Animal Skin Models Research Articles

Skin Structure, Physiology, and Pathology in Topical and Transdermal Drug Delivery.

Several industries are increasingly focused on enhancing the delivery of active ingredients through the skin to optimize therapeutic outcomes. By facilitating the penetration of active ingredients through the skin barrier, these enhancers can significantly improve the efficacy of various formulations, ranging from skincare products to therapeutic agents targeting systemic circulation. As the understanding of skin physiology and the mechanisms of drug absorption deepen, these industries are adopting permeation enhancers more widely, ultimately leading to better patient outcomes and expanded treatment options. However, the structure and physiological function of the skin can vary according to different factors, such as the area of the body and between individuals. These variations, along with external environmental exposures, aging and pathological conditions, introduce complexities that must be carefully considered when designing effective delivery systems. Considering the intricacies of skin structure and physiology, tailoring systems to account for regional differences, individual variability, and changes induced by environmental factors or disease is critical to optimizing therapeutic outcomes. This review discusses the features of skin structure, physiology, and pathologies, as well as the application of permeation enhancers in these contexts. Furthermore, it addresses the use of animal skin models in transdermal delivery and dermatological studies, along with the latest developments in this field.

Radiofrequency Treatment Attenuates Age-Related Changes in Dermal-Epidermal Junctions of Animal Skin.

The dermal-epidermal junction (DEJ) is essential for maintaining skin structural integrity and regulating cell survival and proliferation. Thus, DEJ rejuvenation is key for skin revitalization, particularly in age-related DEJ deterioration. Radiofrequency (RF) treatment, known for its ability to enhance collagen fiber production through thermal mechanisms and increase heat shock protein (HSP) expression, has emerged as a promising method for skin rejuvenation. Additionally, RF activates Piezo1, an ion channel implicated in macrophage polarization toward an M2 phenotype and enhanced TGF-β production. This study investigated the impact of RF treatment on HSP47 and HSP90 expression, known stimulators of DEJ protein expression. Furthermore, using in vitro and aged animal skin models, we assessed whether RF-induced Piezo1 activation and the subsequent M2 polarization could counter age-related DEJ changes. The RF treatment of H2O2-induced senescent keratinocytes upregulated the expression of HSP47, HSP90, TGF-β, and DEJ proteins, including collagen XVII. Similarly, the RF treatment of senescent macrophages increased Piezo1 and CD206 (M2 marker) expression. Conditioned media from RF-treated senescent macrophages enhanced the expression of TGF-β and DEJ proteins, such as nidogen and collagen IV, in senescent fibroblasts. In aged animal skin, RF treatment increased the expression of HSP47, HSP90, Piezo1, markers associated with M2 polarization, IL-10, and TGF-β. Additionally, RF treatment enhanced DEJ protein expression. Moreover, RF reduced lamina densa replication, disrupted lesions, promoted hemidesmosome formation, and increased epidermal thickness. Overall, RF treatment effectively enhanced DEJ protein expression and mitigated age-related DEJ structural changes by increasing HSP levels and activating Piezo1.

A sustainable strategy for generating highly stable human skin equivalents based on fish collagen

Tissue engineered skin equivalents are increasingly recognized as potential alternatives to traditional skin models such as human ex vivo skin or animal skin models. However, most of the currently investigated human skin equivalents (HSEs) are constructed using mammalian collagen which can be expensive and difficult to extract. Fish skin is a waste product produced by fish processing industries and identified as a cost-efficient and sustainable source of type I collagen. In this work, we describe a method for generating highly stable HSEs based on fibrin fortified tilapia fish collagen. The fortified fish collagen (FFC) formulation is optimized to enable reproducible fabrication of full-thickness HSEs that undergo limited contraction, facilitating the incorporation of human donor-derived skin cells and formation of biomimetic dermal and epidermal layers. The morphology and barrier function of the FFC HSEs are compared with a commercial skin model and validated with immunohistochemical staining and transepithelial electrical resistance testing. Finally, the potential of a high throughput screening platform with FFC HSE is explored by scaling down its fabrication to 96-well format.

