Mechanotransduction is defined as the ability of cells to sense mechanical stimuli from their surroundings and translate them into biochemical signals. Epidermal keratinocytes respond to mechanical cues by altering their proliferation, migration, and differentiation. In vitro cell culture, however, utilises tissue culture plastic, which is significantly stiffer than the in vivo environment. Current epidermal models fail to consider the effects of culturing keratinocytes on plastic prior to setting up three-dimensional cultures, so the impact of this non-physiological exposure on epidermal assembly is largely overlooked. In this study, primary keratinocytes cultured on plastic were compared with those grown on 4, 8, and 50 kPa stiff biomimetic hydrogels that have similar mechanical properties to skin. Our data show that keratinocytes cultured on biomimetic hydrogels exhibited major changes in cellular architecture, cell density, nuclear biomechanics, and mechanoprotein expression, such as specific Linker of Nucleoskeleton and Cytoskeleton (LINC) complex constituents. Mechanical conditioning of keratinocytes on 50 kPa biomimetic hydrogels improved the thickness and organisation of 3D epidermal models. In summary, the current study demonstrates that the effects of extracellular mechanics on keratinocyte cell biology are significant and therefore should be harnessed in skin research to ensure the successful production of physiologically relevant skin models.

This research examined the effects of daily application of an oral hygiene regimen on the subgingival microbiome over 24 months. Generally healthy adults (107 enrolled, 87 completed) with early periodontitis used a home-care regimen (stannous fluoride paste, cetylpyridinium chloride rinse, power toothbrush, and floss) or usual care (control). Subgingival plaque samples were analyzed enzymatically for bacterial toxins. TLR ligands were measured using TLR-SEAP and TLR-ATP assays. Proinflammatory cytokines and metalloproteinases were quantified via immunoassays. Subgingival DNA was sequenced using a shotgun approach to assess microbial diversity. Increasing levels of bacteria, toxins, TLR activation, inflammatory cytokines, and MMPs were observed for periodontitis versus gingivitis and gingivitis versus healthy sites. The regimen significantly reduced levels of the critical proinflammatory cytokine IL-1β, as well as MMP-1 and MMP-9, at 24 months. By month 6, TLR ligands within subgingival plaques decreased. The abundance of pathogenic bacteria correlated with levels of virulence factors, proinflammatory cytokines, MMPs, and severity of clinical measures. Two distinct constellations of pathogenic bacteria were identified. Gingival sites were categorized into responders and non-responders per clinical symptoms and biomarkers. The regimen yielded more responder sites (70%) versus the control (47%), p = 0.0002914. The regimen reduced pathogenic bacteria, IL-1β, MMP1, and MMP-9, paralleling clinical reductions in periodontal disease.

This research examined host metabolic processes associated with gingivitis and potential impacts of interventions. First, gingival brush samples from a previously reported clinical study in which participants used an intervention (stannous fluoride (SnF₂) toothpaste/cetylpyridinium chloride rinse/oscillating-rotating electric toothbrush) or control (standard fluoride toothpaste/manual toothbrush) were analyzed with Metabolon metabolomic assessment. Next, hypotheses were generated about mechanisms of metabolic observations and tested in two in vitro models. Human primary blood cells were treated with endotoxins and outer membrane vesicles (OMVs) in the presence of SnF₂. In clinical brush samples, 46 metabolites decreased with intervention at Week 3, including 9- and 13-hydroxyoctadecadienoic acid, citrulline, adenine, and hypoxanthine. Seven metabolites related to nitrogen metabolism and citrulline production were selected for targeted quantification. They were low in health and increased in gingivitis. Targeted quantification demonstrated reduced citrulline, succinate, ornithine and deoxycarnitine (p ≤ 0.001 for all) between baseline and Week 6 with intervention; reductions were greater for intervention vs. control for citrulline, succinate and deoxycarnitine at Week 6 (p ≤ 0.019). In vitro, endotoxin and OMVs increased citrulline and nitric oxide (NO). SnF₂ inhibited elevation of both. Citrulline, deoxycarnitine, succinate, and ornithine were identified as potential novel markers of gingivitis. Elevated NO production is a potential driver of oxidative stress, reduced with intervention.

Objective: Extrinsic, lifestyle stressors can increase oxidative stress and induce premature aging characteristics of the skin. We studied the effects of oxidative stress at the biomolecular level to gain insights into effective stress‐resiliency strategies for skincare. We also investigated if Galactomyces ferment filtrate (GFF) pretreatment could enhance stress resiliency in keratinocytes in vitro. Methods: In vitro single cell Raman spectroscopy was performed to identify an oxidative stress phenotype in keratinocytes exposed to hydrogen peroxide (H ₂ O ₂ ) stress. The gene expression and functional consequences of acute stress were also determined by performing RNA sequencing, mitochondrial metabolism assays, and proliferation assays 24 h after H ₂ O ₂ stress exposure. All experiments were performed with and without GFF pretreatment. Results: Raman spectra revealed a novel stress phenotype which includes the formation of cysteic acid with increasing doses of H ₂ O ₂ , likely from the breaking of disulfide bonds in keratins. GFF pretreatment significantly mitigated this stress phenotype; GFF‐pretreated cells were 11.9 times more likely to exhibit healthy spectra compared to control cells when exposed to high stress. RNA sequencing conducted 24 h after H ₂ O ₂ exposure also demonstrated a stress‐related phenotype characterized by enhanced regulation of apoptosis and reduced keratinocyte differentiation. Conversely, GFF pretreatment increased multiple pathways of skin development at baseline and prevented the stress‐induced dysregulation of gene expression. Keratinocytes pretreated with GFF also had increased stress resiliency in mitochondrial stress and wound healing assays. Conclusion: Our data establishes that oxidative stress induces a distinctive biomolecular stress phenotype in keratinocytes. GFF pretreatment enhances the resilience of keratinocytes, as demonstrated by increased expression across various gene pathways related to keratinocyte differentiation, cell–cell junctions, and lipid metabolism. Following oxidative stress, GFF‐pretreated cells exhibited substantial protection from the stress‐induced biomolecular alterations identified in Raman spectra. Furthermore, GFF pretreatment effectively mitigated the downstream consequences of stress, supporting maintenance of keratinocyte gene‐expression programs, mitochondrial activity, and wound‐closure capacity after oxidative stress.