development of medical devices based on...

UNIVERSIDADE ESTADUAL DE CAMPINAS

FACULDADE DE ENGENHARIA QUÍMICA

Development of Medical Devices Based on

Polyurethane to be Applied in Tissue Engineering

Desenvolvimento de Dispositivos Médicos à Base de Poliuretano para Aplicação na Engenharia de

Tecidos

LAÍS PELLIZZER GABRIEL

Campinas – São Paulo

Agosto/2016

LAÍS PELLIZZER GABRIEL

Development of Medical Devices Based on

Polyurethane to be applied in Tissue Engineering

Desenvolvimento de Dispositivos Médicos à Base de Poliuretano para Aplicação na Engenharia de

Tecidos

Tese apresentada à Faculdade de Engenharia

Química da Universidade Estadual de Campinas como

parte dos requisitos exigidos para a obtenção de título

de Doutora em Engenharia Química.

Orientador: Prof. Dr. Rubens Maciel Filho

Co-Orientadora: Profa. Dra. Carmen Gilda Barroso Tavares Dias

ESTE EXEMPLAR CORRESPONDE À VERSÃO

FINAL DA TESE DEFENDIDA PELA ALUNA

LAÍS PELLIZZER GABRIEL E ORIENTADA

PELO PROF. DR. RUBENS MACIEL FILHO

Campinas – São Paulo

Agosto/2016

A Ata da defesa com as respectivas assinaturas encontra-se no processo de vida

acadêmica do aluno.

“May your efforts to challenge the

impossibilities, remember that the great things of

man were conquered what seemed impossible".

Charles Chaplin

“Out of man's mind in free play

comes the creation Science. It renews itself, like

the generations, thanks to an activity which is the

best game of homo ludens: science is in the

strictest and best sense a glorious

entertainment”.

Jacques Barzun

ACKNOWLEDGEMENTS

To God.

My research accomplishments are due in large part to the encouragement

and guidance of my supervisor Prof. Dr Rubens Maciel Filho. I appreciate his help

and support throughout the duration of this research.

I am grateful to professor Dr. Carmen Gilda Barroso Tavares Dias who

helped me to make substantial contributions to my work, resulting in a more

applicable research work.

I would like to express my gratitude to Dr. André Luiz Jardini Munhoz for

his encouragement to start my academic career, for his invaluable help, attention,

and friendship.

It has been a pleasure to work as a research fellow at National Institute of

Biofabrication (INCT-Biofabris).

I would like to thank prof. Dr. Thomas Webster and the Northeastern

University, for their assistance, I appreciate the time and attention during the period

in the United States.

I would like to thank prof. Dr. Cecília Amélia de Carvalho Zavaglia for her

help and suggestions on the research project.

I appreciate the time and attention of the committee members Dr. Paulo

Kharmandayan, Dr. Jorge Vicente Lopes da Silva and Dr. Rodrigo Alvarenga

Rezende.

I would like to thank Dr. Aulus Roberto Romão Binelli Ana Amélia

Rodrigues and Dr. Viktor Oswaldo Cárdenas Concha for their suggestions and help

in this project.

I would like to thank my friend Dr. Ana Lívia Chemeli Senedese, thank you

for your friendship, confidence and loyalty.

I appreciate the attention, the confidence and the friendship of Dr. Andrea

Arruda Martins Shimojo.

I also would like to thank Prof. Dr. Éder Sócrates Najar Lopes for

academic assistance and help.

It was a pleasure to work with my colleagues in the Lopca laboratory, Dr.

Ingrid, Dr. Anderson, Dr. Ana Flávia, Dr. Marcelle.

I have enjoyed working at Webster Nanomedicine Lab, especially with

Pelagie Favi who helped me in research. Much of my research was conducted in the

company of Cintia Rosa and Tatiane Venturott, I’ve enjoyed their friendship,

assistance, and good humor.

It was a pleasure to live in the United States with Marion Chassouant and

Alena Khilko, that have helped me during the cold winter, and during the difficulties at

work, thank you for the friendship. It was also a pleasure to meet my special friend

Ione Yamamoto.

It was a pleasure to meet my Portuguese friends Daniela Rocha, Ana

Luisa, Ana Oliveira, David Oliveira, Ana Tojeira.

Especial thanks to my Mexican friend Emmanuel Salazar who helped me

in Portugal during the difficulties at work. I’ve enjoyed the friendship and confidence.

I would like to thank the technical staff at the Chemical Engineering

Department. Special thanks to Mara, Gilson and Cristiano.

I would also like to acknowledge the financial support from CNPq who

funded this project.

I would like to offer my deepest gratitude to my family, for their constant

support, guidance, inspiration and encouragement that have helped me through

times of difficulty.

“At times our own light goes out and is rekindled by a spark from another person. Each of us has cause to think with deep gratitude of those who have lighted the flame within us".

Albert Schweitzer

RESUMO

Durante as últimas décadas, muitos estudos têm sido realizados com o

objetivo de desenvolver materiais bioativos que oferecem potencial para crescimento

e reparo de tecidos. As estratégias atuais são baseadas no desenvolvimento de

dispositivos médicos combinados com células, nanopartículas, biomoléculas e

fatores de crescimento. Uma estratégia é através do uso de scaffolds (arcabouços

tridimensionais) que atuam como uma matriz three-dimensional para adesão,

proliferação e diferenciação, resultando no crescimento do tecido através da

mimetização da matriz extracelular natural (ECM). Desta forma, scaffolds podem

apresentar várias formas, como espumas, esponjas, filmes, fibras, membranas,

tubos e hidrogéis. Dispositivos médicos também estão suscetíveis a infecções,

causando a falha do implante. Bactérias podem aderir ao implante, criando um filme.

