Using elastin-like recombinamers as scaffolds for tissue engineering and regenerative medicine in the context of skin regeneration

Salomé Carneiro Neto de Nóbrega Luís Using elastin-like recombinamers as scaffolds for tissue engineering and regenerative medicine in the context of skin regeneration Using elastin-like recombinamers as scaffolds for tissue engineering and regenerative medicine in the context of skin regeneration Salomé Carneiro Neto de Nóbrega Luís UMINHO I 2022 outubro 2022 +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ Salomé Carneiro Neto de Nóbrega Luís Using elastin-like recombinamers as scaffolds for tissue engineering and regenerative medicine in the context of skin regeneration Dissertação de Mestrado Mestrado Integrado em Engenharia Biomédica Ramo de Biomateriais, Reabilitação e Biomecânica Trabalho realizado sob a orientação do Doutor Artur Jorge Araújo Magalhães Ribeiro outubro 2022 i COPYRIGHTS AND TERMS OF USE OF WORK BY THIRD PARTIES This is an academic work that can be used by third parties as long as the rules and regulations internationally accepted good practices with regard to copyright and related rights, are respected. Thus, the present work can be used under the terms provided for in the license indicated below. If the user needs permission to be able to use the work under unforeseen conditions in the indicated licensing, you should contact the author, through the RepositóriUM of the University of Minho. License granted to users of this work ii ACKNOWLEDGEMENTS This dissertation could not have been completed without the cooperation and assistance of several entities, which I will highlight below. First and foremost, I would like to thank my supervisor, Doctor Artur Ribeiro for agreeing to join me on this adventure, for aligning my professional goals with an ambitious project, and for motivating me to be a professional of excellence. I would also like to express my deepest gratitude to Professor José Carlos Rodríguez Cabello for allowing me to develop part of this project at Technical Proteins NanoBiotechnology, S.L. during my ERASMUS traineeship (Financial Agreement 2020-1-PT01-KA103-077707). This adventure would not have been possible without the support of the Bioforge team (Group of Advanced Materials and Nanobiotechnology), particularly of PhD candidate Diana Juanes, who always clarified my doubts and piqued my curiosity, and research assistant Rocío Lera, who not only assisted me but also served as a professional role model for me to emulate. Furthermore, I acknowledge the Technical Proteins NanoBiotechnology, S.L. team's guidance, particularly PhD candidate Federica Sallustio's teachings and advice, and thank Daniela Guerra, Desiré Bustos and Sara Anzola for all of the laughs and adventures we shared over lunch. I would also like to express my gratitude to the Bioprocess and Bionanotechnology Research Group team for their sound advice and willingness to teach whenever I needed it. I would especially like to thank Doctor André da Costa for his support, for sharing his knowledge and encouraging me to be more critical. Furthermore, I want to recognize and thank my parents for their important role in my life's success, not only as a safe haven, but also for constantly challenging me to grow. I thank my brother for his faith in me and the security he has provided. It is inexplicable how much I needed you to accomplish my goals, Duarte and my friends who make me sun. I applaud you never giving up on me, for always listening and counseling me, and for invariably looking out for my well-being. My heartfelt gratitude goes out to everyone who contributed to the creation of this dissertation. “All my certanties are provisional.” - Oki Sato iii STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. iv RESUMO As lesões crónicas resultam do comprometimento do processo de cicatrização. O envelhecimento da população e o aparecimento de comorbidades que prejudicam a regeneração da pele contribuem para o aumento da incidência de lesões crónicas, sendo agora considerado uma epidemia. Como as terapias atuais apresentam desvantagens relevantes no tratamento de lesões crónicas, existe a necessidade clínica para o desenvolvimento de soluções eficazes que tratem as lesões crónicas a curto prazo, reduzindo a falha e a carga económica destas terapias. Assim, esta dissertação visava a criação de um scaffold de acordo com os princípios da Engenharia de Tecidos (TE) para promover a regeneração da pele utilizando elastin-like recombinamers (ELRs). Estas proteínas são polímeros geneticamente modificados com base na sequência natural da elastina, a qual é uma importante proteína estrutural da pele. Como este biomaterial combina as vantagens das proteínas recombinantes e as propriedades da elastina, foram criados dois polímeros recombinantes, SKS-IKVAV e SKS-PPFLM, os quais resultam de duas modificações diferentes da proteína SKS. Os ELRs foram concebidos e expressos na Technical Proteins Nanobiotechnology (TPNBT) S.L., como parte de um estágio ERASMUS (Contrato Financeiro 2020-1-PT01-KA103-077707). Apesar dos resultados favoráveis em relação à expressão proteica, não foi possível desenvolver um protocolo de purificação destes polímeros durante o estágio ERASMUS na TPNBT, o que inviabilizou a construção de scaffolds e concluir se o SKS-IKVAV e/ou o SKS-PPFLM podem ser utilizados na TE da pele. Uma vez que as proteínas SKS-IKVAV e SKS-PPFLM não puderam ser utilizadas para a construção de scaffolds , uma nova abordagem, utilizando a proteína STAR, foi testada no Centro de Engenharia Biológica da Universidade do Minho para o desenvolvimento de soluções de TE da pele. Esta proteína demonstra características semelhantes às dos ELRs, e foi utilizada para a criação de filmes à base de proteínas. Estes materiais baseados na proteína STAR foram caracterizados fisicamente, quimicamente e in vitro e a potencial utilização dos mesmos na regeneração da pele foi favoravelmente concluída. As perspetivas futuras dos filmes à base de STAR incluem aprofundar o conhecimento do seu comportamento in vitro e a incorporação de substâncias bioativas com propriedades anti-inflamatórias, anti-oxidantes e antibacterianas, para combater as principais causas do desenvolvimentos de lesões crónicas. Palavras-chaves: Cicatrização de lesões, Elastin-like-recombinamers , Filmes à base de proteínas, Regeneração da pele. v ABSTRACT Chronic wounds result from a disruption in the wound healing process. The aging of population and the appearance of comorbidities that jeopardize skin regeneration contribute for an increase on chronic wounds incidence being now considered as an epidemic. Since current therapies present significant drawbacks in the treatment of chronic wounds, there is a clinical need to develop effective solutions that treat chronic wounds in the short term, reducing failure and the economic burden of these therapies. As a result, the goal of this dissertation was to design a scaffold based on Tissue Engineering (TE) principles to promote skin regeneration using elastin-like recombinamers (ELRs). These proteins are genetically modified polymers based on the sequence of natural elastin, which is a structural protein found in the skin. Since this biomaterial combines the benefits of recombinant proteins with the properties of elastin, two recombinant polymers, SKS-IKVAV and SKS-PPFLM, were created from two different modifications of the SKS protein. The elastin-like recombinamers were designed and expressed at Technical Proteins Nanobiotechnology (TPNBT) S.L., as part of an ERASMUS traineeship (Financial Agreement 2020-1PT01-KA103-077707). Despite the promising results regarding protein expression, it was not possible to develop a purification protocol for these polymers during the ERASMUS trainee in TPNBT, making the construction of the scaffolds and determining whether SKS-IKVAV and/or SKS-PPFLM can be used in skin TE impossible. Since the SKS-IKVAV and SKS-PPFLM proteins could not be used for scaffold design, a new approach, using the STAR protein, was tested at the University of Minho's Centre of Biological Engineering, for the development of solutions for skin TE. This protein shows similar properties to ELRs and was used to create protein-based films. These STAR-based materials were physically, chemically, and in vitro characterized, and their potential use in skin regeneration was concluded favorably. The future prospects for STAR-based films will include greater understanding of their in vitro behavior, as well as the incorporation of bioactive substances with anti-inflammatory, antioxidant, and antibacterial properties to combat the primary causes of chronic injury development. Keywords: Elastin-like recombinamers, Protein-based films, Skin regeneration, Wound healing. xii Figure 5-6 — A) Preparative agarose gel (1 %) electrophoresis of commercial plasmid :: SKS-IKVAV 2 and commercial plasmid :: SKS-PPFLM 1 digested with Sap I endonuclease (Image Lab™). B) Excision of the bands corresponding to the linearized SKS-IKVAV and SKS-PPFLM inserts (Image Lab™). ........ 51 Figure 5-7 — Analytical agarose gel (1 %) electrophoresis of A) pD :: SKS-IKVAV 1x and B) pD :: SKSPPFLM 1x. The DNA samples were digested with Ear I and EcoR I endonucleases (Image Lab™). C) Theoretical restriction map of pD :: SKS-IKVAV 1x and pD :: SKS-PPFLM 1X vectors digested with Ear I and EcoR I (SnapGene®). ............................................................................................................... 53 Figure 5-8 — Analytical agarose gel (1 %) electrophoresis of A) pD :: SKS-IKVAV 6x and B) pD :: SKS-PPFLM 6x. The DNA samples were digested with EcoR I and Ssp I endonucleases (Image Lab™). C) Theoretical restriction map of pD :: SKS-IKVAV 6x and pD::SKS-PPFLM 6x vectors digested with EcoR I and Ssp I (SnapGene®). ................................................................................................. 54 Figure 5-9 — A) Preparative agarose gel (1 %) electrophoresis of p7 vector digested with Sap I endonuclease and pD :: SKS-IKVAV 6x 1 plasmid and pD :: SKS-PPFLM 6x 3 plasmids digested with Ear I endonuclease (Image Lab™). B) Excision of the bands corresponding to the linearized p7 vectors, SKS-IKVAV and SKS-PPFLM inserts (Image Lab™). C) Theoretical restriction map of p7 vector and pD :: SKS-IKVAV/SKS-PPFLM plasmids digested with Sap I and Ear I, respectively (SnapGene®). .............. 56 Figure 5-10 — Analytical agarose gel (1 %) electrophoresis of A) p7 :: SKS-IKVAV 6x and B) p7 :: SKSPPFLM 6x. The DNA samples were digested with EcoR I and Nde I endonucleases (Image Lab™). C) Theoretical restriction map of p7 :: SKS-IKVAV 6x and p7::SKS-PPFLM 6x vectors digested with EcoR I and Nde I (SnapGene®). ....................................................................................................... 57 Figure 5-11 — SDS-Page (10 %) of SKS-IKVAV and SKS-PPFLM proteins (expression screening). The gel was stained with cooper chloride solution (0.3 M) for visualization (Image Lab™). The inoculum grown overnight in TB medium and ampicillin served as the negative control. .................................... 58 Figure 5-12 — SDS-Page (10 %) of SKS-IKVAV protein after purification. The gel was stained with cooper chloride solution (0.3 M) for visualization (Image Lab™). ........................................................ 59 Figure 5-13 — SDS-Page (10 %) of SKS-PPFLM protein production in the fermenter and purification. The gel was stained with cooper chloride solution (0.3 M) for visualization (Image Lab™). The inoculum grown overnight in TB medium and ampicillin served as the negative control. .................................... 60 Figure 6-1 — Optical density (600 nm) measured for specific elapsed fermentation time-points for the culture conditions evaluated.............................................................................................................. 73 xiii Figure 6-2 – SDS-Page (12.5 %) of STAR protein expression samples A) after 8 h of inoculum’s incubation and TB manual 1:5 after induction and B) after 24 h of inoculum’s incubation and TB manual 1:5 after induction. Gel stained with Coomassie Blue. ........................................................... 74 Figure 6-3 – Macroscopical image of a 3 % STAR-based film, a 5 % DTT STAR-based film and a 5 % DTT methanol STAR-based film. ........................................................................................................ 75 Figure 6-5 — SEM micrographs of 5 % STAR-based films: A) general view of the film surface and B) cross-section view. ....................................................................................................................... 80 Figure 6-4 — SEM micrographs of 3 % STAR-based films: A) general view of the film surface and B) cross-section view. ....................................................................................................................... 80 Figure 6-7 — SEM micrographs of 5 % DTT STAR-based films: A) general view of the film surface and B) cross-section view. ....................................................................................................................... 