Skin Tissue Models

Skin Tissue Models provides a translational link for biomedical researchers on the interdisciplinary approaches to skin regeneration. As the skin is the largest organ in the body, engineered substitutes have critical medical application to patients with disease and injury - from burn wounds and surgical scars, to vitiligo, psoriasis and even plastic surgery. This volume offers readers preliminary description of the normal structure and function of mammalian skin, exposure to clinical problems and disease, coverage of potential therapeutic molecules and testing, skin substitutes, models as study platforms of skin biology and emerging technologies. The editors have created a table of contents which frames the relevance of skin tissue models for researchers as platforms to study skin biology and therapeutic approaches for different skin diseases, for clinicians as tissue substitutes, and for cosmetic and pharmaceutical industries as alternative test substrates that can replace animal models. Offers descriptions of the normal structure/function of mammalian skin, exposure to clinical problems, and more Presents coverage of skin diseases (cancer, genodermatoses, vitiligo and psoriasis) that extends to clinical requirements and skin diseases in vitro models Addresses legal requirements and ethical concerns in drugs and cosmetics in vitro testing Edited and authored by internationally renowned group of researchers, presenting the broadest coverage possible. © 2018 Elsevier Inc. All rights reserved.(undefined)info:eu-repo/semantics/publishedVersio

Hypergranulatory tissue in breast implant surgery and skin scar formation

In this thesis hypergranulation tissue in both the breast after breast implant surgery, as well as in the skin (scar formation) is explored. The histological composition of capsular contracture after breast implant surgery and the role of silicone in this tissue is investigated in multiple papers. Furthermore, the classification used to grade capsular contracture complaints is evaluated. In the second part, the role the systemic immune system has in hypertrophic scarring is examined, and how to use these differences to enable prognostic tools

Three dimensional oral mucosa models: development and applications

Animal experimentation has been extensively and for a long time applied in several research fields, but since 2011 it has been substantially limited by the Commission of the European Parliament to ensure people/animals safety and reduce research costs. To respond to these directives, many attempts have been focused on the development and validation of new in vitro 3D systems, bypassing the traditional 2D cell cultures. In this regard, diverse approaches to tissue-engineered bone and oral mucosa have been developed. Despite the promising premises and the cutting-edge results, the used 3D in vitro bone-oral mucosal models still lack interaction between the mucosal and the bone components. Therefore, this project aimed to create 3D models, entirely made with primary human cells (keratinocytes, fibroblasts, and osteoblasts), able to mimic the natural structure and interaction of bone and oral mucosa. In the present work, the regulatory role of the mesenchymal tissue onto epithelia was evaluated. The main results showed that that during the differentiation hMSC produce and secrete factors that induce the keratinization and the expression of the marker of differentiation CK10; in particular in the middle stage of differentiation (OB14). The proteomic analysis revealed that this effect can be ascribable to KGF secretion. This finding may impact the design of new implantable devices able to induce, alone, the epithelial growth and keratinization to improve implant graft avoiding epithelial graft linked to the morbidity of another zone. Moreover, we also showed that OM might have a pro-innervation effect, at least during the last stages of keratinocytes stratification. Finally, we obtained and characterized an innervated mucoperiosteal model that could open new in vitro frontiers for oral biomaterials validation as well as improve knowledge regarding the mesenchymal stem cells roles onto oral mucosa development

Evaluation of the efficacy of bleomycin encapsulated within liposomes as novel, topical treatment for skin cancer in veterinary species and its potential for human medicine: an in vitro, ex vivo and in vivo study

