3D Skin Model Research Articles

Anti-Aging Potential of Substance P-Based Hydrogel for Human Skin Longevity

Skin aging is generally caused by a decline in the components of the extracellular matrix (e.g., collagen and elastin) and due to inflammatory phenomena. Many growth factors and peptides with cell-growth and collagen-synthesis activities have shown promise in their application in anti-aging materials. However, the effect of collagen production, without anti-inflammatory effect, and skin penetration may not be enough for their use in anti-aging agents. Previously, we reported a substance P (SP)-based hydrogel (SP gel) that had potential wound-healing activities via induction of skin cell regeneration and collagen synthesis. Here, we analyzed the anti-aging activities and skin absorption effects of SP gel to extend its characterization. Toxicity tests, performed on human dermal fibroblasts (HDFs) and on a reconstructed 3D human skin model, indicated SP gel to be safe for long-term use, without causing irritation, even at high concentrations. In-vitro analysis revealed that SP gel elicited stronger collagen production activities than SP alone, and promoted anti-inflammatory effects with increased skin absorption properties. Moreover, SP gel did not induce melanin synthesis in a keratinocyte-melanocyte co-culture system. Together, the results suggest that SP gel has potential cosmetic effects and applicability as a novel ingredient in anti-aging products.

591 A New 3D Skin Model Containing Autologous Immune Cells Reconstructed by Tissue Engineering

The majority of current in vitro skin models lack an inflammatory system, vasculature, and other characteristics of native skin, making them less relevant for the study of biological or pathological processes related to the human skin. To fill this gap, we developed a skin model containing immune and endothelial cells. Skin biopsies were enzymatically treated to isolate epidermal and dermal cells. Following isolation, the epidermal fraction contained keratinocytes, melanocytes, lymphocytes T and Langerhans cells while the dermal fraction contained fibroblasts, endothelial cells and immune cells (macrophages, lymphocytes, dendritic cells). Fibroblasts were first cultured for 3 weeks with ascorbic acid to stimulate the production of an extracellular matrix surrounding cells. The dermal fraction, that contained immune cells of autologous origin to avoid activation of the immune system, was then seed onto one sheet while the epidermal fraction was seeded on another sheet. These two sheets were then superimposed and raised at the air-liquid interface to stimulate epidermis differentiation. After 2 weeks, a 3D skin model containing immune cells isolated from the same donor was obtained. Histological studies revealed a stratified epidermis found on top of a dermis composed of matrix and cells. Immunofluorescence staining showed viable autologous myeloid cells, mast cells and T lymphocytes as well as an autologous capillary network (CD31+) in the dermis. The creation of this first 3D model containing autologous immune cells paves the way to increase knowledge into skin homeostasis as well as in diseases.

227 The effect of endothelial precursor cell-conditioned media on keratinocytes and 3D skin models

227 The effect of endothelial precursor cell-conditioned media on keratinocytes and 3D skin models

Air-liquid interface cell culture: From airway epithelium to the female reproductive tract.

The air-liquid interface (ALI) approach is primarily used to mimic respiratory tract epithelia in vitro. It is also known to support excellent differentiation of 3D multilayered skin models. To establish an ALI culture, epithelial cells are seeded into compartmentalized culture systems on porous filter supports or gel substrata. After an initial propagation period, the culture medium is removed from the apical side of the epithelium, exposing the cells to the surrounding air. Therefore, nutritive supply to the cells is warranted only by the basolateral cell pole. Under these conditions, the epithelial cells differentiate and regain full baso-apical polarity. Some types of epithelia even generate in vivo-like apical fluid or mucus. Interestingly, the ALI culture approach has also been shown to support morphological and functional differentiation of epithelial cells that are not normally exposed to ambient air in vivo. This review aims at giving a brief overview on the characteristics of ALI cultures in general and ALI models of female reproductive tract epithelia in particular. We discuss the applicability of ALI models for the investigation of the early embryonic microenvironment and for its implications in assisted reproductive technologies.

