Avaliação de suportes eletrofiados de PLLA-ECM para regeneração óssea

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UNIVERSIDADE DE SÃO PAULO

INSTITUTO DE QUÍMICA

Programa de Pós‐Graduação em Química

Mariana Carvalho Burrows

Avaliação de suportes eletrofiados de

PLLA‐ECM para regeneração óssea

Versão corrigida da tese de defendida

São Paulo

Data do Depósito na SPG:

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Mariana Carvalho Burrows

Avaliação de suportes eletrofiados de PLLA‐ECM para

regeneração óssea

Tese apresentada ao Instituto de Química da

Universidade de São Paulo para obtenção do

Título de Doutora em Ciências no Programa de

Química.

Orientador: Prof. Dr. Luiz Henrique Catalani

São Paulo

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Mariana&Carvalho&Burrows&

&

Avaliação)de)suportes)eletrofiados)de)PLLA4ECM)para)regeneração)óssea)

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This thesis is dedicated to the ones whom I love most

Almighty God,

My lovely and supportive husband and son,

David John Burrows and David John Carvalho Burrows and

My parents Vicente Carvalho Lima e Lidia M C Carvalho

.

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ACKNOWLEDGEMENTS

Finally, this moment has arrived! I can not express in words all the feelings and moments that come to mind when I think about the idea of finally being awarded my Ph.D. degree. The following quote is the closest description of all the battles I faced throughout my journey: “Ph.D. is an exhausting, emotional struggle. You are forced to confront all your fears, insecurities and doubts you have about yourself and somehow overcome them. It is terrifying. A lot of bravery is required, which often goes unrecognised and unrewarded” ‐ Anonymous.

God, my Saviour, the God who created me and chose me before I was born and even before I was conceived, gave me this victory, and all the Honour is to His name.

God has been very merciful in my life; He gave me the blessing of finding on my academic path such good people from all around the world. The people I met on my journey helped me in many different ways; furthermore, they have my deepest admiration. I will try to mention by name all those people that were so important to make this dream come true. Each of you taught me something that built the person and professional that I am now. How many times have I thought of giving up? Many! How many times have I thought my PhD was a higher mountain than I had the energy to climb? Thousands! However, all the following people were there to show me that I was not alone on this journey, and this gave me the resilience I needed.

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My husband was the person that knew about my dream of doing my MSc. and my PhD since the day we first met. He did everything to inspire me and make it possible for my dream to become reality. He took my dream as his own, everything related to my studies came first and for all the efforts he made, that’s why this thesis also belongs in my heart to David John Burrows.

My son grew up understanding why I was doing a PhD and what I was doing every day at University. The happiest days for my son were when he was able to come and visit the lab for a short time to see for himself where mummy used to go every day. He understood why academic research is so important and so close to everyone’s life. He understood so well that today at 8 years old, he tells people that he wants to be a chemistry researcher.

I cannot express how I felt after some experiments failed after months of preparation... Your biggest experiment has failed and nothing that you can think of would make it work, but then you come home and you find two awesome smiles and many comfort hugs to cheer you up. They thell you that they love you and that they will support you in every task you need to accomplish no matter how many more big experiments you need to conduct, and tell you to never give up. My husband and my son, my two Davids, my two beloveds, were the forces that God put in this world to remind me that I should never give up. They are my angels.

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I am also very proud of, and thankful to, my brothers, Antônio Felipe and Vicente, both aeronautical engineers; they showed me how it is possible to have dreams come true. They are such an inspiration to me, in addition, they always were there supporting me.

I am extremely thankful to my supervisor Dr. Luiz Henrique Catalani, who was responsible for me during my MSc. and my PhD, and who advised, guided and encouraged me on my way. I would like to say that he was not only a boss but also a great mentor. He taught me how to be critical about my data, how to think as a researcher, how to raise questions. In addition to this I can say that he was like a father to me, that took actions always thinking of the best for me.

I am also extremely thankful to my supervisor Dr. Eileen Gentleman, a brilliant researcher, from who I have learned so much, She always believed in my academic potential, always encouraged me, and always had great ideas that solved my problems in five minutes. I am very grateful and proud to have been one of your students.

My research would never have been possible without the support of the Universidade de São Paulo, specifically the Instituto de Química, and King’s College London, specifically the department of Craniofacial Development and Stem Cell Biology. It was a great honour being on the student roll of two such renowned Universities in my research area.

Thanks to Prof. Dr. Frederic Festy who helped me with the SHG and Micro‐Raman analysis and supported me with the Clustering Analysis Program and interpretation of the data.

Thanks to Prof. Dr. Cynthia Andoniadou and Prof. Dr. Agi Gregoriardis that helped me through discussion of my data, and for all the raised questions during our weekly lab meetings. I am very thankful because you gave me the opportunity to learn about different research fields, approaches and techniques.

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de Biomateriais Poliméricos. THANKS to: Vânia Bueno, Ana Paula Immich, Renata Fogaça, Ricardo Bentini, Alliny Naves, Vitor Zamarion, Diego Clemente, Fernando Luengo, Janaína Barros, Daniel Minatelli, Flávia Cara, Mayara Sguerra, Danielle Juais, Patrícia Ponce, Antônio Caldeira and Flávia Gonçalves. They were there every time for whatever I needed and I always knew that. They helped with my experiments and analysis; furthermore, they always had friendly words, hugs and smiles on a daily basis. So much time spent together, these unforgettable friends are far away the best group I could find during such a hard time as post‐graduate studies. I will never forget our songs, our jokes, our happy hours and thesis celebrations, our lunchtimes, our breakfasts, our pizza nights for night work shifts, and how good we were working together. Thanks for your friendship! I wish you the best that life can give to you. God bless you all! Today we are all apart in different parts of the world but our memories keep us together and I wish that wherever you are that you can each shine as a professional and as a person, as you shone on my pathway.

My unforgettable friend, Silvia Oliveira de Paula, this thesis is also dedicated to you, we had such happy moments together, our trips, our lunch times, your hugs, your advice. Every time we met since I went to the UK, we cried out of happiness. You were like a mum to me ‐ I remember your voice, your smile, your hug. I know how much you wanted to see me finishing my PhD, and I do it thinking about how proud you would be of me.

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As I said before, this thesis would never have been possible If God did not put in my path the many angels that I found on my way. My best friend and my sister, Aline Ferro, was always amazing, supporting me in every moment, many times cheering me up, giving me a bed in her house, sometimes making me a hot dinner when I was miles and miles away from my family. I have no words to acknowledge you! You have such a big and pure heart. I know everything you did is because you love me a lot but I can say to you that I love you a lot too and I hope that one day I can give you back everything that you gave to me, not expecting to get it back. Thanks Aline because You gave me the gift of knowing your husband Ricardo Ferro and your son Samuel Ferro. Thanks Ricardo for all the times you looked after me, taking me to the airport, moving house, for all days I have spent in your house, for being so friendly... God bless you! The Burrows family loves the Ferro family, and your family is the clear expression of love and blessings of God in my life.