Bleomycin is a potent anticancer agent that is able to induce single and double stranded breaks in DNA. It is effective against several types of cancer, including cutaneous skin tumours, in both human and veterinary medicine. However, due to its molecular characteristics, including polar charge and high molecular weight, it is unable to effectively penetrate the skin barrier and freely cross the plasma membrane. Bleomycin can be administered systemically but is known to carry severe side effects such as fatal pulmonary fibrosis, which is the most concerning one and represents the main limiting factor to its potential use in clinical applications. Furthermore, several studies claim that bleomycin causes cell death in a cell-type dependent manner, although to date, the actual mechanism of action is not fully elucidated. Advances in cancer therapy aim to minimise the side effects of treatments and improve the quality of life of patients. Within the context of developing minimally invasive treatments, liposomes have become highly valued for their ability to deliver payload drugs to the target tumour tissues and to be applied topically therefore decreasing systemic toxicity. Bleosome is a novel formulation of bleomycin where the cytotoxic agent bleomycin has been encapsulated in ultra-deformable (UD) liposomes. In this study, we evaluated Bleosome as a topical, non-invasive treatment for non-melanoma skin cancer (NMSC). This PhD project also aimed to evaluate the mechanism of action and efficacy of Bleosome ex vivo, in vitro and in vivo. We hypothesised that encapsulation of bleomycin within nanoparticles, namely UD liposomes, enhances the penetration of the drug through the skin, and we tested this in three model systems: canine, equine, and human skin explants. Firstly, we optimised an imaging technique that allowed us to directly visualise the liposomes using the transmission electron microscope, and effectively fluorescently labelled bleomycin, prior to encapsulation within liposomes, to visualise penetration through skin and equine patient-derived sarcoids, using multiphoton microscopy. We concluded that lipid nanoparticles could act as penetration enhancers, carrying the entrapped bleomycin through the channels of the outermost stratum corneum and subsequently releasing the drug and allowing it to penetrate deeper in the skin. At the cellular level, we determined the effect of Bleosome on cell viability on a panel of canine, feline and human cancer cell lines in vitro. Using live cell imaging, we showed that Bleosome is taken up by cancer cells more efficiently than free bleomycin. We also compared the variation in protein and gene expression in two canine cancer cell lines after 8 and 24 hours post-Bleosome treatment. Preliminary data showed that the response to Bleosome is cell type dependent, and the transcriptomic profiling of a canine cancer cell line treated with a time course of Bleosome revealed that these specific cells are unable to repair the DNA lesions, produced by Bleosome treatment. Lastly, we administered Bleosome to equine patients bearing cutaneous sarcoids, in an adjuvant setting, following laser excision. After surgical excision of the sarcoids, Bleosome was applied topically on the site of the scar in 12 horses, twice a day, for a mean length of 4 weeks. To date, after an overall follow-up of 12 months, 5 patients were in complete remission (41.6%), 4 experienced recurrences (33.3%), and 3 failed to follow up (25%). The treatment was overall very well tolerated and the study was still on going at the time of writing. Overall, the findings of my PhD provide compelling evidence of the mechanism of action, efficacy and clinical benefit of Bleosome as topical, non-invasive treatment for NMSC

