Development of a natural origin, mineralizable, fit-to-shape, nanocomposite and tissue-adhesive hydrogel

Universidade de Aveiro 2018

Departamento de Engenharia de Materiais e Cerâmica

Ana Rita Bandarra

Nunes

Desenvolvimento de um hidrogel nanocompósito de

origem natural, mineralizável, fit-to-shape e adesivo

em tecidos

Development of a natural origin, mineralizable,

fit-to-shape,

nanocomposite

and

tissue-adhesive

Universidade de Aveiro 2018

Departamento de Engenharia de Materiais e Cerâmica

Ana Rita Bandarra

Nunes

Desenvolvimento de um hidrogel nanocompósito de

origem natural, mineralizável, fit-to-shape e adesivo

em tecidos

Development of a natural origin, mineralizable,

fit-to-shape, nanocomposite and tissue-adhesive hydrogel

Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Materiais e Dispositivos Biomédicos, realizada sob a orientação científica da Doutora Mariana Oliveira e do Professor Doutor João Mano, Professor Catedrático do Departamento de Química da Universidade de Aveiro

o júri

presidente Professora Doutora Maria da Piedade Moreira Brandão Professora Adjunta, Universidade de Aveiro

Doutora Paula Alexandrina de Aguiar Pereira Marques Equiparada a investigadora Principal, Universidade de Aveiro

Doutora Mariana Braga de Oliveira

agradecimentos Antes demais, gostava de agradecer ao Prof. Dr. João Mano pela oportunidade de trabalhar neste enorme grupo de investigação (COMPASS), por me dar a oportunidade de aprender mais e com os melhores.

À minha orientadora, Dra. Mariana Braga de Oliveira, por toda ajuda dada, por ser sempre incansável, pelo conhecimento transmitido, pela paciência, e pela boa disposição, um grande obrigado.

Agradeço também a todos os membros do grupo COMPASS por todo o apoio e ajuda, em particular, à Dra. Sónia Oliveira, Dr. João Rodrigues, Dra. Sónia Patrício e Márcia Tavares por todo o tempo despendido. Para além disso um enorme obrigado às minhas colegas Jéssica Antunes e Elisa Fernandes pela grande amizade criada e pelo apoio incondicional, foram um grande pilar durante este ano.

Um obrigado também ao Dr. Nuno Silva e Dra. Carmen Freire, bem como ao André Girão e Dra. Paula Maques por toda a disponibilidade manifestada para usar os seus equipamentos.

Um agradecimento especial aos meus pais e avô, porque sem eles nada disto era possível. Obrigada por serem os melhores e me darem sempre o melhor, por confiarem em mim, por me apoiarem incondicionalmente e relembrarem-me que “cada degrau é uma conquista”.

Finalmente, gostava de agradecer a todos os meus amigos que de alguma maneira me ajudaram a chegar a este momento tão importante para mim, um agradecimento especial à Andreia Pereira, Beatriz Rocha e Beatriz Bandeira por seguirem o meu percurso académico e por me ouvirem incansavelmente.

palavras-chave Hidrogéis, Bioatividade, Bioadesão, Auto-recuperação, Shape-fitting, Shape fixation.

resumo Uma grande variedade de hidrogéis são atualmente sugeridos como suportes eficazes para regeneração de tecidos. Sistemas bioadesivos com capacidade de unir partes de tecido são muito populares na comunidade científica, em particular, biomateriais inspirados na forte adesão do mexilhão, estes mostram grande adesividade em ambientes aquáticos e por isso têm sido sugeridos para aplicações como selantes de tecido, adesivos cardíacos e agentes de fixação de osso. No entanto o desenvolvimento de sistemas com propriedades físicas ajustáveis e bioatividade em células e tecidos ainda é escasso. Considerando as limitações atuais associadas aos bioadesivos, esta dissertação tem como objetivo o desenvolvimento de um biomaterial fit-to-shape com bioadesividade, bem como a capacidade de adquirir uma forma fixa com o tempo, aumentando a estabilidade dos tecidos a reparar. Além disso, este sistema, visa também atingir a bioatividade para permitir tanto a integração óssea in situ da cola bioativa como liberação iônica localizada e citocompatibilidade.

Quitosano modificado com o domínio peptídico 3,4-dihydroxyphenyl-L-alanine (DOPA) foi combinado com nanopartículas de sílica produzidas pelo método de Stöber e funcionalizadas com iões cálcio e fosfato para formar um hidrogel compósito multifuncional.

Biomateriais preparados com diferentes quantidades de nanopartículas foram caracterizados quanto ao seu conteúdo de água, capacidade de retenção de água e soluções fisiológicas, comportamento reológico (incluindo rigidez, módulo de perda, comportamento shear-thinning e recuperação após destruição estrutural), capacidade de aderir a tecidos biológicos e capacidade de mineralização em fluido corporal simulado (SBF). Elevando o pH do polímero dissolvido para 7 foi obtido um hidrogel estável com auto-recuperação parcial após a destruição e comportamento shear-thinning. A adição de nanopartículas ao hidrogel levou a um aumento do módulo de armazenamento, e à inalteração do comportamento shear-thinnning. A oxidação dos grupos catecol do quitosano levou a um aumento na rigidez dos hidrogéis, devido à formação de ligações covalentes entre os grupos quinona. Os hidrogéis também mostraram a capacidade de unir tecidos de pele e osso, sendo que o aumento da quantidade de nanopartículas melhora as propriedades de adesão dos materiais. Hidrogéis imersos em SBF promoveram a formação de fosfato de cálcio com rácio Ca/P próximo ao da hidroxiapatite (~1,67). Em geral, o biomaterial aqui apresentado pode ser usado como um adesivo de tecido biológico com propriedades osteointegrativas e de fixação de forma-dependente do tempo, promissor na regeneração de tecidos. Embora direcionado para a fixação e regeneração de defeitos ósseos, o hidrogel proposto nesta dissertação pode encontrar aplicação na regeneração de uma multiplicidade de tecidos, incluindo pele e músculo.

keywords Hydrogels, Bioactivity, Bioadhesion, Self-Healing, Shape-fitting, Shape fixation.

abstract A wide plethora of hydrogels are currently suggested as effective tissue regeneration supports. Aa recent trend concerns bioadhesives with the ability to joint tissue parts together. In particular, biomaterials based on mussel-inspired principles have shown high underwater adhesiveness. Applications as tissue sealants, cardiac patches and bone fixating agents have been suggested. However, the development of systems with tuneable physical properties overtime and bioactivity towards cells and tissues is still scarce. With the current limitations associated to bioadhesives in mind, this thesis targets the development of a fit-to-shape biomaterial with bioadhesiveness as well as the ability to acquire a fixed shape overtime, increasing the mended tissues stability. Moreover, the design of this hydrogel-based system also aims at reaching bioactivity to enable both the in situ osseointegration of the bioactive glue (tackling the regeneration of bone defects), localized ionic release and cytocompatibility.