Uma vez que a bactéria aderiu ao dispositivo, é difícil de removê-la. Desta forma,

existe uma necessidade especial no desenvolvimento de materiais bioativos que

possam reduzir a contaminação hospitalar, o crescimento de microorganismos, a

transmissão de diferentes infecções, e destruir agentes patogênicos e organismos

resistentes a drogas. Uma estratégia é a modificação da superfície dos filmes

poliméricos adicionando nanopartículas que apresentam propriedades

antibacterianas. Nanopartículas também podem ser aplicadas em dispositivos

médicos com o intuito de promover propriedades osteocondutivas e melhorar as

propriedades de resistência mecânica. O uso de membranas poliméricas na área

médica tem crescido consideravelmente. A partir de membranas, é possível obter

pele artificial, bandagens para reparo de feridas, balões de angioplastia, scaffolds e

conexões neurais. Diferentes polímeros têm sido usados para essa aplicação.

Poliuretanos (PUs) têm sido considerados excelentes materiais para aplicações

biomédicas devido possuírem várias propriedades controláveis e apresentarem alta

biocompatibilidade. Mesmo que biocompatíveis, uma grande variedade de

nanopartículas tem sido adicionada aos PUs afim de promover propriedades

desejadas específicas. Atualmente, a maior desvantagem da síntese de PUs é a

dependência de produtos baseados em petróleo. Levando tudo em consideração,

esta tese de doutorado foca no desenvolvimento de diferentes tipos de PUs para

serem aplicados como dispositivos médicos. Nanocompósitos de poliuretano

dopados com nanopartículas de óxido de zinco, prata e hidroxiapatita foram

produzidos e estudados com o objetivo de conferir propriedades antibacterianas aos

nanocompósitos. Também foi proposta a produção de scaffolds de bio-poliuretano

derivado de fonte renovável dopados com a adição de nanopartículas de

hidroxiapatita para numerosas aplicações, incluindo ortopédica. Finalmente, foi

realizada a produção de poliuretano eletrofiado para a obtenção de membranas. Os

dispositivos desenvolvidos nesta tese foram caracterizados por meio de

propriedades morfológicas, estruturais, térmicas e biológicas. Em resumo, esse

estudo suporta a investigação de PUs como dispositivos médicos biocompatíveis

para numerosas aplicações na Engenharia de Tecidos.

Palavras-chave: Biomateriais, nanotecnologia, poliuretano, engenharia de tecidos.

ABSTRACT

During the past decades, many studies have been done in order to

develop bioactive materials offering the potential for tissue growth and repair. Current

strategies are based on the development of medical devices in combination with

cells, nanoparticles, biomolecules and growth factors. One strategy is to use

scaffolds to act as a three-dimensional matrix for cell adhesion, proliferation and

differentiation and tissue ingrowth by mimicking the natural extracellular matrix

(ECM). In this way, scaffolds may present various forms such as foams, sponges,

fibers, membranes, tubes and hydrogels. Medical devices are also susceptible to

infections, causing the implant failure. Bacteria can adhere to the implant, creating a

film. Once the bacteria adhered to the device, it is difficult to remove. In this way,

there is a special need in the development bioactive materials in order to reduce

hospital contamination, microorganism growth, the transmission of different

infections, and destroy pathogens and multi-drug resistant organisms. One possible

strategy is to modify the surface of polymer films using nanoparticles that present

antibacterial properties. Nanoparticles can also been applied in medical devices in

order to promote osteocondutivity properties and improve mechanical properties.

The use of polymeric membranes in the medical field has grown considerably. From

the membranes, it is possible to obtain artificial skin, bandages for wounds,

angioplasty balloons, scaffolds and neural connections. Several polymers have been

used for this application. Polyurethanes (PUs) have been considered excellent

materials for biomedical applications due to their various controllable properties and

high biocompatibility properties. Even though they are biocompatible, a wide range of

nanoparticles have been added to PU to promote select properties. The major

disadvantage in the synthesis of the PUs nowadays is the dependence of petroleum-

based products. Bearing all this in mind, this PhD work focuses on developing

different types of polyurethanes to be applied as a medical device. Nanocomposites

of polyurethane doped with the addition of either zinc oxide (ZnO), silver (Ag) and

hydroxyapatite (HA) nanoparticles in order to impart nanocomposites antibacterial

properties were synthesized and studied. It was also proposed the production of bio-

based polyurethane scaffolds derived from renewable resource dopped with

hydroxyapatite nanoparticles for numerous TE applications, including orthopedic

applications. Lastly, it was proposed the synthesis of electrospun polyurethanes in

order to obtain membranes. The medical devices developed in this project were

successfully characterized by morphological, structural, thermal and biological

properties. In summary, this study supports the investigation of polyurethanes as

biocompatible medical devices for Tissue Engineering applications.

Keywords: Tissue Engineering, Nanotechnology, Biomaterial, Polyurethane.

LIST OF FIGURES

Figure 1.1: Layout of the thesis. ............................................................................ 23

Figure 2.1: Types of scaffolds applicable in TE. ................................................... 26

Figure 3.1: Segmented structure of linear polyurethane polymerized by two-

step method. .................................................................................................... 33

Figure 3.2: PU distribution by area of the Brazilian market. ................................ 34

Figure 3.3: Reactivity of the isocyanate group. .................................................... 35

Figure 3.4: Example of diisocyanates currently applied. ..................................... 36

Figure 3.5: Chemical structure of phosgene and nitrobenzene, respectively. .. 40

Figure 3.6: Graph created from Table 3.3 showing the percentage that some

isocyanates have been applied in TE. ........................................................... 43

Figure 4.1: TEM images of the nanoparticles: a) ZnO, b) Ag, and c) HA. Scale

bars represent 100 nm. ................................................................................... 51

Figure 4.2: SEM of the plain PU. ............................................................................ 52

Figure 4.3: EDS and SEM of PU/ZnO nanocomposite. Scale bar represents 500

nm. ................................................................................................................... 52

Figure 4.4: EDS and SEM of PU/Ag nanocomposite. Scale bar represents 50

µm. .................................................................................................................... 52

