Glycolipid Biosurfactants in Skincare Applications: Challenges and Recommendations for Future Exploitation.

The 21st century has seen a substantial increase in the industrial applications of glycolipid biosurfactant technology. The market value of the glycolipid class of molecules, sophorolipids, was estimated to be USD 409.84 million in 2021, with that of rhamnolipid molecules projected to reach USD 2.7 billion by 2026. In the skincare industry, sophorolipid and rhamnolipid biosurfactants have demonstrated the potential to offer a natural, sustainable, and skin-compatible alternative to synthetically derived surfactant compounds. However, there are still many barriers to the wide-scale market adoption of glycolipid technology. These barriers include low product yield (particularly for rhamnolipids) and potential pathogenicity of some native glycolipid-producing microorganisms. Additionally, the use of impure preparations and/or poorly characterised congeners as well as low-throughput methodologies in the safety and bioactivity assessment of sophorolipids and rhamnolipids challenges their increased utilisation in both academic research and skincare applications. This review considers the current trend towards the utilisation of sophorolipid and rhamnolipid biosurfactants as substitutes to synthetically derived surfactant molecules in skincare applications, the challenges associated with their application, and relevant solutions proposed by the biotechnology industry. In addition, we recommend experimental techniques/methodologies, which, if employed, could contribute significantly to increasing the acceptance of glycolipid biosurfactants for use in skincare applications while maintaining consistency in biosurfactant research outputs.

Toxicity Profiling of Biosurfactants Produced by Novel Marine Bacterial Strains.

Surface active agents (SAAs), currently used in modern industry, are synthetic chemicals produced from non-renewable sources, with potential toxic impacts on humans and the environment. Thus, there is an increased interest for the identification and utilization of natural derived SAAs. As such, the marine environment is considered a promising source of biosurfactants with low toxicity, environmental compatibility, and biodegradation compared to their synthetic counterparts. MARISURF is a Horizon 2020 EU-funded project aiming to identify and functionally characterize SAAs, derived from a unique marine bacterial collection, towards commercial exploitation. Specifically, rhamnolipids produced by Marinobacter MCTG107b and Pseudomonas MCTG214(3b1) strains were previously identified and characterized while currently their toxicity profile was assessed by utilizing well-established methodologies. Our results showed a lack of cytotoxicity in in vitro models of human skin and liver as indicated by alamar blue and propidium iodide assays. Additionally, the use of the single gel electrophoresis assay, under oxidative stress conditions, revealed absence of any significant mutagenic/anti-mutagenic potential. Finally, both 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) cell-free assays, revealed no significant anti-oxidant capacity for neither of the tested compounds. Consequently, the absence of significant cytotoxicity and/or mutagenicity justifies their commercial exploitation and potential development into industrial end-user applications as natural and environmentally friendly biosurfactants.

Microfluidic skin chip with vasculature for recapitulating the immune response of the skin tissue.

There is a considerable need for cell-based in vitro skin models for studying dermatological diseases and testing cosmetic products, but current in vitro skin models lack physiological relevance compared to human skin tissue. For example, many dermatological disorders involve complex immune responses, but current skin models are not capable of recapitulating the phenomena. Previously, we reported development of a microfluidic skin chip with a vessel structure and vascular endothelial cells. In this study, we cocultured dermal fibroblasts and keratinocytes with vascular endothelial cells, human umbilical vascular endothelial cells. We verified the formation of a vascular endothelium in the presence of the dermis and epidermis layers by examining the expression of tissue-specific markers. As the vascular endothelium plays a critical role in the migration of leukocytes to inflammation sites, we incorporated leukocytes in the circulating media and attempted to mimic the migration of neutrophils in response to external stimuli. Increased secretion of cytokines and migration of neutrophils was observed when the skin chip was exposed to ultraviolet irradiation, showing that the microfluidic skin chip may be useful for studying the immune response of the human tissue.

In vitro skin irritation assessment using EpiDerm™: applicability for updating toxicity information of oxybenzone and N,N-diethyl-m-toluamide.