Phototoxic interactions of tattoo pigments with laser and natural light in vitro

Tattooing, a process in which colorants are permanently embedded in human skin to generate long-lasting images, enjoys a high popularity nowadays. Today, every fifth to fourth person is tattooed. This popularity seems not to be affected by the high number of tattoo related side effects, which especially occur when tattooed skin encounters light. Light induced side effects make up to about 60 % of tattoo associated side effects. Consequently, the interactions of ultraviolet light and laser light on tattoo pigments and the resulting effects on human skin cells were investigated in this thesis to increase the understanding of photo-induced side effects found in tattoos. Firstly, we treated postmortem tattooed pig skin with laser used in tattoo removal. We consequently analyzed cleavage products using gas chromatographic separation coupled to mass spectrometric detection. 3,3’-dichlorobenzidine, a carcinogen produced through cleavage of Pigment Orange (P.O.)13, was found to be cytotoxic and genotoxic in terms of DNA double strand breaks in human fibroblast and keratinocyte cell lines even beneath identified concentrations. While photo-induced cleavage is a key event that leads to tattoo clearing during laser removal, it can also occur on a smaller scale when tattooed skin is exposed to natural light, particularly in the ultraviolet (UV) range. Since light-induced effects are the most common side effects associated with tattoos, our goal was to investigate the underlying pathomechanisms accordingly. However, no in vitro model that accurately resembles the architecture of tattooed human skin has been described in the literature so far. Therefore, we established a 3D in vitro ‘tattooed’ full thickness skin model (TatSFT) as an animal replacement model for tattoo research. The uptake of the tattoo pigments used in this study, titanium dioxide (TiO2) anatase, TiO2 rutile, Pigment Orange (P.O.)13 and carbon black by dermal fibroblasts was proven by electron microscopy in TatSFT. Despite this uptake, the pigments showed no effect on the viability of TatSFT nor its dermal compartment (TatSDE). Concordantly, cytokine secretion, general histology, and the expression of important skin homeostasis markers was unaffected by tattoo pigments in TatSFT. Contrary to the absence of toxicity in 3D, TiO2 anatase significantly decreased cell viability and increased interleukin-8 release in 2D monolayer cultured fibroblasts. Due to the inherent differences in toxicity sensitivity of 2D monolayered cultured fibroblasts and 3D cultured models, we investigated phototoxicity of tattoo pigments in both, 2D and 3D. Concordantly with particle toxicity, phototoxic effects were bigger in monolayer cultured fibroblasts than in 3D. While TiO2 showed strong phototoxic effects in 2D, these effects were absent in TatSFT. However, UVB induced DNA damage marker levels in the dermis of TatSFT were reduced by pigments. Combined with photoprotective effects found in TatSDE concerning viability, these data suggest photoprotective properties of tattoo pigments for the tattooed dermis and its underlying tissue. Contrary to these results, we found P.O.13 to alter cytokine secretion upon UV irradiation in both, 2D and TatSDE. While minor amounts of cleavage products of P.O.13 were identified after UV irradiation, we were unable to proof that these cleavage products might have resulted in adverse effects. The data in this thesis not only highlight the need for 3D test systems for tattoo phototoxicity research, but also present a highly modifiable 3D in vitro test system for this purpose. This work also strengthens concerns regarding TiO2 anatase and azo pigments like P.O.13 and their use in tattoo inks.Mit dem Tätowieren erfreut sich heutzutage ein Verfahren größter Beliebtheit, bei dem Farbstoffe dauerhaft in die menschliche Haut eingebettet werden, um permanente Bilder in dieser zu erzeugen. Momentan ist jeder fünfte bis vierte Mensch tätowiert. Dieser Beliebtheit scheint die hohe Zahl an tätowierungsbedingten Nebenwirkungen keinen Abbruch zu tun. Viele Nebenwirkungen treten insbesondere dann auf, wenn die tätowierte Haut mit Licht in Berührung kommt. Insgesamt machen lichtinduzierte Nebenwirkungen etwa 60 % der tätowierungsassoziierten Nebenwirkungen aus. In dieser Arbeit wurden daher die Wechselwirkungen von ultraviolettem Licht und Laserlicht auf Tätowierpigmente und die daraus resultierenden Effekte auf menschliche Hautzellen untersucht, um das Verständnis für lichtinduzierte Nebenwirkungen in tätowierter Haut zu erhöhen. Zunächst behandelten wir postmortem tätowierte Schweinehaut mit Lasern, die zur Tattooentfernung verwendet werden. Anschließend analysierten wir die Spaltprodukte mittels gaschromatographischer Trennung gekoppelt mit massenspektrometrischer Detektion. 3,3'-Dichlorbenzidin, ein Karzinogen, welches durch die Spaltung von Pigment Orange (P.O.)13 entsteht, erwies sich in menschlichen Fibroblasten- und Keratinozyten-Zelllinien bereits unterhalb identifizierter Konzentrationen als zytotoxisch und genotoxisch in Form von DNA-Doppelstrangbrüchen. Während die photoinduzierte Spaltung ein Schlüsselereignis ist, das bei der Laserentfernung zum Verblassen der Tätowierungen führt, kann sie auch in kleinerem Umfang auftreten, wenn tätowierte Haut natürlichem Licht, insbesondere im ultravioletten (UV) Bereich, ausgesetzt wird. Da lichtinduzierte Effekte die häufigsten Nebenwirkungen im Zusammenhang mit Tätowierungen sind, war es unser Ziel, die zugrunde liegenden Pathomechanismen entsprechend zu untersuchen. Allerdings wurde in der Literatur bisher kein in vitro Modell beschrieben, das die Architektur tätowierter menschlicher Haut genau nachbildet. Daher etablierten wir ein 3D in vitro "tätowiertes" Vollhautmodell (TatSFT) als Tierersatzmodell für die Tätowiermittelforschung. Die Aufnahme der in dieser Studie verwendeten Tattoo-Pigmente Titandioxid (TiO2) Anatas, TiO2 Rutil, Pigment Orange (P.O.)13 und Carbon Black durch dermale Fibroblasten wurde elektronenmikroskopisch in TatSFT nachgewiesen. Trotz dieser Aufnahme zeigten die Pigmente keinen Einfluss auf die Lebensfähigkeit von TatSFT oder dessen dermales Kompartiment (TatSDE). Auch die Zytokinsekretion, die allgemeine Histologie und die Expression wichtiger Marker der Hauthomöostase wurden durch die Tattoo-Pigmente in TatSFT nicht beeinflusst. Im Gegensatz zur fehlenden Toxizität in 3D verringerte TiO2-Anatas signifikant die Zellviabilität und erhöhte die Interleukin-8-Ausschüttung in 2D-monolayer kultivierten Fibroblasten. Aufgrund der inhärenten Unterschiede in der Toxizitätsempfindlichkeit von 2D-monolayer kultivierten Fibroblasten und 3D kultivierten Modellen, untersuchten wir die Phototoxizität von Tattoo-Pigmenten sowohl in 2D als auch in 3D. Übereinstimmend mit der Partikeltoxizität waren die phototoxischen Effekte in Einzelschicht-kultivierten Fibroblasten größer als in 3D. Während TiO2 starke phototoxische Effekte in 2D zeigte, waren diese Effekte in TatSFT nicht vorhanden. Allerdings wurden die UVB-induzierten DNA-Schadensmarker in der Dermis von TatSFT durch die Pigmente reduziert. In Kombination mit den photoprotektiven Effekten, die in TatSFT hinsichtlich der Lebensfähigkeit gefunden wurden, deuten diese Daten auf photoprotektive Eigenschaften von Tätowierpigmenten für die tätowierte Dermis und das darunter liegende Gewebe hin. Im Gegensatz zu diesen Ergebnissen fanden wir, dass P.O.13 die Zytokinsekretion bei UV-Bestrahlung sowohl in 2D als auch in TatSDE verändert. Obwohl geringe Mengen an Spaltprodukten von P.O.13 nach UV-Bestrahlung identifiziert wurden, konnten wir nicht nachweisen, dass diese Spaltprodukte nachteilige Effekte hervorgerufen haben könnten. Die Daten in dieser Arbeit unterstreichen nicht nur die Notwendigkeit von 3D-Testsystemen für die Forschung zur Phototoxizität von Tätowierungen, sondern stellen auch ein hochgradig modifizierbares 3D in vitro Testsystem für diesen Zweck dar. Zusätzlich stärkt diese Arbeit auch die Bedenken bezüglich der Verwendung von TiO2 Anatas und Azopigmenten, wie P.O.13, in Tätowiertinten