Thanks to Prof. Dra. Ana Campa and her group for all their patience while giving me the induction training on cell culture, all the professionalism and friendship, and additional special thanks to Silvana, Fabíola, Franciele and Edson.

Thanks to Prof. Dra. Célia Regina da Silva Garcia and her group for the scientific contributions.

Thanks to Prof. Dr. Roberto Torresi and Prof. Dra. Suzana Torresi and their group, that always were kind and ready to cover my SEM samples with gold.

Thanks to my PhD lecturers, Prof. Dr. Jivaldo do Rosário Matos, Prof. Dra. Denise Freitas Siqueira Petri, Prof. Dr. Luiz Fernando da Silva Júnior, Prof. Dr. Josef Wilhelm Baader, Prof. Dr. Yoshio Kawano.

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Thanks to the post‐graduation officers at the Instituto de Química (USP): Cibelle, Milton, Emiliano, Ale e Marcelo. In addition to them, thanks to the post‐graduation officers from King’s College London: Angela Gates and Christopher Healy. Thank you all for your friendship and all your kindness.

Thanks to the lab technicians from Instituto de Química (USP): Luiz, Marcos (Bigão) and Alfredo, you are amazing!!! Always solving my problems, finding and sorting out machines I needed to use, finding and buying my reagents, trying to keep the lab tidy and on top of this for being so very friendly. Thanks to the lab technician from King’s College London: Susmitha Rao, that helped a lot on the cell culture and have being always kind and friendly to me, and Edgar Alasdair that helped me with tissue processing and histology staining.

Thanks to my Brazilian friends that I met in the UK during my PhD, Tathy, Renata, Isabella and Rodrigo Moura, Sara and Eduardo Dias, Mika and Pedro, Dezyree e Pablo Pereira, Katia Reis and Jose Delgado, You guys are fabulous and amazing friends, thanks for the friendship and all the support you gave me, it was very nice to have a piece of Brazil and feeling part of a family in the UK.

Thanks to my in‐laws Suzanne and Donald Burrows. Thanks for every time you supported your son and grandson while I was at University either in São Paulo or in London. Furthermore, I am thankful because you raised the boy who is now my husband and is such a gentleman and honourable man.

Thanks to FAPESP because the financial support for my PhD at USP and for my research sponsorship at King’s College London was essential. Thanks because FAPESP always believed in my potential as a researcher and supported me with funding. In addition, thanks to CNPQ and Capes for the financial support.

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“You cannot connect the dots looking forward; you can only connect them

looking backward. So you have to trust that the dots will somehow connect in

your future. You have to trust in something — your gut, destiny, life, karma,

whatever. This approach has never let me down, and it has made all the

difference in my life.”

Steve Jobs

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Resumo

Burrows, M. C., Avaliação de suportes eletrofiados de PLLA‐ECM para regeneração óssea. 2016. 219. Tese de Doutorado‐ Programa de Pós‐Graduação em Química. Instituto de Química, Universidade de São Paulo, São Paulo.

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malha menos porosa. As análises de TGA e DSC confirmaram a incoporação das ECMs nas malhas eletrofiadas, e através da técnica de TPEF‐SHG observou‐se a distribuição das proteinas no polímero. O potencial dos materiais para a regeneração óssea foi avaliado através da cultura de células tronco mesenquimais de medula óssea sobre os suportes eletrofiados durante 21 dias, e em seguida, medidas de ALP, quantificação de coloração com vermelho de alizarina, imunofluorescência com anticorpo col1a2, e expressão de gênica foram analisadas para a avaliação de como os materiais eletrofiados de PLLA‐ECM induzem a osteodiferenciação.

Comparando‐se materiais produzidos por co‐solução e os materiais imobilizados foi possível observar que a resposta osteogênica é maior nos materiais híbridos devido a liberação de fatores solúveis dos suportes eletrofiados. No entanto, comparando‐se o efeito da digestão enzimática na capacidade de mineralização dos suportes , é possível observar que o efeito da digestão enzimática é dependente do tipo de ECM. Em geral, foi possível observar que os suportes eletrofiados de PLLA‐ECM exibem potencial para uso em engenharia de tecidos, em específico, regeneração óssea, uma vez que apresentaram‐se regulados o conjunto de genes bglap, RunX2, Osx, sparc e col1a2.

Palavras‐chave: Matriz extracelular, PLLA, eletrofiação, engenharia de tecidos, regeneração óssea

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Abstract

Burrows, M. C., Evaluation of electrospun PLLA‐ECM scaffolds as biomaterials for bone regeneration.

2016. 219. Tese de Doutorado ‐ Programa de Pós‐Graduação em Química. Instituto de Química, Universidade de São Paulo, São Paulo.

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bone regeneration, bone marrow mesenchymal stem cells (BMMSCs) were cultured on the scaffolds over 21 days. Osteogenic markers such as ALP activity, mineral nodule formation by ARS staining, col1a2 immunostaining, and gene expression were analysed to access how the materials could induce BMMSCs osteodifferentiation. Comparing NCLK to CLK scaffolds the key factor for osteogenesis is the release of soluble factors, showing NCLK scaffolds with a higher ability to induce mineralization than CLK scaffolds. However, when comparing the effect of the enzymatic digestion on the mineralization of the scaffolds over the days, it is possible to establish that the effect of the enzymatic treatment is also related to the type of ECM. Despite all those differences, some PLLA‐ECM scaffolds exhibited potential to induce earlier mineralization, observed by the analysis of bglap, RunX2, Osx, sparc and col1a2 genes as osteogenic markers.