A skin irritation test using in vitro reconstructed human epidermis (RhE) models was established for hazard identification of irritant chemicals in accordance with UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) category. In this study, EpiDerm™ was used to assess skin irritation by oxybenzone and N,N-diethyl-m-toluamide (DEET), which are widely used sunscreen and insect repellent components, respectively. EpiDerm™ was applied with oxybenzone and DEET, combined and sequentially with each single dose. Epidermal morphology and differentiation/proliferation were examined microscopically. Oxybenzone and sequential administration groups were determined as nonirritant with cell viability >50% and the morphology was comparable to the human epidermis. Contrastingly, the DEET and coadministration groups exhibited cell viability <50% and poor epidermal morphology. Interleukin (IL)-1α release from substance-treated EpiDerm™ increased inversely to cell viability, suggesting the pro-inflammatory reaction was initiated by DEET. CK-10, E-cadherin, Ki-67, laminin, and ceramide were identified as relevant markers to assess oxybenzone- or DEET-induced epidermal injury. In conclusion, these results may indicate to be aware of the possible skin irritation by indiscriminate use of oxybenzone and DEET without animal testing.

Construction of 3D multicellular microfluidic chip for an in vitro skin model.

Current in vitro skin models do not recapitulate the complex architecture and functions of the skin tissue. In particular, on-chip construction of an in vitro model comprising the epidermis and dermis layer with vascular structure for mass transport has not been reported yet. In this study, we aim to develop a microfluidic, three-dimensional (3D) skin chip with fluidic channels using PDMS and hydrogels. Mass transport within the collagen hydrogel matrix was verified with fluorescent model molecules, and a transport-reaction model of oxygen and glucose inside the skin chip was developed to aid the design of the microfluidic skin chip. Comparison of viabilities of dermal fibroblasts and HaCaT cultured in the chip with various culture conditions revealed that the presence of flow plays a crucial role in maintaining the viability, and both cells were viable after 10 days of air exposure culture. Our 3D skin chip with vascular structures can be a valuable in vitro model for reproducing the interaction between different components of the skin tissue, and thus work as a more physiologically realistic platform for testing skin reaction to cosmetic products and drugs.

The Impact of Temperature and the Duration of Freezing on a Hydrogel Used for a 3D-Bioprinted In Vitro Skin Model.

Skin bioprinting has the potential to revolutionize treatment approaches for injuries and surgical procedures, while also providing a valuable platform for assessing and screening cosmetic and pharmaceutical products. This technology offers key advantages, including flexibility and reproducibility, which enable the creation of complex, multilayered scaffolds that closely mimic the intricate microenvironment of native skin tissue. The development of an ideal hydrogel is critical for the successful bioprinting of these scaffolds with incorporated cells. In this study, we used a hydrogel formulation developed in our laboratory to fabricate a 3D-bioprinted skin model. The hydrogel composition was carefully selected based on its high compatibility with human skin cells, incorporating alginate, methyl cellulose, and nanofibrillated cellulose. One of the critical challenges in this process, particularly for its commercialization and large-scale production, is ensuring consistency with minimal batch-to-batch variations. To address this, we explored methods with which to preserve the physicochemical properties of the hydrogels, with a focus on freezing techniques. We validated the pre-frozen hydrogels' printability, rheology, and mechanical and surface properties. Our results revealed that extended freezing times significantly reduced the viscosity of the formulations due to ice crystal formation, leading to a redistribution of the polymer chains. This reduction in viscosity resulted in a more challenging extrusion and increased macro- and microporosity of the hydrogels, as confirmed by nanoCT imaging. The increased porosity led to greater water uptake, swelling, compromised scaffold integrity, and altered degradation kinetics. The insights gained from this study lay a solid foundation for advancing the development of an in vitro skin model with promising applications in preclinical and clinical research.

Study on percutaneous penetration of representative cosmetic ingredients in a baby wipe product in an in vitro diaper rash skin model.