Surfactant nanovesicles for augmented antibacterial activity against carbapenemase resistant enterobacteriaceae and extended spectrum beta-lactamases producing bacteria: in vitro and in vivo evaluation

The ubiquitous emergence of bacterial resistance is a challenging problem in infectious diseases treatment. Recently, new research lines employed nano-drug delivery systems to enhance antibacterial activity of the existing antibiotics. Accordingly, the objective of this study is to optimize surfactant nanovesicles to improve the antimicrobial effect of meropenem, ertapenem and tigecycline against Carbapenemase Resistant Enterobacteriaceae (CRE) and extended spectrum beta-lactamases producing bacteria (ESBL). Klebsiella pneumoniae and Escherichia coli were used as the test organisms. In vivo and in vitro evaluations were conducted to prove the efficacy of niosome-encapsulated drugs formulations. The results revealed that surfactant vesicles were able to reduce the MIC values of the tested drugs by nine-fold change compared to their free forms. Scanning Electron Microscope (SEM) showed possible adhesion/fusion of the vesicles encapsulated drugs on the bacterial cells compared to its solution. In vivo investigations using animal skin model confirmed the superiority of nanovesicles drug encapsulation regarding both wound size and histopathological examination. Wound surface area was reduced from 24.6mm2 in absence of drug to reach 13.9, and 6.2mm2 in presence of ertapenem solution or niosomes, respectively. Nanovesicular formulations can be considered as effective drug delivery systems that can diminish bacterial resistance against β-lactams antibiotics.

Validation and testing of a new artificial biomimetic barrier for estimation of transdermal drug absorption

Human skin remains the most reliable model for studying the transdermal permeation of active compounds. Due to the limited source, porcine skin has been used extensively for performing penetration tests. Performing penetration studies by using human and animal skin, however, would also involve a series of ethical issues and restrictions. For these reasons, new biomimetic artificial barriers are being developed as possible alternatives for transdermal testing. If appropriately optimized, such products can be cost-effective, easily standardized across laboratories, precisely controlled in specific experimental conditions, or even present additional properties compared to the human and animal skin models such as negligible variability between replicates. In this current work we use the skin mimicking barrier (SMB) for drug permeability tests. The aim was to evaluate the suitability of the new barrier for studying the percutaneous absorption of the lipophilic extract of the plant Zingiber officinale Roscoe in vitro and compare its permeability ability with the artificial membrane Permeapad® and porcine skin. Our results showed that the permeability values obtained through the SMB are comparable are comparable to those obtained by using the porcine skin, suggesting that the new barrier may be an acceptable in vitro model for conducting percutaneous penetration experiments.

A Human Ex Vivo Skin Model to Study Candida auris Biofilms.

Candida auris can persist for long periods on hospital surfaces and on the skin. C. auris has the ability to form drug-resistant biofilms, which can substantially impact on patient outcome. In comparison to Candida albicans, C. auris has a lower capacity to form biofilms in in vitro models and a higher capacity when tested on animal skin models. Intraspecies variation is shown to exist, with some clinical isolates having greater biofilm capabilities than others. There is a need for models that closely mimic the real niches where infection occurs on human patients. This protocol describes, in detail, a human skin model to study C. auris biofilm formation using catheterized and non-catheterized skin.

Upconversion nanoparticle incorporated oleogel as probable skin tissue imaging agent

Although upconversion nanoparticle (UCNP) has shown promising role as a near infra-red (NIR) excitation triggered theranostic agent, its application in dermatology is limited due to skin permeation issues. Herein, we report the delivery of UCNP to the inner skin structure in transdermal fashion using oleogel based topical formulation. For this purpose, we have developed a series of oleylamine coated UCNP (OA-UCNP) incorporated oleogel through a simple two-step heat-cool procedure by varying the weight % of OA-UCNP and keeping the weight ratio of soybean oil:stearic acid constant. The UCNP-oleogels were characterized by various microscopic techniques as well as XRD and FTIR in order to understand the changes occurring in the microstructures of the oleogels with the incorporation of OA-UCNP in a concentration-dependent manner. The photoluminescence (PL) spectroscopy studies revealed that the UCNP-oleogel with the highest concentration of OA-UCNP (UG4) showed maximum luminescence among all the other compositions. The skin tissue penetration ability of UCNP was validated using UG4 as a topical agent through in vitro PL experiments involving agarose gel and animal skin models. The permeation of UCNP across the full thickness of intact pig ear skin was evident when UCNP was incorporated in oleogel, whereas UCNP itself in the form of OA-UCNP did not show any detectable skin permeation. The inner layer of animal skins were imaged by capturing the PL of the UCNP under NIR (980 nm) laser excitation.

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.