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List of acronyms and abbreviations

α‐MEM: alpha‐minimal essential medium

α‐SMA: smooth muscle alpha actin

AB: alamar blue

ABAM: antibiotic‐antimycotic ADAMTS: A desintegrin and metalloproteinase with thrombospondin motifs AKT: protein kinase B Ala: alanine ALP: alkaline phosphatase activity Arg: arginine ARS: alizarin red staining Asn: asparagine Asp: aspartic acid B‐ECM‐collagenase: extracellular matrix from demineralized bovine cortical bone digested by collagenase B‐ECM‐pepsin: extracellular matrix from demineralized bovine cortical bone digested by pepsin B‐ECM‐trypsin: extracellular matrix from demineralized bovine cortical bone digested by trypsin B‐ECM: extracellular matrix from demineralized bovine cortical bone Bglap: bone gamma‐carboxyglutamic acid‐containing protein, also known as osteocalcin BMMSCs: bone marrow mesenchymal stem cells BMP: bone morphogenic protein‐2 (BMP‐2); bone morphogenic protein‐4 (BMP‐4) BSA: bovine serum albumin

CBFα‐1: core‐binding factor alpha 1

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Col1a1: type I collagen, alpha 1 chain

Col1a2: type I collagen, alpha 2 chain

CS: chondroitin sulphate

Ct: threshold cycle

DAPI: 4',6‐diamidino‐2‐phenylindole DDR2: discoidin domain containing receptor 2 DS: dermatan sulphate DSC: differential scanning calorimetry DTG: differential thermogravimetric ECM: extracellular matrix EDC: 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide EGF: epidermal growth factor ERK: extracellular signal‐regulated kinase EthD‐1: ethidium homodimer‐1 FACITs: fibril‐associated collagen with interrupted triple helix FBS: fetal bovine serum FGF: fibroblast growth factor FN: fibronectin FWHM: full width half maximum Ile: isoleucine GAGs: glycosaminoglycans GASGER sequence: glycine‐alanine‐serine‐ glycine‐glutamic acid‐arginine sequence GFOGER sequence: glycine‐phenylalanine‐hydroxyproline‐glycine‐glutamic acid‐arginine sequence

GLA: γ‐carboxyglutamic acid

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PLLA/P‐ECM‐trypsin: electrospun scaffolds produced from eletrospinning of PLLA and trypsin‐digested extracellular matrix from bovine pericardium pNPP: p‐nitrophenylphosphate PPII: polyproline II q‐PCR: real‐time polymerase chain reaction RGD: arginine‐glycine‐aspartic acid amino acid sequence RNA: ribonucleic acid ROS: reactive oxygen species RT buffer: reverse transcription buffer RunX2: runt‐related transcription factor 2 SDS‐page: sodium dodecyl sulphate polyacrylamide gel electrophoresis SEM: scanning electron microscopy Ser: serine SIBLING: small integrin‐binding N‐glycosylated SLRPs: small leucine‐rich proteoglycans Sparc: secreted protein acidic and cysteine‐rich, also known as osteonectin Sp7: specific protein 7, also know as osterix Spp1: secreted phosphoprotein 1, also known as osteopontin TES: N‐[Tris(hydroxymethyl)methyl]‐2‐aminoethanesulfonic acid TG: thermogravimetric TGA: thermogravimetric analysis

TGF‐β: transforming growth factor‐beta

Thr: threonine

Tonset: onset temperature of the thermal event

Tpeak: temperature at the peak of the thermal event

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List of Figures

Figure 1. ECM representation and signalling molecules that direct the cell fate. Adapted from Lutolf &

Hubbell 2005) ... 33

Figure 2. Scaffolds produced to mimic the tissue environment; Adapted from (Dvir et al. 2011): 1, (Kim et al. 2010): 2, (Feng et al. 2009): 3, (Roohani‐Esfahani et al. 2010). ... 35

Figure 3. Collagen triple helix stabilization; Adapted from Hunter 2010 ... 38

Figure 4. Collagen fibrillogenesis reproduced from Klug and Cummings, 1997 ... 40

Figure 5. GAGs disaccharide units and features; Adapted from Gandhi and Mancera, 2008 ... 42

Figure 6. Representation of the potential of biomaterials composed by decelullarized ECM on tissue regeneration. Adapted from Reing, J.E. et al., 2009. ... 50

Figure 7. Protein quantification using Bradford assay for comparative analysis of the digestion promoted by collagenase, pepsin and trypsin on B‐ECM), P‐ECM and Col ... 75

Figure 8. Quantification of primary amines through ninhydrin assay for comparative analysis of the digestion promoted by collagenase, pepsin and trypsin on B‐ECM, P‐ECM and Col ... 76

Figure 9. GAGs quantification after digestion of B‐ECM, P‐ECM and Col promoted by collagenase, pepsin and trypsin ... 78

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Figure 11. Type I collagen structure showing triple helical and non‐triple helical domains; Adapted from Fan et al., 2012. ... 80

Figure 12. CD spectra of digested B‐ECM, P‐ECM and Col by collagenase, pepsin and trypsin ... 82

Figure 13. TPEF‐SHG showing images from B‐ECM (A‐C), P‐ECM (D‐F) and Col (G‐I); on the left, autofluorescence in green (A, D, G), on the middle SHG in red (B, E, H), on the right, composed images (C,F,I) ... 85

Figure 14. TPEF‐SHG showing images for B‐ECM digested by collagenase (A‐C), P‐ECM digested by pepsin (D‐F) and P‐ECM digested by trypsin (G‐I); on the left, autofluorescence in green (A, D, G), on the middle SHG in red (B, E, H), on the right composed images (C, F, I) ... 86

Figure 15. AB measurements (average ± standard deviation) for BMMSCs cultured with non‐osteogenic medium supplemented with digested ECMs over 21 days (n=3) ... 94

Figure 16. ALP activity normalized by metabolic activity of BMMSCs (average ± standard deviation) cultured with non‐osteogenic medium supplemented with digested ECMs on day 7, 14 and 21 (n=3) ... 96

Figure 17. ARS for BMMSCs cultured with non‐osteogenic medium supplemented with digested ECMs on day 14 ... 98

Figure 18. ARS for BMMSCs cultured with and non‐osteogenic medium supplemented with digested ECMs on day 21 ... 98

Figure 19. ARS quantification (average ± standard deviation) for BMMSCs cultured with non‐osteogenic medium supplemented with digested ECMs on days 14 (left) and 21 (right) (n=3) ... 99

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Figure 21. ALP activity normalized by metabolic activity (average ± standard deviation) of BMMSCs cultured with osteogenic medium supplemented with digested ECMs on day 7, day 14 and day 21 (n=3) ... 104

Figure 22. ARS images for BMMSCs cultured with osteogenic medium supplemented with digested ECMs on day 14 ... 106

Figure 23. ARS images for BMMSCs cultured with osteogenic medium supplemented with digested ECMs on day 21 ... 106

Figure 24. ARS quantification (average ± standard deviation) for BMMSCs cultured with osteogenic medium supplemented with digested ECMs on day 14 and day 21 (n=3) ... 108

Figure 25. SEM images from electrospun scaffolds (A) PLLA and (B) PLLA‐col 80:20; parameters: 5% (w/v)

solutions in HFP, flow rate (2.0 mL.h‐1), applied voltage (25 kV), distance (18 cm), temperature (22 o