Studying percutaneous penetration of various cosmetic ingredients through intact and compromised skin can provide insight on quantitative exposure assessment for baby products intended for diapered skin. We developed an in vitro model (tape-stripped human skin) designed to achieve the Trans-Epidermal Water Loss values measured in babies with various degrees of diaper dermatitis. Six reference compounds showed the impact of physicochemical properties on absorption through this "diaper rash" skin model. Under simulated diaper conditions, dermal absorption of cosmetic ingredients (phenoxyethanol, sodium benzoate, benzyl alcohol, disodium EDTA, and propylene glycol) was different, but <100%. Additionally, the effect of diaper rash on dermal absorption of well-absorbed ingredients (phenoxyethanol, sodium benzoate, and benzyl alcohol) was limited (enhancement of 1.1-1.3), while the enhancement for moderately absorbed compounds (disodium EDTA and propylene glycol) was 1.8-3.3. Absorption via skin with "diaper rash" is specific to individual ingredients and exposure conditions, so a fixed uncertainty factor is not appropriate for safety assessment. The data support that the default 100% dermal absorption commonly used in first-tier risk assessments for diapered skin is conservative. This diaper rash skin model provides a practical tool of estimating absorption of various ingredients in baby products intended for diapered skin.

Human epidermal keratinocytes and human dermal fibroblasts interactions seeded on gelatin hydrogel for future application in skin in vitro 3-dimensional model.

Introduction: Plenty of biomaterials have been studied for their application in skin tissue engineering. Currently, gelatin-hydrogel is used to support three-dimensional (3D) skin in vitro models. However, mimicking the human body conditions and properties remains a challenge and gelatin-hydrogels have low mechanical properties and undergo rapid degradation rendering them not suitable for 3D in vitro cell culture. Nevertheless, changing the concentration of hydrogels could overcome this issue. Thus, we aim to investigate the potential of gelatin hydrogel with different concentrations crosslinked with genipin to promote human epidermal keratinocytes and human dermal fibroblasts culture to develop a 3D-in vitro skin model replacing animal models. Methods: Briefly, the composite gelatin hydrogels were fabricated using different concentrations as follows 3%, 5%, 8%, and 10% crosslinked with 0.1% genipin or non-crosslinked. Both physical and chemical properties were evaluated. Results and discussion: The crosslinked scaffolds showed better properties, including porosity and hydrophilicity, and genipin was found to enhance the physical properties. Furthermore, no alteration was prominent in both formulations of CL_GEL 5% and CL_GEL8% after genipin modification. The biocompatibility assays showed that all groups promoted cell attachment, cell viability, and cell migration except for the CL_GEL10% group. The CL_GEL5% and CL_GEL8% groups were selected to develop a bi-layer 3D-in vitro skin model. The immunohistochemistry (IHC) and hematoxylin and eosin staining (H&E) were performed on day 7, 14, and 21 to evaluate the reepithelization of the skin constructs. However, despite satisfactory biocompatibility properties, neither of the selected formulations, CL_GEL 5% and CL_GEL 8%, proved adequate for creating a bi-layer 3D in-vitro skin model. While this study provides valuable insights into the potential of gelatin hydrogels, further research is needed to address the challenges associated with their use in developing 3D skin models for testing and biomedical applications.

Layer-by-Layer Analysis of In Vitro Skin Models.

In vitro human skin models are evolving into versatile platforms for the study of skin biology and disorders. These models have many potential applications in the fields of drug testing and safety assessment, as well as cosmetic and new treatment development. The development of in vitro skin models that accurately mimic native human skin can reduce reliance on animal models and also allow for more precise, clinically relevant testing. Recent advances in biofabrication techniques and biomaterials have led to the creation of increasingly complex, multilayered skin models that incorporate important functional components of skin, such as the skin barrier, mechanical properties, pigmentation, vasculature, hair follicles, glands, and subcutaneous layer. This improved ability to recapitulate the functional aspects of native skin enhances the ability to model the behavior and response of native human skin, as the complex interplay of cell-to-cell and cell-to-material interactions are incorporated. In this review, we summarize the recent developments in in vitro skin models, with a focus on their applications, limitations, and future directions.