Chitosan modified with the DOPA peptide domain was combined with Stöber silica nanoparticles enriched with calcium and phosphate ions to form a composite multifunctional hydrogel. Biomaterials processed with different contents of nanoparticles were tested for their water content, swelling in water and physiological-like solutions, rheological behavior (including stiffness, loss modulus, shear thinning behavior and recovery after structural destruction), ability to adhere biological tissues, and mineralization capability in simulated body fluid (SBF). A stable hydrogel with partial self-recovery upon destruction and shear thinning behavior was obtained by raising the pH of the dissolved polymer to 7. The addition of nanoparticles to the hydrogel led to an increase in storage modulus, and the maintenance of the shear-thinning behavior. Time-dependent oxidation of chitosan’s catechol groups led a time-driven increase of the stiffness of the hydrogels due to the formation of covalent bonds between quinone groups. Hydrogels also showed the ability to bind skin and bone tissues together and increasing amounts of nanoparticles improved the adhesion properties of the materials. While immersed in SBF, the hydrogels promoted the formation of calcium phosphates with Ca/P ratios close the one of hydroxyapatite (~1.67). Overall, the biomaterials developed herein may be used as a biological tissue adhesive with osseointegrative and shape-fixation/time dependent stiffening properties, which may make them promising tissue regeneration devices. Although directed to fixation and regeneration of bone defects, the hydrogel proposed in this thesis may find application in the regeneration of a multiplicity of tissues, including skin and muscle.

i

Contents

Page

List of figures _vi

List of tables _x

List of abbreviations and acronyms _xi

CHAPTER I – Introduction

Page I.1. Summary: Definition of Current Challenges and Statement of Purpose ₁

I.2. State of the Art ₃

I.2.1. Structural and physiological aspects of human bone and classical repair

approaches 3

I.2.2. Tissue engineering strategies: the versatility of hydrogels ₅

I.2.3. Hydrogels source ₉

I.2.3.1. Polysaccharide-based hydrogels ₁₁

I.2.4. Hydrogel cross-linking mechanisms ₁₁

I.2.4.1. Physically crosslinked hydrogels ₁₁

I.2.4.2. Chemically crosslinked Hydrogels

13 I.2.4.3. Interpenetrating Network Hydrogels

15

I.2.4.4. Double Network Hydrogels ₁₆

I.2.5. Adhesive hydrogels ₁₇

I.2.5.1. Bioadhesiveness ₁₈

I.2.5.2. Mussel-Inspired hydrogels: overcoming underwater adhesion

ii

I.2.6. Composite hydrogels ₂₂

I.2.6.1. Bioactive glass nanoparticles ₂₄

I.2.7. Interesting properties explored on hydrogels ₂₆

I.2.7.1. Self-Healing ₂₆

I.2.7.2. Drug Delivery Capability ₂₈

I.2.7.3. 3D-printing of bioadhesive hydrogels ₂₈ I.2.7.5. Moldable and time-dependent stiffening hydrogels ₂₉

I.2.7.6. Injectability potential ₃₀

I.2.8. Chemistries used to prepare bioadhesive hydrogels ₃₀

I.2.9. Final Remarks ₃₃

I.3. Definition of the Hypothesis ₃₃

I.4. References ₃₄

Chapter II – Materials and methods

II.1. Synthesis of DOPA-CHT ₄₉

II.1.1. Characterization of DOPA-CHT derivate ₅₀

II.2. Stöber nanoparticles preparation ₅₂

II.3. Preparation of hydrogels ₅₂

II.4. Water content assessment ₅₃

II.4.1. Water, PBS and cell culture medium content assessment ₅₄

II.5. Rheological properties of the hydrogel ₅₄

iii

II.5.2. Flow Behavior ₅₅

II.6. Adhesion behavior studies ₅₆

II.6.1. Test tack ₅₇

II.7. Hydrogel mineralization ₅₈

II.7.1 Characterization of mineralized hydrogel ₆₀

II.8. Cytotoxicity assays ₆₀

II.8.1. Cell expansion ₆₁

II.8.2. Experimental Design ₆₁

II.8.3. Cell viability ₆₂

II.9. Statistical analysis ₆₂

II.10. References ₆₂

CHAPTER III - A Natural Origin Mineralizable Fit-to-Shape Nanocomposite and Tissue-Adhesive Hydrogel

III.1. Abstract ₆₆

III.2. Introduction ₆₇

III.3. Materials and methods ₆₉

III.3.1. Synthesis and characterization of DOPA-CHT ₆₉ III.3.2. Stöber nanoparticles preparation and characterization ₇₀

III.3.3. Preparation of composite hydrogels ₇₁

III.3.4. Water content assessment ₇₁

iv

III.3.5. Rheological properties of the hydrogel ₇₂

III.3.6. Adhesion strength studies ₇₂

III.3.7. Hydrogel Mineralization ₇₃

III.3.8. Cytotoxicity assays ₇₄

III.3.8.1. Viability analysis ₇₄

III.3.9. Statistical analysis ₇₄

III.4. Results and discussion ₇₅

III.4.1. Composite hydrogels formation ₇₅

III.4.2. Water content assessment ₇₆

III.4.2.1. Water, PBS and cell culture medium content assessment ₇₇

III.4.3. Rheological properties of the hydrogel ₈₀

III.4.4. Adhesion behavior studies ₈₅

III.4.5. Hydrogel Mineralization ₈₇

III.4.6. Cytotoxicity assays ₈₉

III.4.6.1. AlamarBlue Assay ₈₉

III.5. Conclusion ₉₀

III.6. References ₉₁

CHAPTER IV – Conclusion and future work

IV.1. General conclusions

97 IV.2. Future work

v

SUPPORTING INFORMATION

A - Characterization of DOPA-CHT ₁₀₂

B- Multiple comparison test between water content after processing (day 0) and water content after immersion in water, PBS and cell culture medium 104

C- Adhesion studies ₁₀₅

D- Tack test: optimized skin bonding protocol

105

D- Hydrogels mineralization 107

F – References

vi

List of Figures

CHAPTER I – Introduction

Page Figure I.1 – Bone macro-, micro- and nanostructure. Figure adapted from 1. ₄ Figure I.2 – The tissue engineering triad. The combination of cells, growth factors, and biomaterials allow tailoring systems that dictate tissue neo-formation for the development of biological substitutes that restore, maintain, or improve body

functions. 6

Figure I.3 – Applications of hydrogels in several fields. ₈ Figure I.4 – Several crosslinking mechanisms, namely physically and chemically crosslinking mechanisms and hydrogels with enhanced toughness, namely, interpenetrating and double network hydrogels. a) hydrogel’s polymeric network formed by ionic interactions, b) hydrogel’s polymeric network formed by radical polymerization, c) interpenetrating network hydrogels, and d) double network

hydrogels. 15

Figure I.5 - Hydrogel adhesiveness on human skin, over torsion, picture adapted

from 85. 20

Figure I.6 - Illustration of how mussels attach to substrates, illustration adapted

from 92. 21

Figure I.7 - Silica nanoparticles structures produced from different techniques. ₂₅

Chapter II – Materials and methods

Figure II.1 – Synthesis of CD polymer. ₅₀

Figure II.2 – Chemical structure of chitosan with numbered protons to calculate

vii

Figure II.3 – Hydrogels photographs in an inverted vial test at: a) 0 hours, right

after their preparation and b) 72 hours before. 53

Figure II.4 - Schematic representation of the plate system used. ₅₄ Figure II.5 – A typical graph of viscosity in function of shear rate, with typical Newtonian and non-Newtonian fluids curves. Figure adapted from 4. 56 Figure II.6 – Qualitative tests for comparison of adhesion property between hydrogels. a) Adhesion to the porcine skin; b) Adhesion to a chicken bone. 57 Figure II.7 - Representation of the systems used for tack test. ₅₈ Figure II.8 – Schematic representation of the experimental design used. ₆₁