Figure 4.5: EDS and SEM of PU/HA nanocomposite. Scale bar represents 5 µm.52

Figure 4.6: In vivo cell viability assay after 1, 3 and 5 days of incubation with

control, PU/ZnO, PU/Ag and PU/HA. ............................................................. 53

Figure 4.7: (a) S. epidermidis and (b) P. aeruginosa bacteria growth after 16

hours on the nanocomposites of interest to the present study. ................ 54

Figure 5.1: Polyphenolic compounds found in açaí seed.................................... 59

Figure 5.2: Abstract graph of the proposed research. ......................................... 60

Figure 5.3: Reaction formation of the natural, green chemistry, polyurethane. 64

Figure 5.4: EDS of the composite. ......................................................................... 64

Figure 5.5: SEM image of the composite. .............................................................. 64

Figure 5.6: FTIR spectra of polyurethane and composites. ................................. 66

Figure 5.7: Thermal degradation of PU and PU-HA by TGA/DTG. ....................... 67

Figure 5.8: MRC-5 viability of the PU and composite after 24 hours. ................. 68

Figure 5.9: Negative control. .................................................................................. 69

Figure 5.10: PU after 7 days. .................................................................................. 69

Figure 5.11: Composite after 7 days. ..................................................................... 69

Figure 5.12: Representative histological sections of tissue after 7 days of

implantation for control (a-c), for PU (d-f) and for the composites (g-i)..... 70

Figure 6.1: Structure of the polyurethane applied in this work. .......................... 79

Figure 6.2: Electrospinning apparatus and electrospun fiber, respectively. ..... 80

Figure 6.3: FTIR-ATR spectra of polyurethane. .................................................... 84

Figure 6.4: TGA/DTG of polyurethane. .................................................................. 85

Figure 6.5: Cell adhesion of human cardiomyocytes monitored by fluorescent

microscopy. ..................................................................................................... 86

Figure 6.6: SEM images of the plain PU and the fibroblasts after 24, 48 and 72

hours of culture. .............................................................................................. 88

Figure 6.7: Cell viability monitored by MTT assay following 24, 48 and 72 hours

exposure of fibroblasts cells. ........................................................................ 89

Figure 6.8: Live-dead assay showing fibroblasts incorporated in PU

membranes, after 24, 48 and 72 hours. ......................................................... 90

Figure 6.9: Live-dead assay showing fibroblasts incorporated in PU

membranes, after 24, 48 and 72 hours. ......................................................... 90

Figure 6.10: Mass loss during in vivo degradation study. ................................... 91

LIST OF TABLES

Table 2.1: Scaffolds desired properties................................................................. 28

Table 2.2: Different fabrication techniques of scaffolds. ..................................... 29

Table 3.1: Main reactions between diisocyanate groups and different

compounds. ..................................................................................................... 37

Table 3.2: Relative reaction rate related to the active hydrogen compound. ..... 37

Table 3.3: Some isocyanates applied as monomers to biomedical

polyurethanes. ................................................................................................ 42

Table 5.1: Functional groups and its absorption peaks (cm-1) that confirm the

formation of urethane. .................................................................................... 65

Table 6.1: Currently monomers and their applications in TE. ............................. 76

LIST OF REACTIONS

Reaction 3.1: Reaction of Wurtz. ............................................................................ 37

Reaction 3.2: Phosgenation of amines to produce isocyanates. ........................ 38

Reaction 3.3: Classic process for MDI production (Vilar, 2004). ......................... 38

Reaction 3.4: Classic process of TDI production (Vilar, 2004). ........................... 39

Reaction 3.5: Oxidation of isonitriles to isocyanates. ......................................... 39

LIST OF ABBREVIATIONS

Ag Silver AFM Atomic Force Microscopy ATR Attenuated Total Reflection BDI 1,4-Butane diisocyanate BDO Butanediol CASE Coating, adhesives, sealants and elastomers CFU Colony Forming Units DBDI 4,4-Dibenzyl Diisocyanate DMEM Dulbecco’s Modified Eagle Medium EDS Energy-dispersive Spectroscopy ECM Extracellular matrix FBS Fetal Bovine Serum FDA Food and drug administration FTIR Fourier Transform Infrared Spectroscopy HA Hydroxyapatite HDF Human dermal fibroblast HDI 1,6-Hexamethylene diisocyanate HMDI 4,4-Dicyclohexylmethane diisocyanate H12MDI 4,4-Methylene dicyclohexyl diisocyanate IPDI Isophorone Diisocyanate MDA Diphenylmethane dianilines MDI 4,4-Diphenylmethane diisocyanate MTT 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide NCT Negative control of toxicity NIPUs Non-Isocyanate Polyurethanes PBS Phosphate-buffered saline PCL Polycaprolactone PCT Positive control of toxicity PEG Polyethylene glycol PGA Poly (glycolic acid) PHA Polyhydroxyalkanoate PHB Polyhydroxybutyrate PLA Poly (lactic acid) PLGA Poly (lactic-co-glycolic acid) POSS Polyhedral oligomeric silsesquioxane PU Polyurethane PUs Polyurethanes PTMG Poly(tetramethylene glycol) S.E.M Standard error of the mean SEM Scanning Electron Microscopy TDI Toluene Diisocyanate TE Tissue Engineering TEM Transmission Electron Microscopy TGA/DTG Thermogravimetric Analysis/Derivative Analysis US United States UV Ultraviolet ZnO Zinc oxide 3D Three-dimensional

TABLE OF CONTENTS

ABSTRACT ................................................................................................................. 1

CHAPTER 1 .............................................................................................................. 20

INTRODUCTION ....................................................................................................... 20

1.1 OBJECTIVE OF RESEARCH ..................................................................... 21

1.2 LAYOUT OF THESIS ................................................................................. 21

1.3 MAIN CONTRIBUTIONS OF THIS WORK ................................................. 24

CHAPTER 2 .............................................................................................................. 25