366 Blocking the IL-22 receptor represents a novel treatment option for atopic eczema

Atopic eczema (AE) is a chronic inflammatory skin disease that presents with elevated levels of type 2 cytokines but also elevated IL-22 expression in lesional skin. The objective of this study is to evaluate a novel IL-22 receptor blocking antibody for AE and demonstrate a difference in mechanism of action to IL-4RA inhibition. Expression of IL-22RA was identified in healthy and AE skin samples by in situ hybridization (ISH). In vitro, primary human keratinocytes were cultured in an air liquid interface (3D) and stimulated with supernatant derived from activated AE lesional T cells (SN) in presence or absence of an IL-22RA1 or an IL-4RA blocking antibody as comparator. Furthermore, fresh AE biopsies were cultured ex vivo in presence or absence of these blocking antibodies. All models were analyzed by real time PCR, RNA sequencing and histology. ISH of IL-22RA1 mRNA revealed only sporadic expression in stratum granulosum predominantly of healthy skin. In contrast, in AE skin IL-22RA1 expression was denser and sporadically expressed in the stratum spinosum and stratum basale. Histological evaluation of 3D human skin models revealed that blocking IL-22RA1 led to a reduction of IL-22 induced acanthosis. On gene expression level, IL-22RA1 inhibition of AE T cell SN stimulated models, significantly reduced expression of DEFB4 but had no effect on CCL2 levels. In contrary, IL-4RA inhibition significantly reduced CCL2 expression, whereas DEFB4 was increased. RNA sequencing of 3D models and in vitro stimulated AE skin revealed different mode of actions of IL-22RA1 and IL-4RA inhibition highlighting the distinct pathways both cytokines use to contribute to disease pathology. In conclusion, IL-22R inhibition shows a different mode of action compared to IL-4RA blocking and could be a powerful alternative therapeutic option to Dupilumab to treat AE.

Patient-derived in vitro skin models for investigation of small fiber pathology.

ObjectiveTo establish individually expandable primary fibroblast and keratinocyte cultures from 3‐mm skin punch biopsies for patient‐derived in vitro skin models to investigate of small fiber pathology.MethodsWe obtained 6‐mm skin punch biopsies from the calf of two patients with small fiber neuropathy (SFN) and two healthy controls. One half (3 mm) was used for diagnostic intraepidermal nerve fiber density (IENFD). From the second half, we isolated and cultured fibroblasts and keratinocytes. Cells were used to generate patient‐derived full‐thickness three‐dimensional (3D) skin models containing a dermal and epidermal component. Cells and skin models were characterized morphologically, immunocyto‐ and ‐histochemically (vimentin, cytokeratin (CK)‐10, CK 14, ki67, collagen1, and procollagen), and by electrical impedance.ResultsDistal IENFD was reduced in the SFN patients (2 fibers/mm each), while IENFD was normal in the controls (8 fibers/mm, 7 fibers/mm). Two‐dimensional (2D) cultured skin cells showed normal morphology, adequate viability, and proliferation, and expressed cell‐specific markers without relevant difference between SFN patient and healthy control. Using 2D cultured fibroblasts and keratinocytes, we obtained subject‐derived 3D skin models. Morphology of the 3D model was analogous to the respective skin biopsy specimens. Both, the dermal and the epidermal layer carried cell‐specific markers and showed a homogenous expression of extracellular matrix proteins.InterpretationOur protocol allows the generation of disease‐specific 2D and 3D skin models, which can be used to investigate the cross‐talk between skin cells and sensory neurons in small fiber pathology.

LB1121 Prodifferentiative and anti-inflammatory effects of fisetin in 2D and 3D human skin model of psoriasis are associated with inhibition of PI3K/Akt/mTOR and MAPK signaling

LB1121 Prodifferentiative and anti-inflammatory effects of fisetin in 2D and 3D human skin model of psoriasis are associated with inhibition of PI3K/Akt/mTOR and MAPK signaling

A comprehensive topical antioxidant prevents ozone-induced damage in a 3D human skin model

A comprehensive topical antioxidant prevents ozone-induced damage in a 3D human skin model

Anti‑photoaging and anti‑oxidative activities of natural killer cell conditioned medium following UV‑B irradiation of human dermal fibroblasts and a reconstructed skin model.