Pharmaceutical Particulates and Membranes for Delivery of Drugs and Bioactive Molecules

This book is a collection of papers published in the Special Issue of Pharmaceutics, entitled "Pharmaceutical Particulates and Membranes for Delivery of Drugs and Bioactive Molecules". A drug release profile is a consequential factor for nanoparticle application, directly related to drug stability and therapeutic results, as well as formulation development. Pharmaceutical particulates of different sizes and shapes (e.g., liposomes, oil-in-water emulsions, polymeric nano- and microspheres, metallic nanoparticles (NPs) such as gold, silver and iron oxide crystals, and core-shell hybrid NPs) offer many diagnostic and therapeutic applications. Membranes are also extensively utilized in many applications. They are especially beneficial to the distribution of macromolecular drugs and biopharmaceutical drugs (peptides, proteins, antibodies, oligonucleotides, plasmids, and viruses) with physicochemical and pharmacokinetic vulnerability. The delivery of drugs and bioactive molecules using particulates and membranes has gained a great deal of attention for various applications, such as the treatment of secondary infections, cancer treatment, skin regeneration, orthopaedic applications, and antimicrobial drug delivery. In addition, several production techniques have been utilized for the fabrication of particulates and membranes in the last decade, which include lyophilisation, micro-emulsion, nano-spray dryer, nano-electrospinning, slip casting and 3D printers. Therefore, pharmaceutical particulates and membranes possess excellent prospects to deliver drugs and bioactive molecules with the potential to improve new delivery strategies like sustained and controlled release