81 Figure 6-6 — SEM micrographs of 3 % DTT STAR-based films: A) general view of the film surface and B) cross-section view. ....................................................................................................................... 81 Figure 6-8 — SEM micrographs of 3 % methanol STAR-based films: A) general view of the film surface, B) cross-section view and C) EDS image highlighting the crystals formed on the film’s surface. ........................................................................................................................................................ 82 Figure 6-9 — SEM micrographs of 5 % methanol STAR-based films: A) general view of the film surface and B) cross-section view. ................................................................................................................ 83 Figure 6-10 — SEM micrographs of 3 % DTT methanol STAR-based films: A) general view of the film surface and B) cross-section view. .................................................................................................... 84 Figure 6-11 — SEM micrograph of 5 % DTT methanol STAR-based films: A) general view of the film surface and B) cross-section view. .................................................................................................... 84 Figure 6-12 – FTIR spectra of STAR protein and STAR-based films. ................................................ 85 Figure 6-13 — Secondary structural conformations of the STAR-based films elucidated by deconvolution of amide I spectral region. STAR protein corresponds to the control. The 310 and α-helix are referred as helical. ...................................................................................................................... 88 Figure 6-15 — BJ-5ta cell viability at 48 h of incubation with pre-conditioned culture media. ............ 89 Figure 6-14 — BJ-5ta cell viability at 24 h of incubation with pre-conditioned culture media. ............ 89 Figure Annex I-1 — Cloning plasmid pDrive All map. Created with SnapGene® software. ............. 108 Figure Annex I-2 — Expression plasmid p7RARE map. Created with SnapGene® software. .......... 108 xiv LIST OF TABLES Table 2-1 — Summary of local and systemic factors that impair the normal wound healing process, adapted from [5,60] ......................................................................................................................... 18 Table 3-1 — Clinically available skin grafts, adapted from [66] ......................................................... 22 Table 3-2 — Results of in vitro testing of ELPs, adapted from [94] ................................................... 30 Table 5-1 — List of chemical reagents employed ............................................................................. 34 Table 5-2 — List of buffer solutions ................................................................................................. 37 Table 5-3 — Relation between fragment size and agarose final percentage in TAE 1X....................... 40 Table 5-4 — Correspondence between target size range and percentage in separation gel ............... 44 Table 5-5 — SDS-PAGE resolving and stacking gel composition (one gel) ......................................... 45 Table 5-6 — Concentration (ng/µL) and degree of purity of pD plasmids after purification ................ 47 Table 5-7 — Concentration (ng/µL) and degree of purity of linearized pD 2 vector ............................ 49 Table 5-8 — Concentration (ng/µL) and degree of purity of commercial plasmids with the SKS-IKVAV or SKS-PPFLM insert ........................................................................................................................ 50 Table 5-9 — Concentration (ng/µL) and degree of purity of SKS-IKVAV and SKS-PPFLM inserts after purification ....................................................................................................................................... 51 Table 5-10 — Concentration (ng/µL) and degree of purity of pD vector harboring the SKS-IKVAV or SKS-PPFLM inserts ........................................................................................................................... 52 Table 5-11 — Concentration (ng/µL) and degree of purity of p7, pD :: SKS-IKVAV and pD :: SKSPPFLM plasmids after purification ..................................................................................................... 55 Table 5-12 — Concentration (ng/µL) and degree of purity of p7 plasmid, SKS-IKVAV 6x and SKSPPFLM 6x genes after purification ..................................................................................................... 57 Table 5-13 — Fermentation conditions for the SKS-PPFLM protein .................................................. 59 Table 6-1 — List of chemical reagents employed ............................................................................. 62 Table 6-2 – List of buffer solutions ................................................................................................. 64 Table 6-3 — Inoculum media and conditions tested to assess the optimal protocol for the production of Star protein .................................................................................................................................. 65 Table 6-4 — List of the STAR-based films prepared .......................................................................... 67 Table 6-5 — Thickness measurements of the STAR-based films with the MPO Dualscope Thickness Gauge (Fischer) ................................................................................................................................ 76 xv Table 6-6 — Contact angle of the STAR-based films performed at room temperature with type I water as test liquid ..................................................................................................................................... 77 Table 6-7 — Swelling degree performed at 37 °C for 24 h with distilled water and culture media and in vitro degradation of the STAR-based films ...................................................................................... 79 Table 6-8 — Substances present in the 3 % methanol film’s crystals and their atomic concentration 83 Table 6-9 — Resulting discrete peaks, respective contribution to the FTIR-derived curves and corresponding structural assignments of STAR polymer and STAR-based films ................................... 86 xvi LIST OF ABBREVIATIONS AND ACRONYMS A aa Amino acid APS Ammonium persulfate ATCC American Type Culture Collection B BBRG Bioprocess and Bionanotechnology Research Group bp Base pairs BPB Bromophenol blue C CEAs Cultured epidermal autographs CEB Centre of Biological Engineering D DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl sulfoxide DTT Dithiothreitol E ECM Extracellular matrix E. coli Escherichia coli EDS Energy-dispersive X-ray spectroscopy EDTA Ethylenediamine tetraacetic acid EFT Elapsed fermentation time xvii ELPs Elastin-Like polypeptides ELRs Elastin-like recombinamers F FBS Fetal bovine serum FTIR Fourier-Transform Infrared Spectroscopy G GAGs Glycosaminoglycans GFs Growth factors I ITT Inverse temperature transition L LB Luria-Broth LCST Lower critical solution temperature M mf final dry mass mi initial dry mass MTS 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide MW Molecular weight marker N NDA Non-disclosure agreement O OD600nm Optical density of a sample measured at a wavelength of 600 nm xviii P p7 p7RARE PBS Phosphate buffered saline PCL Polycaprolactone pD pDrive All PLA Polylactic acid R RM Regenerative Medicine ROS Reactive oxygen species S S.A.P. Shrimp alkaline phosphatase SD Standard deviation SDS Sodium dodecyl sulphate SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SELPs Silk-elastin-based polypeptides SEM Scanning electron microscope SKS Serine-Lysine-Serine T TAE Tris acetate ethylenediamine tetraacetice acid TB Terrific Broth TB AIM Terrific Broth Autoinduction TE Tissue Engineering xix TEDTA Tris ethylenediamine tetraacetic acid TEMED Tetramethylethylenediamine TPNBT Technical Proteins Nanobiotechnology S.L. Tris Tris(hydroxymethyl)aminomethane Tt Transition temperature U UV Ultraviolet V VPGVG Valine, proline, glycine, valine, glycine W Wd Initial dry mass Ws Mass of the swollen material 1 1. INTRODUCTION As the skin protects the human body from external agents and maintains homeostasis, one of its most important properties is wound healing, whereby various molecular and cellular factors enable the recovery of the integrity and function of this organ [1-4]. Despite skin's high regenerative capacity, chronic wounds result in inadequate repair [3,5]. These injuries disrupt one or more phases of the normal healing process, most commonly because of prolonged inflammation and/or infection, the formation of drug-resistant microbial biofilms, and the inability of skin cells to respond to restorative stimuli [5,6]. Hence, these wounds do not recover fully in the expected time, and their treatment usually includes surgical intervention [3,7]. Because of the global epidemic of chronic wounds, research efforts have been focused on developing Tissue Engineering (TE) solutions, which involve cultivating cells on three-dimensional support structures capable of serving as temporary molds for the extracellular matrix (ECM), allowing regeneration of a final functional tissue [8]. These constructs can have antibacterial properties and aid wound healing in one or more layers of affected skin by providing an appropriate mechanical support for cell growth, allowing this organ to regenerate at the same rate as these structures degrade [9,10]. To that end, their design must consider healthy skin's ECM characteristics, such as architecture, fibrous constitution, high water content, and physical, chemical, and mechanical properties [8]. Polymers are the most commonly used materials to create biodegradable structures, and natural polymers, specifically proteins, are widely used in skin TE because they are the main constituents of human tissues, and the organism has established metabolic pathways to process them [11]. Recombinant proteins, in particular, stand out, as it is possible to design natural-like proteins with target characteristics [12]. 1.1. Context and motivation It is estimated that (1 – 2) % of the population in developed countries will suffer from chronic wounds at any given time [6]. Specifically, in Portugal, a study in a continuing care unit 2 demonstrated that 82 % of all wounds identified among their patients were chronic [13]. The worldwide incidence of chronic wounds has been increasing globally in recent years, owing to an ageing population, i.e., an increase in people suffering from diseases and comorbidities that impair wound healing, such as diabetes, obesity, venous hypertension, and peripheral vascular diseases [6]. Furthermore, due to the high rate of cell proliferation in epithelial tissue and frequent exposure to physical and chemical damage, skin cancer is one of the most common cancers in humans and, as the standard treatment consists in the excision of the affected area, the resulting lesions are similar to chronic wounds [1,14]. Because of the numerous types of chronic wounds, existing solutions are developed for specific injuries. However, the most common treatment for chronic wounds is skin graft transplant, which consists of transferring healthy skin from one site to the wounded area [15,16]. Skin grafts are distinguished in autografts (the host is the donor), allografts (harvested from a cadaver), or xenografts (the donor is not of the same species as the host), being the first the current gold standard treatment [16]. Whilst autografts are less likely to provoke an immune response than heterologous ones, they are not exempt from disadvantages, the most relevant being the creation of a deep wound in the donor site that could become chronic [15,17]. There may also be a shortage of suitable donor tissue and/or the patient may not be fit for this treatment due to the presence of diseases/comorbidities that impair wound healing [17]. Additionally, skin grafts show inefficiency or ineffectiveness to completely heal some of these lesions due to the incapacity of overcoming the increase of bacterial resistance inherent to the infiltration of external pathological agents [18]. In addition, the continuous professional care required in these treatments has become a challenge in the COVID-19 pandemic [19]. This sudden change in daily life has reinforced the need to find solutions that effectively treat chronic injuries in the short term, reducing the overall economic/clinical burden of these therapies and the creation of multi-resistant bacterial biofilms, minimizing the failure of the treatment [6,20]. Hence, new skin regeneration therapies are focused on TE solutions, as the technology employed allows the development of off-the-shelf regenerative models customized for each patient with similar shape and function to the healthy native tissue [20,21]. 