Conditioned media from various sources comprise numerous growth factors and cytokines and are known to promote the regeneration of damaged tissues. Among these, natural killer cell conditioned medium (NK-CdM) has been shown to stimulate collagen synthesis and the migration of fibroblasts during the wound healing process. With a long-term aim of developing a treatment for skin photoaging, the ability of NK-CdM to prevent ultraviolet-B (UV-B) damage was assessed in neonatal human dermal fibroblasts (NHDFs) and an in vitro reconstructed skin model. The factors present in NK-CdM were profiled using an antibody array analysis. Protein and mRNA levels in UV-B exposed NHDFs treated with NK-CdM were measured by western blotting and quantitative reverse transcription-PCR, respectively. The total antioxidant capacity of NK-CdM was determined to assess its ability to suppress reactive oxygen species. The anti-photoaging effect of NK-CdM was also assessed in a 3D reconstituted human full skin model. NK-CdM induced proliferation of UV-B-treated NHDFs, increased procollagen expression, and decreased matrix metalloproteinase (MMP)-1 expression. NK-CdM also exhibited a potent antioxidant activity as measured by the total antioxidant capacity. NK-CdM inhibited UV-B-induced collagen degradation by inactivating MAPK signaling. NK-CdM also elicited potential anti-wrinkle effects by inhibiting the UV-B-induced increase in MMP-1 expression levels in a 3D reconstituted human full skin model. Taken together, the suppression of both UV-B-induced MMP-1 expression and JNK activation by NK-CdM suggests NK-CdM as a possible candidate anti-skin aging agent.

Topical Esomeprazole Mitigates Radiation-Induced Dermal Inflammation and Fibrosis.

Radiation therapy is a mainstream strategy in the treatment of several cancer types that are surgically unresectable. Unfortunately, cancer patients often suffer from unintended consequences of radiotherapy, including the development of skin inflammation (dermatitis), which may progress to fibrosis. These morbid complications often require interruption of radiotherapy and threaten the relapse of underlying cancer. Current treatment options for radiation dermatitis are suboptimal and compel the need to develop safer, more effective therapies. In this study, we assessed the biophysical properties of topically-formulated esomeprazole (here referred to as dermaprazole) and performed proof-of-concept studies to evaluate its efficacy in vitro and in vivo. We found that dermaprazole induced nuclear translocation of erythroid 2-related factor 2 (Nrf2) and significantly upregulated heme oxygenase 1 (HO1) gene and protein expression in a 3D human skin model. Our animal study demonstrated that dermaprazole improved macroscopic appearance of the irradiated skin and accelerated healing of the wounds. Histopathology data corroborated the photographic evidence and confirmed that both prophylactically and therapeutically administered dermaprazole conferred potent anti-inflammatory and antifibrotic effects. Gene expression data showed that dermaprazole downregulated several pro-oxidant, pro-inflammatory and profibrotic genes. In conclusion, topical formulation of the FDA-approved drug esomeprazole is highly effective in attenuating dermal inflammation and fibrosis.

In Vitro Skin Models for the Evaluation of Sunscreen-Based Skin Photoprotection: Molecular Methodologies and Opportunities.

Identifying and understanding the biological events that occur following ultraviolet (UV) exposure are mandatory to elucidate the biological and clinical consequences of sun exposure, and to provide efficient and adequate photoprotection strategies. The main UVinduced biological features (markers related to sunburn, cancer, photoaging immunosuppression, pigmentation), characterized in human skin in vivo, could be reproduced in adapted models of reconstructed skin in vitro, attesting their high relevance in the field of photobiology. In turn, 3D skin models were useful to discover precise biological pathways involved in UV response and were predictive of in vivo situation. Although they did not follow a strict validation process for the determination of protection factors, they enabled to evidence important concepts in photoprotection. Indeed, the use of reconstructed skin model highlighted the importance of broad spectrum sunscreen use to protect essential cellular functions, and biologically proved that SPF value was not predictive of the level of protection in the UVA wavelength domain. New biological approaches, such as transcriptomic or proteomic studies as well as quantitative and qualitative determination of DNA damage, will indisputably increase the added value of such systems for sunscreen efficiency evaluation.