Quantifying the role of burn temperature, burn duration and skin thickness in an in vivo animal skin model of heat conduction

To determine the extent to which heat conduction through skin is affected by skin thickness, burn temperature, and burn duration, we perform a suite of experiments using an in vivo porcine (pig) model. Fourteen different burn conditions are considered, and each burn condition is replicated at least four times, giving a total of sixty four individual experimental burns. The subdermal temperature within the skin is recorded as a function of time during each experiment. To quantitatively interpret the experimental data, we develop an exact solution of a simplified, depth-averaged, heat equation. Calibrating this solution to the experimental data provide estimates of the effective thermal diffusivity of the skin, α, and the effective thermal loss rate, k. Estimates of α and k are obtained for the fourteen different, clinically relevant, burn conditions. Overall, we find α=0.03±0.02mm2/s (to one significant figure), and is approximately independent of the burn duration, burn temperature, and skin thickness (H). This estimate implies that the time required for thermal energy to diffuse vertically down, through the skin of thicker (H=2.27mm) and thinner (H=1.40mm) skinned animals is approximately 170 and 70s, respectively. We find that k=0.002±0.002/s (to one significant figure). In summary, our results provide contemporary estimates for the thermal properties of in vivo porcine skin, which has broad application to heat transfer modelling investigations of thermal injury prevention and thermal therapy studies.

CUTANEOUS TOLERANCE OF SEA BUCKTHORN OIL EMULSION

Introduction: Sea buckthorn oil ( Hippophae rhamnoides L.) is medically used both externally and internally, but the external application is unsutable due to its liquid, lipophylic and highly colored nature. These difficulties could be overbriged by formulation of semisolid emulsion with sea buckthorn oil. Previous research on this formulation showed that it has higher wound healing potential then sea buckthorn oil, possesing enhanced structure of liquid crystals, stability and suitability for topical use. Aim: The aim of this investigation was to complete characterization of proposed emulsion, by testing its potential for causing skin irritation. Methods: The emulsion was prepared by standard emulsifying techniques using a combination of surfactants that form an enhanced structure of liquid crystals. Amount of 40% of sea buckthorn oil was incorporated. Since results of previous research on this formulation show wound healing effect on an animal model, we also tested skin tolerance of it on animal skin model. Evaluation was done by Draize test which measures level of skin changes of rabbit skin. Results: There was absence of edema or erythema type of irritation after 2h, 24h, 48h, 72h and 7 days of application of semisolid emuslion with sea buckthorn oil. Conclusion: This study confirmed that combination of the proposed ingredients is adequate and the concentrations of surfactants used to form stable emulsion were appropriate also for skin application. The proposed formulation of emulsion with sea buckthorn oil exerted percutaneous tolerance and could be safely applied on the skin. Key words: sea buckthorn oil, emulsion, topical use, irritation

Characterization of Transcutaneous Protein Delivery by a Hydrogel Patch in Animal, Human, and Tissue-Engineered Skin Models

The development of a simple, easy-to-use, and noninvasive vaccination system is in high demand. For transcutaneous immunization (TCI), we previously reported that a hydrogel patch was an effective TCI device that accelerates antigen penetration through the stratum corneum in mouse and rat models. The present study was performed to characterize the transcutaneous protein delivery induced by the hydrogel patch in mouse, guinea pig, LWD pig, human, or tissue-engineered skin models, and to assess the activity of proteins delivered into the skin. The hydrogel patch promoted protein penetration through the stratum corneum in all skin models, indicating that our original hydrogel patch might have practical application for use in humans. In addition, proteins delivered into the skin by the hydrogel patch retained their activity, suggesting that the hydrogel patch is applicable for the delivery of therapies for other diseases as well. On the basis of these results, translational research in human is now in progress.

Potential photocarcinogenic effects of nanoparticle sunscreens

Titanium dioxide and zinc oxide nanoparticles are being increasingly formulated in sunscreens. While the same compounds, in larger particle form, work by reflecting UV radiation, in nanoparticle form, they absorb UV radiation, resulting in photocatalysis, releasing reactive oxygen species. These reactive oxygen species are known to have the capability to alter DNA. Previous studies suggest that this photocatalytic process may not be significant, because the nanoparticles do not penetrate below the level of the stratum corneum. However, some recent studies suggest that nanoparticles may, under certain circumstances, breach that barrier. The majority of those studies have used animal skin models rather than human skin.