C) and humidity (<40%) ... 114

Figure 26. SEM images from electrospun scaffolds obtained via electrospinning of the co‐solution PLLA/digested ECM 80:20 (w/w); NCLK scaffolds (A) PLLA/B‐ECM‐collagenase, (B) PLLA/B‐ECM‐ pepsin, (C) PLLA/B‐ECM‐trypsin, (D) PLLA/P‐ECM‐collagenase, (E) PLLA/P‐ECM‐pepsin, (F) PLLA/P‐

ECM‐trypsin. Parameters: 5% (w/v) solutions in HFP, flow rate (2.0 mL.h‐1), applied voltage (25 kV) and distance (18 cm), temperature (22 oC) and humidity (<40%) ... 114

Figure 27. SEM Images from electrospun PLLA‐col scaffolds CLK with digested ECMs (A) PLLA‐col/B‐ECM‐ collagenase, (B) PLLA‐col/B‐ECM‐pepsin, (C) PLLA‐col/B‐ECM‐trypsin, (D) PLLA‐col/P‐ECM‐ collagenase, (E) PLLA‐col/P‐ECM‐pepsin, (F) PLLA‐col/P‐ECM‐trypsin; parameters: 5% (w/v)

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Figure 28. Protein released from the electrospun scaffolds over 21 days quantified by Micro BCA protein assay (n=4). Control for NCLK scaffolds: PLLA; and control for CLK scaffolds: PLLA‐col ... 123

Figure 29. TPEF‐SHG images for control samples (A) PLLA and (B) PLLA‐col; on the left, in green, autofluorescence; on the middle, in red, SHG; and on the right composed images ... 124

Figure 30. TPEF‐SHG for scaffolds composed by B‐ECM‐collagenase (A) NCLK (PLLA/B‐ECM‐collagenase) and (B) CLK (PLLA‐col/B‐ECM‐collagenase); on the left, in green, autofluorescence; on the middle, in red, SHG; and on the right composed images ... 126

Figure 31. TPEF‐SHG for scaffolds composed by B‐ECM‐pepsin (A) NCLK (PLLA/B‐ECM‐pepsin) and (B) CLK (PLLA‐col/B‐ECM‐pepsin); on the left, in green, autofluorescence; in the middle, in red, SHG; and on the right composed images ... 126

Figure 32. TPEF‐SHG for scaffolds composed by B‐ECM‐trypsin (A) NCLK (PLLA/B‐ECM‐trypsin) and (B) CLK (PLLA‐col/B‐ECM‐trypsin); on the left, in green, autofluorescence; in the middle, in red, SHG; and on the right composed images ... 127

Figure 33. TPEF‐SHG for scaffolds composed by P‐ECM‐collagenase (A) NCLK (PLLA/P‐ECM‐collagenase) and (B) CLK (PLLA‐col/P‐ECM‐collagenase); on the left, in green, autofluorescence; on the middle, in red, SHG; and on the right composed images ... 127

Figure 34. TPEF‐SHG for scaffolds composed by P‐ECM‐pepsin (A) NCLK (PLLA/P‐ECM‐pepsin) and (B) CLK (PLLA‐col/P‐ECM‐pepsin); on the left, in green, autofluorescence; in the middle, in red, SHG; and on the right composed images ... 128

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Figure 36. Alive (green) and dead (red) Staining for P‐ECM‐pepsin electrospun scaffolds (A‐B) NCLK (PLLA/P‐ECM‐pepsin) and (C‐D) crosslinked (PLLA‐col/P‐ECM‐pepsin) ... 130

Figure 37. Cytotoxicity of the fragments released from NCKL and CLK scaffolds. (A‐B) control – cells on medium, (C‐D) cells cultured on medium containing molecules released from NCLK scaffold (PLLA/P‐ECM‐pepsin), (E‐F) cells cultured on medium containing molecules released from CLK scaffold (PLLA‐col/P‐ECM‐pepsin) ... 131

Figure 38. Collagen functional binding domains are shown in four microfibrils modelling fibril surface and their availability after proteolysis. Glycoprotein VI (GPVI), C‐telopeptide (C‐telo), von Willebrand's Factor (vWF), small leucine‐rich proteoglycan (SLRP), Metalloproteinases (MMPs), peptide sequences (GFOGER, GMOGER, GASGER), secreted protein acidic and cysteine‐rich (sparc also known as osteonectin), imino rich repeat sequence (GPO)5, discoidin domain‐containing receptor 2 (DDR2). Adapted from Zeltz et al., 2014. ... 134

Figure 39. AB measurements (average ± standard deviation) to access BMMSCs attachment on NCLK and CLK scaffolds composed by collagenase‐digested ECM (n=3) ... 135

Figure 40. AB measurements (average ± standard deviation) to access BMMSCs attachment on NCLK and CLK scaffolds composed by pepsin‐digested ECM (n=3) ... 136

Figure 41. AB measurements (average ± standard deviation) to access BMMSCs attachment on NCLK and CLK scaffolds composed by trypsin‐digested ECM (n=3) ... 136

Figure 42. Metabolic activity (average ± standard deviation) of BMMSCs accessed by AB assay on non‐ crosslinked and crosslinked scaffolds composed by collagenase digested ECM (n=3) ... 139

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Figure 44. Metabolic activity (average ± standard deviation) of BMMSCs accessed by AB assay on non‐ crosslinked and crosslinked scaffolds composed by trypsin digested ECM (n=3) ... 141

Figure 45. ALP activity normalized by AB measurements (average ± standard deviation) on day 14 for BMMSCs cultured on NCLK and CLK scaffolds composed by collagenase digested ECM (n=3) ... 144

Figure 46. ALP activity normalized by AB measurements (average ± standard deviation) on day 21 for BMMSCs cultured NCLK and CLK scaffolds composed by collagenase digested ECM (n=3) ... 144

Figure 47. ALP activity normalized by AB measurements (average ± standard deviation) on day 14 for BMMSCs cultured NCLK and CLK scaffolds composed by pepsin digested ECM (n=3) ... 145

Figure 48. ALP activity normalized by AB measurements (average ± standard deviation) on day 21 for BMMSCs cultured on NCLK and CLK scaffolds composed by pepsin digested ECM (n=3) ... 145

Figure 49. ALP activity normalized by AB measurements (average ± standard deviation) on day 14 for BMMSCs cultured NCLK and CLK scaffolds composed by trypsin digested ECM (n=3) ... 146

Figure 50. ALP activity normalized by AB measurements (average ± standard deviation) on day 21 for BMMSCs cultured NCLK and CLK scaffolds composed by trypsin digested ECM (n=3) ... 146

Figure 51. ARS quantification (average ± standard deviation) on day 14 (left column) and day 21 (right column) for BMMSCs cultured on NCLK and CLK scaffolds composed by collagenase‐digested ECM (n=3) ... 149