Ag modified mesoporous bioactive glass nanoparticles for enhanced antibacterial activity in 3D infected skin model

Bioactive glasses (BG) are versatile materials for various biomedical applications, including bone regeneration and wound healing, due to their bone bonding, antibacterial, osteogenic, and angiogenic properties. In this study, we aimed to enhance the antibacterial activity of SiO2-CaO mesoporous bioactive glass nanoparticles (MBGN) by incorporating silver (Ag) through a surface modification approach. The modified Ag-containing nanoparticles (Ag-MBGN) maintained spherical shape, mesoporous structure, high dispersity, and apatite-forming ability after the surface functionalization. The antibacterial activity of Ag-MBGN was assessed firstly using a planktonic bacteria model. Moreover, a 3D tissue-engineered infected skin model was used for the first time to evaluate the antibacterial activity of Ag-MBGN at the usage dose of 1 mg/mL. In the planktonic bacteria model, Ag-MBGN exhibited a significant antibacterial effect against both Pseudomonas aeruginosa and Staphylococcus aureus in comparison to non-engineered (Ag-free) MBGN and the blank control. Moreover, Ag-MBGN did not show cytotoxicity towards fibroblasts at the usage dose. However, in the 3D infected skin model, Ag-MBGN only demonstrated antibacterial activity against S. aureus whereas their antibacterial action against P. aeruginosa was inhibited. In conclusion, surface modification by Ag incorporation is a feasible approach to enhance the antibacterial activity of MBGN without significantly impacting their morphology, polydispersity, and apatite-forming ability. The prepared Ag-MBGN are attractive building blocks for the development of 3D antibacterial scaffolds for tissue engineering.

Direct 3D bioprinted full-thickness skin constructs recapitulate regulatory signaling pathways and physiology of human skin

Currently available tissue engineered skin-equivalents fail to replicate anatomically relevant features, e.g. undulated morphology of the dermal-epidermal junction, resulting in inadequate recapitulation of biologically meaningful cellular signaling pathways. This study reports fabrication of 3D bioprinted, human cell-based full thickness skin model possessing anatomically relevant structural, mechanical and biochemical features akin to human skin. The unique undulated feature of the epidermis-dermis junction could be recapitulated in the 3D bioprinted skin-equivalents. Extensive migration of the keratinocytes within the construct along with differentiation events reminded reepithelialisation. Cell-cell interaction along with diffusible factors promoted expression of differentiation and cornification markers in region-specific manner. Architectural features and silk bioink microenvironment triggered deposition of basement membrane specific-proteins at the interface. Most interestingly, extensive transcriptomics and proteomics analysis accentuated striking similarity of the 3D bioprinted full thickness skin model to the native human skin, with involvement of a number of pathways related to skin development and physiology, such as skin development, extracellular matrix organization, keratinization/cornification and collagen fibril organization. Such bioprinted in vitro human skin models would offer tremendous potential for screening cosmetic products and drugs, as well as to understand the complex physiological processes relevant to human skin thereby bridging the gap between conventional monolayer or 3D cultures and animal models.

