One of the primary drivers of the Sensitive Skin Syndrome (SSS) is the Exposome, a comprehensive term encompassing all exogenous factors and individual encounters throughout their lifespan. Physical stressors like ultraviolet (UV) radiation, temperature fluctuations, and mechanical friction, alongside chemical stressors such as pollutants, water quality, and cosmetic formulations, act as potent triggers.
As cosmetics brands need science-based products, the industry faces an imperative need to develop highly predictive in vitro assays to evaluate ingredients and finished products performance on the exposome protection. This article explores the current landscape of in vitro methodologies, moving from traditional 2D cell systems to sophisticated, innervated 3D models and microfluidic platforms, with a focus on barrier integrity and neurogenic inflammation.
There are various methods which enable to evaluate anti-pollutant efficacy on cell supports, skin model or skin 3Dprint using many biomarker analyses: Cell Proliferation, Lipid metabolism, Carbonylated proteins, Antioxidant defenses markers, DNA damage, various Proteins (barrier function, pigmentation or mitochondrial), Inflammation mediators, or Protease activity.
Technological advances in in vitro assays
The evolution of in vitro testing has moved toward increasing physiological complexity to better reflect the in vivo environment. While 2D cell cultures and co-cultures remain cost-effective for initial screenings, they cannot replicate the 3D cellular architecture or the complex barrier function of human skin. Consequently, 3D reconstructed human epidermis (RHE) and fullthickness Skin model have become the standard for 3D skin models. These models, whether bioprinted or manually reconstructed, can be vascularized, augmented with neurons cells or capillaries. They also can be specifically designed from aged skin and specific skin types and allow for a more accurate assessment of barrier integrity and pharmacological penetration.
The emergence of Skin-on-a-Chip technology offers promising prospects for exposome research. These microfluidic models closely reproduce physiological conditions, such as cytokine gradients and spatially organized cellular interactions. They provide a dynamic environment that mimics the flow of interstitial fluids, offering a more robust platform for screening ingredients against complex urban pollution or fluctuating environmental conditions.
The Physiological Triad of Sensitive Skin Evaluation
To design effective therapeutic or cosmetic solutions, one must understand the three interrelated mechanisms governing the Sensitive Skin Syndrome: barrier function disruption, immune activation, and neurogenic inflammation.
The skin barrier, whose function is directly related to the Stratum corneum integrity, serves as the body’s first line of defense. Utilizing the “brick and mortar” model, the SC consists of corneocytes (the “bricks”) ultra-differentiated, metabolically inactive keratinocytes, embedded in a lipid-rich intercellular matrix (the “mortar”). When this barrier is damaged by external stressors, the penetration of irritants and pathogens increases, fueling the phenomena of inflammation (inflammaging).
In the presence of stressors, resident skin cells, keratinocytes, fibroblasts, and dendritic cells, initiate an inflammatory cascade. This involves the release of key mediators such as IL-1α/β, IL-6, TNF-α, and IFN-γ. In more specific allergic or pruritic reactions, the Th2/Th17 axis is activated, with the release of cytokines like IL-4, IL-13, IL-17, and IL-31. This immune activation is a crucial step in the transition from localized irritation to systemic sensitivity.
Neurogenic Inflammation: The Nervous System’s Role
A distinguishing feature of the Sensitive Skin Syndrome is the involvement of the peripheral nervous system. Sensory nerve fibers respond to mechanical, thermal, or chemical stimulus by releasing neuropeptides, such as Substance P (SP), Calcitonin Gene-Related Peptide (CGRP), and Neurokinin A. These mediators act directly on skin cells and immune cells (mast cells), amplifying the inflammatory response. Furthermore, SSS is often associated with small-fiber neuropathies involving receptors like TRPV1 (Transient Receptor Potential Vanilloid 1), which mediate the perception of heat, acidic pH, and chemical irritants like capsaicin or histamine.
By incorporating sensory neurons derived from induced Pluripotent Stem Cells (iPSCs) in cells co-cultures, researchers can now investigate the neuronal contribution to inflammation with innervated models. These innervated models provide a unique opportunity to study how sensory neurons release neuropeptides under stimulation, driving inflammation and discomfort, and how active ingredients may modulate these pathways.
The Extracellular Matrix (ECM) a key element of the pollution protection
The skin’s ability to withstand the exposome is also dependent on the dermis and the conditions of the extracellular matrix. The skin behaves as a viscoelastic material, a property primarily conferred by the dermis. The Extracellular Matrix (ECM) of the skin is a complex, three-dimensional network that provides structural support, regulates cell behavior, and maintains hydration. It is primarily divided into the fibrous framework and the ground substance:
Fibrous Proteins « The Scaffold »
- Collagens (70-80%): Provide tensile strength. They include fibril-forming (Types I (~80%), II, III (~15%), network-forming (Type IV), and fibril-associated types.
- Elastin (2-4%): Forms a network of elastic fibers and allows the skin to “snap back” after stretching (resilience). Without it, skin develops sag and loss of elasticity.
Ground Substance, the gel-like environment that fills the space between fibers and cells (fibroblasts).
- Glycosaminoglycans (GAGs): Polysaccharides like hyaluronic acid that maintain hydration and shock absorption and Sulfated GAGs
- Proteoglycans (PGs): These are GAGs attached to a protein core: Decorin regulates the assembly of collagen fibers. Versican: bind water and provides the viscoelasticity. Lumican: Crucial for regulating collagen fibril diameter.
- Laminins and Fibronectin: Act as “biological glue,” facilitating cell attachment and migration.