Engineered-Skin of Single Dermal Layer Containing Printed Hybrid Gelatin-Polyvinyl Alcohol Bioink via 3D-Bioprinting: In Vitro Assessment under Submerged vs. Air-Lifting Models.

Three-dimensional (3D) in vitro skin models are frequently employed in cosmetic and pharmaceutical research to minimize the demand for animal testing. Hence, three-dimensional (3D) bioprinting was introduced to fabricate layer-by-layer bioink made up of cells and improve the ability to develop a rapid manufacturing process, while maintaining bio-mechanical scaffolds and microstructural properties. Briefly, gelatin-polyvinyl alcohol (GPVA) was mixed with 1.5 × 106 and 3.0 × 106 human dermal fibroblast (HDF) cell density, together with 0.1% genipin (GNP), as a crosslinking agent, using 3D-bioprinting. Then, it was cultured under submerged and air-lifting conditions. The gross appearance of the hydrogel's surface and cross-section were captured and evaluated. The biocompatibility testing of HDFs and cell-bioink interaction towards the GPVA was analyzed by using live/dead assay, cell migration activity, cell proliferation assay, cell morphology (SEM) and protein expression via immunocytochemistry. The crosslinked hydrogels significantly demonstrated optimum average pore size (100-199 µm). The GPVA crosslinked with GNP (GPVA_GNP) hydrogels with 3.0 × 106 HDFs was proven to be outstanding, compared to the other hydrogels, in biocompatibility testing to promote cellular interaction. Moreover, GPVA-GNP hydrogels, encapsulated with 3.0 × 106 HDFs under submerged cultivation, had a better outcome than air-lifting with an excellent surface cell viability rate of 96 ± 0.02%, demonstrated by 91.3 ± 4.1% positively expressed Ki67 marker at day 14 that represented active proliferative cells, an average of 503.3 ± 15.2 µm for migration distance, and maintained the HDFs' phenotypic profiles with the presence of collagen type I expression. It also presented with an absence of alpha-smooth muscle actin positive staining. In conclusion, 3.0 × 106 of hybrid GPVA hydrogel crosslinked with GNP, produced by submerged cultivation, was proven to have the excellent biocompatibility properties required to be a potential bioinks for the rapid manufacturing of 3D in vitro of a single dermal layer for future use in cosmetic, pharmaceutic and toxicologic applications.

Candida auris Phenotypic Heterogeneity Determines Pathogenicity In Vitro.

Candida auris is an enigmatic yeast that provides substantial global risk in health care facilities and intensive care units. A unique phenotype exhibited by certain isolates of C. auris is their ability to form small clusters of cells known as aggregates, which have been to a limited extent described in the context of pathogenic traits. In this study, we screened several nonaggregative and aggregative C. auris isolates for biofilm formation, where we observed a level of heterogeneity among the different phenotypes. Next, we utilized an RNA sequencing approach to investigate the transcriptional responses during biofilm formation of a nonaggregative and aggregative isolate of the initial pool. Observations from these analyses indicate unique transcriptional profiles in the two isolates, with several genes identified relating to proteins involved in adhesion and invasion of the host in other fungal species. From these findings, we investigated for the first time the fungal recognition and inflammatory responses of a three-dimensional skin epithelial model to these isolates. In these models, a wound was induced to mimic a portal of entry for C. auris We show that both phenotypes elicited minimal response in the model minus induction of the wound, yet in the wounded tissue, both phenotypes induced a greater response, with the aggregative isolate more proinflammatory. This capacity of aggregative C. auris biofilms to generate such responses in the wounded skin highlights how this opportunistic yeast is a high risk within the intensive care environment where susceptible patients have multiple indwelling lines.IMPORTANCECandida auris has recently emerged as an important cause of concern within health care environments due to its ability to persist and tolerate commonly used antiseptics and disinfectants, particularly when attached to a surface (biofilms). This yeast is able to colonize and subsequently infect patients, particularly those that are critically ill or immunosuppressed, which may result in death. We have undertaken analysis on two different phenotypic types of this yeast, using molecular and immunological tools to determine whether either of these has a greater ability to cause serious infections. We describe that both isolates exhibit largely different transcriptional profiles during biofilm development. Finally, we show that the inability to form small aggregates (or clusters) of cells has an adverse effect on the organism's immunostimulatory properties, suggesting that the nonaggregative phenotype may exhibit a certain level of immune evasion.