1041 Development of human skin spheroid system for psoriatic disease modeling and drug development

Psoriasis is a complex chronic immune-mediated inflammatory cutaneous disease that affects 2–3% of the global population. Various 3D skin models have been developed to mimic psoriatic pathogenesis and help develop drugs, but these models are very low throughput and expensive. Here, we established a cost-effective, high throughput system for disease modeling and drug development, using spheroid approach. We plated 1x105 cells primary fibroblasts in 96-well plates for 2-5 days before addition of 5x104 primary keratinocytes. The culture medium contained DMEM/Ham’s F12, (3 : 1) 10% FBS, 0.18 mM adenine , 0.5 μg/ml hydrocortisone , 5 μg/ml insulin, 10-10 M cholera toxin, and10 ng/ml EGF. Similarly, we generated spheroids with iPSC-derived fibroblasts and keratinocytes. Immuno-staining and fluorescent dyes showed that fibroblasts formed a spheroid core surrounded with keratinocytes on the outer layers. More importantly, RT-PCR revealed that treatment with 30 ng/ml of IL-17 for 2-5 days upregulated a panel of psoriatic signature genes such as kRT16, S100A7, S100A12, DEFB4A and IL-8. To facilitate high throughput screening, DEFB4A/GFP reporters will be incorporated into keratinocytes with CRISPR technology. The availability of such cost-effective high throughput systems will greatly accelerate drug development to treat patients. Similar approaches can be used to generate models of other diseases, such as atopic dermatitis.

902 A new 3D immunocompetent skin model reconstructed by tissue engineering

902 A new 3D immunocompetent skin model reconstructed by tissue engineering

Effect of Resveratrol-Enriched Rice on Skin Inflammation and Pruritus in the NC/Nga Mouse Model of Atopic Dermatitis.

Resveratrol-enriched rice (RR) was developed using genetic engineering to combine the properties of resveratrol and rice. To evaluate the effect of RR on pruritic skin inflammation in atopic dermatitis (AD)-like skin lesions, we used dinitrochlorobenzene (DNCB)-induced NC/Nga mice and an in vitro 3D skin model. Normal rice (NR), resveratrol, and RR were topically applied to mice dorsal skin, following which the dermatitis index and scratching frequency were calculated. Histological examination was performed by hematoxylin and eosin and immunohistochemistry staining of IL-31 level. The level of immunoglobulin E (IgE) and IL-31 in the serum was determined by enzyme-linked immunosorbent assay (ELISA). The cytotoxicity of RR and the expression levels of pro-inflammatory cytokines were also determined in cultured human keratinocytes and a 3D skin model. RR significantly reduced scratching frequency, decreased the dermatitis severity and trans-epidermal water loss (TEWL) and improved skin hydration in DNCB-induced NC/Nga mice. RR also significantly decreased serum IL-31 and IgE levels and suppressed the production of IL-6 in human keratinocytes and the 3D skin model. Our study indicates that the synergistic effect of rice and resveratrol manifested by the topical application of RR can serve as a potential alternative therapy for chronic skin inflammatory diseases such as AD.

Quantification and characterization of radical production in human, animal and 3D skin models during sun irradiation measured by EPR spectroscopy

Sun radiation is indispensable to our health, however, a long term and high exposure could lead to erythema, premature skin aging and promotion of skin tumors. An underlying pathomechanism is the formation of free radicals. First, reactive oxygen species (*OH, *O2-) and then, secondary lipid oxygen species (C centered radicals, CCR) are formed. A high amount of free radicals results in oxidative stress with subsequent cell damage. In dermatological research different skin models are used, however, comparative data about the cutaneous radical formation are missing. In this study, the radical formation in porcine-, (SKH-1) murine-, human- ex vivo skin and reconstructed human skin (RHS) were investigated during simulated sun irradiation (305–2200 nm), with X-band EPR spectroscopy. The amount of radical formation was investigated with the spin probe PCA exposed to a moderate sun dose below one minimal erythema dose (MED, ~25 mJ/cm2 UVB) in all skin models. Furthermore, the *OH and *CCR radical concentrations were measured with the spin trap DMPO within 0–4 MED (porcine-, human skin and RHS).The highest amount of radicals was found in RHS followed by murine and porcine, and the lowest amount in human ex vivo skin. In all skin models, more *OH than CCR radicals were found at 0–4 MED. Additionally, this work addresses the limitations in the characterization with the spin trap DMPO.The measurements have shown that the most comparable skin model to in vivo human skin could differ depending on the focus of the investigation. If the amount of radial production is regarded, RHS seems to be in a similar range like in vivo human skin. If the investigation is focused on the radical type, porcine skin is most comparable to ex vivo human skin, at an irradiation dose not exceeding 1 MED. Here, no comparison to in vivo human skin is possible.