9 Collagen is a structural protein that presents variations as it consists of three polypeptide subunits and has a fibrillar appearance due to the highly frequent glycine-proline-hydroxyproline tripeptide, also referred to as α chain, which lacks intrachain hydrogen bonds in its triple helix arrangement [35-37]. Hence, collagen protects the dermis against stress and strain due to the combination of its high tensile strength and woven architecture, making up to 70 % of dermis dry weight [28,30]. Elastin is an amorphous protein that endows the skin with its characteristic elasticity, allowing it to stretch and compress to a certain limit without deformation [30]. Thus, this protein is presented in the center of the elastic fibers, which have a fibrillary structure since they consist of microfibrils intertwine in crosslinked tropoelastin [31,38]. Figure 2-4 shows the elastin and collagen network in the ECM of the dermis. Tropoelastin is defined as a polypeptide with two domains, the hydrophobic region with of any duo up to quintets of glycine, valine, proline, and alanine repeats and the hydrophilic consists of intermittent lysine in alanine or enriched proline domains [40,41]. Hence, the constitution of tropoelastin is responsible for its characteristics, as proline residues enable the stabilization of this protein secondary structures, such as β-turns and β-sheets, and the glycine content favors hydrogen bonds with water. The latter increases hydration, flexibility, and the adoption of organized structures by the focus protein [42]. The production of elastic fibers (elastogenesis) is complex, starting with the secretion and coacervation (self-aggregation by liquid-liquid phase separation due temperature increase [43]) of tropoelastin on the cell surface. After these microstructures’ growth, they migrate into the intercellular space, being covalently crosslinked by enzymes and, with the repetition of this Figure 2-4 — Schematic representation of the dermis. Created with Biorender.com and adapted from [39]. 10 process, the elastic fibers mature. Thus, the flexibility and elasticity displayed by elastin is conferred by its precursor, tropoelastin [44,45]. Similarly, laminin participates in the formation of tissue’s ordered networks and in signaling, laminin 5 or 332 being essential for the anchoring of epidermal keratinocytes in the dermoepidermal junction. Additionally, laminin accelerates the assembly of basement membranes, which hastens the recovery of injured skin [46-48]. Another protein mentioned is fibronectin, which consist of long fibrils of ECM stabilized by disulfide bonds, turning into fibers by self-assembly. In interaction with other ECM proteins, fibronectin participates in cell adhesion, migration, and differentiation via integrin receptors [49-51]. The stated proteins are continuously broken down and replaced, however age and intensive UV exposition affect this process, resulting in loss of skin flexibility [27]. Additionally, the dermis has salts, water, and a viscous gel constituted mainly by proteoglycans (glycosaminoglycans – GAGs - associated with a protein backbone [31]), which are negatively charged macromolecules that intervene in cellular adhesion, migration, differentiation, and proliferation [1,51-53]. Specifically, the proteoglycans-based gel, also referred to as ground substance, allows nutrients, waste products, hormones, and other specialized molecules pass through the dermis, lubricating the collagen and elastin network. Moreover, being a dense bulk, the ground substance also acts as a shock absorber [27,31]. The dermis is divided into two, the papillary dermis and the deeper reticular dermis (Figure 2-5) [26,27]. Figure 2-5 — Schematic representation of the internal dermal layers. Adapted from [54]. 11 The papillary dermis is the more superficial and the thinner of the two, while the reticular is internally bounded to subcutaneous fat, has denser connective tissue, larger capillary vessels, more interwoven elastic fibers, ticker collagen bundles and fewer cells. Nonetheless, as the papillary dermis presents a more random collagen and elastic fibers network and has a higher quantity of gel, withstands greater frictional forces [26-28]. The types of cells present in the dermis are mast cells, immune system cells, Langerhans cells, and fibroblasts. Mast cells have an oval or spindle shape and are scattered throughout the connective tissues of the human body, being particularly present in the papillary layer of the dermis since they detect pain, itch, and temperature [1,55]. Immune system cells, such as leukocytes, can enter the structure of the dermis in response to several stimuli, such as the formation of a wound [1,55]. Moreover, fibroblasts are a relevant constituent of the dermis since in their mature state (as fibrocytes [31]) synthesize elastin, collagen, and the constituents of the viscous gel, playing a crucial role in the wound healing process [27]. Due to its anatomical location and anchorage to the epidermis, one of the main functions of the dermis is to sustain and support nutritionally and physically the epidermis, also having a protective role giving its pliability and tensile strength, preventing mechanical trauma to the underlying structures. However, excessive friction or shearing forces can separate these two layers [27,30]. 2.3. Subcutaneous tissue The subcutaneous tissue (Figure 2-6), also known as the panniculus or hypodermis, is considered the deepest layer of the skin and contains aggregates of lipid cells known as lipocytes, which are separated by fibrous septa of collagen and sizeable capillary vessels. Hence, lipocytes have an insulating effect and are intertwined with blood vessels and nerves to further influence thermoregulation and synthesize leptin, a body weight regulating hormone. This layer also includes mast cells, although in less quantity than the papillary layer of the dermis [1]. 12 Therefore, the panniculus serves not only to store energy, absorb shock, as it also provides buoyancy to the skin and supports the dermal and epidermal layers [1,27]. 2.4. Biomechanical properties of skin The biomechanical properties of the skin are defined by its cellular and molecular components, as well as the architectural organization they take. Although each individual layer endows the skin with different properties and its characteristics subtly change between different anatomical sites, the overall arrangement of this organ can be interpreted as a multi fibrillary and vacuolar system with a balance of tension-compression forces, having the dermis as its main mechanical component [26,31]. Therefore, this complex organ demonstrates anisotropy, indicating that its mechanical properties change depending on the orientation in which the tissue is being analyzed. Specifically, the rich constitution of collagen, elastic fibers, and the presence of the ground substance capacitate the skin with extraordinary elasticity and flexibility, which varies depending on the direction in which the force is applied [26]. This natural behavior was recognized by Langer in the 19th century, who first mapped the naturally occurring tension lines in the skin [57]. Skin exhibits viscoelastic behavior when deformed, usually under stretch, thus its response when stress is applied is non-linear, as represented by its typical stress-strain graph in Figure 2-7 [26]. This graph shows skin’s behavior under an executed force (stress) plotted against the change in length (strain). Figure 2-6 — Schematic representation of the subcutaneous tissue. Created with Biorender.com and adapted from [56]. 13 Skin deformation is a dynamic process characterized by three phases. In the first phase, also referred to as the “loading phase”, the smallest force produces the largest alteration in length, meaning that at a strain of up to 0.3 %, the skin does not offer great resistance. This limited isotropic behavior is due to the alignment of elastic fibers and the entanglement of collagen in the dermis, which enables a linear relationship between stress and strain. Thus, the skin behaves as an elastic material, which is also verified by its low Young’s Modulus (0.1 - 2) MPa [26,57,58]. In the second phase, a greater force is required to deform the skin since the collagen fibers are reoriented and some of skin ECM are forced between them. Accordingly, as the collagen fibers begin to stretch, a non-linear behavior in the stress-strain curve is presented [26,58]. Ultimately, in phase three, for strains higher than 0.6 %, a return to the linear stress-strain relationship can be verified, as the dermis fibers are at maximal length due to the disappearance of their characteristic spindle architecture. The collagen fibers are responsible for this effect since they are stiffer than the elastic. Thus, the force required to produce more strain increases notably, with collagen fibers breaking down on application of a deformation equal to or greater than 0.7 % [26,57,58]. In conclusion, it is possible to classify skin as a quasi-incompressible material since its volume changes are negligible when considerable deformation is applied. However, the effect of deformation on the skin depends on the duration of its application since a hyperplastic response Figure 2-7 — Typical skin stress-strain curve. Created with Biorender.com and adapted from [58]. 14 can be verified when a slowly constant stress is applied, this property being named creep. The phenomenon described occurs due to the decrease in the force required to maintain the skin with a certain length over time [26,57]. Contrarily, if the stress is applied too rapidly to enforce rapid stretch, rupture of the collagen fibers occurs, resulting in dermal injuries [26]. 2.5. Wound healing process As a result of the important functions of the skin, one of its most crucial features is wound healing, i.e., the regeneration process of the skin after the development of a wound. A wound occurs due to the disruption of the epithelial layer or mucosa of the skin caused by thermal or physical trauma and its regeneration involves four overlapping and well-orchestrated stages [5,20,59], as observed in Figure 2-8. Figure 2-8 — Wound healing process. Created with Biorender.com. 15 2.5.1. Hemostasis Hemostasis is immediately triggered by the microvascular disruption and extravasation of blood that occurs from the injury, as vasoconstriction is activated by the endothelium to prevent further blood outflow and the clotting cascade initiates. The latter process aims to form a clot of fibrin, fibronectin, and other proteins with platelets trapped to act as a temporary matrix for cell migration while preventing blood loss [5,20]. Specifically, platelets in the clot and surrounding wound tissue lead to secretion of growth factors and pro-inflammatory cytokines, attracting and activating fibroblasts, endothelial cells and macrophages, key actors of subsequent phases. Additionally, these blood system cells contain vasoactive molecules that heighten microvascular permeability, allowing fluid to pass into the extravascular area [5,20,60]. Furthermore, epidermal cells from the wound edges migrate to the injured area to form a thin cover, this process being known as epiboly [5]. 2.5.2. Inflammation The second phase of the wound healing process has an overall duration of 3 – 5 days. Once the bleeding is under control, an intensive migration of inflammatory cells (chemotaxis) occurs and immune cells such as neutrophils, macrophages, and lymphocytes arrive at the wounded area mainly to cleanse the injured region of external microorganisms, cellular debris, enzymes, and highly reactive species as reactive oxygen species (ROS) that are detrimental to the wound healing process [5,60]. Particularly, macrophages participate in two important roles in this phase. At the beginning, these cells release cytokines, which will recruit and activate leukocytes, inducing further inflammation. Conversely, at the end of this phase, macrophages terminate the induction of apoptotic cells, such as neutrophils, thus promoting the beginning of the proliferative phase [59,60]. Additionally, the inflammatory cells secrete growth factors (GFs) that boost fibroblast migration and proliferation of vascular endothelial cells in the subsequent phase [59,60]. 16 2.5.3. Proliferation The third phase of the wound healing process initiates 3 days after the wound formation and lasts 2 - 4 weeks. It is characterized by fibroblast migration, deposition of provisional ECM components to reconstruct it, and formation of granulation tissue, the latter consisting of new connective tissue and capillary vessels formed from old ones of the injured area initiated by endothelial progenitor cells [5]. At the beginning, fibroblasts migrate to the wound area, followed by endothelial cells, which are attracted by GFs released previously and the enzymatic conversion of fibrinogen to fibrin by thrombin. Hence, the primary goal of these cells is to synthesize proteins that are used to reconstruct the damaged ECM. Additionally, after ECM reformation, the fibroblasts maintain interactions to regulate its further formation and remodeling [5,59]. As the proliferative phase progresses, the temporary matrix of fibrin and fibronectin is progressively replaced by type III collagen produced by dermal skin fibroblasts accompanied by angiogenesis to create the granulation tissue [5,59]. The end of this phase is marked by the re-epithelization of the wound, process enabled by the migration and proliferation of epithelial cells that requires specific conditions, such as high humidity, nutrition, and control of external organisms, being modulated by several GFs [5,60]. Following a wound, the structures of all skin appendages are also capable of reepithelialization through the migration of keratinocytes from the epithelium into the stratum corneum [1]. 