The decellularized human chorion membrane as a new biomaterial: characterization and applications

Laura Passos Morgado Franco FrazãoThe Decellularized Human ChorionMembrane as a New Biomaterial:characterization and applicationsUniversidade do MinhoI3Bs - Instituto de Investigação em Biomateriais, Biodegradáveis eBiomiméticosJunho de 2021 Laura FrazãoThe Decellularized Human Chorion Membrane as a New Biomaterial:characterization and applicationsMinho | 2021U Universidade do Minho I3Bs - Instituto de Investigação em Biomateriais, Biodegradáveis e Biomiméticos Laura Passos Morgado Franco Frazão The Decellularized Human Chorion Membrane as a New Biomaterial: characterization and applications Tese de Doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células Estaminais Trabalho efetuado sob a orientação do Professor Nuno João Meleiro Alves das Neves Junho de 2021 ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição CC BY https://creativecommons.org/licenses/by/4.0/ iii ACKNOWLEDGMENTS This PhD was a long journey, almost impossible to finish without the help, support, and guidance of several people. Not only in the scientific but also in the personal area. I would like to acknowledge the financial support to the Portuguese Foundation for Science and Technology, not only for my individual grant, but also for the project funding which allowed me to conduct the experimental studies. Moreover, I would like to thank to the director of the 3B’s Research Group, for giving me the opportunity to join the research group. I would also like to express my gratitude to my supervisor, Prof. Dr. Nuno M. Neves that accompanied the work developed during these four years with an appeasing attitude when everything seemed to go wrong. All my co-authors were essential in the production of the works presented in this thesis. I would like to thank them all for their valuable insights and for the support in the projects that were developed. I have to thank especially to Dr. Joana Vieira de Castro. Additionally, I would like to acknowledge the professionals of the Hospital of Braga that helped in obtaining the placentas used in this thesis and, without knowing, were crucial for the development of this thesis. To the friends that I made in 3Bs, thank you for your friendship and for making this four-year journey lighter and happier! I cannot express in words how much you helped me with all the beers and fries. But I’m sure it would have been impossible without you! At last, I would like to thank to my family. Thank you for continuing to help me grow, thank you for the love and support, and thank you for telling me the things that are difficult to ear! iv 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. v The decellularized human chorion membrane as a new biomaterial: characterization and applications ABSTRACT Decellularized inner body membranes have been studied for several different tissue engineering applications. However, its availability is limited, except in the case of placental membranes. Placental membranes are composed by the amnion membrane and the chorion membrane. While the human amnion membrane has been widely studied for tissue engineering and regenerative medicine applications, the human chorion membrane was poorly studied, only being subject of research interest in recent years. Envisioning the potential of decellularized human chorion membrane (dHCM) to be used in tissue engineering applications, a decellularization protocol was developed for the human chorion membrane. The dHCM was characterized regarding its composition and mechanical properties. Additionally, the dHCM was used as a substrate to support cell cultures for the creation of a blood brain barrier (BBB) in vitro model, as a surgical patch for the creation of a vascular graft and also for the treatment of congenital diaphragmatic hernias. dHCM showed cytocompatibility with human and mice endothelial cells (EA.hy296, HUVECs, and b.End3), human fibroblasts (MRC5), and mice astrocytes (C8-D1A). The angiogenic properties of the dHCM were also investigated. Moreover, dHCM demonstrated hemocompatibility and antibacterial activity against S. aureus . After in vivo implantation, dHCM integrated well in the host tissue after 28 days when implanted subcutaneously in mice. Nevertheless, when sutured into a pig diaphragm, dHCM was fully integrated by the host tissue after 3 weeks. Altogether, the works developed in this thesis, allowed for a detailed characterization of dHCM for the first time. It was demonstrated that dHCM has unique characteristics that make it a valuable new biomaterial to be use as substrate to support cell cultures and as a surgical patch. Keywords: Chorion membrane; Decellularization; Blood brain barrier; Vascular graft; Diaphragmatic hernia; Biomaterial; Surgical patch vi A membrana do córion humano descelularizada como um novo biomaterial: caracterização e aplicações RESUMO As membranas de órgãos internos descelularizadas têm sido estudadas para diferentes aplicações em engenharia de tecidos. No entanto, a disponibilidade destas membranas é limitada, com exceção das membranas da placenta. A placenta é composta por duas membranas, a membrana amniótica e a membrana do córion. Enquanto a membrana amniótica humana descelularizada foi extensivamente estudada para aplicações em engenharia de tecidos e medicina regenerativa, a membrana do córion só recentemente despertou interesse na comunidade científica para estas aplicações. Para estudar o potencial da membrana do córion humano descelularizada (dHCM) em engenharia de tecidos, foi optimizado um protocolo de descelularização eficiente para esta membrana. A dHCM foi caracterizada em relação à sua composição e propriedades mecânicas. Posteriormente, a dHCM foi utilizada como substrato de suporte ao crescimento de células para a criação de um modelo in vitro da barreira hemato-encefálica, como um enxerto cirúrgico para a criação de enxertos vasculares e também para o tratamento de hérnias diafragmáticas. A dHCM mostrou ser citocompatível com células endoteliais de humano e ratinho (EA.hy296; HUVECs, e b.End3), fibroblastos humanos (MRC5) e astrócitos de ratinho (C8-D1A). As propriedades angiogénicas da dHCM foram investigadas e foi demonstrado que a dHCM é compatível com o sangue humano e tem atividade antimicrobiana contra S. aureus . Após implantação in vivo , a dHCM começou a ser integrada pelo tecido hospedeiro após 28 dias de implantação subcutânea em ratinhos. No entanto, quando suturada ao diafragma de porcos, a dHCM mostrou capacidade de integração no tecido hospedeiro após 3 semanas. Os trabalhos desenvolvidos nesta tese permitiram obter uma caracterização detalhada da dHCM, previamente inexistente na literatura. Adicionalmente, foi demonstrado que a dHCM tem características únicas, tornando-a um novo biomaterial promissor como substrato de suporte ao desenvolvimento de culturas de células e como enxerto cirúrgico. Palavras-chave: Membrana de córion; Descelularização; Barreira hemato-encefálica; enxerto vascular; hérnia diafragmática; biomaterial; enxerto cirúrgico vii TABLE OF CONTENTS ACKNOWLEDGMENTS ................................................................................................................ III STATEMENT OF INTEGRITY ........................................................................................................ IV ABSTRACT .................................................................................................................................. V RESUMO .................................................................................................................................... VI TABLE OF CONTENTS ............................................................................................................... VII LIST OF ABBREVIATIONS .......................................................................................................... XIV LIST OF FIGURES .................................................................................................................... XVII LIST OF SUPPLEMENTARY FIGURES ........................................................................................ XXI LIST OF TABLES ...................................................................................................................... XXII SHORT CURRICULUM VITAE .................................................................................................. XXIII LIST OF PUBLICATIONS ......................................................................................................... XXIV INTRODUCTION TO THE THESIS FORMAT .............................................................................. XXVI SECTION 1 .................................................................................................................................. 1 GENERAL INTRODUCTION ........................................................................................................... 1 CHAPTER I - THE APPLICATION OF DECELLULARIZED INNER BODY MEMBRANES IN TISSUE ENGINEERING: CURRENT STATUS AND FUTURE PERSPECTIVES• ...................................................... 3 Abstract ...................................................................................................................................... 3 I-1. Introduction ................................................................................................................... 4 I-2. Decellularized Inner Body Membranes ............................................................................ 4 I-2.1. Connective Tissue Membranes ............................................................................... 7 I-2.2. Epithelial Membranes ............................................................................................ 9 I-2.3. Placental Membranes .......................................................................................... 11 I-3. The Availability of Inner Body Membranes ..................................................................... 12 I-4. Decellularized Inner Body Membranes in Tissue Engineering ........................................ 13 I-4.1. Cardiovascular applications .................................................................................. 18 I-4.2. Musculoskeletal applications ................................................................................ 23 xiv LIST OF ABBREVIATIONS # ºC – Degrees Celsius µL – Microliter µm – Micrometer µg – Microgram % - Percentage 3D – 3-Dimensional A α-MEM – Alpha-minimum essential medium Abs – Absorbance ANG2 – Angiopoietin 2 ANOVA – Analysis of variance ATCC – American type culture collection B BBB – Blood brain barrier b.End3 – Mouse brain capillary endothelial cell line bFGF – Basic fibroblast growth factor BSA – Bovine serum albumin BM – Basement membrane BMP – Bone morphogenetic protein bp – Base pairs C CaCl2 – Calcium chloride CAM – Chorioallantoic membrane CDH – Congenital diaphragmatic hernia CDHSG – Congenital diaphragmatic hernia study group CFU – Colony forming units cm - Centimeter cm2 – Centimeter square CNS – Central nervous system COL I – Collagen type I CO2 – Carbon dioxide CTR – Control C8-D1A – Mouse cerebellum astrocyte cell line D DAB – 3, 3 -diaminobenzidine DAPI – 4,6-diamidino-2-phenylindole DGAV – Portuguese general directorate of food and veterinary dHAM – Decellularized human amniotic membrane dHCM – Decellularized human chorion membrane DMB – Dimethylmethylene blue DMEM – Dulbecco’s modified Eagle’s medium DNA – Deoxyribonucleic acid DNase – Deoxyribonuclease D-PBS – Dulbecco’s Phosphate-Buffered Saline dsDNA – Double strained DNA E EA.hy926 – Human umbilical vein cell line ECM – Extracellular matrix EDC – 1-ethyl-3-(3-dymethylaminopropy) carbodiimide EDTA – Ethylenediamine tetraacetic acid EGF – Epidermal growth factor ELISA – Enzyme-linked immunosorbent assay EMT – Epithelial to mesenchymal transition ERDF – European regional development fund EU – European Union F FBS – Fetal bovine serum FCT – Portuguese foundation for science and technology FDA – U.S. Food and Drug Administration FITC – Fluorescein Isothiocyanate FGF – Fibroblast growth factor G g – Grams g – Times gravity GAG – Glycosaminoglycan GFP – Green fluorescence protein GLUT – Glucose transporter H h – Hour HA – Hyaluronic acid HAM – Human amniotic membrane HCl – Hydrochloric acid HCM – Human chorion membrane hCMEC/D3 – Human cerebral endothelial cell line HIV – Human immunodeficiency virus HMD – Hexamethyldisilane HUVECs – Human umbilical cord vein endothelial cells H&E – Hematoxylin-eosin I xv IM – Intramuscular IPPROM – Iatrogenic preterm premature rupture of membranes iPSC – Induced pluripotent stem cell ISO – International organization for standardization IV – Intravenous K kDa – Kilodalton kg – Kilogram kN – Kilonewton kV – Kilovolt L LDH – Lactate dehydrogenase LESC – Limbal epithelial stem cell LSCD – Limbal epithelial stem cell deficiency M mg – Milligram MgCl2 – Magnesium chloride min - Minute mL – Milliliter mm – Millimeter mM – Millimolar mmHg – Millimeter of mercury MMP – Matrix metalloproteinase MPa – Megapascal MRC-5 – Human fetal lung fibroblast cell line MT – Masson’s trichrome MTT/MTS – Tetrazolium Mw – Molecular weight N n – Number of samples NaCl – Sodium chloride NADH – Nicotinamide adenine dinucleotide NADPH – Nicotinamide adenine dinucleotide phosphate NaOH – Sodium hydroxide NFM – Nanofibrous mesh ng – Nanograms nm – Nanometer NSAID – Non-steroidal anti-inflammatory NVU – Neurovascular unit P p – Statistical level of significance PATH – Doctoral Program on Advanced Therapies for Health PBS – Phosphate-buffered saline PCL – Polycaprolactone PCLem – Polycaprolactone electrospun meshes PET – Polyethylene terephthalate pg – Picogram PGG – Penta-galloyl glucose PLA – Poly(lactic acid) PLACL – Poly(L-lactic acid)-co-poly(e-caprolactone) PLGA – Polylactic-co-glycolic acid PPP – Platelet-poor plasma PR – Patch repair PRP – Platelet-rich plasma PTFE – Polytetrafluorethylene R RBC – Red blood cells rpm – Rotations per minute RT – Room temperature S s – seconds S. aureus – Staphylococcus aureus SDS-PAGE – Sodium dodecyl sulphatepolyacrylamide gel electrophoresis SCID – Severe combined immunodeficient SD – Standard deviation SDS – Sodium Dodecyl Sulfate SEM – Scanning electron microscopy SIS – Porcine small intestinal submucosa SMA – Smooth muscle actin S1P – Sphingosine 1-phosphate T TBS – Tris buffered saline TEER – Transendothelial electrical resistance Tie – Tyrosine-protein kinase TL – Trophoblast layer TNF-α – Tumor necrosis factor alpha TGF-ß – Transforming growth factor beta TRITC – Tetramethylrhodamine TSB – Tryptic soy broth media U UV – Ultraviolet light V v –- Volume xvi VEGF – Vascular endothelial growth factor xvii LIST OF FIGURES Figure I-1 Representation of human inner body membranes which have been used in decellularization-related studies, divided in three groups 1) Epithelial membranes (pericardium, pleura, and peritoneum that includes mesenthery and omentum); 2) Connective tissue membranes (fascia that includes periosteum, and synovial membrane); 3) Placental membranes (amniotic and chorion membranes). ................................................................................................................. 6 Figure III-1 The human chorion membrane (HCM). (A) Scheme and (B) Hematoxylin/Eosin staining of the native HCM in a transversal cut. The different layers of the HCM – reticular layer, basement membrane, and trophoblast layer - are identified in the figure. ................................... 83 Figure III-2 Separation of the HCM from the human placenta ............................................... 85 Figure III-3 Scheme of the HCM decellularization protocol. .................................................. 87 Figure III-4 dHCM characterization. (A-B) Transversal sections of DAPI staining in native HCM (A) and dHCM (B). (C) SEM micrograph of dHCM in a transversal cut. (D) Content of double-stranded DNA in native HCM and dHCM. (E) Electrophoresis in agarose gel of DNA extracted from both native HCM and dHCM. ...................................................................................................................... 88 Figure IV-1 Human chorion membrane decellularization. Representative transversal sections of Hematoxylin/Eosin (H&E) staining of HCM (A) and dHCM (B). Representative SEM micrographs of dHCM from the reticular layer side (C) and the trophoblast side (D). Representative transversal sections of DAPI staining from HCM (E) and dHCM (F). Double-stranded DNA (dsDNA) quantification in HCM and dHCM (G). Agarose gel electrophoresis of DNA extracted from HCM and dHCM (H). Thickness of air-dried HCM and dHCM measured with a picometer in, at least, three different sites (I). Top view of HCM (J) and dHCM (K) cut into pieces of 2 cm x 2 cm. Swelling behavior of dHCM in culture medium and D-PBS (L). Where, ** p £ 0.01 and *** p £ 0.001. .............................. 106 Figure IV-2 dHCM protein composition. Representative lanes of two different samples from an SDSPAGE gel of the digested HCM and dHCM (A). Representative dot blot results for collagen type I, collagen type IV, fibronectin and laminin, in both native and dHCM tissues. Digestion buffer without samples was used as a negative control, while the native protein was used as a positive control (B). Immunolocalization of collagen type I, collagen type IV, fibronectin and laminin in representative transversal sections of HCM and dHCM (C). Quantification of soluble collagen (D), insoluble xviii collagen (E), and sulfated glycosaminoglycans (GAGs) (F) content in HCM, dHCM, and TL in µg/mg of dry tissue. Where ** p £ 0.01; *** p £ 0.001 and **** p £ 0.0001. .................................... 108 Figure IV-3 Mechanical properties of native and decellularized HCM. Stress-strain curves of HCM (A) and dHCM (B). Ultimate tensile strength (C) and Young’s modulus (D) of both HCM and dHCM. Where **** p £ 0.0001. .............................................................................................. 109 Figure IV-4 Mechanical properties of native and decellularized HCM. EA.hy926 (A) and MRC5 (B) cells’ metabolic activity in commercial inserts (CTR), dHCM trophoblast layer side and dHCM reticular layer side. Representative images of H&E and Masson’s trichrome staining of dHCM subcutaneously implanted on balb/c mice in three different time-points (4, 10 and 28 days) (C). Where, * p £ 0.05; *** p £ 0.001 and; **** p £ 0.0001. ........................................................ 111 Figure V-1 Schematic representation of bEnd3 and C8-D1A cells seeding on both sides of dHCM in mono and co-culture conditions ....................................................................... 129 Figure V-2 The suitability of dHCM for a BBB in vitro model. Metabolic activity (A - B) and phalloidin/DAPI staining (C - D) of bEnd.3 (A and C) and C8-D1A (B and D) cells in dHCM trophoblast side and dHCM reticular layer side in both mono and co-culture conditions at days 3 and 7 of culture. ..................................................................................................................... 131 Figure V-3 BBB model validation. TEER values were obtained for bEnd.3 monolayer (A) and for the system that is composed by bEnd.3 monolayer and dHCM (B), both in mono and co-culture conditions. Diffusion assays with and 70 kDa FITC-dextran (C) were also performed for mono and co-cultures, at two different time-points (days 3 and 7). Where ** p £ 0.01. ............................. 132 Figure V-4 Functional BBB permeability. Different molecules were used to exemplify the different transport systems at the BBB. Caffeine was used for membrane diffusion (A), glucose for carriermediated transport (B), and transferrin for receptor-mediated transport (C). Where * p £ 0.05; ** p £ 0.01. ............................................................................................................................... 134 Figure V-5 GLUT-1 and transferrin receptor expression. GLUT1 (A) and transferrin receptor (B) expression were assessed over time (days 3 and 7) in both conditions (mono and co-culture) by immunofluorescence. ............................................................................................................. 