CHAPTER III - A Natural Origin Mineralizable Fit-to-Shape Nanocomposite and Tissue-Adhesive Hydrogel

Figure III.1 – Schematic representation of the procedure for obtaining hydrogels (a) chemical reaction to obtain the CD polymer through the EDC/NHS chemistry; (b) to this polymer is added distilled water and then the pH is set to 7, here already have hydrogel formation that will be used as control; (c) the composite hydrogels are formed by the addition of silica nanoparticles functionalized with calcium and phosphate (average size of about 57nm), next are present pictures of the formed hydrogels (free-nanoparticles and with incorporations of different nanoparticles contents, namely, 4 and 8 wt% it is also presented a picture of these same hydrogels

after 72h. 76

Figure III.2 – Water content of hydrogels a) after their preparation, b) after 24h of immersion in distilled water, c) after 24h of immersion in PBS, d) after 24h of immersion in cell culture medium – alpha-MEM supplemented with 10% FBS and 1% antibiotic/antimycotic; e) comparison of color between hydrogels used to determine water content. * indicates a statistically significant difference with p <

viii

Figure III.3 – Schematic representation of possible interactions between the

polymer network and bioactive nanoparticles. 81

Figure III.4 – Rheological properties of CD0 and CD72 composite hydrogels. The blue corresponds to hydrogels with 0 wt% of nanoparticles, light grey 4 wt% and dark grey to 8 wt%, a) storage (line) and loss modulus (line with shape) in function of oscillatory shear strain for CD0 hydrogels, b) storage (line) and loss modulus (line with shape) in function of oscillatory shear strain for CD72 hydrogels, c) hydrogels storage modulus at a 0.5% strain, d) hydrogels loss modulus at a 0.5% strain, e) shear strain at each hydrogels with different content of nanoparticles begins to deform, f) step-strain measurements of CD0 hydrogels over five cycles, with a high magnitude strain of 500% and a low magnitude strain of 0.5%, g) step-strain measurements of CD72 hydrogels with the magnitude step-strains applied for CD0 hydrogels, h) recovery percentage of CD0 hydrogels in relation to the previous cycle, i) recovery percentage of CD72 hydrogels in relation to the previous cycle, j) viscosity of the CD0 composite hydrogels at a shear rate from 10 s-1, k) viscosity of the CD72 composite hydrogels at a shear rate from 0.1-10 s-1. * indicates a statistically significant difference with p < 0.05, ** p < 0.01,

*** p < 0.001 and **** p < 0.0001. 83

Figure III.5 - Bioactivity study of CD-NP hydrogels. a) Representative SEM images during 1, 7 and 14 days of immersion in SBF solution, b) Graphs obtained by EDS of hydrogels with 4 wt% of nanoparticles during 1,7 and 14 days of the bioactivity studies, c) Graphs obtained by EDS of hydrogels with 8 wt% of nanoparticles during 1,7 and 14 days of the bioactivity studies, d) Ca/P ratios on hydrogels containing nanoparticles over time, e) FTIR spectra after 0, 1, 7 and 14

days of CD hydrogels immersion in SBF. 88

Figure III.6 - Cell viability after 2 and 7 days of incubation, as determined by Alamar blue assay. Results (mean ± SD) are presented as a percentage of the control cells grown on tissue culture plastic (five replicates). 90

ix

SUPPORTING INFORMATION

Figure S1 - Chitosan 1_{H NMR spectra, in DCl/D2O.}

102 Figure S2 - Chitosan modified with DOPA 1H NMR spectra, in DCl/D2O ₁₀₃ Figure S3 - Preliminary adhesion studies in porcine skin (a) and chicken bones (b) with the CD composite hydrogel between the tissues. 105 Figure S4 – Tack adhesion characterization of the hydrogels. Stress-strain curves of hydrogels (a) hydrogels between pieces of porcine skin, as you can see in the photo below the graph, (b) hydrogels between bovine bone (image below). 105 Figure S5 - Stress-strain curves of CD-NP0 hydrogel, and respective photo of the

system used. 106

Figure S6 – Free-nanoparticles hydrogel SEM images, obtained at 4 kV showing the smooth structure when immersed in SBF for 14 days. 107 Figure S7 – XRD spectra obtained for hydrogels with 0, 4 and 8 wt%, during 1,7

x

List of Tables

CHAPTER I – Introduction

Page Table I.1 - Examples of studies comprising different materials used to prepare

hydrogel with different properties applied in several biomedical applications. 31

Chapter II – Materials and methods

Table II.1 – Differences between ions concentrations in plasma and SBF. Table

adapted from 10. 59

CHAPTER III - A Natural Origin Mineralizable Fit-to-Shape Nanocomposite and Tissue-Adhesive Hydrogel

Table III.1 - Adhesive characteristics of CD composite hydrogels. n = 4 for tack

test with porcine skin and n = 1 for bovine bone test. 86

Supporting Information

Table S1 – Multiple comparison test using one-way ANOVA and Tukey’s test. **** indicates statistically significant difference between the water content of hydrogels with specific NPs amount after preparation and water content after immersion for 24h in the three solutions (p < 0.0001). ns indicates no statistically

xi

List of Abbreviations and acronyms

3D Three-dimensional

1_{H NMR Proton nuclear magnetic resonance}

εmax _{Maximum deformation}

σmax _{Maximum stress}

A

ADH Adipic dihydrazide

APS Ammonium persulfate

C

CaCl2 Calcium chloride CaOH2 Calcium hydroxide

CD Chitosan functionalized with DOPA CD0 Hydrogels right after their preparation

CD0-NP0 Hydrogels right after their preparation with 0 wt% of nanoparticles CD0-NP4 Hydrogels right after their preparation with 4 wt% of nanoparticles CD0-NP8 Hydrogels right after their preparation with 8 wt% of nanoparticles CD72 Hydrogels with 72h of incubation

CD72-NP0 Hydrogels with 72h of incubation and 0 wt% of nanoparticles CD72-NP4 Hydrogels with 72h of incubation and 4 wt% of nanoparticles CD72-NP8 Hydrogels with 72h of incubation and 8 wt% of nanoparticles

xii

CHT Chitosan

D

D2O Deuterium oxide

DCl Deuterium chloride

DHP Diammonium hydrogenphosphate

DMA Dopamine methacrylamide

DMT Dimethyltoluidine

DN Double network

DNA Deoxyribonucleic acid

DOPA 3,4-dihydroxyphenyl-L-alanine

E

ECM Extracellular matrix

EDC 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride) EDS Energy-dispersive X-ray spectroscopy

EPI Epichlorohydrin

F

xiii

G

G’ The storage modulus

G’’ Loss modulus

GelMA Gelatin methacryloyl

H

HCl Hydrochloric acid

I

IPN Interpenetrating network

K

K2HPO4•3H2O Potassium phosphate dibasic trihydrate

KCl Potassium chloride

L

LVER Linear viscoelastic region

M

Md Mass of swollen and dry hydrogel

Mfps Mussel foot proteins

MgCl2•6H2O Magnesium chloride hexahydrate. MMP Matrix metalloproteinase

xiv

Ms Mass of swollen hydrogels

N

Na2SO4 Sodium sulphate

NaCl Sodium chloride

NaHCO3 Sodium bicarbonate

NaOH Sodium hydroxide

NH4OH Ammonium hydroxide

NHS N-hydroxysuccinimide NP Nanoparticles NP0 0 wt% of Nanoparticles NP4 4 wt% of Nanoparticles NP8 8 wt% of Nanoparticles P