2.5.4. Remodeling The last phase of the wound healing process is the remodeling and further synthesis of the ECM initiated in the previous phase, with scar maturation also occurring [5]. Remodeling can last for years, beginning in week 1 of wound formation, and is characterized by constant cycles of collagen II formation and breakdown and its replacement with collagen type I so that the skins’ ECM stabilizes (usually 21 days after injury) and acquires an architecture similar to that of healthy skin. As the scar matures, its tensile strength increases through collagen type I fiber enlargement, accumulation, and cross-liking [5,60]. 17 Moreover, wound contraction may occur, driven by the interaction between fibroblast and the surrounding ECM. This phenomenon happens throughout all healing processes and is regulated by contractile fibroblasts (myofibroblasts), cytokines and growth factors present in the wound area [5,60]. 2.5.5. Chronic wounds Notwithstanding skin's high regenerative capacity, chronic wounds result of inadequate repair. These injuries, by definition, disrupt one or more phases of the normal healing process, most commonly because of prolonged inflammation and/or infection, the formation of drug-resistant microbial biofilms, or the inability of skin cells to respond to restorative stimuli, healing in an unpredictable or long-time frame. Thus, chronic wounds treatment usually includes surgical intervention [3,5,6,59]. As chronic wounds fail to progress beyond the inflammatory phase, they are often characterized by an imbalance of proand anti-inflammatory cytokines and GFs, which impedes the proliferation of vascular endothelial cells, fibroblasts, and collagen matrix deposition. Additionally, high levels of proteases and excessive ROS release by inflammatory cells affect the wound healing process by damaging the cells or the ECM and degrading the GFs [59]. Other characteristics that are commonly associated with chronic injuries and hinder their regeneration process are present in Figure 2-9. Figure 2-9 — General characteristics of chronic wounds. Created with Biorender.com and adapted from [59]. 18 Chronic wounds can be caused by a variety of factors, including burns, trauma, prolonged pressure (pressure ulcers), venous valve malfunction (venous ulcers), or metabolic diseases such as diabetes (diabetic foot ulcers). Hence, several local or systemic factors hinder or prevent normal skin repair [5,59] and a summary of these influential factors is presented in Table 2-1. Table 2-1 — Summary of local and systemic factors that impair the normal wound healing process, adapted from [5,60] Local factors Systemic factors Inadequate blood supply Advancing age and general immobility Increased skin tension Obesity Poor venous drainage Malnutrition Presence of foreign body reaction and/or elements Deficiency of proteins, vitamins, and/or trace elements Presence of slough and/or non-viable tissue Systemic malignancy and terminal illness The continued presence of external microorganisms (e.g., formation of bacterial biofilms) Systemic diseases (e.g., diabetes mellitus , rheumatoid arthritis, connective tissue diseases, metabolic diseases) Infection Peripheral vascular disease and vasculitis Excess local mobility Venous oedema or lymphoedema Underlying osteomyelitis Chemotherapy and radiotherapy Malignant transformation Impaired macrophage activity These factors alter cells and their activity, decrease levels of GFs, and/or lead to abnormal alterations in the ECM, increasing the presence of highly reactive molecules, especially ROS [5]. Thus, deficiency/lack of epithelialization or inadequate blood supply are common compromising factors during the wound healing process, as the regeneration process largely depends on them [5]. 25 biodegradable biomaterials, whose architecture can be customized to the lesion and the stem cells used can be those of the patient. Additionally, the use of the aforementioned active substances used in parallel with skin grafts can be integrated in these constructs in order to exponentiate the success of the therapy [76-78]. Wound healing TE research has been focused on growing cells in three-dimensional scaffolds or other supporting structures, i.e., as films and hydrogels, to engineer constructions capable of serving as temporary molds to the ECM, allowing the regeneration of a final functional tissue while creating a protective barrier that prevents further infection and fluid loss [8,17]. These constructions can aid wound healing in one or in several skin layers affected in chronic wounds, providing an appropriate mechanical support for cell growth, in a manner that the regeneration of this organ occurs at the same rate at which these structures are degraded [9,10]. Accordingly, a requirement of TE is that the constructive models must be biodegradable since the purpose of the therapy is to assist the organism in the regeneration of a certain tissue and not to definitively replace it [76-78]. 3.1.1. General characteristics of regenerative constructs TE combines the use of stem cells, biomaterials, and bioactive substances to improve the success of tissue regeneration. Hence, only support structures associated with the use of cells are considered TE solutions [79]. There are general well-accepted requirements for regenerative constructs as depicted in Figure 3-3. Figure 3-3 — Ideal characteristics of TE solutions. Created with Biorender.com and adapted from [79]. 26 Scaffolds are the most common TE structure because they allow for easier mimicking of the tissue to be regenerated while meeting the above requirements. They should present high porosity, adequate pore size and interconnectivity to enable cell proliferation, migration, adhesion, growth, and differentiation throughout the structure. Also, a high surface area is required to allow easy perfusion of nutrients and gases for the nourishment of the seeded cells or their waste disposal, and biocompatibility [8,11,80], defined as “the ability of a material to perform with an appropriate host response in a specific application” [81]. For the scaffold to be considered biocompatible it should not exhibit cytotoxic response, have good biofunctionality, that can be resumed in the ability to promote ECM deposition, correct gene expression, and not induce changes in cell phenotype [8,11,80]. Moreover, the mechanical properties of the scaffolds should be similar to those of the tissue to be regenerated and the rate of degradation should be adjusted to the rate the new tissue forming, possibly by altering their physical and chemical stability. Also, the degradation products should be non-toxic [8,11,80]. Additionally, these constructs must be efficient, allowing repair of skin structure and function in a timely manner. Moreover, they should be easy to handle, to produce, and obtained using biomaterials that have an unchallenging manufacture to reduce production costs [17]. However, while there are global requirements for a TE solution, it must be developed according to the characteristics of the tissue, such as the specifics of its ECM, cellular content, and biomechanics [81]. 3.1.2. Materials for regenerative constructs Considering the properties of scaffolds, polymeric biomaterials are regarded as ideal for the development of TE devices [82]. This is due to their chemical tunability, which allows scaffolds to be engineered with the properties required for a specific application [11]. Synthetic and natural polymers present different properties and due to their controlled production, synthetic polymers like silicone and polylactic acid (PLA) or PCL are considered more available and tunable than natural ones. These materials are characterized by their carbonic backbone, having identical repeating units, which makes them uniform. Hence, synthetic 27 polymers have very predictable properties that can be highly engineered to mimic the native tissue [11,67,83]. In particular, the potential of PCL has been underlined since it is already used in sutures and drug delivery devices. Still, materials in this category are prone to more unfavorable biological responses since some of the interveners in their manufacturing process may not be biological and/or environmentally friendly [83]. One of the most commonly used natural polymers are proteins, such as collagen, elastin, or keratin, as they are complex macromolecules with one or more aa chains that are substantially present in all human tissues. Additionally, gelatin, derived from denaturation and partial hydrolysis of collagen, silk, a material endowed with more durability, degradability, and strength than others, and fibrin, an important ECM protein, are also extensively referred. Hence, this type of biomaterial with similar but not identical repeating units is less likely to be cytotoxic as they are easily incorporated and expelled into biological systems since there are established pathways for their metabolic processing [11,66]. Nonetheless, the immunogenicity verified in some natural polymers, the complexity of their monomers and their lower availability are disadvantages that need to be overcome. Additionally, natural polymers evidence batch-to-batch variability and unfavorable biomechanical properties due to their rapid biodegradation and low stability [11,12]. Therefore, recombinant proteins stand out since they are natural-like macromolecules, which can be engineered with target characteristics according to the specific requirements of their application. Hence, they are defined as proteins created via heterologous expression, as a result of genetic engineering and examples of them are human-derived proteins, such as collagen and elastin [12,84]. As the manufacture of these proteins is based on the use of different molecular biology tools to design and obtain recombinant genes, it is possible to control the biomaterial at a molecular level with precise size and sequence control, overcoming the low availability and variability of natural polymers. In addition, bioactive sequences or residues can be incorporated to model the physicochemical properties of these proteins [85,86]. The reference to the complexity of natural polymers as a disadvantage is justified by the associated production costs. Hence, as recombinant proteins allow complexity with a low production cost, this characteristic is a relevant advantage of this class of biomaterials [87]. 28 When designing a scaffold for skin TE, the properties and constitution of the skin have to be considered. Among the constituents of the skin, collagen and elastin stand out, mainly due to their functions. Although in lower concentration than collagen, elastin has many relevant functions in the skin, modulating cells-ECM interactions and, as a signaling macromolecule, induces cell adhesion, differentiation, and proliferation in the skin matrix [87]. Additionally, elastin plays a relevant role in angiogenesis, which is crucial for proper wound healing, as deficient blood supply impairs this process [88]. Furthermore, ELRs are a promising class of biologically inspired proteins, their use being widely referred in TE literature since these recombinamers offer the opportunity to biomimicry one of the most important fibrillar proteins of the ECM [88]. 3.1.2.1. Elastin-like recombinamers (ELRs) Elastin-like polypeptides (ELPs) are genetically engineered proteins based on the natural hydrophobic sequence of elastin, the pentapeptide motif valine, proline, glycine, valine, and glycine (VPGVG) [21,43]. This sequence, responsible for the elastic properties of elastin, was first described by Gray and his team [89] and the first type of ELPs was synthesized by Urry's laboratory [90] aiming to elucidate the characteristics of this unit [43]. Physicochemical studies have demonstrated that ELPs have a smart behavior, such as reversible phase transition in response to temperature [22]. The latter phenomenon, also known as inverse temperature transition (ITT) (Figure 3-4), is an inherited feature of elastin/tropoelastin thermoresponsiveness [21]. Hence, ELPs remain soluble below the transition temperature ( Tt ) and aggregate when the temperature is raised above the Tt . Above this temperature the polymer chains hydrophobically fold and lose their clathrate water structures [54], leading to phase separation. Thus, Tt is also known as the lower critical solution temperature (LCST) [21,43]. 