135 Figure VI-1 Endothelial cell compatibility. Phalloidin staining was used to assess the ability of HUVECs to form capillary-like structures (marked with a white triangle) in CTR (Matrigel) condition, dHCM reticular layer side and dHCM trophoblast layer side at 4, 8 and 12 h (A). For the same xix conditions at the same time-points, angiopoietin 2 (ANG 2) production was quantified (B). A reendothelization assay was also performed in three different conditions (CTR (coverslip), dHCM reticular layer side, and dHCM trophoblast layer side). After 48 h HUVECs were stained with phalloidin (red) and for Von Willebrand Factor (green) Where, * p £ 0.05, ** p £ 0.01 and *** p £ 0.001. .................................................................................................................................... 151 Figure VI-2 CAM Assay. Representative photographs of the chorionic and allantoic epithelium of ex ovo CAM after 7 days from implantation in control (CTR) condition (without material) and in both dHCM sides: reticular layer and trophoblast layer (A). Several parameters were quantified in all conditions such as number of blood vessels (B), vascular density (C), and vascular length density (D). ....................................................................................................................................... 152 Figure VI-3 dHCM thrombotic potential. Plasma protein adsorption was quantified in CTR condition (glass surface) and in both sides of dHCM (A). Platelet adhesion was analyzed for the same conditions using isolated platelets (B) and platelet poor plasma (PPP) (C). Isolated platelets on the three different surfaces were stained with phalloidin (D1-D3). Moreover, SEM micrographs were obtained to verify platelet morphology (D4-D6). Where, *** p £ 0.001, **** p £ 0.0001. .......... 153 Figure VI-4 dHCM hemolytic potential. The hemolytic ratio was obtained for the CTR (glass surface) condition and for both sides of dHCM. For the assay positive and negative control, 1% SDS and PBS were added to the RBC suspension, respectively (A). SEM micrographs of RBC seeded into the different surfaces were obtained (B). Where **** p £ 0.0001 .................................................. 155 Figure VI-5 dHCM resistance to infection. dHCM antibacterial activity against S . aureus adherence and attachment, photographs of contact plates in CTR (polypropylene films) (A1), dHCM reticular layer side (A2) and dHCM trophoblast layer side (A3). CFU quantification in all surfaces (B). Where, ** p £ 0.01 and *** p £ 0.001. .............................................................................................. 156 Figure VI-6 dHCM tubular structure. dHCM was rolled creating a tube with 3-4 mm diameter (A) and variable length (B). The walls of the dHCM tube were characterized regarding its thickness (C). Representative SEM micrographs of a transversal cut of the dHCM tube (D), of the inner side of the dHCM tube, the reticular layer side (E), and of the outer side the dHCM tube, the dHCM trophoblast layer side (F). ........................................................................................................................ 157 Figure VI-7 Mechanical characterization of the dHCM tube. dHCM tube was mounted in 3D CultureProTM Bioreactor attached to a peristaltic pump and submitted to a flow rate of 42.7 mL/min xx (A-B). Longitudinal and circumferential Young’s modulus (C) and ultimate tensile strength (D) of dHCM were determined. Despite bending the tubular graft, the flow is not blocked (E-H). ........ 158 Figure VII-1 Experimental timeline. Farm male pigs were acclimatized for 7 days and then submitted to a chest X-ray and thoracoscopic surgery for patch implantation. Seven weeks post-surgery, animals were submitted to a second chest X-ray, thoracoscopy, laparoscopy and were euthanized. .............................................................................................................................................. 172 Figure VII-2 Thoracoscopic defect and repair of the diaphragm. Representation of trocars site (A). Thoracoscopic creation of a diaphragm defect (B). Repair of the diaphragm defect with GoreTexâ (C), PCLem (D) and dHCM (E-H). ................................................................................. 178 Figure VII-3 Outcome Measurements. Descriptive table summarizing different outcome measures, such as surgery duration (min), follow up time (days), recurrence rate (%), weight gain from surgery to euthanasia (kg), and adhesion formation grade at time of death per animal (A). Representative chest x-ray image priori to (B) and post-surgery, at time of euthanasia, in Sham (C) Gore-TexÒ (D), and PCLem (E) groups. Since all animals of dHCM group were submitted to humane endpoints, no chest X-rays were performed post-surgery. .............................................................................. 180 Figure VII-4 Macroscopic and microscopic evaluation of new diaphragmatic tissue. Top view of Gore-TexÒ (A), PCLem (B) and dHCM (C) after follow-up time. Comparison of inflammation, angiogenesis and fibrosis mean scores (score 0-3) between groups, defined by the histological analysis of H&E and MT staining. Two different tissue areas were analyzed, lesion site that corresponds to the patches and transition that represents the native diaphragm at the site of the sutures (D). ........................................................................................................................... 182 Figure VII-5 Immunohistochemistry for a -SMA, CD31 and CD105. Representative sections of a-SMA, CD31 and CD105 staining of the native diaphragm, in both tendon (A, I and O, respectively) and muscle (B, J and R, respectively). Patch samples were divided into two distinct zones: lesion and transition (Figure VII-2C, VII-2D and VII-2H). Representative sections of a-SMA, CD31 and CD105 for Gore-TexÒ (C-D, K-L, S-T, respectively), PCLem (E-F, M-N, U-V, respectively) and dHCM (G-H, O-P, W-X, respectively) groups. .......................................................................... 183 xxi LIST OF SUPPLEMENTARY FIGURES Supplementary Figure IV-1 Representative images of H&E staining of control animals used in the subcutaneous assay performed on BALB/c mice at three different time-points (4, 10 and 28 days). In sham animals, only the surgery was performed (without dHCM implantation). Empty pockets were performed on the contralateral side of animals implanted with dHCM. ..... 120 Supplementary Figure VII-1 Sham animals’ characterization. Thoracoscopic sham surgery without defect creation (A). Representative sections of a-SMA (B), CD31 (C) and CD105 (D) staining of diaphragm section from sham animals. .................................................................. 192 xxii LIST OF TABLES Table I-1 Summary of the clinical tissue engineering applications of the decellularized inner body membranes. ............................................................................................................................. 16 Table VII-1 Scoring system .............................................................................................................. 175 xxiii SHORT CURRICULUM VITAE Laura Passos Morgado Franco Frazão was born on the 3rd of September 1992 in Lisbon, Portugal. She is currently a PhD student at 3B’s Research Group, I3Bs Research Institute on Biomaterials, Biodegradables and Biomimetics, at University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine at Avepark at Caldas das Taipas, Guimarães, Portugal. Laura has graduated in Health Sciences at the University of Lisbon, Portugal with a final grade of 14/20, in 2013. During that time, she did a summer internship in Instituto de Tecnologia Química e Biológica António Xavier (ITQB), at Nova University of Lisbon, Portugal. She proceeded to a master’s degree in Neurosciences at the University of Lisbon, Portugal, which was successfully concluded in 2016, with a final grade of 18/20. The dissertation studies were developed in Portuguese Institute of Oncology of Lisbon where she worked in the genetic profiling of pediatric brain gliomas, which resulted in a publication. Later in 2016, she started her PhD at the 3B’s Research Group, University of Minho, Portugal, under the supervision of Prof. Dr. Nuno Neves in the field of ECM-based biomaterials. Scientifically, Laura has contributed with 2 peer-reviewed papers in international journals (plus 2 submitted), 2 book chapters, and an international patent application (WO/2021/005531). Furthermore, she participated in national and international conferences with 1 oral communication and 4 poster presentations. 2 Chapter I The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 3 Chapter I Chapter I - The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives• ABSTRACT The main objective of tissue engineering and regenerative medicine is to create functional biological substitutes able to maintain, improve or renew the function of a whole organ or a tissue. For that, it is important to mimic the natural environment of cells, the extracellular matrix (ECM). Decellularization is a technique that allows the maintenance of the native ECM while removing its cellular components. So, the use of decellularized matrices in tissue engineering applications is associated with inductive cell invasion, and promotion of remodeling processes including vascularization and innervation. Moreover, they are well tolerated by xenogeneic recipients. Inner body membranes are thin sheets composed by cells and ECM that cover the surface of internal organs and lines body cavities. Decellularized inner body membranes have been explored to create biocompatible and functional tissue constructs for several tissue engineering applications such as cardiovascular, musculoskeletal, and ocular diseases, skin wounds, fetal membrane disorders and even in in vitro models. Nevertheless, there are no standardized decellularization or posdecellularization processing protocols for inner body membranes, limiting their broad use in clinics. In the present review, all these aspects will be explored. •This chapter is based on the following publication: Frazão L. P.; Neves N. M., (2020), “The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives” (submitted). Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 4 I-1. INTRODUCTION The main objective of tissue engineering and regenerative medicine is to use methods of life sciences and engineering to create functional biological substitutes able to maintain, improve or renew the function of a whole organ or a tissue [1–5]. It comprises the involvement of cells, signaling molecules and scaffolds that support and rely upon each other [1]. Scaffolds may be defined as constructs that mimic the natural structure surronding the cells, the extracellular matrix (ECM). Scaffolds may be obtained from synthetic, natural materials or a combination of both [6]. When compared with synthetic, biological scaffolds are associated with a higher biocompatibility, lower cytotoxicity and inflammatory reaction [7,8]. There is a variety of processing methods available to obtain biological scaffolds, such as electrospinning, or 3D printing [1,9]. Nevertheless, given the complexity and incomplete understanding of ECM composition and structure, it is difficult to create an ECM scaffold that fully mimics the native ECM [4]. Decellularization is an approach that allows the maintenance of the native ECM while removing its cellular components. So, decellularized matrices offer additional advantages such as the induction of cell invasion, giving cues for cell modulation and behavior and the promotion of remodeling processes that will allow the formation of a vascularized, innervated, and functional tissue [3,10–13]. Inner body membranes are sheets of cells and ECM that cover the surface of internal organs and line body cavities. Decellularized inner body membranes are commonly used to prepare biocompatible and functional tissue constructs [14]. The aim of this review is to explore the use of decellularized inner body membranes in tissue engineering. Thus, inner body membranes will be characterized and divided in three main groups (epithelial membranes, connective tissue membranes, and placental membranes). Then, inner body membranes will be analyzed for cardiovascular, musculoskeletal, skin, fetal membranes and in vitro models’ applications. I-2. DECELLULARIZED INNER BODY MEMBRANES The main goal of decellularization is to remove all cells and genetic material from the extracellular matrix (ECM), removing potential antigens that can cause an inflammatory response or immune-mediated implant rejection [4,15]. It was established that to avoid host and cell adverse effects, decellularized Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 5 tissues should lack detectable amounts of nuclear material (stained with DAPI), its DNA fragments should have less than 200bp and the amount of double-stranded DNA/mg of dry tissue should be less than 50 ng [16]. Moreover, decellularization aims to maintain the structural, biochemical and biomechanical properties of the ECM [4]. There are several decellularization approaches using mechanical, chemical and enzymatic techniques and/or its combinations. Mechanical procedures comprise agitation, sonication, mechanical pressure, or freeze-drying. They are normally used to disrupt the cell membrane and are usually combined with chemical approaches, generally washing steps using ionic solutions or detergents. For enzymatic techniques, trypsin, dispase, esterases or nucleases are the most used enzymes [9,17]. An extended review of all the decellularized protocols used in decellularized inner body membranes can be found in [18]. Inner body membranes are divided into two main types, connective tissue membranes and epithelial membranes [19]. Since pregnancy is a temporary state, another group of inner body membranes was included, the placental membranes. In this section, inner body membranes will be described regarding its composition and function. Inner body membranes are represented in Figure I-1. Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 6 Figure I-1 Representation of human inner body membranes which have been used in decellularization-related studies, divided in three groups 1) Epithelial membranes (pericardium, pleura, and peritoneum that includes mesenthery and omentum); 2) Connective tissue membranes (fascia that includes periosteum, and synovial membrane); 3) Placental membranes (amniotic and chorion membranes). Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 7 I-2.1. Connective Tissue Membranes Connective tissue membranes are composed by various types of connective tissues and encapsulate organs [19]. Connective tissue membranes include fascia, periosteum and synovial membrane [14,19], that will be described below. I-2.1.1. Fascia Despite the scientific uncertainty regarding “fascia” definition, there is agreement that fascia is a connective tissue membrane that surrounds and holds every organ, blood vessel, bone, nerve fibers, and muscle in place. The fascial tissue has an ubiquitous distribution in the body, creating various layers at different depths and forming a three-dimensional metabolic and mechanical matrix [20]. Four different fascial planes can be distinguished: the meningeal fascia, the visceral fascia, the superficial fascia, and the axial or deep fascia. The meningeal fascia surrounds the central nervous system [21,22]. The visceral fascia extends from the cranial base to the pelvic cavity, covering all organs. This fascia guides the neurovascular and lymphatic packets towards the organs [22]. The superficial fascia or pannicular fascia is the lowermost layer of the skin and it blends with the reticular dermis [22]. The axial fascia, investing fascia or deep fascia is a dense fibrous connective tissue that surrounds muscles, bones, nerves, and blood vessels. This fascia has a high content of collagen fibers and has elastin fibers giving it strength, integrity and extensibility and resilience. The deep fascia can have different nomenclature according with its location, when it encloses the nerves (apineurium, perineurium and endoneurium), the muscles (epimysium, perimysium and endomysium), the blood vessels (tunica externa), the cartilage (perichondrium), or the bones (periosteum, endosteum) [22]. The periosteum will be also discussed below. There are some studies concerning the decellularized fascia. To the best of our knowledge, all of them focus on the deep fascia that surrounds muscles or bones. Excluding the periosteum, most studies focus on the decellularization of human fascia lata (the fascia that surrounds the thigh) [23,24], with two commercial products being available, the Tutoplast® and Suspend [25,26]. There is also a study on the decellularization of porcine anterior rectus fascia (on the rectus abdominal muscles) [27]. Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 8 I-2.1.2. Periosteum The periosteum is a dense connective tissue that adheres to bone surfaces [28]. Anatomically, the periosteum covers the majority of the bony structures with the exception of their intra-articular surfaces and sesamoid bones [29]. Moreover, periosteum is a vital functional structure for bone formation and remodeling [30,31], having a major role in bone growth and repair and an impact on the blood supply of bone and skeletal muscle [29]. Periosteum is structurally divided into two different layers: the outer fibrous layer and the inner layer (or cambium layer) [32]. The fibrous outer layer is primarily composed by collagen and is poor in cells [33]. This layer is subdivided into two parts, the superficial and the deep portions. The superficial part of the outer layer is the most highly vascularized substratum of the periosteum, significantly contributing to the blood supply of bone and skeletal muscle. Moreover, it also presents a rich neural network. On the other hand, the deep portion of the outer layer is fibroelastic and not highly vascularized [29]. In contrast, the inner layer of the periosteum is highly cellular and composed by mesenchymal progenitor cells, differentiated osteogenic progenitor cells, osteoblasts and fibroblasts in a thin collagenous matrix. The thickness of the cambium layer decreases with age, being undistinguishable from the outer layer in adulthood. Consequently, the osteoblastic potential of the periosteum also decreases with age [29]. Some evidences suggest that the osteoblastic potential of periosteum is also influenced by location, showing less osteogenic potential in calvarial periosteum when compared with tibia periosteum [32,34]. As an alternative to periosteal autografts, associated with a limited amount of available healthy periosteum, decellularized periosteum from rabbits have been studied [31,33,35]. I-2.1.3. Synovial Membrane The synovial membrane lines the spaces of diarthrodial joints, tendon sheaths and bursae. It comprises a continuous surface layer of cells (intima) and an underlying tissue (subintima). The intima layer is composed by synoviocytes (fibroblast-like cells) and macrophages embedded in the ECM. The subintima layer contains fibroblasts and lymphatic and blood vessels in a collagenous ECM. Between the intimal surfaces is a small amount of fluid rich in hyaluronic acid, providing cartilage lubrification and a stable non-adherent surface. Other functions of the synovial membrane are arranging the volume of synovial fluid and control the nutrition and composition of chondrocytes in the joints [36]. Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 9 Decellularized synovial membranes were already obtained from the femorotibial and femoropatellar joints of healthy horses as an alternative to cartilage ECM [37,38]. I-2.2. Epithelial Membranes Epithelial membranes are composed by a layer of epithelial tissue and a layer of fibrous connective tissue [19]. Epithelial membranes represent the mesothelium that comprises the pericardium, peritoneum (that includes mesentery and omentum), and pleura [14,19], that will be described below. I-2.2.1. Pericardium The pericardium or pericardial sac is a membrane that surrounds the heart. Its main functions are to hold the heart in place, to prevent overfilling of the heart, to provide lubrication and to function as a physiological barrier, isolating the heart from other organs and avoiding spread of infections. The pericardium is composed by two layers, the inner layer (serous pericardium) and the outer layer (fibrous pericardium) that enclose the the pericardial cavity, that contains a lubrication fluid, minimizing the friction when the heart contracts. The serous pericardium is further divided into two layers, the internal visceral layer that forms the outer layer of the heart (epicardium) and the outer parietal layer that lines the internal surface of the fibrous pericardium (continuous with the central tendon of the diaphragm). It is a rigid structure, composed by tough connective tissue that prevents rapid overfilling of the heart [39,40]. There are several studies involving decellularized pericardium from mice [41], humans [42,43], pigs [44–50], and horses [51]. Nevertheless, bovine pericardium is the most studied [52–68], with two commercial products available Bioripar® and Tutomesh®. I-2.2.2. Peritoneum The peritoneum is the largest and the most complex membrane in the body. It covers the inner lining of the abdominal cavity and supports the internal organs within. The peritoneum is a continuous membrane composed by a basement membrane that contains mesothelial cells and a thin connective tissue carrying blood vessels, nerves, fat, lymphatic ducts, and fibers. It has two layers and a potential space between them (peritoneal cavity). The outer layer, the parietal peritoneum, lines the abdominal and the pelvic walls, while the inner layer, visceral peritoneum, covers the internal organs. The peritoneal Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 10 cavity consists in mesothelial cells that are responsible for the production of a small amount of serous fluid that allows the two peritoneum layers to slide freely over each other [69]. The peritoneum is a highly folded structure and there are several different terms to describe the folds and spaces that are part of the peritoneum. In this work, we will focus on the most relevant decellularized inner body membranes. So, mesentery and omentum will be described below. There are several studies exploring the decellularized peritoneum, normally harvested from pigs [70–74]. I-2.2.2.1 Mesentery The mesentery is a double layer of peritoneum caused by invagination of an organ into the peritoneum. It connects the intraperitoneal organ to the posterior abdominal wall, serving as a conduit for blood vessels, nerves and lymphatic ducts going to and from the organ in question. Moreover, the mesentery is associated with lipid and glycemic metabolism through the production of C-reactive protein. The mesentery from the small intestine is called “mesentery” and prevents the collapse of intestines towards the pelvis when the body posture is standing upright. Nevertheless, mesentery related to other parts of the gastrointestinal system is named according to the viscera it connects to [75]. A limited number of studies were performed using decellularized mesentery from mice [76] and pig [77,78]. I-2.2.2.2 Omentum The omentum is structurally similar to the mesentery since it results of abdominal structures formed from the peritoneum. It is composed by sheets of visceral peritoneum that extend from the stomach and the proximal part of the duodenum to other abdominal organs. The omentum is divided in the greater omentum and in the lesser omentum. The greater omentum consists of 4 layers of visceral peritoneum that descend from the greater curvature of the stomach and proximal part of the duodenum, folding back up and attaching to the anterior surface of the transverse colon. It has a role in immunity since it contains a high number of resident inflammatory cells, such as B and T lymphocytes and it can migrate to infected viscera or to a disturbed site. The lesser omentum attaches from the lesser curvature of the stomach and the proximal part of the duodenum to the liver. It is composed by the hepatogastric ligament and the hepatoduodenal ligament [79]. Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 11 The omentum has been used in surgery over the last three decades. It has been associated to bacteria inactivation and foreign materials absorption. Moreover, due to its high angiogenic capacity and neurotrophic properties, it has been related to the promotion of the recovery of a damaged tissue [79]. So, decellularized omentum from human [80], rat [80] and pigs [81–83] have been studied for different applications. I-2.2.3. Pleura Pleura is a transparent membrane that covers the lung and lines the walls of the thoracic cavity. The main function of pleura is to allow the optimal expansion and contraction of the lugs during breathing. This membrane is composed by two thin layers, the visceral pleura (inner layer) and the parietal pleura (outer layer). The visceral pleura wraps around the lungs and cannot be peeled off while the parietal layer lines the inside of the chest wall. Both layers are composed by a layer of mesothelial cells that secret unsaturated and saturated phospholipids and hyaluronic acid that acts as lubricant, allowing the parietal and visceral pleura to glide over each other without friction [84,85]. Studies on pleura decellularization are scarce. Most studies focus on lung decellularization. Nevertheless, some of these works study pleura as a component of lung. So, there is some available information about decellularized pleura from mice [86], rats [87–89], humans and pigs [90]. I-2.3. Placental Membranes The placenta is an organ that is formed during pregnancy. The human placenta is a discoid organ that has two surfaces, the chorionic plate and the basal plate. The chorionic plate faces the baby and is attached to the umbilical cord, while the basal plate abuts the maternal endometrium [91]. The placenta is the life support system for the fetus, facilitating gas and nutrient exchange between mother and fetus and also acting as barrier, protecting the fetus [92]. During pregnancy, fetus and amniotic fluid are involved by fetal membranes, the amnion and the chorion membranes. These membranes are responsible for protection but also for the exchange of nutrients and metabolic products with the maternal body [93]. The amnion or amniotic membrane is the inner membrane, being in contact with the fetus while the chorion membrane is attached to the amnion membrane in the maternal side [92]. The characteristics and composition of these two membranes will be described below. Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 18 I-4.1. Cardiovascular applications Cardiovascular diseases are the leading cause of death in industrialized countries, and it is rising in developing countries [117,142]. Cardiovascular diseases involve the heart or blood vessels. Symptoms may be alleviated by medication. Nevertheless, this is associated to several side effects. Taking into consideration that is more beneficial to treat the root of the problem, several studies have been focused on the manufacture of new patch materials with potential advantages for vascular and heart repairs [47]. In this section, we will describe the use of decellularized inner body membranes for cardiovascular applications. Decellularized inner body membranes such as pericardium, omentum or amniotic membrane have been described for general cardiovascular applications. Decellularized porcine pericardium was demonstrated to be non-cytotoxic by extract analysis (ISO 10993/5) and mouse subcutaneous assay and to have a good compatibility with human endothelial progenitor cells [47]. Bovine decellularized pericardium was also demonstrated to be biocompatible with bovine aortic endothelial cells and to maintain the biochemical and biomechanical properties of the native tissue [58]. These results were corroborated in a study comparing decellularized human fascia lata, human pericardium, porcine pericardium and porcine peritoneum for cardiovascular applications [23]. On the other hand, five different decellularization protocols of porcine omentum were studied regarding the elimination of cellular components, the maintenance of the native ECM and the biocompatibility with mouse fibroblast and rat cardiomyocytes [81]. Decellularized amniotic membrane was coated with ECM derived from human myocardium, showing in vitro compatibility with murine cardiomyocytes, human cardiac fibroblasts, immune cells and epicardial derived-cells [142]. The decellularized amniotic membrane was also used to mimic the pericardium in rats. The results of the study demonstrated that decellularized amniotic membrane was associated with a low immunogenicity and to a lower occurrence of adhesions and scaring and also to a thicker pericardium [143]. In the next section, the application of decellularized inner body membranes in specific cardiovascular applications, such as myocardial reconstruction, heart valves engineering and vascular grafts, will be described. Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 19 I-4.1.1. Myocardial reconstruction Myocardial infarction is associated with a high mortality, morbidity and economic burden [144]. Fibrotic remodeling, scar formation, and collagen disposition play a key role in infarcted myocardium repair [145]. Moreover, the pool of cardiac progenitor cells is limited and adult cardiomyocytes are unable to proliferate [146]. So, myocardial infarction is associated with myocyte loss and scar formation. Myocardial infarction management has considerably evolved in the past 50 years, with the incorporation of aspirin, ß-blockers, angiostensin-converting enzyme 1, and the use of coronary stents. As innovations emerged, the residual scar has become smaller. However, there is still a portion of the heart that is not fully recovered [110]. Moreover, there is also a shortage of cardiac donors [81]. This scenario motivated the exploration of new technologies for heart regeneration, using tissue engineering and progenitor cells as complementary or alternative to conventional therapies [81,110]. The use of decellularized tissues is one of the strategies to treat damaged myocardium, through the fabrication of myocardial patches [113]. In general, myocardial patches or scaffolds, should be able to mimic the structural architecture of the cardiac muscle, to be deformable to support cardiac contraction, to promote cell migration, and to promote the vascularization of the newly formed tissue [110]. Regarding inner body membranes, due to its location and function, pericardium is the most adequate membrane to be used in myocardial regeneration applications [147]. Along the years, it was developed the concept that three-dimensional (3D) ECM-like scaffolds were crucial for cardiac tissue engineering [81]. Following this tendency, it was developed a scaffold using human decellularized pericardium filled with a mixture of RAD16-I hydrogel and porcine mediastinal adipose tissue-derived progenitor cells, together with an implantable EIS measurement system for in vivo online monitoring of scar formation in a swine model of myocardial infarction [110]. This work, together with another study of the same group [111], allowed the creation of a new myocardial bio prothesis with online monitoring of cardiac repair. This system allows a noninvasive tracking of myocardial scar healing [110,112]. Moreover, it was demonstrated that the same pericardium-based scaffold was associated with the neoformation of vessels and nerves when implanted in the same swine model [111]. Functional vasculature and innervation are important to produce viable and stable contractile function [111]. RajabiZeleti, et al also developed a 3D cardiac scaffold based on human decellularized pericardium. They demonstrated that their 3D macroporous scaffolds enhanced the proliferation, viability, migration and proliferation of human cardiac progenitor cells in vitro and angiogenesis and cell differentiation in vivo , when compared to decellularized pericardium and collagen 3D scaffolds. Moreover, no xenogeneic Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 20 immune reaction was observed one month after subcutaneous implantation in rats [113]. Another strategy proposed to repair the myocardium was the use of an injectable pericardium scaffold. In this work, two injectable pericardium scaffolds (porcine and human) were studied for its biochemical composition, in vitro chemoattractant potential and in situ cellular infiltration, proving the concept of using decellularized pericardium matrix gels as injectable scaffolds for myocardial treatment [148]. Recently, decellularized rabbit pericardium was used in a rabbit myocardial infarction model. In this study, it was hypothesized that the decellularized pericardium alone or seeded with autologous adipose derived mesenchymal stem cells, could be safely used. In fact, it was demonstrated that when compared with the control condition (no intervention), both pericardium alone or seeded, improved left ventricular contractile function and myocardial pathological changes. Nevertheless, pericardium seeded with adipose derived mesenchymal stem cells showed to be better in scar repairing two months after surgery [114]. This was already expected since it was demonstrated that human mesenchymal stem cells and cKit+ cardiac stem cells could reduce scar size and improve the cardiac function after myocardial infarction [149]. Other decellularized inner body membranes have been studied for myocardial repair, such as omentum and amniotic membrane [81,117]. Porcine omentum was shown to support the cultivated rat cardiac cells in vitro. Moreover, due to the dense vascular network that characterized the omentum, a quick vascularization of the patch was achieved in vitro using human umbilical vein endothelial cells [81]. Decellularized human amniotic membrane was used in a mice model of myocardial infarction. The effects of decellularized human amniotic membrane and cellular amniotic membrane with or without epithelial to mesenchymal transition (EMT) induction on postischemic left ventricle were studied. It was found that although the gross infarct size remained unchanged, the decellularized amniotic membrane attached to the surface of the acutely infarcted left ventricle and helped preventing pathologic postinfarct remodeling processes, improving systolic and diastolic function. Moreover, cellular amniotic membrane (with or without EMT) elicited a local immune response [117]. Diverse strategies using different decellularized inner body membranes have been proposed for myocardial repair. Nevertheless, studies comparing different decellularized inner body membranes for myocardial repair would be important to fully understand the advantages and disadvantages of some membranes over others. Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 21 I-4.1.2. Heart valves engineering An ideal tissue-engineered heart valve should accomplish some basic requirements. It should allow for cell migration and tissue development in vivo . Moreover, cells seeded in the prosthetic valve before implantation (preferably autologous), should lead to further growing of a supporting matrix [60]. Decellularized pericardium fulfills many properties required for its use in valvular tissue engineering, including adequate mechanical properties, minimal cytotoxicity, excellent repopulation potential and propensity for matrix remodeling [115]. Moreover, the pericardial tissue is easy to manipulate and shows durability in vivo [49]. Decellularized porcine pericardium is widely used in valve engineering. Collagen-based scaffolds from decellularized porcine pericardium were created. Collagen scaffolds are superior to polymer scaffolds in terms of mechanical and biological properties but also maintain a natural tendency to degenerate rapidly in vivo if they are not stabilized [115]. Different cross-linking protocols to stabilize the collagen were tested. Collagen-based scaffolds derived from decellularized porcine pericardium, were treated with penta-galloyl glucose (PGG) and glutaraldehyde. While, glutaraldehyde cross-linking fully stabilized collagen, it did not allow tissue remodeling and induced calcification when implanted subdermally in rats. On the other hand, PGG cross-linking, was able to stabilize collagen, avoiding calcification in vivo and supporting host cell infiltration and matrix remodeling [115]. Pericardium treated with glutaraldehyde was the material of choice employed in the manufacture of commercial bioprosthetic valves. However, this was associated with severe side effects resulting from the lack of biological compatibility. So, protocols using decellularized porcine pericardium without the use of fixatives appeared, showing compatibility with both porcine and human aortic valve interstitial cells [150]. Following this rationale, other studies exploring methods to seed cells in bioprosthetic valves were developed [48,151]. The impact of porcine pericardium processing, especially sterilization (using supercritical CO2) on the material’s mechanical properties, was reported. Decellularized pericardium, fixed pericardium, and decellularized-sterilized pericardium were compared with native valvular and pericardial tissue. The decellularized tissue showed the lower mechanical properties. However, the decellularized-sterilized condition demonstrated elastic properties similar to the average porcine aortic cusp. Moreover, decellularized-sterilized tissue was able to accept host-derived cells, allowing surface endothelization and long-term structural stability [49]. Religious and cultural practices may limit the use of explanted tissues. For example, one patient may not accept the use of a porcine bioprosthetic. Therefore, there is a need to Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 22 characterize tissues from different sources in order to fit the needs of the patients [152]. So, the behavior of human vascular cells from saphenous vein seeded on bovine decellularized pericardium, both in vitro and in vivo, were studied. In vitro , the seeded cells showed increase of endothelial activity from day 1 to day 7 and remained stable until day 21. For the in vivo assay, the decellularized bovine pericardium seeded with human cells was implanted subcutaneously in nude mice. It was demonstrated that after 30 days, the cells could differentiate and showed all the identities of human endothelial cells, smooth muscle cells, and fibroblasts. These results suggest that human vascular cells from the saphenous vein are an optimal cell source for seeding on decellularized bovine pericardium scaffold for construction of tissueengineered heart valves [60]. Moreover, others reported a case of replacing the mitral valve of a neonate using a cylindrical prothesis fashioned from decellularized equine pericardium. A good performance of the mitral valve protheses at four months of follow up was observed. However, since there is no evidence of the growth potential of cylindrical prothesis using natural materials, a subsequent valve replacement will be necessary in growing children [116]. I-4.1.3. Vascular grafts Vascular deformities are usually caused by vascular diseases, congenital defects, trauma or tumors [153]. The use of autologous blood vessels is limited not only by the availability of the donor but also by the lack of healthy vessels. So, tissue engineered substitutes for blood vessels have been widely studies. Xenograft substitutes have attracted considerable attention. A study used decellularized equine pericardium, implanted it in the descending aorta of juvenile sheep [51]. After 4 months, the grafts were well preserved with a monolayer of endothelial cells covering its luminal side. The outer side of the patch was characterized by a well-developed neo-vascularization. Host fibroblasts were present in all layers of the scaffold. Moreover, there was no evidence of structural deterioration or calcification of the scaffolds [51]. Nevertheless, after vascular graft implantation, it is important to ensure blood supply. A major barrier to a successful vessel graft substitute is the need of a functional vascular network within the engineered tissue. To solve this problem, a novel vascular scaffold was established from decellularized porcine aorta and intact mesentery containing a radiating arterial and venous network [77]. Since the mesentery is rich in small arteries and veins that spread for the outside, it can directly provide blood to an area of ischemic tissue. So, this scaffold provided vascular channels for blood supply. When transplanted into dogs, the aorta of the scaffold was linked to the femoral artery of the dog by arterial anastomoses, creating a rich Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 23 vascular network with several branches. Consequently, this approach was presented as a potential alternative for the xenogeneic transplantation of vessels, compensating the lack of allogenic tissues available for transplantation [77]. Allogenic grafts for vessel replacement are based in the use of human amniotic membrane. This membrane has been used as a single layer with omental microvascular endothelial cells. The use of sphingosine 1-phosphate (S1P) treatment allowed omental microvascular endothelial cells to form a capillary structure on the decellularized amniotic membrane. The scaffold was transplanted into ischemic hindlimbs of mice and the capillary structure significantly improved the blood prefusion 5 days after implantation [118]. Other studies using the human amniotic membrane involve rolling it in a tubular construct. These studies demonstrated that the rolled scaffold provided a 3D microenvironment that resembled natural arteries, promoting an enhanced early cell remodeling [119,120]. The advantage of using human amniotic membrane for the construct of vascular grafts is associated with its greater availability when compared with equine pericardium or porcine mesentery. I-4.2. Musculoskeletal applications The human musculoskeletal system is an organ system that provides shape, support, stability, and articulation of movements to the body. It is composed of bones, muscle, cartilage, tendons, ligaments, and joints. In this section the use of decellularized inner body membranes for musculoskeletal tissue engineering applications will be explored, mainly for bone, cartilage, and tendon. I-4.2.1. Bone Bone defects may be caused by trauma, tumor resection, pathological degeneration, or congenital deformations. An insufficiently treated bone defect can compromise the future mobility of the patient, especially in load-bearing areas. The healing capacity of bone is limited, so critical-sized bone defects may not heal by themselves, resulting in a nonunion [31]. In these cases, bone grafting or bone substitutes are typically used [33]. Autografts are the gold standard for the repair of bone defects. However, its clinical application is limited due to the donor site morbidity and the amount of tissue that can be obtained [31,33]. Periosteum is important for the initiation of bone graft healing and remodeling due to its regenerative capacities [33]. So, various studies explored the use of periosteum for bone regeneration. Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 24 Rabbit decellularized periosteum was shown to allow rabbit periosteum cells to adhere, proliferate and infiltrate in vitro . Moreover, its biocompatibility was demonstrated in an in vivo subcutaneous assay in rabbits [33]. Another study demonstrated the in vitro mineralization using pig decellularized periosteum and that the same scaffold could effectively promote orthotopic regeneration in a critical size bone defect rabbit model [107]. Decellularized rabbit tibial periosteum was compared with decellularized rabbit calvarial periosteum regarding their biological, structural and biomechanical properties, its cytotoxicity, and angiogenic activity (using chicken egg chorioallantoic membrane assay). Osteogenic activity was also studied by the subcutaneous implantation of the decellularized periosteum seeded with mesenchymal stem cells in nude mice. Moreover, angiogenesis and osteogenesis mechanisms were analyzed by seeding human umbilical vein endothelial cells and mouse osteoblast precursor cells, respectively, onto the decellularized scaffolds [35]. In summary, rabbit decellularized calvarial periosteum was associated with a stronger angiogenic activity while rabbit decellularized tibial pericardium was associated with a more marked osteogenic activity [35]. Thus, it was proposed the use of decellularized calvarial periosteum in children with incomplete ossification and the use of decellularized tibial periosteum to promote the repair of bone defects or injuries in adults [35]. In a different approach, a hybrid material combining decellularized rabbit periosteum with chitosan globules was studied [31]. It was expected a synergistic effect between the two materials, maintaining the biocompatibility and biodegradability while boosting the overall mechanical stability. The efficacy of the new scaffold was assessed using a rabbit femoral bone defect model. Chitosan-decellularized rabbit periosteum shown to have a more effective surface for cell adhesion and a superior performance for osteogenesis in the defect area when compared with chitosan globules alone or with the empty defect [31]. Guided bone regeneration was developed to enhance the efficacy of bone repair [52] by the employment of a barrier membrane to protect the space necessary for osteogenesis and to prevent the invasion of fibroblasts. It has been widely used in the dental peri-bone implantation and other bone fractures with large bone loss or poor healing potential. The main goal of applying a barrier membrane is to prevent the invasion of fibroblasts, provide stability for bone grafts and blood clots, and ensure a protected space for osteogenesis to occur [121]. Expanded polytetrafluoroethylene has been extensively studied as a barrier membrane. However, this material is not absorbable, requiring a second surgical procedure for removal, which is time-consuming, expensive and increases patient’s discomfort and risk of secondary infection [154,155]. As an alternative, collagen membranes were proposed as resorbable membranes. Nevertheless, due to in vivo degradation, collagen is structurally unstable [52]. Decellularized inner body membranes were also investigated for this application. Decellularized bovine Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 25 pericardium has shown in vivo efficacy in a rabbit mandibular model. After 16 weeks of in vivo implantation, this membrane was associated with an enhanced deposition of mineralized tissue when compared with defects left empty for spontaneous heal [52]. Moreover, a study using a lyophilized multilayered decellularized human amniotic membrane showed its functionality as a membrane barrier for guided bone regeneration in a rat tibia defect model. After 6 weeks of surgery, the membrane acted as a shield to avoid the invasion of the fibrous tissues, to stabilize bone grafts and to induce massive bone growth (when compared with collagen membranes) [121]. In summary, decellularized periosteum from rabbits and pigs were shown to promote bone regeneration. However, periosteum harvesting site influences its angiogenic and osteogenic properties. For guided-bone regeneration, when compared with decellularized bovine pericardium, decellularized human amniotic membrane presents advantages such as its availability and allogenic source (when for human use). I-4.2.2. Cartilage Cartilage is a connective tissue that covers and protects the ends of long bones at the joints and nerves and is present in the ends of the ribs, separating each vertebra in the spine, ears, nose and bronchial tubes. Cartilage degeneration or injury is mainly caused by osteoarthritis and traumatic injuries and may lead to progressive deterioration and eventual osteoarthritic degeneration. However, cartilage repair is very limited due to is low regenerative capacity [122]. Autologous chondrocyte implantation was previously explored as treatment option for cartilage lesions [108,109]. The use of decellularized periosteum seeded with autologous chondrocytes has shown to be capable of regenerating the articular cartilage of the rabbit patella [108]. Moreover, the use of human chondrocytes with decellularized periosteum demonstrated 73% hyaline-like cartilage regeneration in femoral condylar injury in a cohort of 23 human patients [109]. However, the implantation of autologous chondrocytes with periosteum has been associated with various complications, such as large surgical incisions, peripheral graft hypertrophy, graft delamination, possible calcification and the ability of periosteum to form ectopic bone [156]. These complications stimulated the development of alternative scaffolds for autologous chondrocyte implantation, including membranes of purified porcine collagen type I and III [156–158]. Autologous chondrocyte implantation produces good short-term results, but there are limitations regarding the cost, the need for a two-step procedure (cell collection and re-implantation after cells expansion) and the difficulty of using this technique in larger defects [122]. So, acellular Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 26 scaffolds were studied for cartilage repair. Since cartilage repair mediated by cell-free scaffolds depends on the in situ recruitment of endogenous cells, the acellular scaffold needs to allow host cell recruitment, attachment, growth and differentiation [122]. ECM from mesenchymal stem cells derived from the human amniotic membrane was combined with polylactic-co-glycolic acid (PLGA) to create a scaffold for cartilage repair. This acellular scaffold was implanted into osteochondral defects in the trochlear groove of rat knees and promoted the in-growth of endogenous cells that induced gradual tissue regeneration, resulting in hyaline cartilage repair that was not observed in the empty control group [122]. Another therapeutic strategy for cartilage repair using decellularized inner body membranes is the delivery of anabolic genes locally. To study this hypothesis, decellularized synovial membrane from the femoropatellar and medial femorotibial joints of equine cadavers was seeded with synovial-derived mesenchymal stem cells (from the same sites) [37]. In this work, seeded synovial-derived mesenchymal stem cells were not treated or were cotransduced to enhance the expression of green fluorescent protein (GFP) and human bone morphogenetic protein (BMP)-2. Results demonstrated that equine decellularized synovial membrane seeded with untreated or transduced synovial-derived mesenchymal stem cells resulted in a bioactive scaffold, leading to cell viability, incorporation into the scaffold, and differentiation. Moreover, transduced cells produced greater BMP-2, hyaluronic acid and proteoglycan concentrations, indicating gene expression and enhanced synovial function, promoting cartilage repair [37]. Decellularized inner body membranes such as periosteum or synovial membrane have been used in cartilage repair to support the delivery of cells and/or genes to the injury site. However, further studies are needed to better understand the therapeutic potential of ECM based acellular scaffolds in cartilage regeneration. I-4.2.3. Tendon Tendons are tough fibers that connect muscles to bone. Most tendon injuries occur near the joints at the interface with bone. It may occur suddenly, but it is usually associated with gradual wear and tear to the tendon from overuse or aging. ECM scaffolds and tendon grafts were studied for the repair of tendon ruptures, including Achilles tendon, rotator cuff, and flexor tendons [159,160]. Materials derived from biological sources seem to be more promising for tendon repair since they are associated with a faster and more natural healing of tendon defects using host cells, and with an adequate resorption rate [160]. Decellularized bovine amniotic membrane was used on a rupture Achilles tendon model in rabbit [124]. Biomechanical and Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 27 histological analyses were performed. It was observed that the amniotic membrane enhanced the development of a mature organization of fibroblasts and collagen fibers and improved the properties of the lacerated tendon at the early healing period, when compared with the control group (no treatment). In this study, the long-term effects of the decellularized bovine amniotic membrane on tendon healing were not investigated [124]. However, tendon healing following injury, even when surgically repaired, results in a fibrocartilaginous scar that leads to a decrease in tendon strength and disorganized collagen fibers [123]. In the uninjured tendon, the tendon sheath is a thin synovial membrane that separates the tendon from its surrounding tissue and is composed by synoviocytes and a sheath of fibroblasts. The main purpose of these cells is to produce synovial fluid (mainly hyaluronic acid) that provide nutrition to the tendons and ensure a smooth gliding surface [161]. If the tendon and its sheath are injured, this natural barrier is lost and may lead to postoperative adhesions and scarring. Nevertheless, the human tendon sheath is not possible to be harvested from cadavers due to its brittle consistency and firm attachment to the surrounding bone. Thus, decellularized inner body membranes have been studied for this purpose. The decellularized porcine pericardium was shown to be compatible with human sheath synoviocytes and human adipose derived stem cells. After seeding, adipose-derived stem cells demonstrated a shift in gene expression by down-regulating collagen type I and III and up-regulating the production of hyaluronan synthase 2 . After 8 weeks, a continuous production of hyaluronic acid by both cells was observed [46]. The capacity of the decellularized human amniotic membrane to modulate the inflammatory response in tendon wound healing was also studied. This work combined collagenglycosaminoglycan scaffolds with hyaluronic acid or decellularized amniotic membrane. The combined scaffolds normalized the expression of genes associated with inflammatory response in normal tendon healing ( TNFα , COL I, MMP-3 ). Moreover, the use of decellularized amniotic membrane was associated with increased mechanical properties and increased metabolic activity even when media was supplemented with the pro-inflammatory cytokine interleukin-1ß [123]. Late tendon healing phases are of main importance since they determine the presence of fibrocartilaginous scars associated with a decrease in tendon strength and functionality. Taking into account the anti-inflammatory properties of the decellularized amniotic membrane and its ability to promote tendon regeneration in early healing stages, this may be a good substrate for the development of further tendon healing therapies. Chapter I – The Application of Decellularized Inner Body Membranes in Tissue Engineering: Current Status and Future Perspectives 34 were shown in stress urinary incontinence treatment among both groups, commercial decellularized human fascia lata was associated with a higher recurrence rate [181]. Porcine decellularized pericardium was combined with a rapid-degrading porous collagen matrix and loaded with mice pancreatic islets for beta cell replacement therapy in type 1 diabetes. Scaffolds were transplanted into the epididymal fat pad in a syngeneic mouse model and demonstrated rapid reversal of hyperglycemia with optimal vascularization and integration of islets within the fat tissue [41]. Beyond cardiovascular, musculoskeletal, skin, fetal membranes, ocular, and in vitro applications, there are several more different clinical applications where decellularized inner body membranes may be applied. In this section, studies regarding periodontal regeneration, pharyngocutaneous fistulas and stress urinary incontinence treatment, and beta cell replacement therapy in type 1 diabetes were highlighted. I-5. FINAL REMARKS Decellularized inner body membranes are promising tools for tissue engineering applications and have been studied for a variety of clinical purposes as previously described. Decellularized inner body membranes have been studied for the regeneration of myocardium, bone, cartilage, tendon, fetal membranes, skin, and cornea. Moreover, these membranes were used for the creation of heart valve substitutes, vascular grafts and in vitro models. Standardized decellularization protocols of inner body membranes for specific applications are needed in order to easily compare results and outcomes, and to scale up the production of these membranes for their broad utilization in clinics. Several works crosslink or combine decellularized inner body membranes with synthetic biomaterials to improve their mechanical properties and reduce their biodegradability in vivo . Nevertheless, few works compare these different processing methods for the same application, preventing the understanding of its clinical importance. The availability of the inner body membranes is an important issue. Normally, xenogeneic membranes are used to avoid two surgeries (one for harvesting the material and another to implant the processed material). Another solution is the use of human cadaveric inner body membranes. Regarding placental membranes, this problem does not arise, since placentas are considered a biological waste and are available after full-term pregnancies. This explains the wide interest and study of decellularized amniotic membrane for tissue engineering and regenerative medicine applications. 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Comparison of long-term outcomes of autologous fascia lata slings with Suspend Tutoplast fascia lata allograft slings for stress incontinence. Am. J. Obstet. Gynecol. 2005, 192 , 1677–1681. 51 SECTION 2 EXPERIMENTAL DESIGN 52 Chapter II Materials & Methods Chapter II – Materials and Methods 53 Chapter II Chapter II - Materials and Methods OVERVIEW This chapter provides a detailed description of the materials, methodologies and procedures used to develop the work reported in chapters III to VII of this thesis, complementing the information present in the “Materials and Methods” subsections of those chapters. Firstly, a description of the materials used, namely human chorion membrane and polycaprolactone is presented. In the second part, the methodologies are described providing more details on its fundaments and the rationale that lead to its use. The production techniques, such as decellularization and electrospinning are described as well as the methodology used for the characterization of the decellularized human chorion membrane (dHCM). Moreover, the in vitro and in vivo assays performed to test and validate the developed systems will be presented and justified. In summary, this chapter aims to support the methodologies selected and used in chapters III to VI to answer the specific hypothesis and research questions studied. Chapter II – Materials and Methods 54 II-1. MATERIALS II-1.1. Human chorion membrane (HCM) The human placenta is composed by two fetal membranes, the amniotic membrane and the chorion membrane. The HCM is attached to the amnion membrane in the maternal side [1] and it is composed by three different layers: 1) the reticular layer that contacts with the spongy layer of the amniotic membrane and is composed by collagen type I, III, IV, V and VI; 2) the basement membrane that anchors the reticular and trophoblast layers and is composed by collagen type IV, fibronectin and laminin; and 3) the trophoblast layer that contacts with the maternal decidua and is composed by 2-10 layers of trophoblasts [1,2]. HCM were obtained from human placentas collected from elective and term cesarean sections to limit microorganism’s contamination associated with vaginal delivery. An informed consent was signed by all donors and the collection of human placentas followed a protocol approved by the ethical committee of the Hospital de Braga (where cesarean sections were performed) and of the University of Minho (CESH 030/2016; SECVS 136/2015, respectively). Sterile conditions were maintained during all processing steps. After cesarean section, human placentas were stored in Dubleco’s phosphate buffered saline (DPBS; #21600-044, Alfagene) with 10% antibiotic/antimycotic (#15240062, Thermo Fisher Scientific) at 4 ºC for a maximum of one day. The HCM was obtained from the less vascular part of the human placenta, i.e., the chorionic plate was discarded. The amniotic membrane was separated from the HCM by gently pushing. Then, the HCM was washed with sterile phosphate buffered saline (PBS; #P4417, Sigma-Aldrich) once to remove the blood excess and stored at -80 ºC until further use. In this thesis, the decellularization process of the HCM was optimized and the decellularized human chorion membrane (dHCM) was characterized and studied for different tissue engineering and regenerative medicine applications. II-1.2. Polycaprolactone (PCL) PCL is a biodegradable polymer widely used in tissue engineering and regenerative medicine applications, for long-term implantable devices, such as drug delivery systems, sutures and scaffolds [3]. Chapter II – Materials and Methods 55 Moreover, due to its biocompatibility and ability