PAA Poly(acrylic acid)

PAM Polyacrylamide

PBS Phosphate buffered saline

PDA Polydopamine

PDLA D-lactic acid

PDMA Poly(dimethylacrylamide PEG Poly(ethyleneglycol) PGA Poly(glycolic acid)

xv

p-HEMA Poly(2-hydroxyethyl methacrylate) PLA Poly(lactic acid)

PLGA Poly(lactic-co-glycolic acid)

PLLA L-lactic-acid

PNIPAAm Poly(N-isopropylacrylamine) PVA Poly(vinyl alcohol)

R

RGD Arginylglycylaspartic acid

S

SBF Simulated body fluid

SEM Scanning electron microscopy

T

TEOS Tetraethyl orthosilicate

Tf2N Bis(trifluoro-methane-sulphonyl)imide TFA Trifluoroacetic acid

TRIS Tris(hydroxymethyl)aminomethane

U

xvi

X

1

CHAPTER I – Introduction

Brief description of the chapter

This chapter is divided into three main themes, which target the accomplishment of the following objectives:

i) Definition of the problem/challenges that drove the development of the study presented in this Thesis. A summary of current strategies available in the literature – later justified in more detail in point ii) – are presented, and the need for multifunctional materials as a way to surpass current limitations is presented in the section I.1. Summary: Definition of Current Challenges and Statement of Purpose.

ii) Presentation of an up-to-date literature survey concerning the main topics related to the development of biomaterials targeted at tissue regeneration – with main focus on bone fractures -, emphasizing the role of bioadhesive and bioactive devices. The section I.2. State of the Art justifies the need for the development of the study mentioned in point i).

iii) Establishment of the hypothesis that drove this Thesis, presented in the section I.3. Definition of the Hypothesis.

I.1. Summary: Definition of Current Challenges and Statement of Purpose

Tissue engineering research has received increasing attention for bone damage repair, since other current strategies that target tissue substitution or repair show limitations.1 _Different biomaterials aimed at mimicking the structure, as well as mechanical and biological properties of bone have been developed. Two essential requirements are needed for the

2

implementation of biomaterials in tissues regeneration: biocompatibility and biodegradability.

Biomaterials processed as different structures, including three-dimensional (3D) scaffolds and hydrogels, have been suggested as cell-bearing and modulating structures. Hydrogels are 3D polymeric hydrophilic structures, with viscoelastic and water-insolubility properties.2–5 _{The polymer networks that form stable hydrogels may be formed by natural,} synthetic or polysaccharides; the latter have great attractiveness in the field of tissue engineering, since they are highly biocompatible and degradable by the human body.6 Accordingly to the type of crosslinking mechanisms used to form stable structures – either covalent or physical bonds -, those may be classified as chemical or physical hydrogels.2 Despite the plethora of hydrogels currently suggested as effective tissue regeneration supports, a recent trend concerning bioadhesives with the ability to joint tissue parts together has risen in the last few years. In particular, biomaterials based on mussel-inspired principles have shown high underwater adhesiveness and, for that, promising behavior as tissue adhesives with potential application in tissue regeneration and wound healing, while avoiding additional external support. Applications as tissue sealants, cardiac patches and bone fixating agents have been suggested. Reports of biological glues for tissue regeneration often focus on the rheological, adhesive and mechanical properties of the developed device. However, the development of systems with tuneable physical properties overtime and bioactivity towards cells and tissues is still scarce. Although some bioadhesive materials have been modified with peptide domains to enhance cell adhesion, their integration in tissues and cell modulating properties has never been explored.

With the current limitations associated to bioadhesives in mind, this thesis targets the development of a biomaterial system with bioadhesive capability, as well as the ability to acquire a fixed shape overtime, increasing the mended tissues stability. Moreover, the design of this hydrogel-based system also aims at reaching bioactivity to enable both the in situ osseointegration of the bioactive glue (tackling the regeneration of bone defects), localized ionic release, cytocompatibility, and possible effects on the commitment of surrounding cells. Despite the proof-of-concept directed at bone trauma fixation and regeneration, the system proposed in this thesis may find application in the regeneration of a multitude of tissues, including skin and muscle.

3

I.2. State of the Art

I.2.1. Structural and physiological aspects of human bone and classical repair approaches

Bones perform functions of extreme importance in the human body, such as the enabling of locomotion, protection of the organs and bearing huge loads, due to their exceptional rigidity and strength.1,7,8 _{Long bones, such as the femur, consist of trabecular/spongy and} cortical/compact bone.9 The cortical bone, which corresponds to about 80% of the skeletal bone,7 consists of a cylindrical layer, very dense and hard that surrounds the medullary cavity of the bones, with a porosity between 3-12%.7,9 Trabecular bone, with a porosity of 50-90% is mostly found on the end of long bones, as well as ribs and spine.9 Bone is highly specialized and hierarchical tissue, with a multicellular structure and organization oriented to promote bone function.10 It consists of an organic phase (approximately 30%), responsible for tenacity, viscoelasticity, and stiffness, in which collagen is the main component; it also comprises a mineral phase (about 60%), in which hydroxyapatite is the main element, and this phase is also responsible for rigidity, structural reinforcement, and mineral homeostasis. The remaining 10% of bone composition corresponds to water.7,9 The characteristic hardness and strength of bone derives from its organic matrix interconnected with mineral deposits in a highly organized structure, as shown in Figure I.1.11

Bone regeneration currently represents a major challenge for orthopedics.10 Orthopedic surgical procedures are increasing and this increase has its origin in several parameters, from the fact that there is an aging population, an increase in physical activity9, traumatic injuries and pathological diseases.10 _{About 15% of the population over 60 years of age suffer from} bone damage.12 _{Although the human bone has the capacity to self-healing, due to the high} age, this becomes limited, due to lack of vascularization, innervation, lymphatic networks, and progenitor cells.12

4

Figure I.1 – Bone macro-, micro- and nanostructure. Figure adapted from 1.

In recent years, there has been extensive technological advances in orthopaedic surgeries that promote faster healing and integration,9 such as the use of bone grafts, including autografts, allografts, and xenografts.13 Bone grafts transplantation surgery is considered the second most common after blood transplantation, with an accumulation of 2.2 million surgeries per year in orthopaedics and dentistry.10

Autografts refers to the use of bone tissue by the own individual to be implanted in the damaged site.13 _{This is considered the "gold standard" compared to other bone grafts due to} its intrinsic osteoconductivity - serving as a support for cell growth and integration in the main bone tissue-, osteoinductiveity- promoting cell proliferation and differentiation-, and osteogenicity – due to its natural ability to promote bone formation.9 Despite the interesting results promoted by autografts, several limitations in their application have been described and include their limited quantity, as well as the morbidity of the donor site (about 20-30%

5

of the patients)9 and, in addition, they are not as effective in bone defects with an irregular shape.7

Bone grafts from different sources are also available as allografts and xenografts. Allografts refer to the use of bone tissues from another individual of the same species.13 Although the lack of availability is not as relevant as for autografts, it poses drawbacks that include the possible transmission of diseases.7 _{Furthermore, they may promote immunological} rejection.13 Finally, xenografts, that origin from bone tissues of different species, may trigger exacerbated immune responses, may be a vehicle for transmitting diseases, and pose ethical and religious concerns.13

Tissue engineering strategies have arisen as graft-free promising strategies for the treatment and regeneration of bone defects.10