Microneedle Arrays Permit Enhanced Intradermal Delivery of a Preformed Photosensitizer

Silicon microneedle (MN) arrays were used to puncture excised murine and porcine skin in vitro and transdermal and intradermal delivery of meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP) investigated using topical application of a bioadhesive patch containing 19 mg TMP cm(-2). Animal studies, using nude mice, were then conducted to investigate the in vivo performance of the bioadhesive patch following MN puncture of skin. MN puncture significantly enhanced both intradermal and transdermal delivery of TMP in vitro, though the total amounts of drug delivered (25.22% into porcine skin and 0.07% across murine skin) were still quite small in each case. Notwithstanding this, in vivo experiments showed that MN puncture was capable of permitting a prolonged increase in TMP fluorescence at the site of application. Importantly, fluorescence was negligible at distant sites, meaning systemic delivery of the drug was not sufficient to induce TMP accumulation other than at the application site. In this study we have conclusively demonstrated proof of principle; MN puncture allows true intradermal delivery of a preformed photosensitizer in animal skin models in vitro and in vivo. Importantly, transdermal delivery was much reduced in each case. Increasing MN density would allow increased amounts of photosensitizer to be delivered. However, as MNs create aqueous pores in the stratum corneum, a preformed photosensitizer must possess at least some degree of water solubility in order to permit enhanced intradermal delivery in this way. We believe that use of MN array technology in this way has the potential to significantly improve topical photodynamic therapy of skin tumors.

The validation of in vitro methods for skin irritation

Several alternative in vitro methods for identifying skin irritants have been developed in the last 10 years, the most promising of which use either reconstituted human skin models or animal (e.g. pig or mouse) skin organ cultures. In 1998, the European Centre for the Validation of Alternative Methods (ECVAM) commissioned a pre-validation study of five methods. Two reconstituted human skin methods (Epiderm and EPISKIN) and one animal skin model (the mouse skin integrity function test, SIFT) performed well in Phases I and II of the study (intralaboratory reproducibility and protocol transfer) and proceeded to Phase III which assessed reproducibility and predictive ability in three independent laboratories using a set of 20 coded test chemicals (10 irritants and 10 non irritants). Intralaboratory reproducibility was again acceptable for all three methods but inter-laboratory reproducibility was acceptable only for EPISKIN. The predictive ability of all three methods was also inadequate. Following refinements to the protocols of Epiderm and EPISKIN and changes to the statistical analysis of SIFT, the predictive ability was improved. In 2003, ECVAM concluded that they could proceed to a full validation study. This will be conducted in two phases and is scheduled for completion early in 2005.

Penetration of radionuclides across the skin: Rat age dependent promethium permeation through skin in vitro

The composition and the permeation properties of the skin are dependent on age. In the animal models for permation studies, age affects the mechanical as well as the permeation properties significantly. The time dependence of permeation of 147Pm3+ from aqueous solution was established by the animal skin model and the age dependence of promethium permeation through the skin was examined. The aim was to find the optimum rat skin age model for radionuclide permeation studies and to assess the relative importance of the main permeation pathways: transepidermal and transfollicular permeation. The skin from 5-day-old rats (5DR) was found to represent the optimum animal model to study transepidermal permeation of ions. The skin from 9-day-old rats (9DR) was selected to study transfollicular permeation of ions. Comparison of the permeated amounts of promethium through the skin without hairs (3 DR to 6 DR) and with hairs (7DR to 12DR) showed that the additional permation mode via follicles significantly contributed to the permeation rate and extent.

Transfer of Radionuclides Across Skin Barriers of Animal Skin Models in Vitro

Radioactive skin contamination with radionuclides in ionic forms after the permeation across skin models was studied in vitro. Using animal skin models of 5-day-old rat and 9-day-old rat, either intact or stripped, it was found that besides the transepidermal also the transfolicular flux can be important. Stratum corneum was found to be the principal permeation barrier in all cases. The study of penetration — time profiles revealed that permeated amounts were proportional to time in the case of 137Cs+ and 60Co2+, but they showed a local maximum in the case of 147Pm3+.

Permeation of Cs+ ions across skin barriers

Permeation of metal Cs+ ions from water solutions across the intact, stripped and splitted skin of 5- or 9-day old rat was studiedin vitro using vertical diffusion cells. The results showed that the stratum corneum (horny layer) of this new animal skin model represents the principal pentration barrier. The relative importance of the percutaneous absorption of Cs+ ions through transepidermal and transfolicular routes has been assessed comparing the penetration across skin without hairs and skin just haired. The results have shown that the shunt diffusion along hair follicles can be of preponderant value.