to form blends and copolymers with other polymers, it has been approved by Food and Drug Administration (FDA) for applications in the human body [3]. PCL is prepared by ring opening polymerization of e-caprolactone, it has a melting point around 60 ºC, and presents excellent solubility in common organic solvents. For these reasons, PCL (Mw 80,000, #440744, Sigma-Aldrich) was used on chapter VII of this thesis to produce nanofiber meshes. II-2. METHODS II-2.1. Production Techniques Two main productions techniques were used in this thesis: decellularization of the HCM and electrospinning to produce nanofiber meshes of PCL. II-2.1.1. Decellularization The decellularization process is characterized by cell removal while maintaining the tissue characteristics [4]. In this thesis, the optimized decellularization method of HCM is described in chapter III and comprised a combination of mechanical, detergent and enzymatic techniques. Different pools of various placentas were used in order to ensure the traceability of the results. II-2.1.2. Electrospinning The electrospinning technique is based on the electrohydrodynamic phenomena occurring when a sufficiently high voltage is applied to a liquid droplet, a charge accumulates at the liquid surface. So, when the electrostatic repulsion is higher than the surface tension, the liquid meniscus is deformed into a conically shaped structure (the Taylor cone) and a charged liquid jet is formed and ejected towards a collector. Depending on the solution viscosity, solid fibers will be formed as the solvent evaporates, resulting in a fiber mat deposited on the collector [5]. On chapter VII, electrospinning was used to create PCL electrospun meshes (PCLem). When fibers are produced, they are deposited in the collector. Fibers stack one over another building several interconnections due to the presence of residual solvent. This nanofiber organization resembles the Chapter II – Materials and Methods 56 extracellular matrix (ECM) [5]. So, PCLem were used as a synthetic homologous of dHCM for the treatment of congenital diaphragmatic hernias. A solution of 20% PCL was prepared with a solvent mixture of chloroform (#C/4960/17, ENZYMATIC) and dimethylformamide (#444923, Laborspirit) at 8:2 ratio. The PCL solution was electrospun using a 5 mL syringe (B-Braun), a metallic needle with 0.8 cm of external tip diameter and by applying a voltage of 15.5 kV. The needle tip to ground collector distance was 20 cm and the flow rate of 2 mL/h. After the complete processing of 1 mL of PCL solution (30 min), PCLem were allowed to air dry for 1 day. II-2.2. Decellularized human chorion membrane (dHCM) characterization After decellularization it is important to evaluate the efficacy of this process by characterizing the tissue regarding its DNA content, protein composition and mechanical properties. II-2.2.1. Absence of nuclei, DNA extraction and quantification To avoid the immunogenic reaction caused by cells, the decellularized tissue should have no visible nuclei, and any remaining DNA fragments should have with less than 200 bp and less than 50 ng of dsDNA/mgof dry tissue [4]. To verify the presence of nuclei in decellularized tissue, samples were fixed with 10% formalin and embedded in paraffin. Transverse sections with 5 µm were cut using a microtome and hematoxylin and eosin (H&E) and 4’,6’-diamino-2-fenil-indol (DAPI) staining were performed. H&E is one of the most common histology stains, staining cell nuclei in blue (hematoxylin) and extracellular matrix and cytoplasm in pink (eosin) [6] while DAPI is a fluorescent stain that binds to the adenine-thymine-rich regions of DNA [7]. For H&E staining samples were stained with hematoxylin (#7212, Thermo Fisher Scientific) for 1 min, washed for 30 s and stained with eosin for 10 min (#71204, Thermo Fisher Scientific). Then, slides were washed with water, let to dry and rinsed with alcohol, cleared in xylene and mounted in Entellan rapid (#107960, Merck). Slides were observed under an optical microscope with a coupled digital camera (DM750, Leica). For DAPI staining, samples were incubated with DAPI (#40009, VWR) at 1:10000 for 1-5 min, washed and analyzed using a fluorescence microscope with a coupled camera (Axio Imager Z1m, Zeiss) and processed using Zeiss Zen software. For the DNA extraction, human chorion membranes (native and decellularized) were air dried and weighted. The total DNA was extracted using DNeasy Blood and Tissue Kit (#69504, Qiagen) according to manufacturer’s Chapter II – Materials and Methods 57 instructions. The quantification of dsDNA was performed using the Quant-iT PicoGreen dsDNA Assay Kit (#P7589, Invitrogen) according to manufacturer’s instructions. To assess the size of DNA fragments, electrophoresis was performed using a 1% agarose gel and GeneRuler DNA Ladded Mix (#SM0334, Thermo Fisher Scientific). II-2.2.2. Scanning electron microscopy (SEM) SEM is a type of electron microscopy where the sample is scanned using high energy electrons. The electrons interact with atoms in the sample and the reflected electrons/X-rays are analyzed, giving information about the sample’s surface topography and composition. When analyzing a non-conductive material, the specimen has to be coated with a conductive layer of metal or carbon [8]. In this thesis, SEM was used in Chapter IV and VI to analyze dHCM topography and platelets and red blood cells morphology, respectively. Samples were fixed with 2.5% of glutaraldehyde (#G5882, Sigma-Aldrich) in PBS. After three washes with PBS, samples were dehydrated with increasing concentrations of ethanol (10% - 100%) and treated with hexamethyldisilazane (HMD) (#440191, Sigma-Aldrich). Then, samples were air-dried, mounted in SEM pins using carbon tape, and coated with gold using a Sputter Coater (#EM ACE600, Leica). Micrographs were collected with a scanning electron microscope with energy dispersive spectroscopy (EDS) (#JSM-6010 LV, JEOL) in chapter IV and with a high-resolution field emission scanning electron microscope with focused ion beam (AURIGA COMPACT, Zeiss) in chapter VI. II-2.2.3. Swelling assay The swelling behavior of a material influences its stability in aqueous medium [9]. In chapter IV, the swelling behavior of dHCM was studied in order to characterize its stability during cell culture and storage. Dry samples were weighted and immersed in D-PBS or culture medium. At different time-points (0.5 h, 1.5 h, 2.5 h, 3.5 h, 4.5 h, and 5.5 h), the excess of liquid was removed (using filter paper) and samples were weighted. After each time-point, samples were re-immersed in liquid until the next timepoint. The process was repeated until the equilibrium (stable percentage of water uptake) was reached. Chapter II – Materials and Methods 58 II-2.2.4. Collagen and sulfated glycosaminoglycans (GAGs) quantification The maintenance of collagen and sulfated GAGs in a decellularized tissue is important since these components are important to maintain the integrity and function of the tissue [10]. Nevertheless, the decellularization process is associated with collagen damage and disruption and with GAGs removal, especially when detergents such as SDS and Triton-X are used [4]. So, during decellularization, it is important to maintain a good balance between ECM preservation and complete cellular removal. In chapter IV, collagen and sulfated GAGs were quantified to characterize the dHCM. Sircol Collagen Assay Kit (#S5000, Biocolor) and Sircol Insoluble Collagen Assay Kit (#2000, Bicolor) were used (in accordance with manufacturer’s instructions) to extract and quantify the content of soluble and insoluble collagen (respectively) of HCM, dHCM and trophoblast layer. Sulfated GAGs content from HCM, dHCM and trophoblast layer were extracted and quantified as previously described [11]. Briefly, samples were digested using 0.5 mg/mL papain (#P4762, Sigma-Aldrich) and the supernatant was stained with 1,9dimethylmethylene blue (DMB). Before measurement, samples were diluted 1:10 in distilled water and a dilution series of chondroitin sulfate in distilled water (50 µg/mL) was used as standard solution. Then, using a microplate reader (Synergy HT, Biotek), the absorbance of the mixture of 20 µL of diluted samples and standards with 250 µL of DMB (#341088, Sigma-Aldrich) was read at 525 nm. II-2.2.5. Protein characterization To characterize a decellularized tissue it is important to study its protein composition and distribution. In Chapter IV, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to demonstrate the reproducibility of the decellularized protocol regarding protein composition, DotBlot was used to identify some ECM proteins and protein immunolocalization allowed the study of protein distribution among the tissue. For SDS-PAGE and Dot Blot techniques, protein from HCM and dHCM was extracted using Tissue Extraction Reagent I (#FNN0071, Thermo Fisher Scientific) with a protease inhibitor cocktail (#P8340, Sigma-Aldrich). Then, the soluble protein content was determined using Protein Assay Dye Reagent Concentrate (#5000006, Biorad), according to the manufacturer’s instructions. SDS-PAGE is a common method used to separate proteins according to its molecular weight, since the combination of SDS with polyacrylamide gel allows for the elimination of both the structure and charge influence [12]. Herein, a 4% stacking gel and 9% running gel were prepared using SDS Gel Chapter II – Materials and Methods 59 Preparation Kit (#08091, Sigma-Aldrich). For each sample, 0.5 µg/µL of protein was loaded in the respective well. Following SDS-PAGE, the gel was stained with Coomassie Blue R-250 (HS-604, National Diagnostics) and an image was obtained using a Transilluminator (Biospectrum ACChemi HR 410). Dot blot is a simple technique used to detect proteins. It may be characterized as a simplification of the western blot method, where proteins to be detected do not have to be firstly separated by electrophoresis [13,14]. One drop of soluble protein from each sample was placed in a nitrocellulose membrane. After drying, membranes were blocked for 1 h with 5% BSA (#A2153, Laborspirit) with agitation at room temperature (RT). Membranes were then incubated with primary antibodies overnight: mouse anticollagen type I 1:1000 (#ab90395, abcam); rabbit anticollagen type IV, 1:500 (#ab6311, abcam); rabbit antifibronectin 1:500(#ab45688, abcam); and mouse antilaminin, 1:500 (#L8271, Sigma-Aldrich). After 3 washes for 5 min with Tris Buffered Saline (TBS) with tween 20 (#P1379, Laborspirit), R.T.U. VECTASTAIN® Universal ABC Elite® Kit (#PK-7200, Vector Laboratories) was used as a secondary antibody, in accordance with manufacturer’s instructions. Revelation was performed using Peroxidase Substrate Kit (DAB) (#SK-4100, Vector Laboratories). Extraction buffer was used as a negative control while collagen type I (#sc-136157, Santa Cruz Biotechnology), collagen type IV (#C5533, SigmaAldrich), fibronectin (#FC010, Sigma-Aldrich) and laminin (#L6274, Sigma-Aldrich) were all used as positive controls. Immunolocalization of proteins was performed in paraffin embedded samples sectioned at 5 µm. Samples were incubated with mouse anticollagen type I 1:100 (#ab90395, abcam); rabbit anticollagen type IV, 1:50 (#ab6311, abcam); rabbit antifibronectin 1:300 (#ab45688, abcam); and mouse antilaminin, 1:300 (#L8271, Sigma-Aldrich), overnight at 4 ºC in a humidified atmosphere. Then, R.T.U. VECTASTAIN® Universal ABC Elite® Kit (#PK-7200, Vector Laboratories) was used as secondary antibody and the signal was revealed using DAB (#SK-4100, Vector Laboratories). Samples were counterstained with hematoxylin, mounted in an aqueous mounting medium, and observed under an optical microscope with a coupled camera (DM750, Leica). II-2.2.6. Mechanical properties To study of the biomaterial’s mechanical properties is of great importance, since it allows to predict the material behavior under stress [15]. In chapter IV, dHCM stiffness (Young’s modulus) and tensile strength were studied. HCM and dHCM dry samples were cut and placed in a universal mechanical testing equipment (#5543, INSTRON) equipped with a 1 kN load cell and were hydrated. The strain rate Chapter II – Materials and Methods 66 Cell functionality was assessed by immunofluorescence and phalloidin staining in chapters V and VI, respectively. In chapter V, the expression of receptors important for BBB in vitro model functionality by b.End3 cells, such as glucose transporter 1 (GLUT-1) and transferrin receptor, was studied. In summary, after a permeabilization with 0.5% Triton X-100 (#A16046-0100, Alfa Aesar) for 10 min at RT and a blockage with 5% BSA (#A2153, Sigma-Aldrich) for 1 h at RT, samples were incubated with rabbit anti-GLUT1 1:50 (#ab15309, abcam), and rabbit anti-Transferrin Receptor 5 µg/mL (#ab84036, abcam) antibodies, overnight at 4 ºC. Donkey anti-rabbit 594 1:500 (#A21207, alfagene), used as secondary antibody, was incubated for 1 h at RT. Then, samples were counterstained with DAPI (#40009, VWR) at 1:10000. On chapter VI, the HUVECs ability of forming tube-like structures was also analyzed by phalloidin staining, as described above. A fluorescence microscope with a coupled camera (Axio Imager Z1m, Zeiss) was used to obtain images that were further analyzed with the Zeiss Zen microscope software. II-2.3.4. Transendothelial electrical resistance (TEER) TEER is a measure of barrier tightness, characterizing the impedance across an endothelium layer. A higher TEER is usually associated with stronger tight junctions and consequently with an enhanced resistance to paracellular diffusion. TEER measurement is a commonly criteria to evaluate the quality of BBB in vitro models, especially transwell models [25]. In chapter V, TEER was measured using a Millicell® ERS-2 Electrical System (MERS00002, Millipore) in accordance with manufacturer’s instructions. Each condition was performed in triplicate at three different time-points (3 and 7 days), and five TEER measurements were obtained per sample. At least three independent assays were performed for each condition. II-2.3.5. Permeability assays The study of membrane permeability was further evaluated in combination with TEER measurements [25]. In chapter V, three different transportation mechanisms across the BBB were tested: membrane diffusion (70 kDa FITC-dextran), solute carrier (caffeine), and receptor-mediated (glucose). Permeability assays on transwell models are based on the principle that molecules added in the upper chamber of the transwell insert, will naturally pass to the bottom chamber. So, it is possible to study the influence of a cell monolayer modulating the permeability of the membrane. Chapter II – Materials and Methods 67 At different time-points (3 and 7 days) the inserts were transferred to a new receiver 24-well plate with 1 mL of fresh culture medium. Then, 400 µL of working solution were added to the upper side of the insert. For membrane diffusion 10 µg/mL of 4 kDa or 70 kDa FITC-dextran (#46944 and #46945, Sigma-Aldrich, respectively) in culture medium were added and the inserts were incubated for 2 h at 37 ºC in a humidifier incubator with 5% CO2. Then, 100 µL of culture medium were collected from the receiver plate and transferred to an opaque 96-well plate where fluorescence was read using an excitation of 485 nm and emission of 535 nm, in a microplate reader (SynergyHT, Biotek). For solute carrier and receptormediated transportation, 75 µg/mL of Caffeine (#C0750, Sigma-Aldrich) and 4.41 mg/mL of glucose (#G8270, Sigma-Aldrich) in culture medium (respectively), were used as working solutions. After a 3 h incubation, Caffeine ELISA kit (#DEIA6842, Creative Diagnostics) and Glucose Assay Kit (#ab102517, abcam) were used to quantify caffeine and glucose in the receiver plate, respectively. The permeability coefficient was calculated according to the following equation: Permeability Coefficient = ((VUP /(SA * t))*(CR * CUP) Where: VUP is the volume added to the upper side of the insert (400 µl); SA is the surface area of the insert (0.95 cm2); t is the time in s; CR is the concentration in the receiver plate; and CUP is the initial concentration added to the upper side of the insert. II-2.3.6. Enzyme-linked immunosorbent assay (ELISA) ELISA represents a simple and sensitive assay to quantify specific antigens, such as peptides, proteins, antibodies, and hormones. ELISA is an immunoassay relying on the specific binding between the antibody and the target antigen. Moreover, it is associated with a detection system that allows the quantification of the bound antigen. Generally, a specific primary antibody is attached to the surface of a 96-well plate that will bind to the antigen. Then, a secondary antibody, an enzyme and a substrate that links the enzyme are added. The color change can be measured using a spectrometer. The antigen concentration is determined using a standard calibration curve [26]. ELISA was used in chapters V and VI of this thesis. In chapter V, ELISA was used in permeability assays to assess the functionality of the developed BBB model using dHCM. Basically, after the permeability assay, ELISA was used to quantify the tested molecules present in the collected medium. ELISA kits for caffeine (#DEIA6842, Creative Diagnostics), glucose (#ab102517, abcam), and cholesterol (#STA-384, Cell Biolabs, Inc.) were used in accordance with manufacturer’s instructions. On chapter VI, Chapter II – Materials and Methods 68 ELISA was used as a complement to the tube assay formation for the quantification of angiopoietin 2 (#ab99971, abcam), an angiogenic growth factor secreted by endothelial cells. II-2.3.7. Transferrin uptake assay Transferrin receptor has been exploiting for the delivery of protein therapies to the brain [27]. So, on chapter V the transferrin uptake was evaluated to validate the functionality of the BBB in vitro model. Firstly, cells were starved for 1 h using culture medium without FBS. Then, the culture medium in the upper chamber of the insert was removed and replaced by 400 µL of a working solution of 25 µg/mL of biotinylated transferrin (#T3915, Sigma-Aldrich) in starving medium. Cells were incubated for 30 min and fixed with 10% formalin. After three washes with PBS, biotin signal was revealed using streptavidin 1:500 (#S11227, Thermo Fisher Scientific). Samples were analyzed under a fluorescence microscope with a coupled camera (Axio Imager Z1m, Zeiss). Images were analyzed with Zeiss Zen microscope software. II-2.3.8. Hemocompatibility assays Hemocompatibility is an important criterion that limits the application of blood-contacting biomaterials. When a biomaterial comes in close contact with blood, it should not adversely interact with any blood components and activate or compromise the bioactivity of any blood component [28]. In vitro blood compatibility assays allow for the analysis of a material’s hemocompatibility under controllable conditions, enabling direct comparison of outcomes. Although in vitro assays eliminate disturbing factors related to flow obstruction, surgery, and other tissue effects, they are associated with a more intense blood contact, since the generated products are not cleared [28]. In chapter VI, static and agitated in vitro hemocompatibility assays were performed. Glass surfaces, which show poor hemocompatibility [28] were used as positive controls of cytotoxicity. Fresh blood was collected via venipuncture from healthy human volunteers in BD Vacuntainer tubes (#367704, BD Diagnostics – Prenalytical Systems) containing sodium citrate. Platelet rich plasma (PRP), used in protein absorption assay, was provided by Centro Hospitalar de São João (Porto, Portugal) under an approved institutional board protocol (hospital ethical commission approval number 363/18). Chapter II – Materials and Methods 69 II-2.3.8.1 Protein adsorption assay When a material is in contact with blood, a protein layer mainly composed by plasma proteins (such as fibrinogen, immunoglobulins, fibronectin, vitronectin, factor XI and XII, von Willebrand factor, and plasma prekallikrein) is adsorbed on the material’s surface. This event may trigger adverse reactions, such as the activation of coagulation via intrinsic pathway, the activation of leukocytes, which results in inflammation, and the adhesion and activation of platelets that may lead to thrombus formation [28]. For protein adsorption assay, PRP was centrifuged at 800 G for 20 min at RT in order to obtain platelet poor plasma (PPP). 