29 An ELP Tt can be tuned by salts present in the medium, pH, and pressure, but also by polypeptide hydrophobicity and length [92]. Specifically, ELPs hydrophobicity can be changed through the modification of the fourth aa of the VPGVG pentapeptide, endowing the ELP design with great flexibility since distinct aa in this domain can endow the final polymer with different properties, making their choice depend on the final application of the polymer [43,93]. Hence, the motif began to be described as VPGXG, where X can be any aa except proline since it destabilizes the secondary conformation of the final protein [21,88,93]. Another advantageous characteristic of the pentapeptide sequence is the inability of the host immune system to distinguish between endogenous elastin and ELPs [21]. Table 3-2 shows the favorable biological performance of ELPs in vitro testing conducted by Urry, which demonstrate their promising use in the field of RM [94]. Figure 3-4 — Behavior of ELPs according to temperature. Adapted from [91]. 30 Table 3-2 — Results of in vitro testing of ELPs, adapted from [94] Initially, ELPs were chemically synthesized, hampering the production of long polypeptides. However, with the 1980s advent of recombinant DNA technology, the production of ELPs has begun to be carried out through its expression in heterologous hosts, mainly Escherichia coli ( E. coli ), making also possible to incorporate bioactive sequences into these proteins [43]. Considering the recombinant nature of the ELPs obtained using molecular biology tools, Rodríguez-Cabello et al. [95] proposed a new nomenclature to ELPs, elastin-like recombinamers (ELRs). The use of ELRs in the context of TE is not new. For example, an ELR hydrogel was developed as an experimental model to study and modulate cell behavior in 3D cultures and these recombinamers have already been used in drug delivery systems, vaccines, and gene delivery vectors in the last decades [86,96]. Additionally, other proteins can be recombined with ELRs to alter the properties of the recombinant protein so that they are more suitable for the tissue to be regenerated. An example are silk-elastin-based polypeptides (SELPs) that combine the mentioned advantages of ELRs with additional chemical and thermal stability, mechanical tunability and more physical crosslinking sites due to the tandemly repeated silk units (GAGAGS) [97]. Test Result Ames mutagenicity Non mutagenic Cytotoxicity-agarose overlay Nontoxic Acute systemic toxicity Nontoxic Intracutaneous toxicity Nontoxic Muscle implantation Favorable Acute intraperitoneal toxicity Nontoxic Systemic antigenicity Non antigenic Dermal sensitization Non sensitizing Pyrogenicity Nonpyrogenic Lee-White Clotting Normal In vitro hemolysis Non hemolytic 31 For the design and engineering of ELRs-based scaffolds for skin TE in this dissertation, the final polymer would result from the fusion of different ELRs polymers, these being called SKS, SKS-IKVAV and SKS-PPFLM, the latter two resulting from different modifications of the former [99]. SKS stands for serine-lysine-serine sequence [86] and since a confidentiality agreement has been signed with TPNBT, the complete sequences of the three polymers mentioned will not be presented. Due to intellectual property protection, the STAR protein's aa sequence will also not be disclosed in this dissertation. 3.1.3. Fabrication methods of regenerative constructs The technologies available for the fabrication of scaffolds in TE are immeasurable, as each specific end application and chosen material requires optimized manufacturing to provide the optimal solution. Since relevant characteristics of scaffolds are porosity and pore size, traditional technologies focus on the extrusion of fibers to create porous structures. Using electro/melt/wet spinning, solvent casting, and freeze-drying it is possible to obtain 3D structure on the scaffold ideal to cells to adhere and proliferate [99,100]. Technological advances in several areas have allowed the development of other manufacturing tools, in order to mimic the ECM of several tissues more accurately and biologically functionalize the final structure. For example, manufacturing methods such as 3D printing, which comprises inkjet, micro extrusion, stereolithography and bioprinting, are now being employed to obtain scaffolds with precise architecture [99]. Because the goal of this dissertation was to demonstrate the feasibility of using ELRs in a scaffold to promote skin regeneration, the manufacturing method under study for its construction was based on click-chemistry (Figure 3-5). 32 This is a chemical manufacturing mechanism that allows the covalent cross-linking of two ELRs at a liquid-liquid interface to produce micro-scale, bioinspired scaffolds with tunable diffusion properties. This method, which has already been used for the construction of scaffolds with SKS polymer, is simple to implement and does not require specialized technical knowledge [102]. Figure 3-5 — Schematic representation of the chemical groups involved and resulting from clickchemistry. Adapted from [101]. 33 4. CONTEXT OF THE WORK The construction of SKS-IKVAV and SKS-PPFLM synthetic genes was successfully accomplished at the TPNBT facilities at Valladolid - Spain. However, due to contingencies of time related with the ERASMUS mobility program it was not possible to follow the work with the ELRs, particularly the production and the purification of the polymers. The second stage of the work with the ELRs was expected to be developed at the BBRG at Centre of Biological Engineering (CEB), in University of Minho. However, since none of the polymers were successfully obtained, a new strategy using a new polymer with similar characteristics to those of ELRs – STAR - was used to obtain protein-based films for skin TE. For a better comprehension of the work developed in this dissertation, the materials, methods, results, and discussion of each class of proteins will be presented separately. The first one is related to the ELRs and was developed at TPNBT facilities at Valladolid – Spain and the second part is related with the application of STAR polymer, and was developed at BBRG at CEB, University of Minho. 34 5. ELASTIN-LIKE RECOMBINAMERS: SKS-IKVAV AND SKS-PPFLM FOR SKIN TISSUE ENGINEERING Since ELRs are elastin-based recombinant polymers, they can be potentially used as a biomaterial in TE solutions to promote skin regeneration. Thus, this section of the dissertation was focused on the synthesis of the SKS-IKVAV and SKS-PPFLM recombinant genes using genetic engineering tools and bioproduction of the proteins at TPNBT. 5.1. Materials The materials used for the design and production of the recombinamers are below. Since a confidentiality agreement has been signed with TPNBT, the aa sequences of the SKS-IKVAV and SKS-PPFLM proteins will not be presented. 5.1.1. Chemical reagents The chemical reagents employed in the making of the ELRs are listed in Table 5-1. Table 5-1 — List of chemical reagents employed Reagent and Abbreviation Brand 1 kb Plus Ladder Invitrogen Acrylamide Amresco Agarose Sigma Aldrich Ammonium persulphate (APS) Sigma Aldrich Ampicillin Apollo Scientific Bromophenol blue (BPB) Sigma Aldrich Cooper chloride Sigma Aldrich D (+) glucose Merck Ethanol Merck 41 At the end of the run, the gel was removed from its mold, the DNA bands were stained with a 1X safe DNA stain (40 mL of TAE 1X and 2 µL of SYBR™ Safe DNA Gel Stain) for 30 min at room temperature and visualized on a Bio-Rad Gel Doc EZ Imager, using the Image Lab™ software. The image retrieved was always compared with the theoretical restriction map of the samples obtained with SnapGene® software. 5.2.1.4. DNA fragments purification from an agarose gel To obtain DNA fragments from an agarose gel, a minimum quantity of agarose gel containing the desired band was removed with a scalpel during UV light visualization rapidly to avoid DNA mutations. Then, the recovered gel bands were weighed, and the DNA was purified using the PureLink® Quick Gel Extraction kit (Invitrogen) as per the manufacturer protocol. After, the concentration of the samples was measured and their purity assessed with the Nano Drop 2000c Spectrophotometer (ThermoFisher Scientific), using the NanoDrop2000 software. A 260nm/280nm ratio equal or in between 1.80 and 2.00 indicated that the DNA is pure, and no aromatic compounds, RNA, salts, carbohydrates, or phenolic contaminations are present. 5.2.1.5. DNA insert ligation (sticky-end) into vector DNA The DNA insert ligation (sticky-end) into vector DNA was performed after obtaining the dephosphorylated vector and the gene fragment(s) with compatible sticky ends. Hence, 70 ng of linear vector and the insert gene fragment(s) - with a molar ratio of 1:5 - were incubated in a solution with a final volume of 10 µL of type I water, 1 µL 10X T4 DNA Ligase buffer, and 1 µL of T4 DNA Ligase enzyme. The solution was centrifuged at 11000 rpm for 1 min at room temperature and incubated at room temperature for 3 h 30 min. The T4 DNA Ligase was then inactivated by incubating the mixture for 10 min at 65 °C. 42 5.2.1.6. Transformation of E. coli competent cells The transformation of competent cells was performed in XL1-Blue competent and in XL1-Blue subcloning competent E. coli strains. These strains have different transformation efficiencies, ≥ 1 x 108 and ≥ 1 x 106 transformants per microgram of DNA, respectively, and are used for gene cloning and manipulation. Additionally, BLR (DE3) competent cells, with an efficiency of ≥ 2 x 106 transformants per microgram of DNA, were also transformed for protein expression. For the transformation of the XL1-Blue competent cells was added 5 µL of the ligation reaction solution to 50 µL of bacteria and 1.7 µL of β-mercaptoethanol in a pre-chilled 14-mL BD Falcon polypropylene round-bottom tube (Stratagene). The solution was incubated in ice for 30 min and then submitted to a thermic shock at 42 °C for 45 s, being after placed in ice for 2 min. Subsequently, 450 µL of pre-heated SOC I medium at 42 °C was added to the cells and the solution was putted in an incubator at 37 °C, 250 rpm for 1 h. Finally, a volume of 50 µL or 100 µL of cells were platted on petri dishes with LB-Agar medium, supplemented with kanamycin or ampicillin if the ligation was performed using the pD or the p7 vector, respectively. The plates were then incubated overnight at 37 °C. Some modifications were done when transforming the subcloning and the BLR (DE3) strains. To transform the XL1-Blue subcloning competent cells were only used 10 µL of E. coli , 1 µL of ligation reaction solution, the first incubation on ice was for 20 min and the heat-pulse at 42 °C was for 30 s. To transform the BLR (DE3) competent cells was used 250 µL of cells without β-mercaptoethanol, 400 ng of plasmid DNA and the thermic shock was submitted for 2 min. Additionally, 250 µL of pre-warmed LB medium at 37 °C were used instead of SOC I medium. 5.2.1.7. Plasmid purification from bacteria The day following E. coli strains transformation, the bacteria were incubated overnight in LB medium with the appropriate antibiotic depending on the vector used (kanamycin for the cloning and ampicillin for the expression vector). After, the plasmids were purified using the NucleoSpin® Plasmid kit (Macherey-Nagel) and their concentration and purity were measured 43 with the Nano Drop 2000c Spectrophotometer (ThermoFisher Scientific), using the NanoDrop2000 software. After purification of the plasmids and bacterial gene fragments, the samples were sequenced to compare the DNA sequences of the samples with the theoretical sequences. For sequencing the genes cloned in the pD plasmid, were used primers T7 and Sp6 while for genes cloned in the p7 vector, primers T7 and T7ter were employed. 5.2.1.8. Expression screening After confirmation of the gene sequences cloned in the p7 vector, BLR (DE3) cells harboring the p7 :: ELRs constructs were grown in expression medium (55.85 g/L TB medium, 8 mL/L glycerol and 100 µL of defoamer, 5 mL of the expression medium in a 50 mL falcon) with 5 µL of ampicillin at 37 °C and 250 rpm overnight. The negative control consisted in BLR (DE3) cells harboring the p7 :: ELRs constructs grown in 5 mL of TB medium and 5 µL of ampicillin. 5.2.1.9. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) To assess if the ELRs proteins were expressed, their expression pattern was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a 10 % resolving gel. Samples were prepared by adding 20 µL of cells previously centrifuged at 11000 rpm for 1 min and 5 µL protein loading buffer 5X to an Eppendorf tube. The samples were then boiled for 5 min at 100 °C and were then centrifuged for 5 min at 13400 rpm. The samples and the protein weight marker (Unstained Protein Molecular Weight Marker (ThermoFisher Scientific) in Figure 5-3) were loaded into the polyacrylamide gel and the electrophoresis was ran in a “MiniVe vertical electrophoresis system” from Hoefer (Amersham Pharmacia Biotech) using the following conditions: 300 V (variable) and 25 mA (constant) for 80 – 120 min. 44 To form the resolving gel, the total percentage of acrylamide (% T) was chosen according to the molecular weight of the ELRs to be separated, as shown in Table 5-4. Table 5-4 — Correspondence between target size range and percentage in separation gel Target size range (kDa) % T in separation gel 24 - 205 7.5 14 - 205 10 14 - 66 12.5 14 - 45 15 Furthermore, the resolving and stacking gel compositions are illustrated in Table 5-5. Figure 5-3 — Unstained Protein Molecular Weight Marker. Adapted from [106]. 