However, chronic exposure to the urban exposome and free radicals alter this balance. Collagen fibers can become excessively cross-linked via glycation, leading to the formation of Advanced Glycation End Products (AGEs), which increase rigidity and degrade the ECM architecture.
This incredible network that represents the skin extracellular matrix is modulated by exogenous environment. The own biochemical properties of the ECM can be studied in many ways through the analyse of its various components and their interactions and constitute a “gold” support to substantiate ingredients and finished product claims.
A healthy skin barrier as the best protection to exposome
A healthy barrier requires a balanced cycle of cells proliferation and differentiation.
The first line of biological defense involves antimicrobial peptides (AMPs) such as human cathelicidin LL-37, beta-defensins, and Psoriasin (S100A7). These peptides, alongside the pH and hydration status (driven by Natural Moisturizing Factors from filaggrin degradation), maintain the skin’s biological balance.
1. Evaluation of skin barrier efficiency
Skin barrier normally prevents the passage of various molecules. Its integrity may therefore be assessed by measuring Trans Epidermal Water Loss (TEWL), Transepithelial/transendothelial electrical resistance (TEER), or the entry of various molecules through the epidermis thanks to Franz Cell (OECD 428) or other percutaneous penetration technics.
2. Skin barrier formation
Skin barrier integrity involves an appropriate formation and renewal correlated to keratinocyte proliferation, differentiation, and desquamation. Various biomarkers allow to assess the distribution of undifferentiated keratinocytes (K5, K14), their stemness (K15, K19), their proliferation (Ki67) and their state of differentiation (K1, K10, Loricrin, Involucrin, Filaggrin).
Other markers such as K6, K16 (reinforce the cell-cell and cell-matrix cohesion) transglutaminases 1, 3 and 5 (control involucrin and loricrin covalent-crosslinking), Sirtuin-1 (controls filaggrin synthesis), Caspase 14 (controls filagrin degradation) or kallikreins (involved in desquamation) are also interesting. Filaggrin degradation leads to Natural Moisturizer Factor (NMF), a key factor for skin hydration. Appropriate skin hydration and pH allow the proper functioning of skin enzymes involved in stratum corneum formation and cell cohesion.
Some components of the dermal-epidermal junction (DEJ) such as Laminin 332 (Laminin V), type IV collagen, nidogen-1 & 2 and Perlecan or allowing the fixation of keratinocytes on the DEJ such as Integrin α6 and β4 are not only responsible for the adherence between dermis and epidermis but also have an impact on keratinocyte survival, stemness, proliferation and differentiation and therefore on skin barrier function.
3. Tight junctions and skin integrity
Tight junctions are responsible for the cohesion between the corneocytes and prevent the transfer of various molecules through the SC. Their integrity may be assessed with Corneodesmosin, Zonula Occludens 1 (ZO1), Occludin, E-Cadherin, Desmoglein-1, Claudin 1. SC cohesion also involves proteins such as envoplakin and periplakin which connect intracellular keratins to membrane and cellular junctions.
4. Antimicrobial peptides
The first line of defense against pathogens is formed by the antimicrobial peptides secreted on skin surface. Such antimicrobial peptides are for example human cathelicidin LL-37, types 1-4 β-defensins, psoriasin (S100A7), calprotectin (S100 A8/9), koebnerisin (S100A15) and RNase 7.
5. Stratum corneum lipid barrier
Lipid composition and organization is also highly important for skin barrier function. Epidermal thickness, SC thickness and lipid organisation may be assessed using Raman microspectroscopy while lipid composition is obtained using liquid chromatography coupled to high-resolution mass spectrometry. This may allow to evaluate in particular ceramide synthesis, subclasses, and organization.
Anti-Pollution Specific Protocols
The urban exposome induces chronic stress, leading to carbonylated proteins, DNA damage, and lipid peroxidation. Testing laboratories have developed specific protocols to substantiate anti-pollution claims.
Pollutants, such as cigarette smoke, ozone, heavy metals, volatile organic compounds (VOCs), and particulate matter (PM2.5), can be applied directly to culture media or sprayed in controlled chambers. The choice of model depends on the specific mechanism being studied, such as pollutant adhesion or removal, or the reduction of oxidative stress.
To provide robust data packages, contemporary protocols focus on several critical markers:
- Aryl Hydrocarbon Receptor (AhR) Activation: A primary sensor for chemical pollution. Its modulation indicates a product’s ability to prevent the biological “alarm” triggered by Ozone or PAHs.
- Protein Carbonylation (PC): A stable indicator of long-term oxidative damage to the dermal matrix,
- Lipid Peroxidation (SQ-OOH / MDA): Markers of immediate oxidative stress on the surface.
- Inflamm-aging Mediators: IL-1\alpha and IL-8 quantify the inflammatory response and the soothing efficacy of neuro-cosmetic ingredients.
- Filaggrin & Loricrin Expression: to prove the reinforcement of the physical skin barrier against particle penetration.
The in vitro evaluation of the skin exposome represents a source of constant innovation. By combining the “three pillars” of assay design, relevant biomarkers, advanced analytical methods and complex assay supports, the industry can move toward a more predictive and ethical alternative to human testing. These assays can demonstrate the effects in preventing pollutants from adhering to the skin surface, removing pollutants from the skin or in reducing their oxidative impact.
These advanced tools do not merely assess surface-level interactions; they provide a deep understanding of how external stressors disrupt the balance between barrier integrity, neurogenic signaling, and the biomechanical scaffold of the ECM. As the global beauty market continues to be challenged by evolving environmental stressors, these high-fidelity in vitro models remain the “gold” standard for studying the mechanism of action of protection from the exposome.
REFERENCES
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