THEORETICAL BACKGROUND .............................................................................. 25

2.1 Theoretical Background ........................................................................... 25

2.2 FUTURE TENDENCIES IN TISSUE ENGINEERING ................................. 30

2.3 CONCLUSIONS ......................................................................................... 31

CHAPTER 3 .............................................................................................................. 32

POLYURETHANES – RAW MATERIALS AND PRODUCTS PROCEDURES ........ 32

3.1 POLYURETHANES - THEORETICAL BACKGROUND ............................ 32

3.2. ISOCYANATES ......................................................................................... 35

3.2.1 Isocyanates process description ..................................................... 37

3.2.2 Isocyanates applied In biomedical polyurethanes ......................... 41

3.3 POLYOLS ................................................................................................... 43

3.4 CONCLUSION ............................................................................................ 43

CHAPTER 4 .............................................................................................................. 45

ANTIBACTERIAL PROPERTIES OF THE POLYURETHANE NANOCOMPOSITES45

4.1 INTRODUCTION ........................................................................................ 45

4.2 OBJECTIVE ................................................................................................ 47

4.3 MATERIALS AND METHODS ................................................................... 47

4.3.1 Materials ............................................................................................. 47

4.3.2 Methods .............................................................................................. 48

4.4 RESULTS AND DISCUSSION ................................................................... 51

4.4.1 Nanoparticles characterization ......................................................... 51

4.4.2 PUs nanocomposite characterization .............................................. 51

4.4.3 In vivo cell viability of nanocomposites .......................................... 53

4.4.4 Antibacterial activity of PU nanocomposites .................................. 54

4.5 DISCUSSION .............................................................................................. 56

4.6 CONCLUSION ............................................................................................ 56

CHAPTER 5 .............................................................................................................. 57

THE INFLUENCE OF HYDROXYAPATITE NANOPARTICLES ON THE

STRUCTURE, THERMAL AND BIOLOGICAL BEHAVIOR OF BIO-BASED

POLYURETHANE COMPOSITES ............................................................................ 57

5.1 INTRODUCTION ........................................................................................ 57

5.2 OBJECTIVE ................................................................................................ 59


5.3.1 Materials ............................................................................................. 60

5.3.2 Methods .............................................................................................. 61

5.3.3 Material Characterization .................................................................. 61


5.4.1 Structure and morphology ................................................................ 64

5.4.2 Microphase-separated structure ...................................................... 65

5.4.3 The thermal degradation of polyurethane ....................................... 67

5.4.4 In vivo analysis .................................................................................. 68

5.4.5 In vivo analysis .................................................................................. 68

5.5 DISCUSSION .............................................................................................. 72

5.6 CONCLUSION ............................................................................................ 72

CHAPTER 6 .............................................................................................................. 73

SYNTHESIS AND CHARACTERIZATION OF ELECTROSPUN POLYURETHANE

MEMBRANES FOR TISSUE ENGINEERING APPLICATIONS ............................... 73

6.1 INTRODUCTION ........................................................................................ 73

6.2 OBJECTIVE ................................................................................................ 79


6.3.1 Materials ............................................................................................. 79

6.3.2 Methods .............................................................................................. 80

6.3.3 Material characterization ................................................................... 81


6.4.1 Microphase-separated structure ...................................................... 84

6.4.2 The thermal degradation of polyurethane ....................................... 85

6.4.3 Human cardiomyocytes cell adhesion by fluorescent microscopy86

6.4.4 Evaluation of the fibroblasts cells adhesion of the electrospun

fibers using SEM ......................................................................................... 87

6.4.5 In vivo fibroblasts cell viability assays ............................................ 88

6.4.6 Live/Dead ............................................................................................ 89

6.4.7 In vivo degradation study ................................................................. 91

6.5 DISCUSSION .............................................................................................. 93

6.6 CONCLUSION ............................................................................................ 93

CHAPTER 7 .............................................................................................................. 94

CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK ................................ 94

7.1 CONCLUSIONS ......................................................................................... 94

7.2 SUGGESTIONS FOR FUTURE WORK ..................................................... 95

REFERENCES .......................................................................................................... 96

APPENDIX 1 ........................................................................................................... 110

CHAPTER 1 – Introduction 20

CHAPTER 1

INTRODUCTION

Tissue Engineering has emerged as a technology with potential for repair

of tissues through medical devices. This area of research is motivated by several

factors, including a rise in the elderly population, accidents and diseases such as

cancer, diabetes and osteoporosis. In 2013, the World Health Organization reported

that 347 million people worldwide have diabetes (World Health Organization, 2013).

Patients with diabetes carry a risk of amputation that may be more than 25 times

greater than normal patients (Dieren et al., 2010).

It is estimated that is spent annually about $ 300 million worldwide in

research focused on tissue engineering and cell therapy products before they

become commercially available, being the orthopedic segment the largest market

(Teoh et al., 2015). TE products also include cancer, cardiac and vascular diseases,

reconstructive surgery, providing an alternative to organ transplantation. The skin

wounds market has been considered the most successful market in TE offering

products to heal severe burns and diabetic ulcers (Teoh et al., 2015). Medical

devices have been applied as bone, skin and vascular grafts, and also can be

applied as cartilage, breast implants, knee replacements and artificial hearts.

Surface polymer modifications have been applied in order to improve

interactions between the medical device and the surrounding environment to prevent

biological responses. Nanotechnology can provide the control over biomolecular

interactions, since the biological molecules, structures and interactions in the ECM

occurs in nanoscale. This advantage can be applied in a specific goal, for example,

modifying the medical device behavior in vivo and in vivo and also modify the

medical device surface.

Although they are biocompatible, polyurethanes have been studied due to

the mechanical properties, processability and biodegradability.