I.2.2. Tissue engineering strategies: the versatility of hydrogels

Tissue engineering strategies aim at the full regeneration of a wounded tissue. The final result of this process is aimed at a fully functional and anatomically equal-to-native tissue. The use of permanent and non-biodegradable materials, such as prosthetics, in tissue engineering strategies are not contemplated. In fact, biomaterial-based tissue engineering strategies seek the use of tuned biodegradable biomaterials that serve as a supporting matrix to the growing tissue, and vanish after deposited extracellular matrix (ECM) and a cell-based de novo tissue is established.14

Tissue engineering strategies are a combination of engineering sciences, physics, and biology that involve the sole use or association of cells, biomaterials, and biomolecules, as represented in Figure I.2.10,15,16

These strategies are divided into two large groups scaffold-based and scaffold-free approaches. Scaffold-free approaches consist in the use of cells in a 3D microsphere or cell sheets to be administered, which may contain biomolecules to increase their effectiveness.17 Biomolecules consist of growth factors molecules for surface modification or genetic material that control the different cellular processes, such as migration, differentiation, and proliferation.17,18 In scaffold-based strategies, biomaterials are designed to show specific properties to enhance their adequacy to the regeneration of tissues. Factors that may be

6

controlled are the type of biomaterial (e.g. hydrogels, porous 3D scaffolds, microparticulate systems and fibers17), which may also showcase variable features such as pore size, geometry, permeability, and spatial distribution.15

Figure I.2 – The tissue engineering triad. The combination of cells, growth factors, and biomaterials allow tailoring systems that dictate tissue neo-formation for the development of biological substitutes that restore, maintain, or improve body functions.

Besides their role as cell cue-providers and supports, the biomaterials used in scaffold-based approaches can also be used as bioactive agents/drug delivery devices and may show mechanical features that foster the successful implementation of the tissue engineered system in the defective site. Besides their shape and spatiotemporally-controlled physical properties (e.g. stiffness, relaxation, roughness), the materials used in the fabrication of biomaterials are of utmost importance for their performance; not only they define their processability and post-processing ability (e.g. insertion of cell-interactive domains, such as

7

cell adhesive peptides), but also dictate their chemical features (e.g. wettability, degradability), which are well reported as providers of biological cues.18 Several material types have been suggested to build biomaterials. Those include metals, ceramics, polymers, and combinations thereof in which a high-performance biomaterial is achieved, named composite materials. For tissue engineering, biomaterials must show a biodegradable character, and, for that, most strategies are based on the use of polymers, ceramics and glasses. Due to their low degradability, metallic materials are often excluded from tissue engineering strategies.19

Currently, hydrogels are probably the most popular and versatile biomaterial platforms for tissue regeneration. Those are 3D polymeric hydrophilic structures, with viscoelastic and water-insolubility properties, which may respond to various stimuli such as pH, temperature, ionic strength, electric field, and enzymatic action. They can contain or absorb large amounts of water or physiological fluids, such as serum or plasma, reaching thousands of times their dry weight, without losing their 3D structure, soft and elastic consistency and low surface tension with water and biological fluids.2–5

Hydrogels have an extensive number of applications in tissue engineering. Different types of hydrogels have been developed to show excellent biological integration properties, as well as adequate biological performance.14,20 Biomedical applications of hydrogels have been described since the 1960s, with the development of a synthetic poly(2-hydroxyethyl methacrylate) (p-HEMA) material, which gave rise to the first contact lenses.21 In the last 60 years, the field has been through an outstanding evolution, and hydrogels have been applied to a plethora of fields (Figure I.3) including agriculture, civil engineering, cosmetic products development, pharmaceutics, biomedical engineering, dermatology, orthodontics, oil processing, and textile engineering, among others.2,3

The development of hydrogels with specific physical and chemical optimized compositions, showing functional properties as elasticity, non-toxicity, biodegradability and biocompatibility, tissue alikeness and sensitiveness to stimulus, drove their application and widespread use in the biomedical field.6 _{These materials have served as scaffolds, drug} delivery systems, adhesives or protective/selective barriers between tissue and material surfaces, biosensors, artificial muscles, cancellous bone filling, cartilage replacement, among many others.6,20,22

8

Figure I.3 – Applications of hydrogels in several fields.

Two of the most unanimous properties of biomedical hydrogels targeting implantation into humans as defect filling agents capable of promoting tissue regeneration are biocompatibility and biodegradability.23 Biocompatibility refers to the ability of a material to promote a favorable response to a specific application, i.e., the material should be toxic, non-irritating, non-allergenic, non-carcinogenic, mechanically compatible with the surrounding tissues, and promote a targeted response of the tissue and surrounding tissues (through the coordinated action of physical and biochemical signals).24 Although a foreign body response may cause the rejection of an implanted biomaterials, nowadays the concept of immunomodulatory biomaterials requires the design of surface and bulk materials features that cause controlled inflammatory and immune responses, and may even be used to modulate the correct integration of the biomaterials in the surrounding tissues.23 _The immunomodulatory aspects of biomaterials have been thoroughly reviewed by Shoichet et al.25

Biodegradable hydrogels may degrade by hydrolytic degradation, or in contact with physiological fluids. Hydrolytic degradation occurs in members of polylactide/glycolide

9

family (e.g. PLLA and PLGA), which are eliminated via Krebs cycle, as carbon dioxide and water.26 In contact with physiological fluids, hydrogels can be degraded by enzymatic action, generating soluble products, easily excreted by normal metabolic mechanisms. They present the advantage of not requiring a surgical intervention for their removal.23 Commonly used peptides susceptible to enzymatic cleavage include matrix metalloproteinase (MMP)-sensitive peptides,4 which are cleaved in glycosidic bonds. MMPs are abundant enzymes in disease sites.5 _{Several polymers have been modified with MMP-sensitive peptide domains,} which include hyaluronic acid, 27 _PEG28 _{and alginate}29_.

Hydrogels often consist of mainly polymeric networks containing functional hydrophilic groups, such as hydroxyl OH), carboxylic acid COOH), amine NH2) and sulphate (-SO3H), conferring them with the ability to absorb large amounts of water.5 Hydrogel swelling, water content and uptake of aqueous solutions often happen due to external environment fluctuations, including pH and salt concentration.5,21 However, crosslinking of polymeric chains is the main factor that influences swelling. Highly crosslinked hydrogels have a compact structure and lower swelling than less crosslinked hydrogels. Crosslinking obstructs the mobility of the polymer chain, reducing expansion.21

The interaction between hydrogels/water/biological fluids occurs through capillary, hydration and osmotic forces, which are balanced, causing the expansion of hydrogels.6 When swollen, hydrogels contain a high water volume, have a soft and rubbery appearance and have similar properties to living tissues, mimicking the extracellular matrix.2,20

I.2.3. Hydrogels source

The polymeric network used to establish hydrogels structure may be formed by synthetic, natural or hybrid polymers (natural polymer with a synthetic one), giving rise to 3D networks by molecular tangling or chemical crosslinking.2

Hydrogels from synthetic polymers (e.g. poly(ethyleneglycol) (PEG),30 poly(vinyl alcohol) (PVA),31 poly(acrylic acid) (PAA)32,poly(2-hydroxyethyl methacrylate) (p-HEMA)33 and poly(N-isopropylacrylamine) (PNIPAAm)34) usually have well-defined architecture and mechanical and chemical properties. However, they may not show biocompatibility and degradability under physiological conditions.23 Synthetic hydrogels do not have inherent

