UNIVERSITÀ DEGLI STUDI DI NAPOLI FEDERICO II DEPARTMENT OF CHEMICAL, MATERIALS AND PRODUCTION ENGINEERING (DICMAPI) PHD IN INDUSTRIAL PRODUCTS AND PROCESSES ENGINEERING XXIX CYCLE “DEVELOPMENT AND SCALE-UP OF 3D ENDOGENOUS HUMAN SKIN EQUIVALENT MODELS AS PLATFORM FOR COSMETICS TESTING” Supervisor Prof. Paolo Netti Advisors Giorgia Imparato, PhD Francesco Urciuolo, PhD Costantino Casale, PhD Coordinator PhD Student Prof. Giuseppe Mensitieri Francesca Rescigno 2014/2017 Table of Contents Chapter 1. Skin models for cosmetics testing: an overview 1.1INTRODUCTION 3 1.2 STRUCTURE AND FUNCTIONS OF HUMAN SKIN 4 1.2.1 Epidermis 6 1.2.2 Dermis 9 1.2.3 Dermal-epidermal junction 13 1.3 TISSUE ENGINEERING OF THE SKIN 14 1.3.1. Alternative non-animal testing and european regulations 16 1.3.2. Insights from in vitro skin models for clinical applications 22 1.3.3. Commercially available human skin equivalents for in vitro applications 29 1.4 REFERENCES 39 Chapter 2. A novel approach for skin tissue engineering: endogenous human skin equivalent models 2.1 INTRODUCTION 52 2.2 MATERIALS AND METHODS 56 2.2.1 Porous scaffold preparation 56 2.2.2 Crosslinking of GPMs 56 2.2.3 Cell source 57 2.2.4 Fibroblasts and keratinocytes isolation and culture 57 2.2.5 HD-μTP precursors fabrication 59 2.2.6 3D Human dermis equivalent model: HD-μTP molding 60 2.2.7 3D Human skin equivalent model fabrication 61 2.2.8 Characterization of HD-µTP 63 2.2.9 Characterization of 3D human dermis equivalent models 64 2.2.10 Characterization of 3D human skin equivalent models 65 2.3 RESULTS 66 2.3.1 HD-µTP assembly assessment 66 2.3.2 3D human dermis equivalent characterization 67 2.3.3 3D human skin equivalent characterization 69 2.4 DISCUSSIONS 73 2.5 REFERENCES 75 Chapter 3. Endogenous 3D human skin equivalent as a novel testing platform 3.1 INTRODUCTION 79 3.2 MATERIALS AND METHODS 83 3.2.1 3D Human skin equivalent model realization 83 3.2.2 Quantitative biochemical assay of ECM components in 3D HDE and HSE during the culture time 84 3.2.3 Histological evaluation of ECM components in 3D-HDE and HSE during the culture time 88 3.2.4 Mechanical analysis of 3D HDE and HSE models by means of non- destructive nanoindentations methods 91 3.2.5 Induction of damage on 3D-HDE and HSE samples 93 3.3 RESULTS AND DISCUSSION 98 3.3.1 Biochemical, mechanical and histological analysis of 3D-HDE and HSE during the culture time 98 3.3.2 Reactive Oxygen Species (ROS) production in damaged and protected 3D human dermis and skin platforms 104 3.3.3 Effect of UVA photo-degradation on cellular and tissue senescence in our 3D human skin equivalent models 108 3.4 CONCLUSIONS 121 3.5 REFERENCES 123 Chapter 4. Scale-up strategy for skin models production 4.1 INTRODUCTION 132 4.2 MATERIALS AND METHODS 136 4.2.1 Characterization of commercially available gold standards models and comparison with our endogenous 3D human skin platform 136 4.2.2 Scale-up of 3D human skin equivalent multistep tissue process 140 4.3 RESULTS 148 4.3.1 Characterization of Phenion Full-Thickness skin model by Henkel by SHG imaging 148 4.3.2 EpiDerm 312X FT skin model by MatTek Corporation and comparison with our endogenous 3D human skin platform 150 4.3.3 Industrialization of 3D human skin equivalent models production 157 4.3.4 Organization planning to improve human skin models production 159 4.3.5 Quality control of HD-µTP 162 4.3.6 Quality control of endogenous 3D human dermis equivalent models 165 4.3.7 Quality control of endogenous 3D human skin equivalent models 167 4.3.8 Scale-up of 3D human skin equivalents models 169 4.4 DISCUSSIONS AND FUTURE PROSPECTIVE 172 4.5 CONCLUSIONS 177 4.6 REFERENCES 179 Abstract The current necessity of development new biological in vitro models that mimic the characteristics and the complexity of human tissue arises from the need to find a valid alternative to animal models to test and validate new products, and to screen substances and procedures for tissue repair and regeneration. In particular, in the cosmetics field, big multinational companies have developed and devised methods for the realization of testing platforms in large scale, since the European Regulations (Decree 76/768/EEC and the EU Cosmetic Regulation 1223/2009) ban the putting on the market cosmetic products whose