fibers and supported cellular growth within the scaffolds. Both proteins affected the spatial rearrangement of fibroblasts by favoring packed cell clusters or intermittent cell spreading. These cell arrangements resembled the structural characteristic of dense and soft connective tissues, respectively. Within 3 days of incubation, fibroblast spheroids interacted with the fibers and grew robustly by increasing their thickness compared to monolayers. While the ECM key components, such as fibronectin and MMP2 encoding genes, were expressed in both protein groups, Col1α1 was predominantly expressed in bundled fibroblasts grown on collagen fibers. This enhanced expression of collagen1 is typical for dense connective tissue. Based on results of this study, our gas-blown, collagen- and fibrin-coated PLGA nanofibers are viable candidates for engineering soft and dense connective tissues with the required structural characteristics and functions needed for wound healing applications. Rapid regeneration of these layers should enhance healing of open wounds in a harsh oral environment.

Leachables from dental restoratives induce toxicity in gingival and pulp tissues and affect tissue regeneration/healing. Appropriate testing of these materials requires a platform that mimics the in vivo environment and allows the architectural self-assembly of cells into tissue constructs. In this study, we employ a new 3D model to assess the impact of triethyleneglycol dimethacrylate (TEGDMA) on early organization and advanced recruitment/accumulation of immortalized mouse gingival fibroblasts (GFs) and dental papilla mesenchymal cells (DPMCs) in extracellular matrix. We hypothesize that TEGDMA (1) interferes with the developmental architecture of GFs and DPMCs, and (2) inhibits the deposition of mineral. To test these hypotheses, GFs and DPMCs were incubated with the soluble TEGDMA at concentrations (0-2.5) mmol/L. Diameter and thickness of the constructs were determined by microscopic analysis. Cell differentiation was assessed by immunocytochemistry and the secreted mineral detected by alizarin-red staining. TEGDMA interfered with the development of GFs and/or DPMCs microtissues in a dose-dependent manner by inhibiting growth of inter-spherical cell layers and decreasing spheroid size (four to six times). At low/moderate TEGDMA levels, GFs organoids retained their structures while reducing thickness up to 21%. In contrast, at low TEGDMA doses, architecture of DPMC organoids was altered and thickness decreased almost twofold. Overall, developmental ability of TEGDMA-exposed GFs and DPMCs depended on TEGDMA level. GFs constructs were more resistant to structural modifications. The employed 3D platform was proven as an efficient tool for quantifying the effects of leachables on tissue repair capacities of gingiva and dental pulp.

Dental pulp stem cells (DPSCs) regenerate injured/diseased pulp tissue and deposit tertiary dentin. DPSCs stress response can be activated by exposing cells to the monomer triethyleneglycol dimethacrylate (TEGDMA) and inducing the DNA-damage inducible transcript 4 (DDIT4) protein expression. The goal of the present study was to determine the impact of TEGDMA on the ability of DPSCs to maintain their self-renewal capabilities, develop and preserve their 3D structures and deposit the mineral. Human primary and immortalized DPSCs were cultured in extracellular matrix/basement membrane (ECM/BM) to support stemness and to create multicellular interacting layers (microtissues). The microtissues were exposed to the toxic concentrations of TEGDMA (0.5 and 1.5 mmol/l). The DPSCs spatial architecture was assessed by confocal microscopy. Mineral deposition was detected by alizarin red staining and visualized by stereoscopy. Cellular self-renewal transcription factor SOX2 was determined by immunocytochemistry. The microtissue thicknesses/vertical growth, surface area of the mineralizing microtissues, the percentage of area covered by the deposited mineral, and the fluorescence intensity of the immunostained cells were quantified ImageJ. DDIT4 expression was determined by a single molecule RNA-FISH technique and the cell phenotype was determined morphologically. DDIT4 expression was correlated with the cytotoxic phenotype. TEGDMA affected the structures of developing and mature microtissues. It inhibited the deposition of the mineral in the matrix while not affecting the SOX2 expression. Our data demonstrate that DPSCs retained their self-renewal capacity although their other functions were impeded. Since the DPSCs pool remained preserved, properties effected by the irritant should be restored by a proper rescue therapy.