Polysaccharide-Based Bioink Formulation for 3D Bioprinting of an In Vitro Model of the Human Dermis.

Limitations in wound management have prompted scientists to introduce bioprinting techniques for creating constructs that can address clinical problems. The bioprinting approach is renowned for its ability to spatially control the three-dimensional (3D) placement of cells, molecules, and biomaterials. These features provide new possibilities to enhance homology to native skin and improve functional outcomes. However, for the clinical value, the development of hydrogel bioink with refined printability and bioactive properties is needed. In this study, we combined the outstanding viscoelastic behavior of nanofibrillated cellulose (NFC) with the fast cross-linking ability of alginate (ALG), carboxymethyl cellulose (CMC), and encapsulated human-derived skin fibroblasts (hSF) to create a bioink for the 3D bioprinting of a dermis layer. The shear thinning behavior of hSF-laden bioink enables construction of 3D scaffolds with high cell density and homogeneous cell distribution. The obtained results demonstrated that hSF-laden bioink supports cellular activity of hSF (up to 29 days) while offering proper printability in a biologically relevant 3D environment, making it a promising tool for skin tissue engineering and drug testing applications.

Fabrication of Bioengineered Skin by Injection Molding: A Feasibility Study on Automation.

The use of bioengineered skin has facilitated fundamental and applied research because it enables the investigation of complex interactions between various cell types as well as the extracellular matrix. The predominantly manual fabrication of these living tissues means, however, that their quality, standardization, and production volume are extremely dependent on the technician's experience. Simple laboratory automation could facilitate the use of living tissues by a greater number of research groups. We developed and present here an injection molding technique for the fabrication of bilayered skin equivalents. The tissue was formed automatically by two separate injections into a customized mold to produce the dermal and epidermal skin layers. We demonstrated the biocompatibility of this fabrication process and confirmed the resulting bilayered morphology of the bioengineered skin using histology and immunohistochemistry. Our findings highlight the possibility of fabricating multilayered living tissue by injection molding, suggesting that further investigation into this automation method could result in the rapid and low-cost fabrication of standardized bioengineered skin.

An in vitro model to evaluate the impact of environmental fine particles (PM0.3-2.5) on skin damage.

Exposure to airborne particulate matter (PM) has significant effects on human health mainly leading to cardio-respiratory diseases. However very few data are available regarding the impact of PM on the skin, so to better understand the impact of fine particle (PM0.3-2.5) on both inflammatory response and epidermal structure, we exposed a reconstructed human epidermis (RHE) to several doses of PM collected in Cotonou (Benin, West Africa). After 24 h of exposure, inflammatory response, histological observations, and gene expression related to oxidative stress, antioxidant defense and structural damages were determined. No PM-linked changes in tissue morphology or membrane integrity were observable. PM was however cytotoxic in a dose dependent manner. An inflammatory response appeared as shown by the increase in IL-1α and IL-8 cytokine productions. PM also induced oxidative stress, leading to an increase in 4-HNE immunostaining and to the up-regulation of HMOX1, MT1G and MT1E. Finally, PM had a negative impact on fundamental skin functions such as tissue anchorage, cell differentiation, cornification / skin desquamation and apoptosis. Our data show that airborne fine particles have an adverse effect on skin integrity, most probably leading to accelerated ageing.

Current Strategies and Future Perspectives of Skin-on-a-Chip Platforms: Innovations, Technical Challenges and Commercial Outlook.