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biological properties, yet, they have well-defined structures that can be modified to produce degradability and functionality.21 Despite the lack of degradability, they have also been successfully applied in applications that include the development of contact lenses, coatings and membranes.23 To surpass lack of cell interaction issues, peptide sequences known for promoting cell adhesion have been used to modify several polymers to later fabricate hydrogels.35 The most well-reported sequence is RGD, natively found in fibronectin. Several polymers have been modified by arginylglycylaspartic acid (RGD) motif, such as PEG36,37_, alginate38_{, hyaluronic acid}39_{, chitosan}40_{, among many others.}

Natural polymers (e.g. hyaluronic acid,41 _cellulose,42 _chitosan,43 _{and alginate}44_{) are} structures formed during the growth cycle of living organisms, and often show a degree of resemblance to the components of native tissues. The fact that they contain similar or even identical chemical structures to those found in the organic matrices of organisms reduces the possibility of toxicity of the materials and their degradation products. However, they often present low mechanical resistance and poorly reproducible properties.23 It should be noted that in protein based-polymers, degradation frequently happens via enzymic cleavage, which means that have greater susceptibility to be metabolized by the physiological mechanisms in the body.45 Polymers of natural origin may be extracted from a plethora of sources, including animals, vegetables and microorganisms.14 Those may be of several types, including proteins and polysaccharides. Here, we will address polysaccharide-based hydrogels, since the polymer used in the work described in this thesis is chitosan: a polysaccharide derived from the treatment of chitin – a polymer extracted from diverse sources, including the most common crab shells.

The combination of synthetic and natural polymers in the synthesis of hybrid hydrogels has allowed achieving advanced and tailored properties of these two classes of polymers, resulting in biocompatible, biodegradable hydrogels and at the same time with good mechanical properties.23

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I.2.3.1. Polysaccharide-based hydrogels

Polysaccharides are chains of natural sugar monomers bound together by glycosidic linkages.4,21 These are abundant and obtained from renewable sources, such as the plants, animals, which may be obtained from marine sources.21 For example, chitosan is extracted from chitin (the second most abundant biopolymer after cellulose) that is synthesized by a large number of living organisms, nevertheless the main source are the shells of shrimp and crabs.43,46 There is a variety of polysaccharides-based hydrogels mentioned in literature, including chitosan47_{, dextran}48_{, alginate}49_{, hyaluronic acid}50_{, among others.}

Hydrogels produced from polysaccharides show several advantages, including the abundance of the raw material and possibility to be obtained from renewable sources, including natural kingdoms of algae and plants, cultures of microbial strains.21 Furthermore, polysaccharides have been reported as interesting materials to produce hydrogels because of their high swelling capacity, biocompatibility, biodegradability, non-toxicity, self-healing, mechanical properties and their ability to be eliminated by enzymatic digestion in the human body.6

I.2.4. Hydrogel Cross-linking mechanisms

Hydrogels can be classified as physical- or chemical-based, according to their crosslinking mechanism.2 Hydrogel crosslinking promotes the formation of insoluble 3D structures with tailorable mechanical properties, which allows for the tailored immobilization and effective release of active agents and biomolecules.2,3

I.2.4.1. Physically crosslinked Hydrogels

Physically crosslinked hydrogels can be formed by several types of reversible chemical interactions. Physical crosslinking involves tangled chains and ionic-driven interaction forces hydrophobic interactions and hydrogen bonds (Figure I.4).2,51 Physically crosslinked hydrogels have gained significance because of their relative ease of production and the advantage of not using crosslinking agents during their synthesis.6 Various methods reported in the literature for obtaining physically crosslinked hydrogels, specifically,

freeze-12

thawing,31,52 stereocomplex formation,53,54 ionic interaction,55,56 hydrogen-bonding41,57 and maturation.58. The freeze-thawing technique has an advantage of being able to crosslink the polymer solution without leaving any crosslink remnant which could provoke an inflammatory response after implantation.52,59 In addition, it creates a highly elastic and strong gel.21 _{PVA hydrogels have been cross-linking by this process. Liu et al. describe that} it is possible to produce PVA-based hydrogels with tailored mechanical properties by controlling the number of freeze-thaw cycles. The freezing step forms ice crystals that promote the formation of PVA crystallites. These crystallites act as crosslinking sites for polymer chains. Simultaneous thawing would melt the ice crystals and create one region of polymer-rich gel surrounded by another one of a polymer-poor solution. Additional formation of ice crystals would take place in the region of a polymer-poor solution and the newly generated crystalline sites, during successive freeze-thawing cycles, enhance the polymer crystallization in the gel, resulting in a hydrogel with smaller pore size and higher stiffness.52 Stereocomplexation is described as the interaction between polymers having different tacticities or configurations.60 The advantage of stereocomplex formation is that a hydrogel can be easily formed by dissolving the polymers in water, followed by mixing the solution. A major limitation is the limited range of polymer compositions which can be used.6 Jong et al. proposed a hydrogel which crosslinking is established by stereocomplexes formation between lactic-acid oligomers (PLLA and PDLA, L-lactic-acid and D-lactic acid, respectively), grafted to dextran chains, obtaining fully biodegradable, biocompatible and mechanical properties easily modelled by the way the oligomers bind to dextran.53 Ionic interactions (Figure I.4a) are achieved by the contact of polyelectrolytes with oppositely charged ions.6 Masruchin et al. report a hydrogel by inducing ionic interactions between negative supramolecular cellulose microfibrils and a positive metal ion (Al3+), promoting a swelling and drug delivery behavior of hydrogel.55 Oliveira et al. described a hydrogel based on a first-step polymeric polyelectrolyte complexation from positively charged amine groups (in chitosan) and negatively charged carboxylic groups (in alginate), followed by a neutralization step of polyelectrolyte complexes and further compactation and dehydration leading to hydrogen bonding and stabilization of the material. The hydrogel was cytocompatible with stem cells and showed rapid recovery ability after structural damage, sequential shape-morphism, and moldability, what makes them potential candidates for several applications including cell encapsulation/delivery and cosmetics.61 Hydrogels may

13

also be prepared through hydrogen bonding interactions. Those involve the existence of a highly electronegative functional group and an electron deficient in hydrogen atoms.6,21 Yang et al. reported a hydrogel cross-linking based on multiple hydrogen bonds that features self-recovery ability. Hydrogen bonding is effective for energy dissipation applied when the hydrogel is compressed, restoring its original status.57 _{Finally, maturation is a process of} heat-induced aggregation.6 During the heat treatment, polymers are dissociated into monomers, resulting in exposed hydrophilic groups. This exposure is then translated in a gel formation by the aggregation process.62 _{Erickson et al. produce a hydrogel by maturation} which improved integration and compressive properties of the tissue-engineered cartilage.58