45 Table 5-5 — SDS-PAGE resolving and stacking gel composition (one gel) One gel Resolving gel Stacking gel 7,5 % 10 % 12 % 15 % 4 % Type I H2O 4.10 mL 3.63 mL 3.25 mL 2.69 mL 1.585 mL Tris 1.5 M (pH = 8.8) 1.88 mL 1.88 mL 1.88 mL 1.88 mL - Tris 0.5 M (pH = 6.8) - - - - 0.625 mL Acrylamide 40 % 1.40 mL 1.875 mL 2.25 mL 2.81 mL 250 µL SDS 10 % 75 µL 75 µL 75 µL 75 µL 18.75 µL APS 10 % 37.5 µL 37.5 µL 37.5 µL 37.5 µL 18.75 µL TEMED 3.75 µL 3.75 µL 3.75 µL 3.75 µL 2.35 µL Total Volume 7.5 mL 7.5 mL 7.5 mL 7.5 mL 2.5 mL The resolution gel used was 10 % since the SKS-IKVAV protein is 75946 kDa and the SKSPPFLM protein is 76980 kDa. Following the termination of SDS-PAGE, the gel was stained with copper chloride solution (0.3 M) for 10 min with constant agitation to visualize the proteins and the expression pattern (protein bands) was visualized on a Bio-Rad Gel Doc EZ Imager, using the Image Lab™ software. 5.2.1.10. Glycerol stock preparation After analyzing the expression screening results, the clones that best expressed the ELRs of interest were selected and conserved by preparing glycerol stocks. The positive colony was grown in 5 mL LB medium supplemented with 1 % glucose and 1 % of ampicillin at 37 °C and 250 rpm until reaching an optical density at a wavelength of 600 nm (OD600nm) of 0.6 – 0.8. Subsequently, 0.9 mL of these bacterial suspensions and 0.1 mL of 80 % sterile glycerol were added to a cryovial. The glycerol stock was then conserved at – 80 °C to ensure total preservation. 46 5.2.2. Bioproduction of SKS-IKVAV and SKS-PPFLM polymers The polymer ELRs were bioproduced using two distinct methods. The SKS-IKVAV protein was produced in Erlenmeyer flasks, while the SKS-PPFLM protein was produced in a fermenter. Initially two pre-inoculums were prepared, each in 5 mL of LB medium supplemented with ampicillin, 250 µL of glucose 20 %, and the corresponding transformed cells were grown at 37 °C overnight with constant agitation (250 rpm). After this period, two subcultures were prepared using 30 mL of LB medium, 30 µL of ampicillin, 1.5 mL of glucose 20 % and 100 µL of corresponding pre-inoculum in each. Then, each inoculum was prepared in two 2 L Erlenmeyer flasks, each with 15 mL of subculture, 500 mL of LB-Agar medium, 1 % antibiotic and 1 % glucose. The inoculums were then grown at 37 °C with constant agitation (250 rpm) for 2 h. After the 2 h incubation period, the SKS-IKVAV inoculum was used to inoculate 2 L Erlenmeyer flasks, each with 500 mL of fermenter medium (final volume of 8 L). The SKS-PPFLM protein was produced using a 20 L bioreactor (Applicon bioreactor (ADI 1025, ADI 1032, ADI 1010, USA)). After the 2 h incubation period, the SKS-PPFLM inoculum was used to inoculate the fermenter, which had 10 L of TB medium with glycerol and defoamer and 4 L of LB medium (final volume of 15 L). The higher volume of TB medium in the fermenter medium was justified due to the TB richer nutritional composition when compared to the LB medium. To prepare the negative controls, the inoculums were grown each in 5 mL of TB medium supplemented with ampicillin at 37 °C with constant agitation (250 rpm) overnight. The optimal culture conditions used for SKS-IKVAV protein expression was 37 °C and 250 rpm and for SKS-PPFLM was 37 °C, 500 rpm, 20 L/min air flow and pH 7.0, having both productions been left overnight to induce the maximum of protein expression. The bacterial growth in the fermenter and Erlenmeyer flasks was monitored through absorbance measurements. If the peak of absorbance (600 nm) was reached (two contiguous measurements with similar OD600nm values or the second measure was lower than the first), the bioproduction was immediately stopped. 47 5.3. Results and discussion The first part of this dissertation, developed at TPNBT, was focused on the synthesis of SKS-IKVAV and SKS-PPFLM genes. After gene synthesis, the expression of these proteins was tested. 5.3.1. Genetic engineering of SKS-IKVAV and SKS-PPFLM genes The synthesis of the recombinant SKS-IKVAV and SKS-PPFLM genes was carried out in parallel at each step of the process in order to optimize resources. 5.3.1.1. Cloning vector pDrive All Initially it was necessary to obtain cloning plasmids to insert the first SKS-IKVAV/SKS-PPFLM gene fragment. For this purpose, pD plasmids were inserted in XL1-Blue subcloning competent cells and the concentration and purity of pD plasmid samples were analyzed after their purification from bacteria (Table 5-6). Table 5-6 — Concentration (ng/µL) and degree of purity of pD plasmids after purification All samples presented high DNA concentration and purity. However, for the first insertion of the gene fragments, the sample pD 2 was selected due to its higher DNA concentration. Afterwards, pD 1, 2 and 3 underwent a fast digestion with the Ear I endonuclease and an analytical agarose gel (1 %) electrophoresis was performed to verify if the characteristics bands of the plasmid were obtained after Ear I digestion. The experimental result (Figure 5-4 A)) were Sample Concentration (ng/µL) Purity (260nm/280nm) pD 1 1175.9 1.88 pD 2 1200.3 1.89 pD 3 493.5 1.89 48 compared with the theoretical restriction map of the pD vector obtained with the Ear I restriction enzyme (Figure 5-4 B)). Analyzing the results of the electrophoresis, no bands were detected after digestion with the Ear I enzyme for samples pD 1 and pD 3. These results were most likely related with pipetting problems on the agarose gel (the volume of sample used was not enough for the bands to be noticeable). However, since the use of sample 2 was foreseen (higher DNA concentration) and the restriction map obtained for this sample agreed with the bands expected, the experiment was not repeated for pD1 and pD3 samples. The three samples (pD 1, pD 2, and pD 3) were sequenced with the appropriate primers of pD vector (T7 and Sp6) and the results were analyzed with the SnapGene® software, having been concluded that, due to the irrelevant quantity of mismatches and gaps presented in their sequence, the three samples would be suitable for use in future experiments. Since a confidentiality agreement has been signed with TPNBT, the sequenced samples were not presented in this master dissertation. The pD 2 plasmid was linearized using Sap I endonuclease (Figure 5-5 A)). The digestion of the plasmid was compared to the theoretical restriction map obtained using the SnapGene Figure 5-4 — A) Analytical agarose gel (1 %) electrophoresis of pD 1, pD2 and pD3 vectors digested with Ear I endonuclease (Image Lab™). B) Theoretical restriction map of pD vector digested with Ear I (SnapGene®). 49 software. After confirmation of vector linearization, the band corresponding to the linearized pD vector was excised (Figure 5-5 B)). Despite the band corresponding to the linearized pD vector was present in the experimental agarose gel, another band was observed between 2000 – 3000 bp. This band indicated that the linearization was not complete, corresponding to one of the isoforms of the non-linearized vector. Since a good concentration of linearized vector was obtained, the restriction with Sap I enzyme was not repeated. After DNA purification, the DNA concentration and purity of the sample were determined (Table 5-7). Table 5-7 — Concentration (ng/µL) and degree of purity of linearized pD 2 vector It is important to note that the purity (260nm/280nm = 1.67) of the plasmid was not within the reference values (1.8 - 2.00). However, previous laboratory experience indicated that this value was not reliable for DNA obtained after a purification from an agarose gel. Moreover, data Sample Concentration (ng/µL) Purity (260nm/280nm) pD 2 47.7 1.67 Figure 5-5 — A) Preparative agarose gel (1 %) electrophoresis of pD 2 vector digested with Sap I endonuclease (Image Lab™). B) Excision of the band corresponding to the linearized pD2 vector (Image Lab™). C) Theoretical restriction map of pD vector digested with Sap I (SnapGene®). 50 showed that this degree of purity did not affect the ligation and cloning steps of SKS-IKVAV and SKS-PPFLM gene fragments. Thereafter, the plasmid pD 2 underwent a DNA dephosphorylation and was further used for cloning purposes. 5.3.1.2. SKS-IKVAV and SKS-PPFLM inserts The linearized pD 2 plasmid was then used to clone the SKS-IKVAV and SKS-PPFLM DNA inserts previously obtained with Ear I endonuclease. The cloning strategy was based on compatible sticky ends obtained by using the Sap I enzyme for the pD plasmid and the Ear I for the ELRs inserts. For the preparation of the ELRs inserts, XL1-Blue subcloning competent cells were transformed with the commercial plasmids containing one insert of the SKS-IKVAV or with one insert of the SKS-PPFLM gene. Afterwards, four positive transformants of each ELR were selected, the plasmids were purified, and their concentration and purity were assessed (Table 5-8). Table 5-8 — Concentration (ng/µL) and degree of purity of commercial plasmids with the SKS-IKVAV or SKS-PPFLM insert Sample Concentration (ng/µL) Purity (260nm/280nm) Commercial plasmid :: SKS-IKVAV 1 576.1 1.89 Commercial plasmid :: SKS-IKVAV 2 820.2 1.89 Commercial plasmid :: SKS-IKVAV 3 658.9 1.86 Commercial plasmid :: SKS-IKVAV 4 595.2 1.89 Commercial plasmid :: SKS-PPFLM 1 738.6 1.89 Commercial plasmid :: SKS-PPFLM 2 626.1 1.86 Commercial plasmid :: SKS-PPFLM 3 5.6 2.47 Commercial plasmid :: SKS-PPFLM 4 288.0 1.90 All samples presented good purity and DNA concentration with the exception of sample commercial plasmid :: SKS-PPFLM 3. Commercial plasmid :: SKS-IKVAV 2 and commercial plasmid::SKS-PPFLM 1 were selected for ELR insert purification. 57 Table 5-12 — Concentration (ng/µL) and degree of purity of p7 plasmid, SKS-IKVAV 6x and SKS-PPFLM 6x genes after purification Sample Concentration (ng/µL) Purity (260nm/280nm) p7 1 32.2 1.75 p7 2 19.5 1.53 SKS-IKVAV 6x gene 14.9 1.77 SKS-PFLM 6x gene 17.4 1.99 After cloning of the SKS-IKVAV 6x and SKS-PPFLM 6x genes into the expression plasmids, several positive transformants of XL1-Blue competent cells were obtained for both genes and were analyzed by analytical agarose gel (1 %) electrophoresis after a fast digestion with the EcoR I and Nde I enzymes (Figure 5-10). Figure 5-10 — Analytical agarose gel (1 %) electrophoresis of A) p7 :: SKS-IKVAV 6x and B) p7 :: SKSPPFLM 6x. The DNA samples were digested with EcoR I and Nde I endonucleases (Image Lab™). C) Theoretical restriction map of p7 :: SKS-IKVAV 6x and p7::SKS-PPFLM 6x vectors digested with EcoR I and Nde I (SnapGene®). 58 The results obtained in Figure 5-10 confirmed the successful cloning of the SKS-IKVAV 6x and SKS-PPFLM 6x genes in the p7 expression vector. The gene sequences were further confirmed (data not shown) and the plasmids were used to transform E. coli strain suitable for protein expression. 5.3.2. Bioproduction of SKS-IKVAV and SKS-PPFLM polymers The expression strain E. coli BLR (DE3) was transformed with the p7 vectors harboring the SKS-IKVAV 6x and the SKS-PPFLM 6x ELRs genes. Four transformants of each gene were selected and the expression of the two proteins was performed in expression medium. The expression of SKS-IKVAV 6x (75946 Da) and SKS-PPFLM 6x (76980 Da) proteins was further evaluated in a SDS-Page (10 %) proteins (Figure 5-11). Figure 5-11 — SDS-Page (10 %) of SKS-IKVAV and SKS-PPFLM proteins (expression screening). The gel was stained with cooper chloride solution (0.3 M) for visualization (Image Lab™). The inoculum grown overnight in TB medium and ampicillin served as the negative control. 59 Analyzing the results, both proteins were expressed. A band near the theoretical molecular weight (75.95 kDa for SKS-IKVAV 6x and 76.98 kDa for SKS-PPFLM 6x) was observed for all the tested transformants. However, the transformant for the two proteins that presented a higher expression level was further selected for the production of the SKS-IKVAV and SKS-PPFLM. To reduce the time required to obtain the recombinant polymers, the SKS-IKVAV protein was produced in Erlenmeyer flasks and the SKS-PPFLM protein was produced in a fermenter. The bioproduction of SKS-IKVAV was terminated after the decrease of the OD600nm from 13.48 to 10.91. The Table 5-13 presents the fermentation conditions for the SKS-PPFLM. Table 5-13 — Fermentation conditions for the SKS-PPFLM protein Production samples pH Temperature (°C) Agitation (rpm) Base added (mL) OD600nm SKS-PPFLM 1st 7 37 499 13 mL 9,05 2nd 7 37 499 - 11,596 3rd 7 37 499 - 13,21 4th 7 37 499 - 11,746 After to confirm the success of the bioproduction, SDS-Pages (10 %, Figure 5-12 and 5-13) were performed with the samples collected over time and heated after production. The latter correspond to samples in which purification based on the Tt of the ELRs produced was attempted. Figure 5-12 — SDS-Page (10 %) of SKS-IKVAV protein after purification. The gel was stained with cooper chloride solution (0.3 M) for visualization (Image Lab™). 