Sympathetic modulation of sensory nerve activity with age: human and rodent skin models.

1. Sensory nerves serve an afferent role and mediate neurogenic components of inflammation and tissue repair via an axon reflex release of sensory peptides at sites of injury. Dysfunction of these nerves with age could contribute to delayed tissue healing. 2. Complementary animal and human skin models were used in the present studies to investigate changes in the modulation of sensory nerve function by sympathetic efferents during ageing. Laser Doppler flowmetry was used to monitor neurogenic skin vascular responses. 3. The animal model used skin of the hind footpad of anaesthetized rats combined with electrical stimulation of the sciatic nerve, while the human model comprised capsaicin electrophoresis to the volar surface of the forearm. Sympathetic modulation was effected by systemic phentolamine pretreatment in animals and local application in the human model. 4. The results obtained from the human model confirmed the reported decline in sensory nerve function and showed no change in sympathetic modulation with age. The results from the animal model confirm and expand results obtained from the human model. 5. The use of low (5 Hz) and high (15 Hz) frequency electrical stimulation (20 V, 2 ms for 1 min) revealed a preferential response of aged sensory nerves to low-frequency electrical stimulation parameters with differential sympathetic modulation that is dependent on the frequency of stimulation.

Evaluation of Transdermal Penetration Enhancers Using A Novel Skin Alternative

A novel alternative to animal skin models was developed in order to aid in the screening of transdermal penetration enhancers. The skin alternative consists of a dermal layer containing human fibroblasts dispersed in a collagen matrix and an epidermal layer of differentiated and stratified human keratinocytes. Skin alternatives were placed in modified Franz diffusion cells (receptor volume 12mL, donor area 3.14cm2, n=5) and enhancer solution (0.4M in propylene glycol (PG)) was applied. Following 1h of pretreatment, 10μLof saturated hydrocortisone (He) solution in PG was applied, and the cells were occluded with Parafilm. Samples were removed from the receptor compartment over 24h, replaced with fresh receptor solution, and analyzed for steroid content using HPLC. Skin HC content was also determined. Receptor concentration at 24h (Q24) for full-thickness skin alternative (control) was 28.6±13.7 μΜ and permeability (P) was 8.3 x 10−4±5.5×10−4 cm h−1. Azone (1) produced a Q24 of 105.0±36.1 μΜ and a P of 11.3×10−4±1.8×10−4 cm h−1, while the novel penetration enhancer 1-dodecyl-2-pyrrolidinone (2) produced a Q24 of 164.8±61.2 μΜ and a P of 33.3×10−4±6.6×10−4 cm h−1. Compound 3 produced the highest values for all permeability parameters tested with a Pof 48.0±36.8cmh−1 and a Q24 of 186.1±45.1 μΜ. When compared to the control, compound 1 gave an enhancement ratio (ER) of 3.7 for Q24 and 1.4 for P. Compound 2 gives an ER 5.8 for Q24 and 4.0 for P. These enhancement ratios are similar to those found using HC and human skin.

Effect of novel penetration enhancers on the transdermal delivery of hydrocortisone: an in vitro species comparison

Six novel compounds were examined for enhancer activity using occluded hairless mouse skin (HM), hairless rat skin (HR), human cadaver skin (HC) in vitro with hydrocortisone as the model drug. The compounds investigated included: N-dodecyl-2-pyrrolidinone (DPY), N-dodecyl-2-piperidinone (DPI), N-dodecyl- N-(2-methoxyethyl)acetamide (DMEA), N-dodecyl- N-(2-methoxyethyl)isobutyramide (DMEI), N-dodecyldiethanolamine (DDE), 2-(1-nonyl)-1,3-dioxolane (ND), and Azone®. Controls consisted of no enhancer or vehicle treatment. All enhancers were applied at 0.4 M in propylene glycol 1 h prior to skin application of a saturated suspension of hydrocortisone in the same vehicle. Enhancement ratios (ER) were determined for 24 h diffusion cell receptor concentrations (Q 24), permeability coefficients (P), and 24 h full-thickness skin steroid contents. ER for controls was 1.0. N-dodecyl-2-pyrrolidinone (DPY), an Azone® analog, showed the greatest ER values for permeability coefficient (HM: 21.3, HR: 20.7, HC: 8.0) compared to control (ER: 1.0) and Azone® (HM: 18.0, HR: 13.1, HC: 5.5) in all three animal skin models. All six enhancers exhibited poor skin steroid retention (compared to Azone®) in all three skin models.