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Figure 53. ARS quantification (average ± standard deviation) on day 14 (left column) and day 21 (right column) for BMMSCs cultured on NCLK and CLK scaffolds composed by trypsin‐digested ECM (n=3) ... 150

Figure 54. ARS images for NCLK and CLK scaffolds on produced from B–ECM‐collagenase on day 14 and day 21 ... 151

Figure 55. Histology from cross‐sections of NCLK and CLK (PLLA/P‐ECM) scaffolds cultured with BMMSCs in the osteogenic medium for 21 days stained with alizarin red ... 153

Figure 56. Immunostaining on cross‐sections of electrospun scaffolds cultured with BMMSCs for 21 days. On the left, coloured in blue, DAPI marks nuclei; on the middle primary and secondary antibody, coloured in green, marking the expression of human type I collagen; and on the right, the combined image. ... 154

Figure 57. Immunostaining of mouse tissue and BMMSCs cultured PLLA‐ECM scaffold without primary antibody. On the left, coloured in blue, DAPI marks nuclei; on the middle secondary antibody, coloured in green, marking non‐specific binding of the secondary antibody to the sample; and on the right, the combined image. ... 155

Figure 58. Immunostaining of mouse tissue with primary antibody. On the left, coloured in blue, DAPI marks nuclei; on the middle primary and secondary antibody, coloured in green, marking non‐ specific type I collagen immunofluorescence; and on the right, the combined image. ... 156

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Figure 60. RunX2 expression (average ± standard deviation) for BMMSCs cultured for 21 days with osteogenic media on NCLK and CLK scaffolds produced from PLLA and collagenase‐digested ECM (n=3) ... 160

Figure 61. Osx (sp7) expression (average ± standard deviation) for BMMSCs cultured for 21 days with osteogenic media on NCLK and CLK scaffolds produced from PLLA and collagenase‐digested ECM (n=3) ... 161

Figure 62. Sparc expression (average ± standard deviation) for BMMSCs cultured for 21 days with osteogenic media on NCLK and CLK scaffolds produced from PLLA and collagenase‐digested ECM (n=3) ... 161

Figure 63. Col1a2 expression (average ± standard deviation) for BMMSCs cultured for 21 days with osteogenic media on NCLK and CLK scaffolds produced from PLLA and collagenase‐digested ECMs (n=3) ... 162

Figure 64. Bglap expression (average ± standard deviation) for BMMSCs cultured for 21 days with osteogenic media on NCLK and CLK scaffolds produced from PLLA and pepsin‐digested ECM (n=3) ... 165

Figure 65. RunX2 expression (average ± standard deviation) for BMMSCs cultured for 21 days with osteogenic media on NCLK and CLK scaffolds produced from PLLA and pepsin‐digested ECM (n=3) ... 165

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Figure 67. Sparc expression (average ± standard deviation) for BMMSCs cultured for 21 days with osteogenic media on NCLK and CLK scaffolds produced from PLLA and pepsin‐digested ECM (n=3) ... 166

Figure 68. Col1a2 expression (average ± standard deviation) for BMMSCs cultured for 21 days with osteogenic media on NCLK and CLK scaffolds produced from PLLA and pepsin‐digested ECM (n=3) ... 167

Figure 69. Bglap expression (average ± standard deviation) for BMMSCs cultured for 21 days with osteogenic media on NCLK and CLK scaffolds produced from PLLA and trypsin‐digested ECM (n=3) ... 170

Figure 70. RunX2 expression (average ± standard deviation) for BMMSCs cultured for 21 days with osteogenic media on NCLK and CLK scaffolds produced from PLLA and trypsin‐digested ECM (n=3) ... 170

Figure 71. Osx (sp7) expression (average ± standard deviation) for BMMSCs cultured for 21 days with osteogenic media on NCLK and CLK scaffolds produced from PLLA and trypsin‐digested ECM (n=3) ... 171

Figure 72. Sparc expression (average ± standard deviation) for BMMSCs cultured for 21 days with osteogenic media on NCLK and CLK scaffolds produced from PLLA and trypsin‐digested ECM (n=3) ... 171

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Figure 74. Raman spectrum from demineralized bone, calvaria bone, PLLA and PLLA‐col electrospun scaffolds cultured with BMMSCs in osteogenic media during 21 days. Each spectrum represents one xy area of 20 x24 points (480 scans), where each x and y point was 10 nm and 2.7 nm apart using a 785 nm laser. ... 174

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List of Tables

Table 1. Primers sequences for osteogenic markers ... 72

Table 2. Comparative protein quantification between Bradford and ninhydrin assays ... 77

Table 3. Data obtained from undigested B‐ECM and digested by collagenase, pepsin and trypsin TG/DTG curves ... 88

Table 4. Data obtained from undigested P‐ECM digested by collagenase, pepsin and trypsin TG/DTG curves ... 89

Table 5. Data obtained from Col undigested and digested by collagenase, pepsin and trypsin TG/DTG curves ... 89

Table 6. Data obtained from TG/DTG curves of NCLK scaffolds ... 118

Table 7. Data obtained from TG/DTG curves of CKL scaffolds ... 119

Table 8. Data obtained from DSC curves of NCKL and CKL electrospun PLLA/B‐ECM scaffolds ... 121

Table 9. Data obtained from DSC curves of NCLK and CLK electrospun PLLA/P‐ECM scaffolds ... 121

Table 10. Comparison of mineral matrix ratio and mineral crystallinity for controls ... 176

Table 11. Comparison of mineral matrix ratio and mineral crystallinity for NCLK and CLK electrospun scaffolds from B‐ECM ... 177

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3.7 BMMSCs cultured on scaffolds – Part 2 ... 65 3.7.1 Alive and dead – cytotoxicity of released proteins from scaffolds ... 66 3.7.2 Alive and dead – scaffolds cytotoxicity ... 66 3.7.3 Alamar blue assay (AB) ... 67 3.7.4 Phosphatase alkaline activity (ALP) ... 67 3.7.5 Alizarin red staining (ARS) ... 67 3.7.6 Histology ... 68 3.7.7 Immunohistochemistry ... 68 3.7.8 Real‐time polymerase chain reaction (q‐PCR) ... 69 3.7.9 Micro‐Raman ... 72

3.8 Statistical analysis ... 73

4 Results and Discussion ‐ Part 1 ... 74

4.1 Bradford, ninhydrin and GAGs quantification ... 74

4.2 SDS‐page ... 79

4.3 Circular dichroism (CD) ... 81

4.4 Combined TPEF‐SHG microscopy ... 83

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4.6 Biological assays ... 91 4.6.1 BMMSCs cultured with non‐osteogenic medium supplemented with digested ECMs ... 91 4.6.2 BMMSCs cultured with osteogenic medium supplemented with digested ECMs ... 101