60 Since no SDS-Page was performed on the SKS-IKVAV samples after production, it is not possible to determine which method produced the greater concentration of protein. Nonetheless, due to the noticeable width of the band corresponding to the SKS-PPFLM protein in the gel in the previous figure, it is denoted that the fermenter protocol is suitable for the production of this specific protein. It is expected that if the solution containing the ELRs is heated to a temperature above the Tt , the recombinamer will be present in the pellet; otherwise, if the solution is chilled below the Tt , the protein will be dissolved in the supernatant. At temperatures below Tt , the SKS-IKVAV protein is observed in the supernatant but not in the pellet. Furthermore, at 4 °C, the protein is more noticeable in the solution's supernatant while remaining absent from the pellet. In both the supernatant and the pellet, the SKS-IKVAV protein is undetectable at temperatures above Tt . The previously reported smart behavior was also observed for the SKS-PPFLM protein, this polymer presented in the solution supernatant at temperatures below Tt and not in the pellet. Similarly, the protein was present in the pellet of the heated solution above Tt , and it was also present, although in lower concentration, in the heated supernatant. Figure 5-13 — SDS-Page (10 %) of SKS-PPFLM protein production in the fermenter and purification. The gel was stained with cooper chloride solution (0.3 M) for visualization (Image Lab™). The inoculum grown overnight in TB medium and ampicillin served as the negative control. 61 5.4. Conclusions Regarding the genetic engineering tools used in this section of the dissertation project, it is concluded that the development of the recombinant SKS-IKVAV and SKS-PPFLM genes was successful since the iterative-recursive method allowed for easy clustering of inserts to create the final genes. It is proposed in future projects to optimize the production of both ELRs and, although the promising results, the complete purification of the two ELRs produced was not possible during my ERASMUS. Therefore, it is imperative to develop an optimized purification protocol for each recombinant protein to obtain the ELRs polymers and proceed with their characterization, scaffold manufacturing, and ultimately conclude their utility as biomaterials for skin TE solutions. It is recommended that the purification protocol be based on the Tt of the ELRs polymers, with the protein solubility being altered by varying the salt concentration, acidity, and temperature of the solution, due to the promising results obtained. 62 6. EXPLORING STAR PROTEIN FOR SKIN TISSUE ENGINEERING The use of STAR protein in the second part of this dissertation for the development of skin TE applications is based on its similarity with proteins from the ELR family. Since the genetic engineering process involved in the development of the STAR protein was already developed and the expression confirmed, this section of the dissertation was focused on the optimization of STAR expression and in the development and characterization of STAR-based films for skin TE applications. 6.1. Materials The materials used for the production and purification of the STAR polymer and for the preparation and characterization of STAR-based films are below. The aa composition of STAR will not be disclosed in this dissertation for the sake of intellectual property protection. 6.1.1. Chemical reagents The chemical reagents employed are listed in Table 6-1. Table 6-1 — List of chemical reagents employed Reagent and Abbreviation Brand Acetic acid Sigma Aldrich Acrylamide Plus one Ammonium sulphate Sigma APS Biorad Coomassie Brilliant Blue G-250 Merck Dialysis membrane with a 14 kDa cutoff Sigma Aldrich Dimethyl sulfoxide (DMSO) Sigma Distilled water 63 D - glucose Sigma DTT Sigma Dulbecco's modified Eagle’s medium (DMEM) Sigma Aldrich D(+) Lactose monohydrate Panreac Ethanol Merck EDTA Sigma Fetal bovine serum (FBS) 5 % Sigma Aldrich Glycerol Fisher Bioreagents Hydrochloric acid Fisher Scientific Hygromycin Sigma Aldrich Kanamycin Fisher Chemical L - glutamine Sigma Medium 199 Sigma Aldrich Methanol Carlo Erba Penicillin Sigma Aldrich Phenylmethylsulfonyl fluoride Sigma Phosphate buffered saline (PBS) Biochrom Potassium phosphate dibasic trihydrate Sigma Potassium dihydrogen phosphate Panreac Potassium phosphate dibasic Sigma Sodium bicarbonate Aldrich SDS Biorad Sodium hydroxide Sigma Streptomycin Sigma Aldrich TEMED Biorad Tris Panreac Trypan Blue Sigma Trypsin Aldrich Tryptone Grisp Type I water Yeast extract Grisp β-Mercaptoethanol Sigma Aldrich 64 6.1.2. Culture media for E. coli growth The culture media used for E. coli growth include Luria-Broth (LB) (Lennox, Grisp) 20 g/L and Terrific Broth Autoinduction medium (TB AIM) (Grisp) 35 g/L. The TB manual medium consisted of yeast extract (24 g/L), tryptone (12 g/L), glycerol (5.04 g/L) and 1X solution of K2HPO4 (12.54 g/L) and KH2PO4 (2.31 g/L) dissolved in 1 L distilled water. 6.1.3. In vitro culture medium for immortalized human fibroblasts The BJ-5ta cell line (normal human skin fibroblasts immortalized by overexpression of telomerase) was maintained according to American Type Culture Collection (ATCC) recommendations, i.e., four parts of DMEM (powder) containing 4 mmol/L-glutamine, D-glucose, 4.5 g/L sodium bicarbonate and 1 part of medium 199, supplemented with 5 % (v/v) of FBS, 10 µg/mL hygromycin and 1 % (v/v) of penicillin/streptomycin solution. The cell viability assay was performed with the MTS (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide) cell assay kit (Promega). 6.1.4. Buffer solutions The buffer solutions used are listed in Table 6-2. Table 6-2 – List of buffer solutions Buffer solution Composition or supplier Protein loading buffer 5X 0.3 M tris pH 6.5, 10 % (w/v) SDS, 50 % (v/v) glycerol, 25 % (v/v) β-mercaptoethanol, 2 % (v/v) BPB SDS-PAGE running buffer 25 mM tris-base pH 8.3, 192 mM glycine and 0.1 % (w/v) SDS Tris-EDTA (TEDTA) 50 mM tris pH 8, 1 mM EDTA 65 6.2. Methods The methods used in this section of the dissertation are described further below. 6.2.1. Optimization of STAR polymer production The STAR protein was produced using several media and culture conditions in order to optimize protein expression. Previously, E. coli BLR (DE3) cells transformed with the expression plasmid containing the STAR recombinant gene were incubated for 8 h in 10 mL of LB medium supplemented with 10 µL of kanamycin at 37 °C and 180 rpm. Then, LB medium was added to a final volume of 100 mL and the bacteria were incubated overnight at 25 °C at 180 rpm. The pre-inoculum was centrifuged at 6000 rpm, 4 °C for 10 min and the cell pellet was resuspended in 20 mL of TB manual medium. The cell suspension was then used to inoculate different culture media to an OD600nm of 0.1, and the cells were incubated for 24 h at 37 ºC with constant agitation to induce the expression of STAR protein (Table 6-3). The OD600nm of the samples of each condition was measured at specific fermentation time-points (elapsed fermentation time, EFT) in order to assess the growth of E. coli . Table 6-3 — Inoculum media and conditions tested to assess the optimal protocol for the production of Star protein Type of media Agitation of incubation (rpm) Ratio (flask capacity/culture media volume) Induction TB AIM 180 1:5 Autoinduction LB 180 1:5 Lactose induction at an OD600nm 0.6 – 1.5 TB manual 180 1:5 Lactose induction at an OD600nm 0.6 – 1.5 LB 250 1:10 Lactose induction after 8 h EFT TB manual 250 1:10 Lactose induction after 8 h EFT TB manual 250 1:5 Lactose induction after 8 h EFT TB AIM 250 1:10 Autoinduction 66 6.2.2. STAR polymer purification The STAR polymer was purified using the salting-out effect, which is the precipitation of a protein due to a decrease in its solubility caused by the addition of high concentrations of salt. Ammonium sulfate was used as the salt because it is a preferred reagent for salting-out due to its much higher solubility than any of the phosphate salts [107]. After fermentation, the cells were harvested by centrifugation at 6500 rpm for 10 min at 4 °C and the pellet was resupended in 55 mL of TEDTA with 1 % of protease inhibitor (phenylmethylsulfonyl fluoride). Then, the cell suspension was sonicated (30 min with 3 s ON, 9 s OFF cycles) and placed at 4 °C overnight. The next day, the lysate was centrifuged at 8000 rpm for 20 min at 4 °C and the soluble and the insoluble fractions were collected separately. The soluble fraction was incubated in ice with constant agitation (250 rpm) and ammonium sulphate was added to a final concentration of 30 % to promote protein precipitation. The suspension was then centrifuged at 8000 rpm for 20 min at 4 °C and the pellet was resuspended in cold distilled water at 4 °C overnight. The protein solution was dialyzed against distilled water for 4 days using a dialysis membrane with a 14 kDa cutoff. The pure STAR protein was then freeze dried for 6 days and kept in an exsiccator until further use. 6.2.2.1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) The expression of the STAR protein was verified by SDS-PAGE with a running gel of 12.5 % and stacking gel of 4 %. The samples were prepared with 5X loading buffer, denatured at 100 °C for 5 min, and were loaded onto the gel. The samples were run with variable voltage and constant amperage (20 mA per gel). The gels were stained with Coomassie Blue solution (methanol 50 % (v/v), acetic acid 10 % (v/v), Coomassie Brilliant Blue G-250 2.5 g/L) for 40 min followed by an incubation in distaining solution (methanol 15 % (v/v), acetic acid 10% (v/v)) for 1.5 h. 73 After 2 h of incubation, there were no perceptible differences in E. coli growth. However, after 4 and 6 h of incubation, the TB manual 1:10 had the highest OD600nm values (3.46, 5.55), followed by LB 1:10 (3.27, 4.40) and TB manual 1:5, 250 rpm (3.17, 4.44), respectively. After the incubation, the TB manual 1:5 medium, 250 rpm sample presented the highest OD600nm value (7.37). Generally, the culture medias that presented higher E. coli growth throughout the incubation period were TB manual 1:5, 250 rpm and TB manual 1:10. Samples collected after 8 h (before induction; LB 1:5 and TB manual 1:5 at 4 h) and 24 h of inoculum incubation were analyzed by SDS-Page (12.5%, Figure 6-2) to confirm the correlation between bacterial growth and protein production. Figure 6-1 — Optical density (600 nm) measured for specific elapsed fermentation time-points for the culture conditions evaluated. 74 Given that the STAR protein has a molecular weight at around 41000 Da, it was observable that the LB 1:5 and the TB manual 1:5 conditions did not produce STAR protein after 4 h of incubation, which was expected since the induction had not yet happened. However, after 8 h (Figure 6-2 A)), the referred conditions produced STAR protein due to the lactose induction initiated at EFT = 4 h. The highest productions were verified after 8 h and 24 h of production in TB manual 1:5, 250 rpm, and TB manual 1:10 conditions. Figure 6-2 – SDS-Page (12.5 %) of STAR protein expression samples A) after 8 h of inoculum’s incubation and TB manual 1:5 after induction and B) after 24 h of inoculum’s incubation and TB manual 1:5 after induction. Gel stained with Coomassie Blue. 75 Comparing the highest bacterial productions and the conditions that allowed for higher protein expression, the TB manual 1:5, 250 rpm, was selected for express the STAR protein. 6.3.2. STAR-based films The STAR-based films were obtained by solvent casting of STAR solutions in circular Teflon molds. DTT was added to some solutions to prevent the formation of intramolecular and intermolecular disulfide bonds between cysteine residues of this protein, helping in the filming properties of STAR [109]. Afterwards, some of the films obtained were exposed to a saturated methanol atmosphere to promote their insolubility. The effect of protein concentration, presence of DTT and insolubilization with methanol on the macroscopic properties of the STAR-based films were evaluated (Figure 6-3). Regardless of the condition, all the films were macroscopically translucent, with opaque regions on the periphery of the film. This could be due to the presence of some impurities in the solution and was more noticeable for the highest concentrations of STAR protein (5 %). Therefore, the protein content of the films was the property that most influenced their macroscopic appearance because the film after being physically crosslinked with methanol maintained the same macroscopic structure. In addition, due to the low concentration of DTT, the reducing agent had no influence on the macroscopic structure of the films. Figure 6-3 – Macroscopical image of a 3 % STAR-based film, a 5 % DTT STAR-based film and a 5 % DTT methanol STAR-based film. 76 6.3.3. STAR-based films characterization The developed STAR-based films were characterized in terms of their physical and chemical properties, morphology, and cytotoxic potential. 