ingredients, or parts thereof, have been tested on animal models. in this scenario, researchers have been spent many efforts to develop innovative tissue engineering strategies to create 3D skin equivalent models that faithfully recapitulate the characteristics of human skin in terms of organization, complexity, architecture and responsivity to specific exogenous stimuli. The optimization of the process to produce these skin equivalent represents a crucial step to obtain i) tissue in large scale in order to allow the screening of a large number of molecules/exogenous factor ii) a high-fidelity replica of the native counterpart in order to evaluate the effect of molecules/exogenous factor on the mechanical properties and ECM composition organization and hydration. For this purpose, in my PhD work, after a deep study of the literature, it was developed a method of production of 3D skin equivalent models in large scale with great reproducibility. In the first part of the thesis, there is a description of the principal systems that composed the human skin and a summary about the main arguments of European Regulation related to cosmetics testing, the development of alternatives animal 1 tests and its principal applications. In the second chapter, we exploited a bottom- up tissue engineering approach to build up the skin tissues. Such approach allowed to obtain skin tissues composed of endogenous extracellular matrix (ECM), produced by human dermal fibroblasts and by stratified epithelial cells that constitute a fully differentiated epithelium resembling the human epidermis. In the third chapter, we performed a morphological characterization of our 3D skin tissue by histological, biochemical and mechanical analysis in order to better describe the main features of this human skin equivalent models. Furthermore, to validate the skin produced as testing platform, we induced different kinds of damages (UVA, H O ) and after evaluating the effect on the tissue, with the aim to 2 2 study the effectiveness of molecules having antioxidant and photo-protective action. Finally, in the last part of thesis, we described firstly, a comparison between 3D skin models designed by us with the commercially available gold standard models produced by best international companies, and then a scale-up strategy in order to improve the production process of 3D-skin equivalent models with the prospective of realization of a start-up. In this last part, we described phases and all critical steps of human skin equivalent production passing from the realization of skin tissue in small scale to the large scale and the development of a working plan of all activities to better control step by step the quality and the effectiveness of final product. All results reported in my thesis strongly suggested a possible use of the developed skin tissues as a valid alternative to the use of animal models for the testing of new cosmetic compounds. 2 Chapter 1. Skin models for cosmetics testing: an overview 1.1 INTRODUCTION Over the last years, Tissue Engineering (TE) has become established in biomedical and scientific scenario as a new emerging field consisting of several interdisciplinary applications that combine the principles and methods of life science with those of engineering, aiming at repairing or regenerating portions or of whole biological tissue and organs (bone, cartilage, bladder, skin, cornea, blood cells, muscle, liver, pancreas, intestine, etc.) [1]–[4]. The purpose of TE research is very clear: establishing a new clinical technology that makes possible medical applications for diseases that have been too difficult to be cured by existing methods. Initially, it was thought that the principles of TE could be applied only virtually, but today it is known that many applications are widely used not only in tissue regeneration, but also in the industrial field [5], [6]. The classical approach of TE is based on the paradigm according to which, by three elements, which cells, scaffold and soluble factors, it is possible to induce the regeneration of damaged tissue, in attempting to replace traditional medical treatments that in some cases It cannot be applied [4]. The biological materials are made up of cells, cellular signaling and extracellular matrix (ECM). The