Approaching Inner Ear Hair Cell Regeneration Through Non-Viral Gene Delivery

Tissue engineering traditionally has taken an "outside-in" approach to address deformities, injuries, and wear-and-tear of tissues. The current thesis examines an opposite approach through an "inside-out" strategy using non-viral gene delivery with mesenchymal stem cells to regenerate mechanosensory hair cells and supporting cells of the inner ear responsible for hearing and balance. Primary cells, stem cells, and progenitor cells are often difficult to transfect unless using a viral vector, which may have systemic safety concerns. However, non-viral vectors circumvent the safety issues associated with viral vectors, but commonly exhibit low transfection efficiencies. The work of the current thesis identified and enhanced an effective non-viral gene delivery approach that reprogramed human mesenchymal stromal cells, isolated from Wharton's jelly of human umbilical cords to produce characteristics similar to the hair cell and supporting cell phenotype found in the cochlea and vestibular organs of the inner ear. Studies from the literature highlighted electroporative methods as effective non-viral strategies for difficult-to-transfect cells. In vitro studies demonstrated that human Wharton's jelly cells (hWJCs) that underwent electroporation and were treated with Y-27632 ROCK Inhibitor outperformed untreated cells in transfection efficiency and cell viability by factors of four and three, respectively. The identification and tracking of positively transfected cells was tremendously improved by use of a photo-converting reporter, which greatly increased signal to noise ratios. The up-regulation of atoh1, and down-regulation of hes1 and hes5, in hWJCs produced a complex phenotype that exhibited over an 11-fold increase in gene expression of the critical hair cell marker, myosin VIIa, with visual morphological changes compared to untreated cells. The current thesis has demonstrated that hWJCs are susceptible to non-viral gene delivery methods, and for the first time non-viral genetic reprogramming of hWJCs induced phenotypic changes characteristic of hair cells and neural epithelium. The current thesis has bridged the gap between non-viral gene delivery, stem cell therapy, and tissue engineering, which now presents new opportunities for further investigation utilizing non-viral gene delivery in concert with stem cell therapies for regenerative medicine applications

Minimally invasive clinical monitoring and data transference in cardiac patients

'Wet' electrodes used in electrocardiography (ECG), are applied to the surface of the skin to record cardiac activity. Over time, water-based electrolytic gels between the electrodes and skin dehydrate, reducing signal quality. Microneedle-electrodes negate the need for conductive gels and potentially improve signal fidelity by circumventing the stratum corneum and contacting the underlying conductive epidermal layers. This thesis aimed to assess the wearability and functionality of microneedle-electrodes in cardiac signal acquisition. Epoxy, 500μm-length microneedles were applied to excised skin models to assess insertion performance. Increasing downward application force increased microneedle penetration efficiency from 79%±8.20 (5N) to 87%±13.32 (15N). The microneedle application technique also had an impact on penetration efficiency, with impact insertion (93%±5.75) proving more effective than manual downward force (71%±22.01). Metallised versions of the epoxy microneedles were integrated into a commercial electrode and compared to conventional wet electrodes in human volunteers. Wet electrodes recorded higher quality signals than microneedle-electrodes in healthy human participants (1.6dB difference between the electrode types). This clinical data informed the development of an in vitro laboratory skin model to assess the influence of microneedle-electrode parameters on a simulated ECG signal. Increasing microneedle length from 500μm (25.2dB±3.25) to 600μm (24.3dB±2.31) did not result in a sustained improvement in signal quality (p>0.05). Bespoke second-generation microneedle-electrodes were manufactured allowing an improved signal quality to be maintained over the recording period (17.3dB±2.11 compared to 15.0dB±1.97 for wet electrodes; p>0.05) in the laboratory model. Human participant studies assessed their wearability and functionality. At rest, the metallised epoxy (23.2dB±5.79) and bespoke (22.5dB±7.57) microneedle-electrode performance was comparable to wet electrodes (24.9dB±6.44) (p>0.05). Under active conditions, the signal-to-noise ratio declined for all electrodes and ECG traces highlighted increased motion artifacts. Participants preferred wet electrodes and highlighted seven key wearability themes. Further optimisation of microneedle-electrodes for ECG monitoring is therefore, warranted