300 µL of PPP or PBS (negative control) were added to both sides of dHCM and glass surfaces. After a 1 h incubation at 37ºC, samples were washed three times with PBS. Then, samples were immersed in 1% SDS under agitation for 1 h and sonicated for 20 min. For protein quantification Micro BCATM Assay Kit (#23235, Thermo Scientific) was used according to manufacturer’s instructions. II-2.3.8.2 Platelet adhesion and aggregation assay Blood platelets are the smallest (1-3 µm) and the second most abundant cell type in the blood. In physiological conditions, platelets circulate in the blood in a quiescent state for 7-10 days. However, the exposure of a biomaterial to the blood results in protein adsorption that may lead to an undesired activation of platelets and consequently lead to thrombus formation [28]. Platelet isolation was performed by centrifuge the fresh blood at 200 g for 20 min at RT to obtain PRP. All the centrifugation steps of the protocol were performed with no brake applied. The PRP was then mixed with HEP buffer 1:1 ratio (v/v) with 1 µM prostaglandin E1 (P5515, Sigma-Aldrich) and centrifuged at 100 g for 18 min at RT. The supernatant was transferred to a new plastic tube and centrifuged at 800 g for 18 min. The pellet was washed twice with Platelet Wash Buffer (without resuspension) and the platelet concentrate was resuspended in 20 mL of Tyrode’s buffer containing 5 mM glucose (#G8270. Sigma-Aldrich) and 3% BSA (#A2153, Sigma-Aldrich). Finally, 300 µL of platelet suspension were seeded in both sides of dHCM and in glass surfaces and incubated for 1 h at 37ºC in a humidifier incubator with 5% CO2. After the incubation period, samples were washed 3 times with PBS to remove any non-adherent platelets and fixed with 10% formalin or 2.5% glutaraldehyde for fluorescent imaging and SEM, respectively (as described in sections II-2.3.3.3 and II-2.2.2, respectively). Moreover, samples were also immersed in Chapter II – Materials and Methods 70 2% Triton X-100 (#A16046, Thermo Fisher Scientific) in PBS to be used in lactate dehydrogenase (LDH) assay. Platelets have a high LDH activity. So, platelet adhesion was quantified by measuring LDH released when adherent platelets were lysed using 2% Triton X-100 using the CyQUANTTM LDH Cytotoxicity Assay (#C20301, Invitrogen) according to manufacturer’s instructions. The percentage of platelet adhesion on the different materials was determined from a calibration curve obtained by using different dilutions of the seeded platelets. II-2.3.8.3 Hemolytic assay Erythrocytes or red blood cells (RBC) are the most abundant cells in the blood and are important for the transport of oxygen from the lung to all tissues and of carbon dioxide from tissues to the lung and its damage may lead to a reduced oxygen transport. Moreover, when RBC are ruptured, they release hemoglobin that can induce toxicity or alter the kidney function. One way to assess the material compatibility with RBC is by the quantification of free hemoglobin, since an increased concentration of free hemoglobin is an indicator of erythrocyte disruption [28]. RBC were isolated by centrifugation of the fresh blood at 200 g for 20 min at RT. PRP was used for platelet isolation (as described in subsection II.2.3.8.2), buffy coat was discarded, and RBC (the downer portion) were washed 3 times with PBS. After each wash a centrifugation of 5 min at 3700 rpm was performed. Finally, RBC concentrate was diluted to a final concentration of 5 vol. % and 300 µL of RBC suspension were seeded in both sides of dHCM. For negative and positive controls, PBS (0 % lysis) and 1 % SDS (82 % lysis) were added to the RBC suspension, respectively. After 1 h incubation at 37ºC in a shaking incubator chamber, samples were washed with PBS (3x) and fixed with 2.5% glutaraldehyde (for SEM analysis as described in section II-2.2.2) or centrifuged at 120 g for 10 min to further hemoglobin quantification. Supernatants (50 µL) were transferred into a 96-well plate and the absorbance was measured at 540 nm using a microplate reader (Synergy HT, Biotek). To determine the hemolytic ratio the follow equation was used: Hemolytic ratio = (Sample – Negative control)/(Positive control-Negative control) Hemolysis ratio was interpreted as follow: < 2% non-hemolytic, 2-5% slightly hemolytic, and > 5% hemolytic samples. Chapter II – Materials and Methods 71 II-2.4. In vivo assays In vivo assays are important to assess material biocompatibility and functionality when implanted. Although in vitro models are useful and cost-effective in a primary phase of material characterization, they have limited capacity to recreate the complex in vivo environment. Thus, in vivo models are needed to understand host response to a biomaterial and are useful to predict its clinical behavior, functionality, safety and biocompatibility [29–31]. Laboratory animal protection legislation assumes that in, specific conditions, it is morally acceptable to use animals for scientific purposes when the following objectives are met: use the minimum number of animals, define legitimate purposes for which laboratory animals may be used, ensure the training ability of all laboratory personnel and researchers, avoid animals use when there are practicably available alternatives, avoid unnecessary pain or distress to animals, provide for the inspection of facilities and procedures, and ensure public responsibility [32]. The choice of an appropriate animal model is intrinsically related to the specific goals of the experiment. In this thesis, dHCM was implanted subcutaneously in mice to assess its biocompatibility (chapter IV). To verify dHCM angiogenesis, a chick chorioallantoic membrane (CAM) assay was used (chapter VI). Moreover, an orthotopic model in pigs was used to study the ability of dHCM to close diaphragmatic hernias (chapter VII). All studies were performed following EU Directive 2010/63/EU and approved by the animals’ ethics committee of the institution where the study was performed. Moreover, all personnel involved in the procedures were approved as competent for animal experimentation by DGAV (Portuguese general directorate of food and veterinary). II-2.4.1. Subcutaneous implantation in mice In early phases of research, ectopic models are preferred to orthotopic models because it is easier to identify the tissue response, to compare results with others in the literature, and to obtain the technical skills needed to perform them. Ectopic models are frequently used to assess the foreign-body reaction of a material in vivo , since they are able to provide information about chronic inflammatory response, and integration of the biomaterial within the host tissue [29,30]. One of the most commonly used implantation sites is the subcutaneous implantation. Chapter II – Materials and Methods 72 In chapter IV, a total of 20 BALB/c mice aged 9 weeks old (Charles Rivers, Barcelona, Spain) were used to study dHCM biocompatibility in vivo . Animals were housed five per cage in a limited-access rodent facility. Food and water were available ad libitum and animals were maintained in a climatecontrolled room (22.0 ± 0.5 ºC) in 12/12 h light/dark cycles. Before surgery, animals were exposed to handling twice a day. On surgery day, mice were anesthetized by an intraperitoneal injection of Domitor (medetomidine 0.5 mg/kg) and Imalgene (ketamine 75 mg/kg). The dorsum of the animals (implantation area) was shaved, cleaned and sterilized with Betadine® and two skin incisions were made (each 0.5 cm length) in each mouse. dHCM was implanted subcutaneously in each of the pockets and the incision was closed using 5-0 sutures (15 animals, 5 animals for each time-point). Empty defects were used as controls, either in the control group (5 animals, 2 animals for the first two time-points and 1 animal in the last time-point) and in the contralateral side of the pocket containing the material. After the procedure, the anesthesia was reversed with a subcutaneous injection of antisedan and the animals were monitored until fully awake (eating and grooming). After 4, 10 or 28 days, animals were euthanized using pentobarbital injection, and dHCM membranes and empty pockets were explanted (with the surrounding tissues). Explanted samples were fixed with 10% formalin and embedded in paraffin. Histochemical stains such as H&E and Masson’s trichrome (MT) were performed in 5 µm thick transversal sections. H&E staining was performed as described in subsection II.2.2.1. MT staining is commonly used and stains cartilage and collagen fibers in blue/green, muscle fibers in red, nuclei in black, cytoplasm in red/pink, and RBCs in red [33]. For MT staining, slides were submitted to Azure B solution for 5 min, stained with hematoxylin for 5 min and washed in picric ethanol for 5 min. After that, samples were stained in Biebrich Scarlet-Acid Fuchsin for 15 min, submitted to 1% phosphomolybdic acid for 5 min and to Aniline Blue for 4 min. Then, slides were washed with water, let to dry, rinsed with alcohol, cleared in xylene, mounted in Entellan rapid, and observed under an optical microscope with a coupled digital camera. II-2.4.2. Chick chorioallantoic membrane (CAM) assay CAM assay represents a rapid, simple and cost-effective in vivo test to screen the angiogenic potential of biomaterials, due to the highly vascularized nature of the CAM [34,35]. In chapter VI, white fertilized eggs were incubated for 3 days at 37ºC and a window was open into the eggshell to evaluate embryo viability. After 7 days, dHCM were implanted on the CAM forming two groups: dHCM reticular layer side in direct contact with CAM and dHCM trophoblast layer side in direct contact with CAM. Some Chapter II – Materials and Methods 73 eggs were left empty and were used as control. Eggs were then returned to the incubator and at day 17, embryos were sacrificed by freezing at -80 ºC for 10 min. After fixation with 4% formalin, ex-ovo images were captured for each condition in both sides of the CAM. At least 7 eggs were analyzed per condition (CTR, dHCM reticular layer side and dHCM trophoblast layer side). The macroscopic evaluation of the angiogenic response was carried out as previously described [36]. Moreover, an automated angiogenesis quantification analysis was performed using the Vessel Analysis plugin with Image J, where the measurements of vascular density and vascular length density are expressed as percentage area. II-2.4.3. Congenital diaphragmatic hernia (CDH) model in pigs In chapter VII, dHCM was compared with electropsun nanofiber meshes (PCLem) and GoreTex® for congenital diaphragmatic hernia repair in pigs. So, a novel diaphragmatic hernia animal model was established using thoracoscopy in sexually immature farm pigs, allowing for characterization of the effect of subject growth in defect size and regeneration. Fourteen farm male pigs ( Sus scrofa domesticus ) with approximately 6 weeks of age (6.54 ± 0.57 kg body weight) were used. Animals were housed in a facility with controlled temperature (24 ± 2 ºC) and automatic air renewal in socially stable groups of 2 or 3 animals in each pen. Dry pellet food was provided once a day, and water was available ad libitum . Wood shavings, fresh fruit, ropes and balls were provided for enrichment. Animals were randomly assigned to 3 different experimental groups of 4 animals each, determined by the type of patch used during thoracoscopic diaphragmatic repair (Gore-TexÒ, PCLem, or dHCM). Two additional animals were used as age and weight-matched controls and submitted to thoracoscopy without creation of diaphragmatic defect (sham group). After, at least, 7 days for acclimatization, and at the end of the protocol, animals were submitted to a chest X-Ray evaluation under sedation in order to document lung pathology, hernia recurrence or diaphragmatic eventration, and skeletal deformations such as scoliosis or thoracic wall deformities. All surgical procedures were performed under general anesthesia with endotracheal intubation and mechanical ventilation. An 8 h fasting period was performed, while water was provided until sedation time. Animals were anesthetized with a combination of ketamine (20 mg/kg, intramuscularly (IM)), xylazine (2 mg/kg), and atropine (0.04 mg/kg, IM), followed by propofol (4 mg/kg, intravenously (IV)) and maintained with continuous propofol infusion (20 mg/kg/h, IV) and buprenorphine (0.05 mg/kg). All surgical interventions were performed under sterile conditions and continuous cardiorespiratory monitoring. Animals were positioned in right lateral decubitus. A 5 mm port was placed Chapter II – Materials and Methods 74 in the 6th intercostal space aligning with the shoulder joint, for introduction of a 5 mm 30° telescope. Moreover, two working ports were placed in the 7th intercostal space under endoscopic vision, one 3 mm port along a line passing through the tip of the left scapula and the other 5 mm port approximately 5 cm from the left costal arch, allowing access to the diaphragm. Capnothorax was established with an inflation rate of 1 L/min and maintained at a maximum pressure of 3 mmHg. A left posterolateral diaphragmatic defect of 5 cm diameter was performed using a 3 mm scissor. Each patch was placed intra-thoracic via a 5 mm port and fixed using non-absorbable silk 4/0 or 5/0 simple interrupted sutures. While placing the dHCM and Gore-TexÒ patches, care was taken to place the previously marked side facing the abdominal cavity. Patches were tailored to the size and shape of the diaphragmatic defect. Two dHCM membranes were used in each pig. The dHCM patch was sutured to a plastic o-ring, allowing the membrane to unfold when inside the thorax and adjust to the defect area. Trocar-placement wounds were closed in layers using 2/0 polyglactine suture (Vicrylâ; Ethicon, New Jersey, USA), and a running subcuticular 4/0 polyglecaprone suture (Monocrylâ; Ethicon). Wounds were covered with a t-shirt to prevent scratching and biting during the post-operative period. Moreover, antibiotic (enrofloxacin, 5 mg/kg, IM) was administrated for 8 days. Post-operatively, animals were checked for any signs of distress, behavior changes, anorexia or weight loss. When any of these signs was present, human endpoints were applied in order to avoid suffering. After a post-operative period of 7 weeks, animals were euthanized using an overdose of pentobarbital (2000 mg/kg, IV) after administration of ketamine (20 mg/kg, IM) and xylazine (2 mg/kg, IM). In each animal, both hemidiaphragms were collected for further histologic and immunohistochemistry evaluation. Nevertheless, before euthanasia, all animals underwent a thoracoscopical and laparoscopical exploration in order to evaluate adhesion formation and the macroscopic condition of the patches. For histological and immunohistochemistry evaluation, samples were fixed in 10% formalin, embedded in paraffin and cut to 5 μm sections. H&E and MT staining were performed as previously described in sections II.2.2.1 and II.2.4.1, respectively. Slides were analyzed and scored by an operator blinded to the study groups and outcome at the time of the evaluation. For immunohistochemistry, slides were incubated with rabbit anti-alpha smooth muscle actin 1:50 (#ab5695; Abcam), rabbit anti-CD31 1:50 (#ab28364; Abcam) and mouse anti-CD105 1:100 (#MCA1557F; BioRad) for 2 h at RT. A secondary antibody from the R.T.U. VECTASTAIN® Universal ABC Elite® Kit (#PK-7200, Vector Laboratories) was used in accordance with manufacturer’s instructions and revealed with DAB (#SK- Chapter II – Materials and Methods 75 4100, Vector Laboratories). Sections were counterstained with hematoxylin, mounted in an aqueous mounting medium, and observed in an optical microscope with a coupled camera (DM750, Leica). II-2.5. Microbiology Microbiology tests are important to determine if a material has antibacterial properties. In chapter VI, dHCM antibacterial properties against Staphylococcus aureus ( S. aureus ) were studied, since it is the most prevalent microorganism associated with vascular graft infection [37,38]. For microbiology tests, dHCM were cut in pieces of 2.5 x 2.5 cm2 and polypropylene films were used as positive controls. S. aureus ; (#25923, ATCC) were gown in tryptic soy broth media (TSB; #610053, Frilabo) in a shaking incubator (80 rpm) at 37 ºC overnight. Prior to inoculation, bacterial strains were sub-cultured in fresh TBS at 1:50 dilution and incubated for approximately 2 h in a shaking incubator (80 rpm) at 37 ºC. A bacterial inoculum of 1 x 104 colony forming units (CFU)/mL was used to completely submerge the membranes in the dish for 1 h at RT. To remove non-attached bacteria, the dish was washed three times with sterile PBS, for 10 s with an 80 rpm rotation and surfaces were dried for 1 h. To identify viable bacteria, contact plates were pressed onto inoculated membranes for 20 s and incubated overnight at 37 ºC. After incubation, contact plates were photographed and colonies were counted. The resulting colonies were log transformed and presented as log CFU/ contact plate (2.5 x 2.5 cm2). II-3. STATISTICAL ANALYSIS Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, Inc. 2016). The Shapiro–Wilk test was used to assess data normality. When data followed a normal distribution, parametric tests were used, namely the unpaired t -test, one-way ANOVA followed by Dunn’s multiple comparison test or two-way ANOVA followed by Tukey’s multiple comparison test. When data did not follow a normal distribution, the Mann–Whitney test was used. When it was necessary to compare survival distributions between groups, Logrank test was used. In chapter VII, GPower version 3.1.9.2 (program written by Franz Faul, Universität Kiel, Germany 1992-2014) was used to calculate the minimum number of animals (effect size 0.7), to obtain a statistical power of 95%. Results were considered statistically significant when p < 0.05. Chapter III – Method to decellularize the human chorion membrane 82 III-1. INTRODUCTION Tissue engineering is an interdisciplinary field that aims to develop strategies to replace or regenerate disrupted or injured human tissues or/and organs to restore or establish normal function [1– 3]. It consists in a triad of cells, signaling molecules and scaffolds that support and rely upon each other. In fact, scaffolds can be combined with signaling molecules that will provide structural and biochemical cues for guiding cell behavior and tissue regeneration. Scaffolds may be composed by natural and/or synthetic materials and may be obtained through a variety of processing methods [1]. The therapeutic potential of scaffolds derived from the extracellular matrix (ECM) has been widely studied for the regeneration of a range of tissues [4,5] such as musculoskeletal [6–8], cardiovascular [9,10], gastrointestinal [11,12], central nervous system [13], among others [14]. These scaffolds are typically produced through the decellularization of a variety of mammalian tissues [5,13,15,16]. Decellularized matrices can function as substitutes for traditional biomaterials while offering additional advantages of maintaining important properties of the native ECM structure, giving cues for cell modulation and behavior [3,17–19]. These scaffolds are inductive for cell invasion, promoting a constructive remodeling processes that form vascularized, innervated, functional tissue [5,14,18,19]. Moreover, as ECM components are generally conserved among species, decellularized matrix scaffolds are well tolerated even by xenogeneic recipients [15,20,21]. The human placenta is considered a biological waste, but it is also a rich source of ECM proteins [22]. The human chorion membrane (HCM) is located between the amnion (fetal side) and the maternal decidua. [23,24]. It is formed by three different layers (from the fetal to the maternal side): 1) reticular layer that contacts with the amnion and represents the majority of the chorion thickness, composed of collagens I, III, IV, V and VI; 2) basement membrane, which anchors the reticular layer and the trophoblasts and is composed by collagen IV, fibronectin and laminin; 3) trophoblasts layers (3-5 cell layers) that provide nutrients to the embryo by digesting proteins before passing them into the fetal blood [25,26] (Figure III-1). Although promising, the potential of HCM alone is still largely unexplored as a biological membrane for applications in tissue engineering and regenerative medicine. It has only been used as a reservoir of stem cells [27], pro-angiogenic cells [28] and as a source of small-diameter vascular grafts [22]. Chapter III – Method to decellularize the human chorion membrane 83 Here we describe, for the first time, the decellularization process of the human chorion membrane that results in a compact and handleably membrane. Figure III-1 The human chorion membrane (HCM). (A) Scheme and (B) Hematoxylin/Eosin staining of the native HCM in a transversal cut. The different layers of the HCM – reticular layer, basement membrane, and trophoblast layer - are identified in the figure. III-2. METHODOLOGY III-2.1. Overview Human placentas are collected from elective and term cesarean sections. An informed consent must be signed by all donors. Sterile conditions are maintained during all processing steps and all solutions are sterilized by filtration (0.2 µm - #99500, Techno Plastic Products) or autoclaving. In summary, after collection the placentas are stored in Dulbecco’s phosphate-buffered saline (D-PBS - #21600, Gibco) with 10% antibiotic/antimycotic (#15240062, Thermo Fisher Scientific) at 4 ºC for a maximum of 1 day. The HCM is removed and separated from the amnion, washed with phosphate buffered saline (PBS - #P4417, Sigma-Aldrich) once to remove the blood and stored at -80ºC (Section 4) until the decellularization process (Section 5) for a maximum of 1 year. Pools of different placentas should be created in order to ensure traceability of the results. The decellularization protocol was optimized by quantifying and analyzing the presence and distribution of cell nuclei and dsDNA by DAPI (#40009, VWR) staining, PicoGreen (#P7589, Invitrogen) and electrophoresis. Chapter III – Method to decellularize the human chorion membrane 84 III-2.2. Materials • Human Placenta; • 350 x 250 x 70 mm Tray (#BOCH8736, VWR); • Glass container • Forceps, scissor and scalpel; • 3 L Buckets (#216-0793, VWR); • Cell scrapper (#99002, TPP); • Suture line (#01075503, Minhomedica) • Filtration system (#99500, TPP) NOTE: all materials used are previous sterilized III-2.3. Solutions • 70% Ethanol; • D-PBS (#21600, Gibco); • PBS (#P4417, Sigma-Aldrich); • 0.5% sodium dodecyl sulfate (SDS) (#MB18101, NZYTech) in ultra-pure water; • 0.1% SDS in ultra-pure water; • 1% Triton X-100 (#A16046, Alfa Aeasar) in PBS; • 0.001 mg/mL DNase (#A3778 PanReac AppliChem ITW Reagents) in DNA Reaction Buffer; • DNA Reaction Buffer: 10 mM Tris-HCl, 2.5 mM MgCl2, 0.5 mM CaCl2, pH = 7.6, RT; • 1-2% and 10% antibiotic/antimycotic (#15240062, Thermo Fisher Scientific) in PBS. III-2.4. Equipment • Flow chamber; • -80 ºC freezer; • 4 ºC fridge; • Water bath at 37 ºC; • Orbital shaker at 110 rpm; • Weighing machine. Chapter III – Method to decellularize the human chorion membrane 85 III-2.5. Separate the chorion membrane from human placenta 1 - Collect human placentas from elective and term cesarean sections. 