cells are the core of the tissue that need an adequate systems of signal transduction and/or ECM to exert its functions. The ECM is a of collagen and elastin fiber network synthetized by the cells themselves and secreted in the extracellular space. This complex structure supports biological processes and cell proliferation as well as guarantees resistance and support to the tissue. Single 3 cells, without a three-dimensional guide, are indeed not able to organize a 3D tissue with its complex architecture, since the only cell proliferation is not sufficient. Single cells need a 3D support, defined “scaffold” whose main function is to support and control cellular processes, as well as the proliferation, differentiation and the synthesis of organic molecules and mediators, known as growth factors, which act in an autocrine and paracrine pathways on cellular processes [7]. As it is known from the literature, the first 3D tissue models made through the application of TE principles were skin equivalent, bone, cornea and cartilage, all tissues more frequently damaged [8]. In parallel with the new methods of regenerative medicine, several companies and industries, from the ‘90s, started off methods suitable for the implementation of tissue equivalent models as in vitro test systems in order to verify the safety of substances, active ingredients and cosmetic products [9]. The in vitro models became important tools in the pharmaceutical and cosmetics industries for research and development. The best model useful for these studies is the human skin, but the difficulties linked to samples availability, legal and bioethical issues related to the use of cadaver skin, biopsy material or cosmetic surgery, they have increased the need to develop innovative methods aimed to the realization of in vitro tissue equivalent models. Furthermore, the availability of tissues is limited not only by the sources, but also by the current regulations. Until the early 2000s in fact, all the tests performed by cosmetic companies before putting a finished product on the market, were carried out on animal models [10]. 1.2 STRUCTURE AND FUNCTIONS OF HUMAN SKIN The skin is named also integumentary system and it is the continuous largest organ of the body and it performs many vital functions, including protection 4 against external physical, chemical, and biologic assailants, as well as prevention of excess water loss from the body and a role in thermoregulation [11]. The integumentary system is formed by the skin and its derivative structures (Figure 1). The skin is composed of three layers: the epidermis, on the upper part, the dermis, and subcutaneous tissue [11]. The outermost level, the epidermis, consists of a specific population of cells known as keratinocytes, which synthesize keratin, a long, threadlike protein with a protective role. The middle layer, the dermis, is fundamentally made up of collagen, while the subcutaneous tissue contains small lobes of fat cells known as lipocytes. The thickness of these layers varies considerably, depending on the geographic location on the anatomy of the body. The palms and soles of the feet for example, have the thickest epidermal layer, measuring approximately 1.5 mm. The dermis is thickest on the back, where it is 30–40 times as thick as the overlying epidermis (Figure 1) [12]. Figure 1. The structure of integumentary apparatus (Mayo’s Foundation). 5 1.2.1 Epidermis The epidermis is a stratified, squamous epithelium layer that is composed primarily of two types of cells: keratinocytes and dendritic cells. [13]. The epidermis includes also a number of other cell populations, such as melanocytes, Langerhans cells, and Merkel cells, but the keratinocyte cell type comprises the majority of the cells. The epidermis commonly is divided into four layers according to keratinocyte morphology and position as they differentiate into horny cells, including the basal cell layer (stratum germinativum), the squamous cell layer (stratum spinosum), the granular cell layer (stratum granulosum), and the cornified or horny cell layer (stratum corneum) [12], [13]. The lower three layers that constitute the living, nucleated cells of the epidermis (Figure 2) [13]. Figure 2. Epidermal layers of human skin (McGrow and Hill Companies). 6