1.1 OBJECTIVE OF RESEARCH

This research project aims to develop medical devices based on

polyurethanes to be applied in Tissue Engineering. In this way, films, scaffolds and

electrospun fiber membranes of polyurethanes were developed and characterized

through the structure, thermal and biological behavior of polyurethane composites.

1.2 LAYOUT OF THESIS

This PhD thesis supports the investigation of polyurethanes as medical

devices for tissue engineering applications carried over a period of four years. It

consists of seven chapters and one appendix. These are as follows:

Chapter one introduces the purpose of this research project by giving the

background of the current polyurethane applications in TE and describes the

objective and specific objectives, and main research contributions. This section

outlines the aims of this PhD research.

Chapter two describes a brief background to polyurethanes, isocyanates,

polyols and medical devices focusing on the availability and properties.

Chapter three presents the polyurethane theoretical background, raw

materials and products procedures to be applied in Tissue Engineering. The aim was

to show the current progress and advantages of research in this field.

Chapter four covers the incorporation of nanofeatures onto polyurethane

films in order to impart antibacterial properties. This involves the following specific

objectives:

4.1. – Stock solution preparation of polyurethane, zinc oxide (ZnO),

silver (Ag) and hydroxyapatite (HA).

4.2. – Polyurethane films preparation doped with the addition of either

ZnO, Ag and HA nanoparticles;

4.3. – Nanoparticles evaluation using Transmission Electron

Microscopy (TEM);

4.4. – Morphology and distribution of nanoparticles evaluation using

Scanning electron microscopy (SEM) and energy-dispersive spectroscopy

(EDS);


4.5. – In vivo cell viability of nanocomposites determined by MTS

assay.

4.6. – Antibacterial properties evaluation using bacterial assays against

the Gram-positive bacterium S. epidermidis and the Gram-negative bacterium

P. aeruginosa;

Chapter five concentrates on the production of bio-polyurethane scaffolds

by the addition of hydroxyapatite nanoparticles in order to evaluate the influence of

nanoparticles on the structure, thermal and biological behavior of polyurethane

composites. This covers the following specific objectives:

5.1. – Polyurethane preparation in a batch reactor;

5.2. – HA nanoparticle preparation;

5.3. – Composite (PU-HA) preparation;

5.4. – Morphological and structural characterization using Scanning

electron microscopy (SEM) and energy-dispersive spectroscopy (EDS);

5.5. – Chemical and structural characterization using Fourier

transform infrared (FTIR) spectroscopy;

5.6. – Thermal characterization using thermogravimetric analysis

(TGA);

5.7. – In vivo cell viability of nanocomposites determined by MTT

assay.

5.8. – In vivo and histological studies.

Chapter six presents the production of polyurethane fibers using the

electrospinning apparatus in order to evaluate the influence of fibers on the structure,

thermal and biological behavior of electrospun polyurethane. This involves the

following specific objectives:

6.1. – Polyurethane electrospinning stock solution preparation;

6.2. – Electrospinning of the polyurethane;

6.3. – Morphological and structural characterization using Scanning

electron microscopy (SEM);

6.4. – Chemical and structural characterization using Fourier

transform infrared (FTIR) spectroscopy;

6.5. – Thermal characterization using thermogravimetric analysis

(TGA);


6.6. – In vivo adhesion of human cardiomyocytes determined by

fluorescent microscopy;

6.7. – Evaluation of the fibroblasts cells adhesion and proliferation of

the electrospun fibers using Scanning electron microscopy (SEM).

6.8. - In vivo cell viability of electrospun polyurethane determined by

MTT assay;

6.9. – Live/Dead of the fibroblasts cells adhered in the PU membrane;

6.10 – In vivo degradation tests of the membrane.

Chapter seven lists the conclusion derived from this study as well as

suggestions for future work.

Appendix 1 presents the approved Ethics Committee in Animal

Experimentation for the in vivo studies presented in Chapter 5.

Figure 1.1: Layout of the thesis.


1.3 MAIN CONTRIBUTIONS OF THIS WORK

This PhD work focused on developing different types of polyurethane

scaffolds to be applied in Tissue Engineering. In this way, the main contributions of

this are summarized below:

PU films incorporated with zinc oxide (ZnO), silver (Ag) and

hydroxyapatite (HA) nanoparticles were successfully prepared in order to impart

antibacterial properties. This study showed evidences of bacteria inhibition of films

current applied as medical devices due to the possibility of surface modification using

different nanoparticles;

A bio-based green chemistry composite scaffold was also prepared and

the influence of hydroxyapatite nanoparticles was evaluated through morphology,

thermal and biological studies. This study showed that hydroxyapatite nanoparticles

promoted the fibroblasts cell adhesion and tissue adherence. In summary, the

medical device developed in this work is biocompatible and could be applied for

numerous TE applications;

Lastly, it was prepared polyurethane membranes by the

electrospinning method. The membranes developed in this work showed fibroblasts

cell adhesion and proliferation. This study supports the application of polyurethane

membranes as biocompatible wound healing.

CHAPTER 2 – Theoretical Background 25

CHAPTER 2

THEORETICAL BACKGROUND

Trauma, injure or disease can damage tissues in the human body, which

available treatments are based on autografts or allografts transplantation, among

others. Currently, the main obstacle to the organ transplantation is the lack of donors.

In 2014, 29,534 patients received an organ transplant, while 133,828 patients were

on the waiting list, in the United States (OPTN / SRTR Annual Report, 2015), but

there are risks of immune rejection and transmission of pathogens and further

infection. Furthermore, the increase in life expectancy, malfunctioning of organ

tissues or tissue loss caused by injury or disease, have led to reduced quality of life

for many patients and increased socioeconomic costs worldwide.