2 - Store human placentas in Dulbecco’s phosphate-buffered saline (D-PBS) with 10% antibiotic/antimycotic for a maximum of 1 day. NOTE: Ideally maintained at 4 ºC or store at RT in an ice box. 3 - Put human placenta in a sterile tray and separate the less-vascular part of HCM from the placenta with a scissor (Figure III-2A-E). 4 - Remove the amnion membrane from the HCM by gently pushing it (Figure III-2F). 5 - Wash HCM with PBS once to remove the blood excess (Figure III-2G). 6 - Freeze the chorion membrane at -80 ºC until further use. Figure III-2 Separation of the HCM from the human placenta Chapter III – Method to decellularize the human chorion membrane 86 III-2.6. Decellularization protocol 1 - Submit HCM to two freezing/thawing cycles (- 80 ºC and 37 ºC, respectively - for 20 min each). 2 - Place the membranes in a sterile bucket and perform three washes with 0.5% SDS in ultra-pure water for 2 h at 4 ºC in an orbital shaker at 110 rpm. Change the buckets in each wash. 3 - Treat HCM with 0.1% SDS in ultra-pure water overnight, at 4 ºC in an orbital shaker at 110 rpm. 4 - Preform three washes with 1% Triton X-100 in PBS for 15 min at 4 ºC in an orbital shaker at 110 rpm. Change the buckets in each wash. 5 - Wash the membranes with PBS to remove the detergent for 2 h at 4 ºC in an orbital shaker at 110 rpm. Repeat this step at least three times. 6 - Scrape the membranes in both sides (Figure III-3). In order to identify each side of the membrane, knots with a suture line are performed. NOTE: In this step the volume of the membranes is considerably lower, due to the removal of the trophoblast layer. Membranes can be switched to a smaller compartment. 7 - Treat the membranes with 0.001 mg/mL DNase I in DNA reaction buffer at 37 ºC for 30 min. NOTE: DNase is maintained in sterile conditions. Only the DNA reaction buffer needs to me filtered. 8 - To block the DNase action, wash the membranes with 0.1% SDS in ultra-pure water for 30 min at 4 ºC in an orbital shaker at 110 rpm. 9 - Wash the membranes with PBS to remove the detergent for 2 h at 4 ºC in an orbital shaker at 110 rpm. Repeat this step at least three times. 10 - Store the membranes in PBS with 1 - 2% antibiotic/antimycotic at 4 ºC. Chapter III – Method to decellularize the human chorion membrane 87 Figure III-3 Scheme of the HCM decellularization protocol. III-2.7. DNA content measurement 1 – Use air-dry decellularized and native membranes. 2 – Weight 5 mg of each membrane type. 3 – Extract the DNA from both native and decellularized membranes using the DNeasy Blood and Tissue kit (#69504, Qiagen), according to the manufacturer’s instructions. 4 – Quantify the double-strand DNA (dsDNA) using Quant-IT PicoGreen dsDNA Assay kit (#P7589, Invitrogen) according to the manufacturer’s instructions. 5 – Perform an electrophoresis, to assess the size of the DNA fragments, using a 1% agarose gel and GeneRuler DNA Ladder Mix (#SM0334, Thermo Fisher Scientific) (Figure III-4). Chapter III – Method to decellularize the human chorion membrane 88 Figure III-4 dHCM characterization. (A-B) Transversal sections of DAPI staining in native HCM (A) and dHCM (B). (C) SEM micrograph of dHCM in a transversal cut. (D) Content of double-stranded DNA in native HCM and dHCM. (E) Electrophoresis in agarose gel of DNA extracted from both native HCM and dHCM. III-3. PITFALLS AND LIMITATIONS As all biological tissues, there is some variability between chorion membranes from different donors. Thus, it is very important to use pools of chorion membranes that were obtained from different donors. This variability is observed right from the beginning of the protocol, when the chorion membrane is separated from the human placenta since the less-vascular chorion size may vary between donors (section 4, Figure III-2). Moreover, the consistency of the chorion membranes is not always the same. This is particularly critical during the scrapping step (section 5, Figure III-3). In this step, less consistent membranes, can rupture. This may be a problem if large areas of tissue are needed, forcing the use of bigger placenta pools. Chapter III – Method to decellularize the human chorion membrane 89 III-4. DISCUSSION An appropriate decellularization process consists in the removal of cells and cell remnants while retaining the three-dimensional ultrastructure and composition of the ECM [13,14]. In this way it is possible to minimize the immune and inflammatory response to the ECM scaffolds [13]. In fact, there are established criteria for decellularized tissues to avoid host and cell adverse effects that consists in: 1) lack of visible nuclear material (stained with DAPI); 2) DNA fragments with less than < 200 bp; and 3) amount of dsDNA/mg of dry tissue less than 50 ng [29]. Importantly, all these criteria are achieved in the dHCM. Decellularization methodologies comprise mechanical, chemical, detergent and enzymatic techniques and/or combinations. The method presented in this work comprises a combination of these procedures. These techniques invariably cause a disruption of the ECM architecture, composition and consequently surface ligands [14]. In fact, the dHCM loses the trophoblast layer during the decellularization process. This protocol starts with two freezing/thawing cycles. This temperature change will form intercellular ice crystals, disrupting the cell membrane and leading to cell lysis [29]. Detergent combinations increase the ECM protein loss [30] and also promote better detergent removal from the decellularized tissue, preventing an adverse immune response in vivo [31] and cytotoxicity [32,33] In this protocol, the ionic detergent SDS is used to solubilize cell and nucleic membranes. Although SDS is associated with the removal of nuclear remnants and cytoplasmatic proteins, it is also related with the removal of glycosaminoglycans and growth factors and with collagen damage. So, it is expected a decrease of this ECM components in the dHCM. In addition to SDS, triton X-100 is also used. Triton X100 is a non-ionic detergent that disrupts protein-DNA, lipid-lipid, lipid-protein interactions as well as protein-protein interactions, in a milder degree. Like SDS, this detergent removes cellular components and is associated with the disruption and removal of GAGs [29]. The damage and elimination of ECM proteins and DNA from decellularized tissues increases with the exposure time to detergents [34,35] and also with the type of tissue and donor age [29]. In the protocol presented herein, the exposure times to detergents are relatively short but sufficient for cell removal, probably due to the small thickness of the tissue. Moreover, all solutions are used under agitation, facilitating tissue exposure and removal of cellular material. The agitation used in this protocol (110 rpm) is not considered aggressive, avoiding ECM disruption [29]. Chapter III – Method to decellularize the human chorion membrane 90 A physical step consisting in membrane scrapping is also applied in this protocol. In this step the trophoblast layer is removed. By membrane scrapping it is possible to decrease the exposure time to detergents and agitation, preserving the original ECM. After this step, suture knots are important to identify the sides of the membrane without being toxic and harmful for the decellularized membrane. This is a way to control and, eventually, decrease the variability associated with biological tissues. A treatment with DNase is also performed in order to have a DNA content in accordance with the established criteria for decellularized tissues, as previously described [29]. Nucleases catalyze the hydrolysis of ribonucleotide and deoxyribonucleotide chains, being useful for nucleotides removal after cell lysis [36,37]. However, they are difficult to remove from the tissue and could invoke an immune response. In this protocol a small concentration (0.001 mg/mL) of DNase is used in a reaction buffer to increase its action. In order to successfully remove it from the decellularized tissue, washes with 0.1% SDS (to inhibit its action) and PBS are performed. Traditionally, the decellularized tissue aims to mimic the original tissue. So, the application of the decellularized tissue is aimed at substituting or to regenerate the original tissue [16,38–42]. It was shown that ECM from different tissues has different compositions, architecture and ligands that will influence cell behavior differently [13]. For example, Crapo and colleagues showed that different cellular responses (including chemotactic and differentiation effects) to central nervous system (CNS) versus non-CNS ECM scaffolds were observed when using PC12 cell line. However, the mitogenic effects were similar in the different scaffolds, suggesting that both scaffolds could support site-appropriate cell phenotype [43]. Oppositely, the purpose of the dHCM is not to mimic the chorion membrane of the human placenta. The dHCM is aimed to mimic biological barriers in vitro (such as endothelium and epithelium) by providing a structural support but also chemical cues. This is achieved since an ECM compact and handleably membrane is obtained through the decellularization process. Moreover, we envision the application of this membrane in other fields of tissue engineering and regenerative medicine such as a scaffold to support and/or deliver stem and differentiated cells as well as a surgical patch for tissue regeneration (for example in hernias, wounds, burns and skin defects). Furthermore, ECM scaffolds are intended to facilitate remodeling of injured tissues in vivo [13]. The advantage of using placenta-derived tissues is its privileged immune tolerance [23], allowing a human body implantation without severe immune response. Chapter III – Method to decellularize the human chorion membrane 91 III-5. CONCLUSIONS This work describes, for the first time, a decellularization protocol for the HCM alone that results in a compact and handleably membrane. The decellularization protocol was successful since there were lack of visible nuclear material; the DNA fragments had less than 200 bp; and the amount of dsDNA was less than 50 ng/mg of dry tissue. The application of the dHCM is promising to mimic biological barriers in vitro, as substrate to support cell growth and/or deliver, and as a surgical patch. III-6. FUNDING This work was supported by the Portuguese Foundation for Science and Technology (FCT) (grant PD/BD/128103/2016 and FSE/POCH/PD/169/2013), by the European Regional Development Fund (ERDF) (grant NORTE-01-0145-FEDER-0000232) and by the project Cells4_IDs (PTDC/BTMSAL/28882/2017). The authors declare no conflict of interests. III-7. ACKNOWLEDGEMENTS The authors would like to acknowledge the donors and the professionals of Hospital of Braga. III-8. REFERENCES 1. Gilpin, A.; Yang, Y. Decellularization Strategies for Regenerative Medicine: From Processing Techniques to Applications. Biomed Res. Int. 2017, 2017 , 1-13. 2. He, M.; Callanan, A. Comparison of Methods for Whole-Organ Decellularization in Tissue Engineering of Bioartificial Organs. Tissue Eng. Part B 2013, 19 , 1-15. 3. Hortensius, R.A.; Harley, B.A.C. Naturally derived biomaterials for addressing inflammation in tissue regeneration. Exp. Biol. Med. 2016, 1015–1024. 4. Badylak, S.F. The extracellular matrix as a scafold for tissue reconstruction. Semin. Cell Dev. Biol. 2002, 13 , 377–383. 5. Badylak, S.F. The extracellular matrix as a biologic scaffold material. Biomaterials 2007, 28 , 3587–3593. Chapter IV – Decellularized Human Chorion Membrane as a Novel Biomaterial for Tissue Regeneration 98 IV-1. INTRODUCTION The human placenta is usually considered a biological waste. Nevertheless, it is consistently available from full-term births and it is also a source of mesenchymal stem cells, growth factors and extracellular matrix (ECM) proteins [1,2]. Some placenta-derived products are already used for tissue regeneration. Nevertheless, when nondecellularized they are only used in poorly vascularized sites, such as some wounds, tendon, ligament and cartilage [3]. So, one way to obtained ECM proteins and avoid a host adverse immune response when implanted in vivo , is through tissue decellularization [4]. In fact, the human placenta has been fully decellularized trough the creation of ECM suspensions to further create membranes [5] or sponges [6]. In these works, the various components of the human placenta are not differentiated, such as the amnion (HAM) and the chorion membrane (HCM) [7]. Moreover, the native tissue architecture is not maintained. Nevertheless, some works maintained the native tissue architecture after decellularization, for the whole placenta [8] and for the HAM [9–17] and HCM alone [18]. Decellularized HAM alone is widely characterized and studied and has been shown to induce osteogenic differentiation of human dental apical papilla cells [9], to be a good chondrocyte substrate/carrier [10], and to promote epithelization [11–13]. Moreover, decellularized HAM was used in cell-matrix adhesion studies, to produce skin equivalents, and as a pericardial substitute [12,14–17]. On the other hand, HCM alone as a biomaterial for tissue regeneration is still largely unexplored. Although, HCM has been used as a reservoir of stem [19] and pro-angiogenic [20] cells, and as a source of small-diameter vascular grafts [1], only one study uses a decellularized HCM (dHCM) with native tissue architecture (i.e. without ECM suspension creation) as a substrate for cell differentiation [18]. However, in that study, the dHCM is poorly characterized for the presence of ECM components, mechanical properties, in vitro cytocompatibility and in vivo biocompatibility. The purpose of this study is to fully characterize the dHCM, for the presence and distribution of cell nuclei, DNA, and ECM components. Finally, the mechanical properties, the in vitro and in vivo biocompatibility of dHCM were characterized envisioning its use as an ECM-based scaffold able to guide tissue regeneration, particularly when a membrane is needed to separate tissues, organs or other biologic compartments. Chapter IV – Decellularized Human Chorion Membrane as a Novel Biomaterial for Tissue Regeneration 99 IV-2. MATERIALS AND METHODS IV-2.1. Human Placentas Human placentas used in this study were collected from elective and term cesarean sections performed on the Obstetrics Department of the Hospital de Braga. The study follows a protocol approved by the ethical committee of the Hospital de Braga and of the University of Minho (CESH 030/2016, SECVS 136/2015, respectively) and following all the international best practices for research with human biological samples. An informed consent was signed by all the donors. Sterile conditions were maintained during all processing steps. Placentas were stored in Dulbecco’s phosphate-buffered saline (D-PBS) with 10% antibiotic/antimycotic (#15240062, Thermo Fisher Scientific) at 4 ºC for a maximum of 1 day. HCM was separated from the amnion, washed with phosphate buffered saline (PBS) to remove any blood and stored at - 80ºC until further use. IV-2.2. Decellularization of human chorion membrane (HCM) As previously described by us [21], HCM were submitted to two freezing/thawing cycles (- 80 ºC and 37 ºC, respectively) and, subsequently, were treated with different concentrations (0.5% and 0.1%) of sodium dodecyl sulfate (SDS) (#MB18101, NZYTech) solution in ultra-pure water. HCM were submitted to three washes with 0.5% SDS during 2 h each. Then, the membranes were washed overnight with 0.1% SDS. Three washes of 15 min were performed with 1% triton-X100 (#A16046, Thermo Fisher Scientific) in PBS. All treatments were done at 4 ºC in an orbital shaker at 110 rpm. The membranes were then scrapped in both sides and the trophoblast layer (TL) was removed and further used for collagen and glycosaminoglycans (GAGs) quantification. In order to identify both membrane sides (trophoblast and reticular layer sides), the membranes were marked with a knot with suture line. Subsequently, membranes were treated with 0.001 mg/mL DNase I (#A3778 PanReac AppliChem ITW Reagents), for 30 min at 37 ºC, followed by a 30 min wash with 0.1 % SDS at 4 ºC. Finally, the membranes were washed with PBS at least three times, during 2 h each, at 4 ºC. All the process was done under sterile conditions. After decellularization, the membranes were stored in PBS with 1-2% antibiotic/antimycotic at 4 ºC. Chapter IV – Decellularized Human Chorion Membrane as a Novel Biomaterial for Tissue Regeneration 100 IV-2.3. DNA extraction and quantification Membranes were air dried and weighed and total DNA from both native and decellularized HCM (dHCM) was extracted using the DNeasy Blood and Tissue kit (#69504, Qiagen), according to manufacturer’s instructions. The quantification of double-strand DNA (dsDNA) was performed using Quant-IT PicoGreen dsDNA Assay kit (#P7589, Invitrogen) according to manufacturer’s instructions. Electrophoresis was performed to assess the size of the DNA fragments, using a 1% agarose gel and GeneRuler DNA Ladder Mix (#SM0334, Thermo Fisher Scientific). Four independent samples were used in each condition. IV-2.4. Histological analysis The samples were fixed in 10% neutral-buffered formalin at 4 ºC (for at least 24 h), embedded in paraffin and 5 μm thick transverse sections were cut with a microtome. Histochemical stains such as hematoxylin and eosin (H&E) and Masson’s trichrome were performed in the samples. For H&E staining, samples were stained with hematoxylin (#7212, Thermo Fisher Scientific) for 1 min, washed for 30 s and stained with eosin for 10 min (#71204, Thermo Fisher Scientific). For Masson’s trichrome staining, slides were submitted to Azure B solution for 5 