I.2.4.2. Chemically crosslinked Hydrogels

Chemical crosslinking involves the formation of covalent bonds that are established between polymer chains. These type of interactions presents high stability, and usually give rise to mechanically stiff hydrogels.2 Several methods reported in the literature for obtaining chemically crosslinked hydrogels include the use of chemical crosslinkers,6 _grafting,63 radical polymerization,4 _{condensation reaction,}64 _{enzymatic reaction}65 _{and high-energy} radiation.33 _{To initiate chemical crosslinking of hydrogels low molecular weight crosslinkers} are often applied to natural and synthetic polymers. Such crosslinkers include glutaraldehyde, genipin, epichlorohydrin, adipic dihydrazide (ADH), polyaldehydes, 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride) (EDC) / N-hydroxysuccinimide (NHS), etc.6 Genipin has been widely investigated as a crosslinking agent for chitosan (as an alternative to glutaraldehyde, a commonly used crosslinker), due to its natural origin, low toxicity (less toxic than glutaraldehyde) and high biocompatibility.23 Genipin and glutaraldehyde react especially with amines.66 Epichlorohydrin is a crosslinker widely used to react with chitosan. Epichlorohydrin and polyaldhydes react with hydroxyl groups to form a epichlorohydrin and polyaldehydes crosslinked product.67 On the other hand, ADH and EDC/NHS reacts with carboxylic acid groups.68 Hydrogels prepared by polymerization may have a weak structure so, in order to improve mechanical properties, graft-based hydrogels could be prepared. This technique implies the formation of free radicals in a strong surface and then polymerizing monomers onto the backbone of a pre-formed polymer.6,63 Talaat et al. reported a hydrogel synthesis by granting technique.69

14

Radical polymerization is frequently used to assemble covalently crosslinked hydrogel networks. For example, PEG-modified with reactive double bonds, have been used to produce hydrogels via photo- or redox-initiated polymerization. Photoinitiators undergo homolytic cleavage for generating radicals to initiate the radical polymerization, upon exposure to visible or ultraviolet (UV) light. Hydrogels produced by radical polymerization (Figure I.4b) are suitable for the use as injectable formulations.4 The chemically crosslinked hydrogels can be obtained from low molecular weight monomers in the presence of a crosslinking agent, by radical polymerization. This is one of the most widely used methods because it is efficient and relatively fast.6 Kim et al. describe a hydrogel cross-linked by free radical polymerization using ammonium persulfate (APS) and dimethyltoluidine (DMT) as free radical initiator and accelerator, respectively.70 Cyclodextrin-based hydrogels are frequently fabricated by a condensation reaction as building blocks and by a crosslinking agent (e.g. epichlorohydrin (EPI)).64 Enzymatic reaction is used to identification of systems. For example, Ebrahimi et al. report a chitosan based-hydrogel, which serves as a bacteria marker.65 High energy radiation was the most extensively applied technique for hydrogels fabrication.33 High energy radiation facilitates the formation of hydroxyl groups in water molecules, which interact with the polymer chains, resulting in the formation of covalent bonds.6 Radiation polymerization crosslinking has many advantages over conventional chemical methods. It occurs at room temperature and physiological pH, is a simple, environmentally friendly and additive-free process.33 At which point, adjusting irradiations conditions the crosslinking degree can be controlled. The advantage of preparing hydrogels by radiation have the absence of toxic additives (e.g. crosslinkers), having the potential for use in biomedical applications.33

The type and degree of crosslinking used to process different hydrogels modulate a range of properties, such as swelling, elasticity modulus and transport of molecules throughout the polymeric network.2 _{In physically crosslinked hydrogels, variables as gelation time, pore} size, the rate of degradation and dissolution may be difficult to control. Moreover, due to their usually poor mechanical properties and, because they are formed by reversible bonds, are susceptible of being easily dissolved. In contrast, chemical crosslinking improves the mechanical properties of the hydrogel.23 _{However, due to the internal fracture of the fragile} network, a structural rupture of these hydrogels causes the impossibility of the hydrogels to return to their initial form.47

15

I.2.4.3. Interpenetrating Network Hydrogels

Interpenetrating network hydrogels (IPN) are described as the combination of two independent cross-linked synthetic or/and natural polymers, in which, at least one is synthesized or crosslinked in the immediate presence of the other. This is typically done by immersing a pre-polymerized hydrogel into a solution of monomers and an initiator.63,71 The individual polymer chains are fully entangled (Figure I.4c), and there may or may not be chemical bonds between the combined networks.49 _{Crosslinked polymers with groups} that can act as hydrogen -donor or - acceptor, develop IPN via reversible complexes through hydrogen bonding.34

Figure I.4 – Several crosslinking mechanisms, namely physically and chemically crosslinking mechanisms and hydrogels with enhanced toughness, namely, interpenetrating and double network hydrogels. a) hydrogel’s polymeric network formed by ionic

16

interactions, b) hydrogel’s polymeric network formed by radical polymerization, c) interpenetrating network hydrogels, and d) double network hydrogels.

In literature, is described that the IPN encompasses the advantages of both the conventional hydrogels and improve the hydrogel mechanical, biological e physicochemical properties and an effective drug loading compared to conventional hydrogels.71–73 _{This design allows} the modulation of the IPN properties, according to the type and percentage of each component.73

Liu et al., reported an IPN hydrogel promising for vascular tissue engineering. They used gelatin mixed with bifunctional dextran modified with methacrylate and aldehyde groups, the results obtained shown superior elastic properties as well as increased compressive modulus and strength.48

Zhang et al., develop an IPN hydrogel based on polymer PNIPAAm with different composition ratios, the results reveal improved mechanical properties in comparison with non-IPN hydrogels, and a controllable response rate that depended on the composition ratio of the PNIPAAm.34

I.2.4.4. Double Network Hydrogels

A double network hydrogel (DN) is prepared from several pairs of different polymers. It consists of a first fragile network and a second ductile network covalently crosslinked (Figure I.4d).47,74,75 This system has been demonstrating to accomplish high mechanical strength and toughness.76

The first network is densely cross-linked and highly swells in water, which allows it to absorb a large amount of the second network monomers, after swelling, the first network becomes rigid and brittle, promoting an adequately dissipating energy and thus achieving high toughness.76

If the first network is too soft and ductile, internal fracture does not occur, and become a low toughness system and thus the high toughness is mainly derived from the fracture of the first network upon deformation, because relatively large damage zones formed in the first

17

network allow for more accumulated damage before macroscopic crack propagation occurs throughout the whole network.76

On the other hand, the second network is soft and ductile, provided by the chemically poor crosslinking (usually by copolymerization of monomers and cross-linkers). An increase in the second network concentration leads to an improvement of the mechanical strength, however, a second network highly crosslinked results in a decrease of fracture stress and fracture energy, due to the break of mechanical balance between the networks.76

Also, the interaction between the first and second network contribute to the mechanical properties enhanced of DN gels. This can be formed by either covalent or noncovalent interactions and with or without chemical crosslinkers. Being that, if covalent bonds are formed between the two networks, there are an increase of the strength as well as the entanglement between the networks them.76 This chemistry is used to obtain hard materials that exhibit high resistance to fracture, being able to sustain a higher compression than the others.47,77

Azevedo et al., report a DN hydrogel formed by a first network composed by DOPA-CHT, genipin and Fe3+ ions and a second network of medium molecular weight CHT, forming a hydrogel with superior compressive strength and stiffness, toughness, with self-healing, injectability and swelling behavior, besides, cytocompatible.47 Liu et al., prepared a nanocomposite DN hydrogel with a first network that consists of dopamine methacrylamide (DMA) and laponite, and a second network formed by polyacrylamide (PAAm). This DN hydrogel show improved mechanical properties and high dissipated fracture energy ability, as well as self-recovery capability and stiffness and architecture practically the same after compression.78 All these DN hydrogel systems exhibit increased mechanical strength and toughness compared to individual components.74