2.1 Theoretical Background

Tissue engineering (TE) has emerged in the early 90s as a technology

that involves the study of the repair of organs or tissues that have suffered injury to

restore, maintain or improve the tissue function (Langer and Vacanti, 1993), using a

combination of procedures, cells, materials and growth factors. Researches directed

to effective routes for the fabrication of medical devices through reactions of

manufacturing processes are gaining attention nowadays. It is an interdisciplinary

field, where scientists are applying the principles of cell biology, engineering,

materials science, computer science and medicine to create medical devices that will

restore and maintain the function of injured or diseased tissues.

It is already known that after the injury, an inflammatory response appears

and the extracellular matrix (ECM) is replaced by a blood clot and platelet stimulate

the cellular activity. After that, the hemostasis starts and inflammatory cells as

macrophages, lymphocytes and neutrophils start a process to eliminate bacteria and

dead tissue, and growth factors are released during inflammation. At the end of the

inflammatory stage, different cells, such as fibroblasts and endothelial cells, migrate

and proliferate to the injury site, to start a new tissue formation. The last stage is a

process known as angiogenesis, when occurs the remodeling of new tissue, re-


establishment of the blood and nutrients and oxygen necessary for normal cellular

activity (Dreifke et al., 2015).

The tissue consists of a matrix and different cell types. Along these lines,

in order to repair a tissue, the TE proposes a network scaffold matrix to allow cell

growth, proliferation and differentiation to promote tissue growth. They provide an

initial biochemical substrate to the cell proliferation, to create a new ECM (Bártolo et

al., 2008). In vivo, the ECM provides the attachment of cells with molecules and

proteins and thus creates an appropriate physical and biochemical environment

(Kausar e Kishore, 2013).

The TE usually requires the cell culture seeded on biodegradable

scaffolds, under a controlled environment, including temperature, pH, biochemical

and mechanical stimulation.

Current TE strategies are based on the use of three-dimensional scaffolds.

Three-dimensional biomimetic scaffolds have been applied in TE because of their

architecture, similar to ECM (Lu et al., 2013). However, the synthesis of scaffolds

adequate to a three-dimensional cell culture is a challenge to be overcome. They act

as extracellular matrix that provides support to cells, stimulate rapid blood vessel

ingrowth, and the reconstruction of the injured tissue. Scaffolds can be classified in

solid and injectable. They are produced in accordance with the desired application,

and may present various forms such as foams, sponges, fibers, membranes, tubes,

films and hydrogels, as shown in Figure 2.1.

Figure 2.1: Types of scaffolds applicable in TE.


Solid scaffolds are produced with greater control of the three-dimensional

architecture and are quite stable. They have ability to adequately support the

transport of nutrients, adhesion and migration of cells. However, the main

disadvantage is in need of surgery for implantation. Moreover, injectable scaffolds do

not require surgical intervention only minimally invasive application, which reduces

the risk of infection, scarring training and the cost of treatment.

Scaffolds should have a porous structure, appropriate pore size,

interconnectivity, good mechanical properties and sufficient biocompatibility that

allows the vascularization process and tissue ingrowth, to make them implantable

into the patient (Li et al., 2014). Usually, it is necessary a porosity around 90% and

pore size between 100 and 500 micrometers, to promote the cellular functions of

ECM (Bártolo et al., 2011). Tissues with porosity less than 20% have a mechanical

support function, and tissues with porosity around 90% have the protection function

and blood cell production (Salinas et al., 2013).

In addition, pore sizes directly affect the cell seeding in the center of the

scaffold and in the inner surfaces, when the pore size is too small, on the other hand,

large pores affect the stability of the scaffold and the ability to provide physical

support for the seeded cells (Levenberg & Langer, 2004). Cell seeding on bi-

dimensional scaffolds surface is easy to perform, however the construction of 3D

scaffolds is more complex. Adequate pore size that allows cells migration and

adherence to the scaffolds surface, but interconnecting pores are necessary to

permit cell growth into the scaffold interior (Chang & Wang, 2011). Specific cells

require different pore sizes for optimal cellular activity. DeGroot et al. (1996) reported

that the PU pore sizes for meniscal implants ranging from 150 to 300 µm induced the

ingrowth of fibrocartilaginous tissue.

Nowadays, one challenge to be overcome is to control the degradation

rate of the polymer matrix, because while the matrix degrades, occurs the formation

of the newly tissue. These depend upon the type and characteristic of the tissue to

be considered. Table 2.1 shows the desired properties of the scaffolds.


Table 2.1: Scaffolds desired properties.

Scaffolds desired properties

Description

Biocompatibility

Scaffolds must be biocompatible and demonstrate satisfactory performance in order to produce adequate response to the host tissue without producing cytotoxic or immune response. No by-product degradation can cause inflammatory or toxic reactions.

Biodegradability

Scaffolds must be biodegradable and possessing controlled degradation rate, in order to match the tissue repair rate (Olsson et al., 2008). The degradation of the scaffolds may occur by mechanisms involving physical processes or hydrolysis or biological processes, such as enzymatic cleavage.

Porosity

Scaffolds should exhibit high porosity with cell-scaffold interactions, in order to control the adequate diffusion of nutrients and oxygen to cells, metabolite dispersal, local pH stability and cell signaling.

Pore size

Pore size is an important feature due to cell penetration and tissue vascularization. The scaffolds must satisfy the condition of providing an empty volume of pores, and directly influence the amount of space the cells have for 3D organization (Chang & Wang, 2011), in order to occur cell adhesion, vascularization, and new tissue formation.

Surface chemical

properties

Scaffolds surface can control the effect of cell adhesion and proliferation, due to be the primary site of interaction between cell-scaffold. After implantation, the cells migrate to the scaffold surface and a series of physical and chemical reactions occurs, in order to fix the cells to the matrix.

Mechanical resistance

Scaffolds must have adequate mechanical properties for manipulation in vivo and in vivo. Also scaffolds must provide adequate mechanical resistance, since the processes of release of bioactive agents and degradation have direct effect on the scaffolds. The mechanical properties must satisfy the mechanical resistance of the tissues to be regenerated. Hollister (2005) proposed a mechanical resistance of soft tissues between 0,4 – 350 MPa and hard tissues between 10 – 1.500 MPa.