min, stained with hematoxylin for 5 min and washed in picric ethanol for 5 min. After that, samples were stained in Biebrich Scarlet-Acid Fuchsin for 15 min, submitted to 1% phosphomolybdic acid for 5 min, and to Aniline Blue for 4 min. After each staining, all slides were washed with water, let to dry, and rinsed with alcohol, cleared in xylene, and mounted in entellan rapid (#107960, Merck). Slides were observed under an optical microscope with a coupled digital camera (DM750, Leica). At least, three independent samples were used in each condition. IV-2.5. Scanning electron microscopy (SEM) Samples were fixed with 2.5% glutaraldehyde in PBS. After three PBS washes, samples were dehydrated with increasing concentrations of ethanol (10% to 100%). Samples were air dried and mounted in SEM pins using carbon tape. The samples were coated with gold using a Sputter Coater (#EM ACE600, Leica). Micrographs were collected with a Scanning Electron Microscope with EDS (#JSM-6010 LV, JEOL). At least, three independent samples were used in each condition. Chapter IV – Decellularized Human Chorion Membrane as a Novel Biomaterial for Tissue Regeneration 101 IV-2.6. Swelling assay Initially, dry samples were weighted and then were immersed in D-PBS or culture medium. At different time-points (0h30 min; 1h30 min; 2h30 min; 3h30 min; 4h30 min; 5h30 min), samples were weighted immediately after the excess of liquid was removed by putting them between two pieces of filter paper. Samples were re-immersed in liquid until the next time-point. The process was repeated until the equilibrium was reached (stable percentage of water uptake). Six samples were used in each condition. IV-2.7. Collagen quantification The collagen content of HCM, dHCM and trophoblast layer (TL) was extracted and quantified using Sircol Collagen Assay Kit (#S5000, biocolor) for soluble collagen and Sircol Insoluble Collagen Assay kit (#2000, bicolor) as described before [22] and according to manufacturer’s instructions. Three independent samples were used in each condition. IV-2.8. Sulfated glycosaminoglycans quantification Sulfated glycosaminoglycans (GAGs) content of HCM, dHCM and TL was quantified as previously described [23]. Samples were digested with 0.5 mg/mL papain (#P4762, Sigma-Aldrich) and the supernatant was stained with 1,9-dimethylmethylene blue (DMB). A dilution series of chondroitin sulfate in distilled water (50 μg/mL) was used as standard solution. Samples were diluted 1:10 in distilled water before measurement. Twenty μL of standards and diluted samples were mixed with 250 μL of DMB in a 96-well plate and absorbance was measured immediately at 525 nm using a microplate reader (SynergyHT, Biotek). Three independent samples per condition were analyzed. IV-2.9. Protein extraction and quantification Soluble protein was extracted from HCM and dHCM using Tissue Extraction Reagent I (#FNN0071, Thermo Fisher Scientific) with a protease inhibitor cocktail (#P8340, Sigma-Aldrich). The soluble protein content was determined using Protein Assay Dye Reagent Concentrate (#5000006, Biorad) according to manufacturer’s instructions. Chapter IV – Decellularized Human Chorion Membrane as a Novel Biomaterial for Tissue Regeneration 102 IV-2.10. SDS-PAGE The SDS Gel Preparation Kit (#08091, Sigma-Aldrich) was used to prepare the 4% stacking gel and 9% running gel. For each sample, 0.5 µg/µL of protein were loaded in the respective well. Following SDSPAGE, the gel was stained with Coomassie blue R-250 (HS-604, National Diagnostics) and image was obtained using a Transilluminator (Biospectrum ac chemi hr 410, UVP). At least, three independent samples were used in each condition. IV-2.11. Dot-Blot One drop of each sample of soluble protein was placed in a nitrocellulose membrane. After drying, membranes were incubated for 1 h with 5% BSA, with agitation, at RT. Subsequently, membranes were incubated with primary antibody: mouse anti-collagen type I 1:1000 (#ab90395, abcam); rabbit anticollagen type IV, 1:500 (#ab6311, abcam); rabbit anti-fibronectin 1:500 (#ab45688, abcam); and mouse anti-laminin, 1:500 (#L8271, Sigma-Aldrich). After overnight incubation, membranes were washed 3x for 5 min with TBS-tween 20, and then R.T.U. VECTASTAIN® Universal ABC Elite® Kit (#PK-7200, Vector Laboratories) was used as a secondary antibody, in accordance with manufacturer’s instructions. Finally, incubation was revealed using Peroxidase Substrate Kit (DAB) (#SK-4100, Vector Laboratories). Extraction buffer without samples was used as a negative control. Collagen type I (#sc-136157, Santa Cruz Biotechnology), collagen type IV (#C5533, Sigma-Aldrich), fibronectin (#FC010, Sigma-Aldrich), and laminin (#L6274, Sigma-Aldrich) were used as positive controls. At least, three independent samples were used in each condition. IV-2.12. Immunolocalization of proteins Immunolocalization of different proteins, such as collagen type I, collagen type IV, fibronectin and laminin, was performed in paraffin-embedded samples sectioned at 5 μm, as previously described for collagen type II [24]. Briefly, samples were incubated with mouse anti-collagen type I 1:100 (#ab90395, abcam); rabbit anti-collagen type IV, 1:50 (#ab6311, abcam); rabbit anti-fibronectin 1:300 (#ab45688, abcam); and mouse anti-laminin, 1:300 (#L8271, Sigma-Aldrich), overnight at 4 ºC in a humidified atmosphere. As a secondary antibody, R.T.U. VECTASTAIN® Universal ABC Elite® Kit (#PK-7200, Vector Laboratories) was used, in accordance with manufacturer’s instructions. Incubation was revealed using Peroxidase Chapter IV – Decellularized Human Chorion Membrane as a Novel Biomaterial for Tissue Regeneration 103 Substrate Kit (DAB) (#SK-4100, Vector Laboratories). Samples were counterstained with hematoxylin and mounted in an aqueous mounting medium. Slides were observed in an optical microscope with a coupled camera (DM750, Leica). At least, three independent samples were used in each condition. IV-2.13. Mechanical properties Static mechanical properties were assessed using Universal mechanical testing equipment (#5543, INSTRON) equipped with a 1 kN load cell. Nine samples of HCM and dHCM were cut in pieces of 20 mm x 5 mm and mounted in specific cassettes (to prevent the clamping system from damaging the samples) and the specimens were hydrated. The strain rate was defined at 5 mm/min and a 10 mm gauge length was used in tensile tests. Tests were finished when the specimens were ruptured. IV-2.14. Cytotoxicity analysis Human umbilical vein endothelial cell line EA.hy926 and human lung fibroblast cell line MRC-5 were cultured in complete medium (DMEM with 10% fetal bovine serum (FBS; #A3160801, Thermo Fisher Scientific) and 1% Penicillin/Streptomycin (#15240062, Thermo Fisher Scientific)) in T150 flasks at 37 ºC in a humidifier incubator with 5% CO2. After reaching 80% of confluence, the cells were seeded into inserts. dHCM were mounted in cell crown inserts for 24-well plates (#Z742380-12EA, Sigma). Millicell hanging cell culture inserts, PET 0.4 µm for 24-well plates (#MCHT24H48, Millicell) were used as control. Both inserts were immersed in culture medium overnight before cell seeding. EA.hy926 and MRC-5 cells were seeded on the inserts with a density of 20x103 cells/cm2. Metabolic activity was evaluated by MTS assay (CellTiter 96 AQueous One Solution, Promega). Three independent assays for each cell line were performed. IV-2.15. In vivo studies IV-2.15.1. Animals and ethical issues The experiments were performed in male Balb/c mice with 9 weeks old (Charles Rivers, Barcelona, Spain) and a total of 20 animals were used. Animals were housed five per cage in a limited-access rodent facility. Food and water were available ad libitum and animals were maintained in a climate-controlled room (22.0 Chapter IV – Decellularized Human Chorion Membrane as a Novel Biomaterial for Tissue Regeneration 104 ± 0.5 °C of temperature) in 12/12 h light/dark cycle with lights on at 8:00 am. The experimental protocol was approved by the Institutional Ethical Commission and followed the European Community Council Directive concerning the use of animals for scientific purposes. Before surgery, animals were exposed to handling twice a day. All efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data. IV-2.15.2. Subcutaneous implantation The main goal of this in vivo study was the assessment of the biocompatibility of dHCM membranes in healthy animals. Balb/c mice were anesthetized by an intraperitoneal injection of Domitor (Medetomidine 0.5 mg/Kg) plus Imalgene (Ketamine 75 mg/kg). The dorsum of the animals (implantation area) was shaved, cleaned and sterilized with betadine®, and two skin incisions were made (each 0.5 cm length) in each mouse. A sample of the dHCM was implanted subcutaneously in each of the pockets and the incision was closed using 5-0 sutures (15 animals, 5 animals for each time-point). Empty defects were used as controls, either in the control group (5 animals, 2 animals for the first two time-points and 1 animal in the last time-point) and in the contralateral side of the pocket containing the material. After the procedure, the anesthesia was reversed with a subcutaneous injection of anti-sedan and the animals were monitored until fully awake (eating and grooming). After 4, 10, or 28 days, animals were euthanized by pentobarbital injection, and dHCM membranes and surrounding tissues were explanted for subsequent histological analyses. IV-2.16. Statistical analysis Statistical analysis was performed using Graph Pad Prism 7. Shapiro-Wilk test was used to assess data normality. When data followed a normal distribution, parametric tests were used, namely unpaired t-test and two-way ANOVA test followed by Turkey’s multiple comparisons test. When data did not follow a normal distribution, the Mann-Whitney test was used. A p < 0.05 was considered statistically significant. Chapter IV – Decellularized Human Chorion Membrane as a Novel Biomaterial for Tissue Regeneration 105 IV-3. RESULTS IV-3.1. HCM decellularization and ECM protein retention The efficiency of the decellularization protocol was assessed and the results are summarized in Figure IV-1. In the H&E stainings no nuclei can be observed in the dHCM when compared with native tissue (Figure IV-1A, IV-1B). Moreover, the chemical and physical processes used in the decellularization process completely removed the HCM’s trophoblast layer and the nuclei from the reticular layer (Figure IV-1A, IV-1B). SEM images revealed that the dHCM is composed by nanofibers in the reticular layer side (Figure IV-1C), however, a thin compact layer covered the dHCM in the trophoblast layer side of the membrane (Figure IV-1D). Cellular removal in the dHCM (Figure IV-1F) was also corroborated by DAPI staining in which no signal is detected compared with the native membrane (Figure IV-1E). Additionally, DNA quantification and length of DNA fragments showed a significant removal of DNA ( p = 0.0002) in dHCM when compared with HCM (Figure IV-1G). The dHCM presented a DNA content around 10 ng of dsDNA/mg of dry tissue (Figure IV-1G). DNA content of dHCM was so low that no DNA was visible on the agarose gel in lanes loaded with dHCM, compared with the large amounts of DNA in lanes loaded with native tissue (Figure IV-1H). Thickness measurement of air-dried HCM and dHCM was also performed. After the decellularization protocol the membrane became 5 times thinner (119.50 ± 34.32 µm of HCM compared to 24.50 ± 3.11 µm of dHCM) ( p = 0.0150; Figure IV-1I). Although, having large differences in tissue thickness between native and decellularized tissue, a compact and easy to manipulate membrane was obtained (Figure IV-1J, IV-1K). The swelling behavior of dHCM in culture medium and PBS is shown in Figure IV-1L. In the first 30 min, an increase of 230 – 240% of dHCM’s weight was observed. After this time, the swelling behavior stabilized around 300 – 350%. Moreover, when wet, dHCM’s thickness increased to 36.00 ± 8.03 µm ( p = 0.0159; data not shown). Chapter IV – Decellularized Human Chorion Membrane as a Novel Biomaterial for Tissue Regeneration 106 Figure IV-1 Human chorion membrane decellularization. Representative transversal sections of Hematoxylin/Eosin (H&E) staining of HCM (A) and dHCM (B). Representative SEM micrographs of dHCM from the reticular layer side (C) and the trophoblast layer side (D). Representative transversal sections of DAPI staining from HCM (E) and dHCM (F). Double-stranded DNA (dsDNA) quantification in HCM and dHCM (G). Agarose gel electrophoresis of DNA extracted from HCM and dHCM (H). Thickness of air-dried HCM and dHCM measured with a picometer in, at least, three different sites (I). Top view of HCM (J) and dHCM (K) cut into pieces of 2 cm x 2 cm. Swelling behavior of dHCM in culture medium and D-PBS (L). Where, ** p £ 0.01 and *** p £ 0.001. Chapter IV – Decellularized Human Chorion Membrane as a Novel Biomaterial for Tissue Regeneration 107 The dHCM’s composition was assessed using SDS-PAGE, dot blot and immunolocalization of proteins. SDS-PAGE gel showed similarity among dHCM samples (Figure IV-2A), suggesting that the decellularization protocol is reproducible. Moreover, when compared to HCM, dHCM seems to preserve the higher molecular weight species (Figure IV-2A). By dot blot, it was possible to confirm the presence of some ECM high molecular weight proteins, such as collagen type I, collagen type IV, fibronectin, and laminin, in both native and decellularized tissues (Figure IV-2B). To assess the distribution of these proteins, tissue sections of HCM and dHCM were used. As observed in Figure IV-2C, while collagen type I was spread along the dHCM, collagen type IV, fibronectin, and laminin were focused on dHCM’s trophoblast layer side. Quantitative analysis was performed to detect the presence of soluble and insoluble collagen and sulfated glycosaminoglycans (sGAG) content in HCM, dHCM, and trophoblast layer (TL). Regarding soluble collagen (Figure IV-2D), significant differences were observed between the HCM, dHCM and TL ( p = 0.0045 (HCM vs. dHCM); p < 0.0001(HCM vs. TL); p = 0.0006 (dHCM vs. TL)). Soluble collagen content was 6.33 ± 0.92 µg/mg of dry tissue in HCM, 3.70 ± 0.46 µg/mg of dry tissue in dHCM and, 0.80 ± 0.06 µg/mg of dry tissue in TL. This tendency was not observed in insoluble collagen content (Figure IV-2E), where dHCM was associated with a higher quantity of insoluble collagen (137.90 ± 19.68 µg/mg of dry tissue) when compared with HCM (68.51 ± 18.51 µg/mg of dry tissue, p < 0.0001) and TL (55.09 ± 20.42 µg/mg of dry tissue, p < 0.0001). No statistically significant differences were observed between HCM and TL ( p = 0.2850). The total collagen (soluble and insoluble) amount was significantly higher in dHCM (142.30 ± 20.07 µg/mg of dry tissue) when compared with HCM (74.78 ± 17.60 µg/mg of dry tissue, p < 0.0001) and TL (54.66 ± 20.92 µg/mg of dry tissue, p < 0.0001). No differences were observed between HCM and TL ( p = 0.1406). For sulfated GAG quantification (Figure IV-2F), dHCM presented the lowest content (2.64 ± 0.19 µg/mg of dry tissue) when compared with HCM (17.07 ± 1.08 µg/mg of dry tissue, p < 0.0001) and TL (6.45 ± 2.09 µg/mg of dry tissue, p = 0.0040). Sulfated GAGs content in HCM was also significantly higher than in TL ( p < 0.0001). Chapter IV – Decellularized Human Chorion Membrane as a Novel Biomaterial for Tissue Regeneration 114 4 days after implantation, suggesting a rapid recovery of the pro-inflammatory phase associated with biomaterial implantation. This also demonstrates that dHCM final washes with PBS are sufficient and efficient since, it is free of DNase and detergents (such as SDS) associated with adverse immune responses in vivo [50]. Importantly, on day 28, it is possible to observe that dHCM membranes are being integrated by the host tissue revealing the beginning of a tissue regeneration stage. In contrast with SIS that is almost completely infiltrated by host cells after 1 week subcutaneously [49]. However, SIS has been associated with higher recurrent rates in hernia repairs [51–53] that may be associated with rapid degradation and integration. So, the longer time until integration by host tissue observed with dHCM might be a positive point, allowing a sustained tissue regeneration and eventually decreasing unwanted/adverse effects such as hernia recurrence. Moreover, in general, dHCM and dHAM have a similar behavior in vivo [34]. Altogether, these results support the hypothesis that dHCM may be used as a biomaterial for promoting tissue regeneration in case of hernias, barrier defects, wounds, burns and/or skin defects. IV-5. CONCLUSIONS In this work, the dHCM was fully characterized, for the first time, for the presence and distribution of ECM components, mechanical properties and in vivo and in vitro behavior. It was demonstrated that the dHCM is in accordance with the established criteria for decellularized tissues to avoid cell and host adverse reactions [4]. As SIS and dHAM, dHCM preserved some important ECM proteins such as collagen type I, collagen type IV, fibronectin and laminin and showed to be biocompatible in vitro and in vivo in subcutaneous implantation. Nevertheless, as a differentiator characteristic, dHCM has two different surfaces (reticular layer side and trophoblast layer side) that have different composition and topography. These results, together with the ability to suture dHCM and its mechanical properties, support the hypothesis of dHCM to be used as a biomaterial for tissue regeneration applications, particularly when a membrane is needed to separate tissues, organs or other biologic compartments. Chapter IV – Decellularized Human Chorion Membrane as a Novel Biomaterial for Tissue Regeneration 115 IV-6. FUNDING This work was supported by the Portuguese Foundation for Science and Technology (FCT) (grant PD/BD/128103/2016 and FSE/POCH/PD/169/2013) and by the European Regional Development Fund (ERDF) (grant NORTE-01-0145-FEDER-0000232). The authors declare no conflicts of interest. IV-7. ACKNOWLEDGMENTS The authors would like to acknowledge the donors and the professionals of the Hospital of Braga that helped in obtaining the placenta samples used in this study. IV-8. REFERENCES 1. Schneider, K.H.; Aigner, P.; Holnthoner, W.; Monforte, X.; Nürnberger, S.; Rünzler, D.; Redl, H.; Teuschl, A.H. 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SUPPLEMENTARY MATERIAL Supplementary Figure IV-1 Representative images of H&E staining of control animals used in the subcutaneous assay performed on BALB/c mice at three different time-points (4, 10 and 28 days). In sham animals only the surgery was performed (without dHCM implantation). Empty pockets were performed on the contralateral side of animals implanted with dHCM. 121 SECTION 4 DECELLULARIZED HUMAN CHORION MEMBRANE (DHCM) APPLICATIONS 122 Chapter V A functional Blood-Brain Barrier in vitro model using decellularized human chorion membrane Chapter V – Decellularized human chorion membrane as a substrate to create a BBB in vitro model 123 Chapter V Chapter V - Decellularized human chorion membrane as a substrate to create a BBB in vitro model † ABSTRACT The creation of functional blood-brain barrier (BBB) in vitro models is of great interest for drug screening, to study diseases’ mechanisms, and to create valid alternatives to animal testing. In this work, we demonstrated the suitability of the decellularized human chorion membrane (dHCM) to create an in vitro model of the BBB. For that, cerebral endothelial (bEnd.3) and astrocytic (C8-D1A) cell lines were used. Our results demonstrated that dHCM can mimic the BBB basement membrane and that the transendothelial electrical resistance (TEER) values were higher in our dHCM-based BBB in vitro model than the ones described in literature. Moreover, we determined the apparent permeability coefficient (Papp) of three different transport routes across BBB (membrane diffusion, solute carrier, and receptor-mediated) by using caffeine, glucose, and transferrin, respectively. The expression of glucose (GLUT1) and transferrin receptors by bEnd.3 cells was also demonstrated. In this work, we demonstrated that dHCM is a suitable substrate to create an in vitro model of BBB. † This chapter is based on the following publication: Frazão, L. P.; Vieira de Castro, J.; Nogueira-Silva, C.; Neves, N.M. Decellularized human chorion membrane as a substrate to create a BBB in vitro model (submitted). Associated with the international patent application WO/2021/005531.