I.2.5. Adhesive hydrogels

The pre-historic man used grass, leaves and other natural agents as wounding dressing, the search for devices with the ability to control bleeding and showing wound closure properties dates to primordial times. Nowadays, the most common procedure to stop bleeding and close tissue defects are based on suturing. However, competitor techniques, such as the use of

18

staples, ribbons, and tissue adhesives are also on the rise.79 Rapid closure of wounds is of major importance in most surgeries.30 A fixation element should hold the tissues together to enable proper healing, stop leakage of biological fluids and, simultaneously, impart the tissues with resistance to multiple loads.80

The use of bioadhesives and sealants for wound closure and convalescence is becoming increasingly popular, especially as a way to avoid prone-to-infection staples, tacks, and sutures.24,30,81,82 In addition, some bioadhesives were reported to improve tissue repair, interrupt body fluid leaks, and promote wound healing. Their use allows withdrawing local anesthetics and needles, which minimizes trauma, reduces surgery time, and potentiates their use in geometrically irregular places. Moreover, the use of biodegradable agents or easy-to-retrieve materials may simplify complex procedures and do not require secondary procedures for removal.24,30,82,83

Surgical adhesives should present the following properties: i) safety and non-toxicity, ii) absorbability and degradability, iii) rapid solidification under physiological conditions to minimize bleeding time and surgery, iv) adherence – they should keep the tissues together without additional support until the wound has healed enough, v) healing ability, vi) hydrophilicity, to form strong adhesive bonds with living tissues, and vii) equivalent or lower surface tension as compared to the adherent.

I.2.5.1. Bioadhesiveness

Bioadhesion is defined as the state in which two materials, at least one of which is from biological nature, are held together as a result of interfacial forces.24,81 Adhesion refers to the ability of the adhesive to flow, wet the substrate and maintain certain physical-chemical intermolecular forces.24 The main mechanisms that characterize adhesion and bioadhesion are reported in the literature, as follows: electrostatic binding, chemical binding, wetting, adsorption, diffusion and mechanical.24,79

In bioadhesion, the electrostatic mechanism involves the formation of a double layer of electrical charges at the bioadhesive/living tissue interface. Opposite charge interplay induces the formation of electrostatic forces, which may play a significant role in the

19

adhesion of two surfaces. This adhesion system is mainly efficient on metallic systems, however, it shows limitations on the adhesion of non-metallic materials.24,79

Intermolecular bonding is the most commonly described adhesion mechanism between adhesive substrates and materials. These interactions are driven by weak forces that include hydrogen bonds, dipole-dipole interactions, London dispersion and van der Waals forces.79 The adsorption theory declare that the binding force between the bioadhesive and the tissue arises from the van der Waals and hydrogen bond interactions.24,84 Wettability results from intermolecular interactions between two surfaces. Interfacial tensions partially dictate the bioadhesive's ability to spread and contact with living tissue. Adhesion-promoting wettability implies that the contact angle between the bioadhesive and the tissue is, or is very close to zero.24

Diffusion theory describes how the interpenetration and entanglement of the bioadhesive polymer chain in the living tissue produce semi-permanent bonds.24 The entanglement explains the adhesion of two polymers, as they diffuse over the polymer-tissue contact interface and form interpenetrating chains.79 The mechanical theory of bioadhesion is directly related to the entanglement. In this mechanism, a mechanical blockade of the polymer chains and living tissue occurs, i.e. the adhesive material infiltrates the pores and irregularities of the surfaces of the adhesives and mechanically blocks the microscopic surface roughness of the adhesives.24,79

An adhesive can bind "objects" when applied to their surfaces and withstand separation by transferring the applied loads from one substrate to another through the junction area.24 Hydrogels are considered good candidates for adhesives (Figure I.5) due to their excellent biocompatibility, fracture strength, tensile strength, shear strength, viscosity, the degree of swelling and degradation, rapid application, less traumatic wound closure, less pain, no removal suture, excellent cosmetic result and localized release of drugs.30,80,81

Bioadhesives are used for many applications, including tissue protection, prevention of fluid leakage (e.g blood), wound closure and wound healing, and drug delivery.24,79 _{In addition,} these are manifesting in applications such as tissue engineering, regeneration, dental and bone.79

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One of the major limitations is the low adhesion resistance of sealants to humid environments. With a view to solving this problem, stickers were created inspired by the strong adhesion of some marine creatures.85

Figure I.5 - Hydrogel adhesiveness on human skin, over torsion, picture adapted from 85_.

I.2.5.2. Mussel-Inspired hydrogels: overcoming underwater adhesion limitations Nature has been a wide source of inspiration in the search for materials with outstanding properties, including underwater effective adhesion.86 Geckos, flies, and spiders can adhere to vertical walls and even walk on them, however, the problem of wet adhesiveness, in a humid environment, persists.86,87 However, some marine species, including mussels47,86,88,89 and the sandcastle worm90 secrete adhesive materials capable of bonding, in a few seconds, to a huge variety of (often irregular) humid substrates, in conditions including a wide range of temperatures, high salinity, in the presence of naturally occurring forces created by waves and currents.91 Inspired by these characteristics, a family of adhesives was developed that intends to mimic the underwater adhesion properties of mussels.

Mussels secrete a sticky glue that works in typical marine environments and does not lose the adhesion properties even in saline solution.85,92 Mussel fixation consists of acellular wires with a disc-like termination, called byssi, that connect the mussel to the substrates (Figure I.6),86 in the attachment mechanism, mussels start by establish close contact with the surface, then their feet emerges from the shell and as they contacts with the seawater, the byssal threads immediately solidify due to the pH increase (from the pH of the produced byssus (pH ≈ 5.5) to the seawater pH ≈ 8.2).93

The proteins present in byssi are named mussel foot proteins (Mfps). Twenty-five to thirty different mussel foot proteins have been identified, in which five of them (mfp-2 to mfp-6)

21

have a high content of 3,4-dihydroxy-L-phenylalanine (DOPA),93 which is known to be one of the key components responsible for adhesion to underwater substrates.86,91

Figure I.6 - Illustration of how mussels attach to substrates, illustration adapted from 92_.

DOPA – the precursor of the neurotransmitter dopamine - is a catechol-containing amino acid formed from post-translational modification of tyrosine residues.86 The catechol side chain of DOPA is able to displace water molecules well bonded to the substrates since they form strong hydrogen bonding interactions.91 In addition, DOPA has two forms: an o-catechol from (unoxidized), and the oxidized o-quinone. Catechol is capable of binding inorganic surfaces and quinone provides binding to organic surfaces.86,94 Catechols react with, for example, metal ions or metal oxide surfaces, creating reversible and strong coordination bonds. In opposite, quinones bind to organic surfaces by reversible covalent attachment.86

Several synthetic and natural polymers, including PEG30,94,95, chitosan47,88,96, hyaluronic acid,97 and alginate83, have been modified with the bioadhesive peptide domain DOPA. These materials presented enhanced mechanical and adhesive properties, which allowed their application as advantageous platforms for regenerative medicine. Besides mechanical adhesion, polymers conjugated with catechol side groups have been ascribed with additional properties. Han et al., designed a hydrogel inspired in the mussel’s ability to maintain the catechol groups protected from oxidation to quinones by secreting a redutive cysteine-rich protein. In a polydopamine–polyacrylamide (PDA–PAM) 3D structure, the overoxidation of catechol groups was prevented and obtain a stretchable, high toughness and repeatable adhesion ability hydrogel.98

Catechol group