Bioactivity

Scaffolds should provide bioactivity properties in order to be able to incorporate bioactive or signaling molecules to promote tissue repair.

Currently, scaffolds have been applied for the repair of soft and hard

tissues, such as bone, cartilage, tendons, ligaments, skin, muscles and blood

vessels, and have been produced using different techniques such as solvent casting,

gas foaming, freeze drying, electrospinning, rapid prototyping and thermally induced

phase separation, using a broad variety of biomaterials. These techniques produced

porous structures, however most of the conventional fabrication methods have

inadequate degree of control over the pore structure (Tan et al., 2013). The main

advantages and disadvantages are shown in Table 2.2.


Table 2.2: Different fabrication techniques of scaffolds.

Medical devices fabrication techniques

Technique Advantage Disadvantage

Solvent casting/

particulate

leaching

Simple method, adequate porous size

and controlled porosity.

Use of organic solvents, structures

generally isotropic. Limited thickness

between 0,5 to 2 mm.

Gas foaming

This technique does not use any

solvent. It can be obtained foams with

pore size of 100μm and porosity of

93%.

Closed porous structure and from 10

to 30% of interconnected porous.

Freeze-drying

The concentration of the polymer

solution and viscosity affected the

porosity and the pore size. Scaffolds

with 90% of porosity, and pore size

from 20 to 200 μm, can be obtained.

Porous without connection.

Electrospinning

High surface area: volume ratio, high

porosity, small pore size,

extraordinary length, low density.

Fibers ranging from nanometers to

micrometers can be obtained.

Slow production rate, inadequate

mechanical strength, possible toxicity

due to the residual solvent.

Additive

manufacture

It is possible to convert designs from

computer directly into solid objects,

fast fabrication with complex

geometries.

High production costs.

Thermally induced

phase separation

Adequate mechanical strength, high

porosities, interconnected pore

structures.

Scaffolds with pore size from 10-100

μm can be produced, inadequate to

the repair of most of tissues small

scale production.

Among the biodegradable synthetic polymers, linear aliphatic polyesters,

as poly (glycolic acid) (PGA), poly (lactic acid) (PLA) and poly (lactic-co-glycolic acid)

(PLGA), have been widely used in TE due to the relatively hydrophilic nature.

Other linear aliphatic polyesters, such as polycaprolactone (PCL) have

also been investigated in tissue engineering particularly for long-term implants, due

to degradation rate significantly slower than PLA, PGA and PLGA. Polyethylene

glycol (PEG) have been applied as injectable and solid scaffolds, however it can

present toxicity.


In recent years, polyurethanes have been also applied in TE mainly

because of the control possibility of their mechanical and morphological properties

(Janik & Marzec, 2015). Furthermore, the surface of polyurethane (PU) can be

biomimetically modified to improve blood compatibility (Hu et al., 2012). PUs are also

susceptible to biodegradation through hydrolysis or catalyzed by enzymes or

oxidation promoting the cleavage of hydrolytically sensitive bonds present in their soft

segments.

Some of the applications of PUs are as vascular grafts (Hou et al., 2014),

drug delivery (Mishra et al., 2014b), shape memory (Peponi et al., 2013), catheters

(Kara et al., 2014), breast implants (Prokopovich & Perni, 2010), cartilage (Podsiadlo

et al., 2009, Laurenti et al., 2014), orthodontic applications (Jung & Cho, 2010),

cardiovascular applications (Kara et al., 2014; Nishi et al., 2014; Sgarioto et al.,

2014) and also for regenerating articular cartilage (Laurenti et al., 2014).

2.2 FUTURE TENDENCIES IN TISSUE ENGINEERING

The TE in the near future achieve to produce a functional human tissue,

using multiple types of living cells under a controlled manufacturing process (Ozbolat

& Yin, 2013). It is intended to facilitate the printing of tissues or organs through the

bioprinter, which will facilitate fusion of the tissues and maintain the printed tissue to

the desired maturation (Rezende et al., 2011). While the concept is simple

considering the complexity and functionality of printing organs, there are still several

limitations.


2.3 CONCLUSIONS

This fascinating area of research demonstrates how multidisciplinary field

of TE is vast, the opportunities for improving human health are immense. Many

challenges are still limited to this multidisciplinary area and the complexity of

biological systems involved in the repair of different tissues. Significant progress has

been made to achieve the TE goals. While simple polymeric scaffolds that were

being developed in the past were not providing adequate interactions between cells

and the ECM, future advances in TE are depending on new systems that will actively

modulate the behavior of cells to a functional TE. The success of TE depends on the

researchers efforts to understand the results of science and clinical applications. The

integration of knowledge of life sciences areas along with materials science,

medicine, chemistry, biology and engineering is crucial for the scaffolds design, and it

will also generate new technologies for application in regenerative medicine.

The possibility to control the processability and properties of

polyurethanes is an exciting and a promising field for TE applications, due to the

possibility to promote some desirable properties in the polyurethane, focusing on the

desired application.

CHAPTER 3 – Polyurethanes – Raw materials and products procedures 32

CHAPTER 3

POLYURETHANES – RAW MATERIALS AND PRODUCTS

PROCEDURES

This chapter is a review of the polyurethanes structure, morphology and

properties, the chemical properties of aliphatic and aromatic isocyanates and their

advantages and disadvantages when used as precursors for polyurethane (PU)

synthesis, and the current polyols that have been applied in biomedical

polyurethanes. Isocyanates react quantitatively with primary hydroxyl groups

producing urethane groups. The presence of urethane groups can promote the tissue

repair. Molecules with two or more hydroxyl groups reacting with molecules

containing two or more isocyanate groups results in PU. PU is biocompatible, with

the condition that have no remaining free isocyanate groups. This Chapter also

focuses on the isocyanates applications in the TE, PU market and the production of

medical devices based on PUs.