6.3.3.1. Thickness measurements The macroscopic structure of a TE device (films, scaffolds…) affect both the mechanical properties and the cell response [112]. Thus, the thickness of the STAR-based films was measured (Table 6-5) to conclude the effect of the different compositions on the films’ threedimensional structure. Table 6-5 — Thickness measurements of the STAR-based films with the MPO Dualscope Thickness Gauge (Fischer). STAR-based films Thickness (µm) 3 % 3.9 ± 2.3 3 % DTT 5.7 ± 3.4 3 % methanol 10.3 ± 1.6 3 % DTT methanol 7.7 ± 2.6 5 % * 7.8 ± 2.9 5 % DTT * 16.3 ± 7.1 5 % methanol * 308.0 ± 259.7 5 % DTT methanol * 170.5 ± 224.6 *P<0.05 The difference in thickness between 3 % and 5 % films was expected based on the higher protein concentration in the latter. In contrast, the increase of the films thickness after methanol physical cross-liking was not predicted. Furthermore, with the addition of DTT, there was an increase in thickness, which could be attributed to a change in the chemical structure of the films. Comparing the unmodified films with those with DTT or physically crosslinked with methanol, only statistically significant differences existed between the films 5 % vs. 5 % methanol, 77 5 % vs. 5 % DTT methanol, 5 % DTT vs. 5 % DTT methanol, and 5 % methanol vs. 5 % DTT methanol. As a result, for the 5 % films, the addition of DTT or physical cross-liking with methanol causes a significant increase in film thickness. The increase in SD values across the table reflects the increasing difficulty encountered during thickness measurement in keeping the films' surface flat, which was related to the concave surface that the films presented due to the extraction process of the films from the respective molds. Furthermore, the surface of the films had a limited diameter in comparison to the thickness gauge, making thickness measurement in different areas of the surface challenging. 6.3.3.2. Contact angle The surface wettability of a material is determined by its surface roughness and chemical composition, and the study of this property determines whether the material is suitable for a particular application. Because differential adsorption is dependent on surface hydrophobicity in TE applications, adequate wettability allows for optimal adhesion or cell release, ensuring the material's biofunctionality [113-115]. Because contact angle is the main parameter used to determine the wettability of a surface [113], the surface wettability of the STAR-based films was measured. Only the films physically crosslinked with methanol were assessed (Table 6-6), as the others dissolved in contact with type I water. Table 6-6 — Contact angle of the STAR-based films performed at room temperature with type I water as test liquid STAR-based films 0’’ 15’’ 30’’ 45’’ 1’ 3 % methanol (93.3 ± 7.4) ° (101.8 ± 11.8) ° (104.9 ± 10.7) ° (115.2 ± 4.6) ° (113.6 ± 9.3) ° 3 % DTT methanol (111.5 ± 14.6) ° (126.6 ± 19.8) ° — — — 5 % methanol (102.8 ± 32.4) ° (129.4 ± 25.4) ° (128.9 ± 26.3) ° (99.7 ± 0.0) ° (99.7 ± 0.0) ° 5 % DTT methanol (101.3 ± 5.7) ° (101.7 ± 7.8) ° (106.8 ± 9.2) ° (111.6 ± 14.8) ° (111.2 ± 18.5) ° Since all contact angles obtained are greater than 90 °, it can be stated that the STARbased films physically crosslinked with methanol had a hydrophobic surface [116]. The surface hydrophobicity of the 3 % methanol and 5 % methanol films increased in the first 15’’ and 45’’ 78 after droplet deposition, respectively, and decreased until 60". Furthermore, the contact angle values for the 5 % methanol films were very similar at 0’’ and 60’’. Moreover, the hydrophobicity of the 3 % and 5 % DTT methanol films increased over time. The changes in contact angle values over time were most likely caused by structural rearrangement of the molecules in the films in contact with water. Because the 3 % and 5 % films were physically crosslinked with methanol under the same conditions, the higher hydrophobic character of the 5 % samples might be related with the higher STAR content. The addition of DTT to the 3 % STAR-based films resulted in an increase in hydrophobicity. However, the opposite was observed for the 5 % films, even though the increase in the hydrophilic character with the addition of DTT could be disregarded as it is not statistically significant. 6.3.3.3. Swelling degree and in vitro degradation The swelling of materials in the presence of fluids is essential to enable absorption and transfer of nutrients or metabolites throughout the film structure in a physiological environment and can be measured through the swelling degree [117]. The ability of a structure to absorb a specific fluid, which is primarily determined by its hydrophobicity and microstructure, influences cell adhesion and proliferation, as well as its mechanical integrity and properties, because biomaterials used in TE are biodegradable. In addition, as water is the primary constituent of human body fluids, it is critical to assess the degradation of a biomaterial caused by contact with water – in vitro degradation [117]. The swelling degree and the in vitro degradation of STAR-based-crosslinked with methanol are shown in Table 6-7. The swelling degree was evaluated at 37 °C using distilled water or culture media, and the in vitro degradation was determined with the samples used to evaluate the swelling degree in distilled water. 79 Table 6-7 — Swelling degree performed at 37 °C for 24 h with distilled water and culture media and in vitro degradation of the STAR-based films STAR-based films Swelling degree (%) In vitro degradation (weight loss %) Distilled water Culture medium 3 % methanol 174.4 ± 24.0 60.0 13.7 ± 14.7 3 % DTT methanol 137.6 ± 1.3 47.6 17.7 ± 2.0 5 % methanol 94.4 ± 7.9 82.6 12.4 ± 1.3 5 % DTT methanol 84.7 ± 27.3 53.3 5.9 ± 0.2 The swelling degree and the in vitro degradation were only determined for the samples incubated in methanol because all the other samples dissolved when incubated in water. Comparing the swelling degree results in distilled water, the highest value was obtained for the 3 % films. This was related with the lower protein content of these films, which led to a less compaction in the polymeric matrix during the methanol physical crosslinking, resulting in a higher capacity to absorb water. The 3 % methanol films also exhibited the highest degradation profiles. When the STAR-based films were incubated in cell culture medium a decrease on the swelling degree was observed for all the conditions. This might have been due to the presence of proteins and other bioactive substance in the culture media. 6.3.3.4. Microstructural morphology The microstructural morphology of TE solutions is primarily responsible for the mechanical properties of a TE solution. Thus, it should resemble the native tissue to be regenerated in order to meet physiological mechanical needs and ensure a favorable environment for cell adhesion, proliferation, and differentiation [118]. SEM was used to examine the microstructural morphology of the STAR-based films at surface and cross-sections (Figures 6-4 and 6-5). 80 The 3 % STAR-based films presented more surface irregularities than the 5 % STAR-based films. Figure 6-5 — SEM micrographs of 3 % STAR-based films: A) general view of the film surface and B) cross-section view. Figure 6-4 — SEM micrographs of 5 % STAR-based films: A) general view of the film surface and B) cross-section view. 81 Figures 6-6 and 6-7 show the microstructural morphology of the 3 % DTT and 5 % DTT films. Figure 6-6 — SEM micrographs of 5 % DTT STAR-based films: A) general view of the film surface and B) cross-section view. Figure 6-7 — SEM micrographs of 3 % DTT STAR-based films: A) general view of the film surface and B) cross-section view. 82 Although the surface roughness of the 5 % films increased slightly with the addition of DTT, the surface of the 3 % DTT presented more clusters of protein aggregates and less clefts. Nonetheless, the addition of DTT did not considerably alter the microstructure of the films. The surface of the films crosslinked with methanol are presented in Figures 6-8 and 6-9. Figure 6-8 — SEM micrographs of 3 % methanol STAR-based films: A) general view of the film surface, B) cross-section view and C) EDS image highlighting the crystals formed on the film’s surface. 89 Analyzing the previous graphs, it was concluded that the degradation products and leachable of the STAR-based films did not interfere with the viability of the BJ-5ta cells. The minimal decrease of cells observed after 48 h of incubation in were due to the build-up of toxic Figure 6-14 — BJ-5ta cell viability at 48 h of incubation with pre-conditioned culture media. Figure 6-15 — BJ-5ta cell viability at 24 h of incubation with pre-conditioned culture media. 90 metabolites and decrease of nutrients and oxygen. In addition, the increase denoted in some STAR-based films conditioned media corresponded to the characteristic growth of the used cells. As a result, the indirect contact assay allowed the assessment of the degradation products and leachable of the films as inert to BJ-ta5 cells. 6.4. Conclusions The optimization of STAR protein expression and the development and characterization of STAR-based films as a new approach for the development of skin TE applications was successfully achieved in the second part of this dissertation. The expression of STAR protein was increased using the following conditions: TB manual medium with a 1:5 flask capacity/culture media volume ration, constant agitation (250 rpm), 37 °C for 24 h. Two sets of STAR-based films were obtained using different protein concentrations (3 % and 5 %). The films were translucent and fragile, particularly the samples with 3 % of protein content, and presented a concave curvature caused by the demolding process. The influence of DTT and methanol on film formation and film properties was evaluated. The amount of protein and the incubation with methanol influenced the thickness of the films. The films with 5 % presented greater thickness when compared to the 3 % films, and the same was observed for the films incubated with methanol when compared with the films without methanol. After measuring the wettability, the films proved to be mainly hydrophobic with contact angles higher than 93.3 ± 7.4 °. Despite the ideal contact angle value for skin TE applications in not yet disclosed, the literature indicates that surfaces with contact angles ranging between 60 ° to 80 ° promote cell adhesion and growth [114]. However, the contact angle measured in vivo on the skin in areas with low sebaceous lipids is between 91 ° and 102 °, which is comparable to the values obtained for the STAR-based films. Hence, although hydrophilic surface materials are generally preferred, since skin contact angles range from 57 ° to 102 °, hydrophobic films can also be advantageous for skin TE applications in anatomical areas with lower sebaceous lipid content [123]. In terms of microstructure, the samples revealed a rough surface, which was most noticeable in the 3 % samples. The presence of irregularities was reduced with the addition of 91 DTT, and the physical crosslinking with methanol revealed crystalline clusters on the surface of the films. It should be noted that the roughness displayed by all of the analyzed films is a promising feature when considering a cell-based application, as it provides a favorable environment for cell adhesion. The evaluation of the cytotoxic potential of STAR-based films degradation products and leachable revealed that all the films could be considered as safe for cells. 92 7. INTEGRATED CONCLUSIONS AND FUTURE PROSPECTS The goal of this dissertation was to create scaffolds for Tissue Engineering and Regenerative Medicine in the context of skin regeneration using ELRs. To that end, two synthetic genes were created that corresponded to different recombinant proteins based on the natural sequence of elastin, SKS-IKVAV and SKS-PPFLM. The ELRs were produced, however, the scaffolds were not obtained because the purification protocols for the proteins were not developed due to the limited time of the ERASMUS internship. Because of the high potential of the proteins developed to serve as biomaterials for skin regeneration, the development of purification protocols to assess the properties of SKS-IKVAV and SKS-PPFLM scaffolds is of paramount importance to continue this project. The second part of this dissertation was dedicated to the production optimization of the STAR polymer, a protein with properties similar to the ELRs, and the development of STAR-based films for skin TE. As the genetic engineering process involved in the development of the STAR protein had already been developed and its expression confirmed, an optimized process for producing the protein was developed. The creation of STAR-based films was a proof of concept of the potential of this protein to be used in the formation of biodegradable films with some properties ideal for Tissue Engineering applications. Since the STAR-based films proved to be fragile when handled, the design of scaffolds with this protein could expand its application in the tissue engineering field. Additional, cell adhesion and proliferation assays should be performed to confirm the potential of STARbased films for skin tissue engineering applications. 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