Unripe peach (Prunus persica) extract ameliorates damage from UV irradiation and improved collagen XVIII expression in 3D skin model.

Collagen type XVIII regulates cellular activities of adjacent cells at the dermal-epidermal junction (DEJ). To investigate its possible changes during aging, we compared its mRNA levels and protein localization in skin samples from female participants aged 20-70years old. In addition, we evaluated the beneficial effects of unripe peach extracts in a 3D skin model. Sun-exposed or sun-protected female skin samples were compared by DNA array or by immunohistochemistry for basement membrane components. To evaluate protective effects of fresh unripe peach extract, UV-B irradiated human 3D skin models were incubated in the presence or absence of the extract, followed by measurements of mRNA levels by real-time PCR, or by immunohistochemistry. In aged skin samples, COL18A1 mRNA levels were lower and the protein localization exhibited less intensive signal by anti-collagen type XVIII immunostaining. As observed in the skin tissues, collagen type XVIII exists at the DEJ in the 3D skin model. Fresh unripe peach extract significantly improved mRNA levels and partially localizations of collagen type XVIII, suggesting that fresh unripe peach extract ameliorates DEJ damages caused by UV-B irradiation. Collagen type XVIII and fresh unripe peach extract can be promising protective cosmetic strategies against skin aging.

Linear viscoelastic and microstructural properties of native male human skin and in vitro 3D reconstructed skin models

The study reports first ever account of measurements of linear viscoelastic moduli under small amplitude oscillatory shear deformations, for commercially available juvenile and aged in vitro 3D reconstructed skin models. The results were compared with those of native male whole human and dermis-only foreskin samples, catering to a wide age group from 0.5 to 68 years, including samples from a 23-year-old male abdomen. In the strain sweep tests, the dermis of the juvenile/young age group assumed a higher intrinsic elastic modulus than the whole skin. A reverse qualitative trend was noted for the adult/aged age group. Confirmed by the histological examination of the stained cross-sections, this is attributed to the nascent epidermal differentiation and the high fiber density of dermal collagen. The oscillation frequency sweeps exposed a greater dependence of the elasticity on the frequency for the native male dermis foreskin samples as compared to the whole skins, irrespective of age. This is anticipated since the extremely structured epidermis confers higher resistance to the whole skins towards intracycle deformations compared to the dermis, thereby storing smaller elastic energy. The 3D skin models examined in this work exhibited a broader linear viscoelastic region, a larger viscoelasticity, and much higher dynamic moduli, compared to the native skin. The rheological trends are a significant addition to the literature and may be used as a reference for the design of next generation of scaffolds.

Evaluation of the Transepidermal Penetration of a Carnosine Complex in Gel Formulation by 3D Skin Models

Carnosine has several physiological roles, from intracellular pH buffering to antioxidant activities, which all depend on bioavailability. This study was conducted in a human skin 3D model and focuses on the effects of the topical delivery of carnosine, from a dermo-cosmetic gel, through the stratum corneum in the presence of a magnesium ion as a complexing agent. To evaluate possible enhancement for small peptide delivery to the skin from simple cosmetic formulations, we discovered that complexation was able to improve the delivery of carnosine into human skin 3D models by application in gel formulation. The concentrations of carnosine released in the underlying media and those that remained in the reconstructed human epidermis (RHE) tissues after 24 and 48 h exposure were measured. Moreover, the influence of magnesium ions was also evaluated comparing the same formulation with and without the salt. The results obtained in this study support hypothesis that magnesium can influence the delivery of small peptides and that the gel formulation based on the carnosine-magnesium complex allows for superior delivery of carnosine in the lower skin layer at a concentration up to 60% more than carnosine alone.