5 Results and Discussion – Part 2 ... 110

5.1 Production of electrospun PLLA‐ECM scaffolds ... 113

5.2 Thermal analysis – TGA and DSC ... 116

5.3 Protein Release from scaffolds ... 122

5.4 Combined TPEF‐SHG microscopy ... 123

5.5 Biological assays ... 129 5.5.1 Alive and dead assay – Scaffolds cytotoxicity ... 129 5.5.2 Alamar blue assay (AB) – BMMSCs attachment and metabolic activity ... 132 5.5.3 Alkaline phosphatase activity (ALP) ... 142 5.5.4 Alizarin red staining (ARS) ... 147 5.5.5 Histology ... 152 5.5.6 Immunohistochemistry ... 153 5.5.7 Real‐time polymerase chain reaction (q‐PCR) ... 156 5.5.8 Micro‐Raman ... 172

6 Conclusions ... 178

7 References ... 180

8 ANNEXES ... 198

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1 Introduction

Tissue engineering combines life sciences and materials engineering with the aim to restore, maintain or improve tissue functions (Langer & Vacanti 1993).

In order to regenerate tissues, 3 main strategies are employed: (1) Cell therapy treatments, (2) Development of systems that carry tissue‐inducing substances, such as growth factors, to the target tissue and (3) Design of 3D scaffolds, acellular or with seeded cells, that allow exchange of nutrients and the integration with the in vivo tissue (Haraguchi et al. 2012; Cancedda et al. 2003; Blackwood et al. 2012; O’Brien 2011).

The better approach to design a biomaterial requires a deep understanding of the tissue that has to be regenerated, including its architecture, mechanical, chemical and physical properties and these properties are linked to its composition and molecular structural organization conferred by the extracellular matrix (ECM) (Dvir et al. 2011).

The ECM is a tridimensional array of protein fibrils and fibres interwoven within a hydrated network of glycosaminoglycan chains. It is composed of proteins such as collagens, which are the major components; glycoproteins such as elastin, laminin and fibronectin; proteoglycans, glycosaminoglycans, growth factors, chemokines and cytokines. These molecules are assembled to form ECM‐complex, composed of proteins with multiple individually folded domains, that contain biological and evolutionary relevance, as shown in Figure 1 (Hynes 2009; Hynes & Naba 2012; Kim et al. 2011).

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Figure 1. ECM representation and signalling molecules that direct the cell fate. Adapted from Lutolf & Hubbell 2005)

The transduction of the ECM signalling to the cells occurs via two different binding mechanisms: (1) direct binding of the adhesion receptors on ECM domains, and (2) binding/release of growth factors (Hynes & Naba 2012).

In the first case, the ECM adhesion proteins, such as fibronectin and laminin bind adhesion receptors on the cells, such as integrins, that provide transmembrane links to the actin cytoskeleton. Furthermore, the bound enables focal adhesion assembly (Mitra et al. 2005) and (Tadokoro et al. 2003) which can transmit/activate signalling cascades within the cells and also regulate ECM affinity of the receptors (called the inside‐out signalling) (Geiger & Yamada 2011).

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proteoglycans and glycosaminoglycans are responsible for binding the growth factors on the ECM, and their release is regulated by proteolysis promoted mainly by matrix metalloproteases (MMPs) (Page‐McCaw et al. 2007; Cawston & Young 2010) and ADAMTS (Kumagishi et al. 2009; Kim et al. 2011). However, other families of enzymes such as heparanase, elastases, cathepsins and various serine esterase protease (Lu et al. 2011) also contribute to regulation of growth factor release.

Although, ECM‐bound growth factor do not have to be released in soluble form to function (Kim et al. 2011), the soluble factors can bind receptors on the cell membrane surface. These are integrated with signalling pathways that regulate gene expression and cell fate processes such as proliferation, survival, differentiation, migration and apoptosis (Schaffer 2008) and (Discher et al. 2009).

Integrins can activate several signalling pathways independently or in conjunction with growth factor receptors while functional activities of growth factor receptors are controlled by integrins. Thus, growth factors require specific integrins present on the particularly interested cell type (Alam et al. 2007). Although it is not clear how this synergy happens, It is known that there is a crosstalk between integrins and growth factors signalling transduction. Nonetheless, some reports suggest that domains within ECM proteins bind to canonical growth factors receptors (Alam et al. 2007; Engel 1989; Kim & Kim 2008).

As is shown in Figure 2, the tissue characteristics are closely dependent on ECM and its cells. Furthermore, it has modulated the morphological, physical and chemical properties of engineered scaffolds.

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Figure 2. Scaffolds produced to mimic the tissue environment; Adapted from (Dvir et al. 2011): 1, (Kim et al. 2010): 2, (Feng et al. 2009): 3, (Roohani‐Esfahani et al. 2010).

The ECM rich composition and perfect architecture brought to the tissue engineering field the possibility of its use as an acellular scaffold (Eitan et al. 2010). However, the decellularization is a key step for this application and involves the cell membrane rupture and subsequently washes. The cell lysis can be promoted by chemical agents (i.e. surfactants, acids and bases), by physical agents (i.e. heating/thawing cycles, ultrasound, pressure), or by biological agents (i.e. enzymes such as nucleases and trypsin) (Crapo et al. 2011). Thus, for decellularization, it is necessary to remove completely the DNA from the host tissue to prevent immune responses when implanted. In addition, the decellularization should attempt to keep most of the chemical and physical properties from the native ECM.

1)

2)

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Although some decellularized ECMs are commercially available (Crapo et al. 2011), they are mainly restricted to skin, pericardium and small intestinal submucosa tissues. Nonetheless, there are reports regarding the decellularization process of many other tissues to obtain the acellular extracellular matrix (Gilbert et al. 2006).

The main issues related to the use of acellular scaffolds are: (1) variations from batch to batch and scale‐up difficulties (Lanza et al. 2011), (2) susceptibility to enzymatic hydrolysis and consequently loss of mechanical strength during its degradation, so (3) collagen crosslink could prevent its degradation and loss of mechanical properties but crosslinking agents can induce an immune response (Parenteau‐Bareil et al. 2010).

Besides their use as acellular scaffold, the decellularized extracellular matrices have been used as components of artificial ECMs, which are basically hybrid materials that combine the advantages of the biomacromolecules present in the ECM to a synthetic polymer. Therefore, the final properties such as porosity, time of degradation, stiffness, free surface energy can be tailored in order to enhance the cell response.