The skin is the largest and most exposed organ in the human body. Not only it is involved in numerous biological processes essential for life but also it represents a significant endpoint for the application of pharmaceuticals. The area of in vitro skin tissue engineering has been progressing extensively in recent years. Advanced in vitro human skin models strongly impact the discovery of new drugs thanks to the enhanced screening efficiency and reliability. Nowadays, animal models are largely employed at the preclinical stage of new pharmaceutical compounds development for both risk assessment evaluation and pharmacokinetic studies. On the other hand, animal models often insufficiently foresee the human reaction due to the variations in skin immunity and physiology. Skin-on-chips devices offer innovative and state-of-the-art platforms essential to overcome these limitations. In the present review, we focus on the contribution of skin-on-chip platforms in fundamental research and applied medical research. In addition, we also highlighted the technical and practical difficulties that must be overcome to enhance skin-on-chip platforms, e.g. embedding electrical measurements, for improved modeling of human diseases as well as of new drug discovery and development.

Improved normal tissue protection by proton and X-ray microchannels compared to homogeneous field irradiation.

The risk of developing normal tissue injuries often limits the radiation dose that can be applied to the tumour in radiation therapy. Microbeam Radiation Therapy (MRT), a spatially fractionated photon radiotherapy is currently tested at the European Synchrotron Radiation Facility (ESRF) to improve normal tissue protection. MRT utilizes an array of microscopically thin and nearly parallel X-ray beams that are generated by a synchrotron. At the ion microprobe SNAKE in Munich focused proton microbeams ("proton microchannels") are studied to improve normal tissue protection. Here, we comparatively investigate microbeam/microchannel irradiations with sub-millimetre X-ray versus proton beams to minimize the risk of normal tissue damage in a human skin model, in vitro. Skin tissues were irradiated with a mean dose of 2 Gy over the irradiated area either with parallel synchrotron-generated X-ray beams at the ESRF or with 20 MeV protons at SNAKE using four different irradiation modes: homogeneous field, parallel lines and microchannel applications using two different channel sizes. Normal tissue viability as determined in an MTT test was significantly higher after proton or X-ray microchannel irradiation compared to a homogeneous field irradiation. In line with these findings genetic damage, as determined by the measurement of micronuclei in keratinocytes, was significantly reduced after proton or X-ray microchannel compared to a homogeneous field irradiation. Our data show that skin irradiation using either X-ray or proton microchannels maintain a higher cell viability and DNA integrity compared to a homogeneous irradiation, and thus might improve normal tissue protection after radiation therapy.

An in vitro skin model to study the effect of mesenchymal stem cells in wound healing and epidermal regeneration.

The development of new wound therapies, such as bioengineered skin equivalents, is an ongoing process. Multi-potent mesenchymal stem cells (MSCs) give rise to many tissue lineages and have been implicated in wound healing making them a potential candidate for cell-based bioengineered products for injured tissue. In this study, we investigated the mesenchymal/epithelial interactions of cultured MSCs in comparison to cultured fibroblasts on epidermal proliferation, differentiation, and extracellular matrix (ECM) protein expression using a de-epidermalized dermis (DED) skin model. We also studied whether MSCs can transdifferentiate to keratinocytes using the same model. Keratinocytes were cultured on unseeded DED or DED populated with fibroblasts or MSCs at an air-liquid interface to induce epidermal differentiation. Fibroblasts or MSCs were also seeded on the papillary surface of the DED alone or on the reticular surface. General histology and immunostaining was performed on the skin equivalents to examine the expression of pan keratin (K) (K1, K5, K6, and K18) and protein markers for epidermal differentiation (K10), hyperproliferation (K6), proliferation (PCNA), ECM component (collagen type IV), and mesenchymal marker (vimentin). Keratinocyte-fibroblast skin model and keratinocyte-MSC skin model both displayed an epidermal phenotype similar to epidermis in vivo. Positive expression of proliferation, differentiation and ECM protein markers was observed. MSCs failed to adopt an epithelial phenotype in the DED skin model. Our findings highlight the potential use of MSCs in bioengineered tissue for the treatment of wounds.