To avoid the disadvantages of the acellular scaffolds and still deliver the benefits from the rich gross composition of the ECMs on biomaterials for tissue engineering, the first step required is its solubilisation, whereby the highly organized structure is submitted to enzymatic digestion. Thus, the proteolytic degradation of the ECM makes possible the incorporation of its major components into biomaterials, such as hydrogel or polymer/ECM hybrid materials (Badylak et al. 2009).

Although there are a high number of published research articles involving the digestion and incorporation of ECMs to produce biomaterials, very few try to understand the importance of the enzymatic digestion step for the final cell response (Hinderer et al. 2016; Reing et al. 2009).

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1.1 ECM composition and signalling

ECM consists of secreted molecules that are immobilized outside the cells that are responsible for tissue‐type specific extracellular architecture, tissue maintenance and regulation (Mouw et al., 2014). ECM exists as an integrated network structure composed of collagens, proteoglycans, glycosaminoglycans, glycoproteins, growth factors, cytokines and cell adhesion molecules (Mouw et al., 2014). These molecules consist of many individual domains that when combined with different arrangements, composition and abundance direct to different ECM‐tissue cell microenvironment (Muow et al., 2014).

The ECM is primarily composed of two main classes of macromolecules: fibrous proteins (including collagens and elastin) and glycoproteins (including fibronectin, proteoglycans (PGs) and laminin) (Mouw et al. 2014).

Collagen is a large family of proteins composed of 28 different collagen types, and is the major component of the ECMs. In general, collagens are regarded as triple helical proteins that have functions in tissue assembly or maintenance, including tissue scaffolding, cell adhesion, cell migration, cancer, angiogenesis, tissue morphogenesis and tissue repair (Kadler et al. 2007).

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The triple helix structure is stabilized by intrachain and interchain hydrogen bonds between the polypeptides chains as shown in Figure 3. Stabilization promoted by interchain hydrogen bonds, involve glycine amide groups and carboxyl groups on the X position (Figure 3a). In addition, they involve Inductive effects promoted by the hydroxyl group in the 4‐hydroxyproline (Y position), that enhance the triple helix stabilization by favouring the trans conformation of the hydroxyprolyl peptide bond (Figure 3b). Gauche conformation is achieved if pyrrolidine ring, in the X position, is puckering down (Cγ endo‐pucker) while pyrrolidine ring, in the Y position, is puckering up (Cγ exo‐pucker), as shown in Figure 3c (Hunter 2010; Bann & Bachinger 2000). Moreover, each alpha‐helix chain is stabilized by intrachain hydrogen bonds, where all the N‐H and C=O groups are involved (Pauling et al. 1951; Hunter 2010).

Figure 3. Collagen triple helix stabilization; Adapted from Hunter 2010

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39 Triple helical collagens can assemble into fibrils that confer the tensile strength in animal tissues. The unique mechanical properties of fibrillar collagens are mainly controlled by its structure; furthermore, it reflects the intrinsic relationship between three‐dimensional protein structure and the function of the resultant ECM (Fratzl et al. 1998). Nonetheless, not all the collagens are fibril‐forming (i.e. collagen I). Collagens can also be classified as fibril‐associated collagens with interrupted triple helix (FACITs) (i.e. collagen IX), or network‐forming collagens (i. e. collagen IV) or transmembrane collagens (i.e. collagen XIII) (Kadler et al. 2007).

Fibrillar collagen formation starts when α‐chains that contain N‐ and C‐propeptides at the end of the triple helical domains are imported into the rough endoplasmatic reticulum. Subsequently, lysine and proline residues are modified by hydroxylation and O‐linked glycosylation (Kadler et al. 2007; Mouw et al. 2014), followed by association of α‐chains to form procollagen initiated by the C‐propeptide domains.

Procollagen is packed in the Golgi complex and secreted into the ECM. The cleavage of C‐propeptide domains by the enzymatic action of MMPs is required for fibrillogenesis. However, N‐propeptide domains are cleaved just on the necessary extension to promote the fibril shape and diameter (Hulmes 2002) as shown in Figure 4a.

Type I collagen has C‐propeptide and N‐propeptide completely removed forming tropocollagen which is arranged to form microfibrils with an axial D‐periodicity of approximately 67 nm. The assembly of the microfibrils into fibrils is mediated by aldol and aldol‐histidine crosslinking within and between microfibrils and provides an additional stability and tensile strength to the collagen fibrils, as shown in Figure 4b.

Aldol crosslinking involves aldehydes and amino groups on lysine, while aldol‐histidine crosslinking, involves a trifunctionality crosslinking derived from Michael addition of a histidine residue to the aldol. Fibril‐associated collagens with interrupted triple helix (FACIT) and small leucine‐rich repeat proteoglycans (SLRPs) form a connective tissue within the fibrils are bundled together to form collagen fibers, which are tissue‐age‐stage specific (Mouw et al. 2014; Siegel 1976; Fairweather et al. 1972).

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Figure 4. Collagen fibrillogenesis reproduced from Klug and Cummings, 1997

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41 As described previously, fibrillogenesis is a process that starts inside the cell and ends in the extracellular space, furthermore a series of ECM components such as proteolytic enzymes (i.e. MMPs), proteoglycans (i.e. SLRPs), and crosslinking enzymes (i.e. lysyl oxidase) are involved. In turn, collagen fibres provide binding sites for other ECM components (Mouw et al. 2014).

Carbohydrate can be covalently attached to proteins to form glycoproteins and proteoglycans; the biggest difference between them relies on the fact that carbohydrate percentage on the macromolecule is much smaller on glycoprotein than on the proteoglycan.

Proteoglycans decorate cell membrane to bind ECM components such as growth factors, enzymes, protease inhibitors and chemokines to play a role in signal transduction. In addition to this, fill the extracellular space to provide a hydrated gel (Berg et al. 2002; Perrimon & Bernfield 2001; Bishop et al. 2007).

Proteoglycans consist of a core protein where one or more glycosaminoglycans (GAGs) are attached to the various serine residues. Proteoglycans properties are dominated by GAGs chemical properties, thus, an important agent that provides a hydrated gel that resists compressive forces (Gandhi & Mancera 2008).

GAGs on proteoglycans work as a docking station providing the interactions necessary for proteoglycan binding functions on the ECM. GAGs‐protein interactions can involve Van Der Walls, hydrogen bonds, carbohydrate backbone and ionic interactions. However, binding affinity results mainly from the ionic interaction of highly acidic sites on the GAG and basic amino acid side chains Asn, Asp, Glu, Gln, Arg, His and Trp(Malik & Ahmad 2007; Shionyu‐Mitsuyama et al. 2003; Taroni et al. 2000).

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Figure 5. GAGs disaccharide units and features; Adapted from Gandhi and Mancera, 2008

Glycoproteins are cell membrane components that play a key role in cell adhesion. Glycoproteins are classified as N‐linked, whereas the carbohydrate is attached to the amide side chain of an asparagine residue in the Asn‐X‐ Ser or Asn‐X‐Thr sequence; or O‐linked, whereas the carbohydrate is linked to the hydroxyl group of a Ser or Thr residue, in addition O‐glycosylation can appear on amino acid sequences (Wang & Amin 2014).

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43 Laminin and fibronectin are multidomain glycoproteins that play an important role connecting the structural ECM network to the cells. Nonetheless, they also can bind to other ECM molecules, growth factors or soluble molecules (Mecham 2012).

Laminins are a family of large mosaic glycoproteins (~900 KDa) composed of globular, laminin‐type epidermal growth factor (EGF)‐like repeats and coil‐coiled domains (Mouw et al. 2014). Laminins are heterotrimers composed of α, β and γ chains, which are characterized by the presence of different domains, that combine via the triple helical coiled‐coil domain in the centre of each chain to form cross‐shaped, Y‐shaped or rod‐shaped laminins. (Aumailley et al. 2005; Miner & Yurchenco 2004).

Laminins are required for basement membrane assembly and cell polarization, however, the differences in the way that isoforms combine, determinate laminin heterotrimers spatial shape, cell‐anchoring properties its interaction with other basement membrane components (Mecham 2012; Li et al. 2003).

Fibronectins (FNs) are cell‐secreted glycoproteins that contain 4‐9% carbohydrate and exist as a dimer of two similar chains (~250 KDa) linked by disulphide bonds at C‐terminal region (Singh et al. 2010). Fibronectin has the ability of binding many of the extracellular matrix components and to Interact with cells because it has multiple binding domains to attach to integrins, collagen, gelatine, glycosaminoglycans, fibrins and integrins (Singh et al. 2010; Hynes 1999). Thus, fibronectin mediates a wide variety of functional activities playing important roles in cell adhesion, migration, growth and differentiation (Schwarzbauer & DeSimone 2011). Furthermore, fibronectin contains RGD sequence, well known for being a motif for cell adhesion.

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Other important molecules on the ECM environment are growth factors; these polypeptides can bind to integrins, activating cell signalling and transcription factors to regulate growth, differentiation and apoptosis. Growth factors are found in the extracellular space in its soluble form or bound to GAGs on proteoglycans. Growth factors can be released from ECM, by degradation of ECM proteoglycans that enable the establishment of a stable gradient of growth factors in the extracellular matrix (Hynes 2009; Canalis et al. 1991).

1.2 ECM materials directing stem cell behaviour

Tissues are constantly remodeled in a dynamic interaction between the cell niche and the extracellular matrix. Furthermore, the ECM must provide the structure and biochemical signaling required by the cells for the tissue function (Badylak 2002). Decellularized materials have been researched due to this specific cell‐ECM microenviroment that is believed to conduct tissue regeneration because of their ability to direct the stem cell differentiation.

The biggest challenge in the use of decellularized tissues to produce biomaterials is the decellularization method. Differences in the cell density and morphology for each tissue make it necessary that each tissue requires an adapted protocol (Keane et al. 2015).

The potential for decellularized tissues, also known as decellularized ECM, as biomaterials, range from their use as an intact whole organ, sections or blocks (Agmon and Chistman 2016). In addition, if the decellularized ECM is digested, materials for cell and/or drug delivery, hydrogels, coatings, electrospun scaffolds or printing/bioprinting scaffolds can be designed for different goals and benefits (Hinderer et al. 2016).

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45 maintenance of the organ architecture and vasculature which is important for large implants. Nevertheless, it needs to be extensively explored to understand clinical success, such as in trachea transplants (Gonfiotti et al. 2014), and failure such as in heart valves transplant (Simon et al. 2003).

ECM hydrogels aims the regeneration of the tissue via injectable biomaterials into injury site (Agmon and Chistman 2016). There are different strategies employed in this approach that range from the ECM as the only component or with other components. The use of crosslinking agents and the variations on the composition allows obtaining hydrogels with different degrees of stiffness that can direct stem cell differentiation through mechanotransduction (Agmon and Chistman 2016). In addition, degradation times and adhesion‐ligand sites for cells can be tailored (Murphy et al. 2014; Agmon and Chistman 2016). These ECM hydrogels have been explored as scaffolds or as drug and/or cell carriers to the injury site (Hinderer et al. 2016).

Digested ECMs can also be used for coatings providing the biochemical cues to direct differentiation. In this case, the molecules that are physically absorbed on the surface promote the enrichment of the surface. The disadvantage of this approach relies on the fact that the biomaterial does not maintain the tissue native architecture (Agmon and Chistman 2016; Hinderer et al. 2016).

Scaffolds composed by ECM can be produced via the electrospinning technique. The electrospinning technique generates 3D fibrous and highly porous scaffolds that have been widely explored. Fiber diameter, porosity, degradation time, stiffness and hydrophylic/hydrophobic are properties that can be controlled to achieve the tissue regeneration through allowing cell attachment, proliferation and differentiation.

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a fibrous mesh is generated on the collector (Hinderer et al. 2016).

Regardless of the technique used to process the decellularized tissues into biomaterials, the biggest challenge is understanding how the stem cell behavior is affected by differences on the matrix. However, it is important to highlight that apart from donor age and health, processing procedures can also be very relevant to the success or failure of the ECM biomaterial. Moreover, Rajabi‐Zeleti and co‐workers were able to demonstrate that lyophilized hydrogels from human pericardium had the interconnectivity of the pores increased and that resulted in improved cell migration (Rajabi‐Zeleti et al. 2014).

Some studies were able to verify that lung ECM used to supplement media could not initiate specific differentiation of MSCs but could enhance differentiation (Daly et al. 2012; Shojaie et al. 2015). Other research was able to demonstrate that certain tissues, such as kidney, have region specific effect on metabolic activity, proliferation and differentiation (O’Neill et al. 2013).

Neural differentiation was observed when solubilized ECMs from optic nerve, spinal cord, brain and urinary bladder were incorporated into culture media. However, it was not possible to confirm that in order to achieve this a tissue specific matrix is necessary. Thus suggesting that the effect of solubilized ECMs could be masked by the use of differentiation media (Crapo et al. 2012).

Interestingly, heart, liver and adipose tissue ECMs were able to direct differentiation on standard media (Cheung et al. 2014; French et al. 2012; Zhang et al. 2015). It is not clearly understood what causes these differences, but some possibilities are the specific tissue microarchitecture, variations in the decellularization process or that some stem cells require less specific biochemical cues to differentiate in a specific lineage (Agmon and Chistman 2016).