Keywords: Isocyanates, polyols and polyurethane market.

3.1 POLYURETHANES - THEORETICAL BACKGROUND

Commercialized since 1954, the global market of PUs was estimated at 14

million tons in 2010 and the projection for 2016 is to produce 18 million tons (Nohra

et al., 2013), which 75% of the projection refers to production of foams. The PUs

holds 7% of the total market demand, representing the 5th position in the global

production of plastics (Cornille et al., 2015).

PUs are produced by the reaction between molecules with two or more

hydroxyl groups reacting with molecules containing two or more isocyanate groups.

According to the nature of the reactants and reactivity of isocyanate, PU can be

obtained with two thermodynamic incompatible phases, originating a phase-

separated structure (Cetina-Diaz et al., 2014; Dempsey et al., 2014). In general, PUs

may be synthesized by one-step and more commonly by a two-step method. Usually

two-step method is more precise, reliable, with a highly controllable reaction, and a

final product is more reproducible. During the two-step method (Figure 3.1) which is


also known as prepolymer method, the excess of diisocyanate reacts with the polyol

to form the NCO-terminated prepolymer. In the second step, the chain extension

occurs, where the prepolymer reacts with a short organic diol to obtain the high

molecular weight PU.

Figure 3.1: Segmented structure of linear polyurethane polymerized by two-step

method.

In 2012, the manufacturer companies and distributors of raw materials and

additives for PU in Brazil earned between R $ 10 and 100 million (Plástico Industrial,

2012).

Figure 3.2 represents the consumption of raw materials and additives

derived the PUs separated by area during 2010 and 2012. CASE is represented by

coating, adhesives, sealants and elastomers. The medical field is still very limited in

the consumption and production of PUs.


Figure 3.2: PU distribution by area of the Brazilian market.

PU is an already established material for the manufacture of orthopedic

prostheses, including approved by the ANVISA and FDA. PU has been applied in the

medical field due to compatibility properties with living tissues, with favorable aspects

of processability, formulation, excellent structural properties, releasing no toxic by-

products in the body, low cost, it is hemocompatible and has excellent physical and

mechanical properties.

PUs are commonly known as versatile polymers in terms of applications

because of a variety of monomers which can be used in its synthesis, such as the

nature of the polyol (polyether, polyester, polycarbonate, polyolefins) and isocyanate

structure (aromatic ou aliphatic). Furthermore, a great variety of chemicals can be

added, for example, catalysts, curing agents, chain extenders, blowing agents, fillers,

among others.

PUs based on aromatic isocyanates are considered less biocompatible

than PUs based on aliphatic isocyanates. This happens because the products of

degradation are toxic, such as the aromatic amines from the rigid segments of PUs.

PU biodegradation has been extensively studied since this material has been applied

in several TE areas (Ates et al., 2014; Kucinska-Lipka et al., 2015; Guo et al., 2014;

Singhal et al., 2014).

Isocyanates are chemical compounds from the isocyanic acid having NCO

(nitrogen, carbon, oxygen) groups in the structure. Due to the molecular orbital

theory, the isocyanic group represents a linear structure with double bonds between

C = N and C = O on the same axis with the respective π electrons double bands

situated in different perpendicular planes. The reaction occurs when a nucleophilic


compound containing an active hydrogen atom attacks the carbon atom from NCO,

the hydrogen atom is added to the nitrogen, thus leading the break of the double

bond. The reactivity is based on the high electronegativity of the atoms of nitrogen

and oxygen, which displaces the electron density of the molecule. In the

polymerization process of the polyurethane, isocyanates containing at least two

functional groups in the structure are required.

The hard segment domain of PUs is based on the diisocyanate and the

chain extender applied during the PU synthesis. Diisocyanates can be divided in two

groups: (1) aromatic (2) aliphatic diisocyanates. In general, aromatic isocyanates are

more reactive than aliphatic ones, and this choice will change the final properties of

the PUs (Cherng et al., 2013). For example, if R is an aromatic group, the negative

charge shifts in the R direction, so aromatic isocyanates are more reactive than

aliphatic or cycloaliphatic. In the case of aromatic isocyanates, the nature of the

substituent also determine the reactivity, an electron which attract substituents in the

ortho position increase the reactivity, and electron donors substituents reduce the

reactivity of the isocyanate group.

The reactivity of diisocyanates is still more complex (Figure 3.3).

Symmetrical diisocyanates have the same reactivity (k1 = k2 = k) and assymmetric

diisocyanates exhibit different reactivities (k1 and k2) (Delebecq et al., 2013),

furthermore, it is necessary to consider the steric factors. Aromatic substituents in

para position are more reactive than ortho substituents due to steric effects.

Figure 3.3: Reactivity of the isocyanate group.

3.2. ISOCYANATES

All of the isocyanates commercially available have two or more functional

groups. The use of a diisocyanate is directly proportional to the amine price and

commercial availability. Aliphatic amines do not exhibit good prices and are more

difficult to be commercialized as 1,6-hexamethylene diisocyanate (HDI) and 1,4-

butane diisocyanate (BDI). Aromatic amines are commercially available and at a low-


price, due to these factors, 95% of commercial diisocyanates are based on toluene

diisocyanate (TDI) and diphenylmethane diisocyanate (MDI) and its derivatives, as

shown in Figure 3.4.

Aromatic isocyanates are mainly represented by MDI and TDI, but they

can undergo photodegradation. Aliphatic isocyanates, such as HDI and isophorone

diisocyanate (IPDI) are more resistant to ultraviolet irradiation (UV).

Figure 3.4: Example of diisocyanates currently applied.

Isocyanates are highly reactive compounds, and can react with different

compounds such as alcohols, amines, water, urethane and urea. The main reactions

are shown in the Table 3.1. The relative reaction rates related to the active hydrogen

compound are presented in the Table 3.2.


Table 3.1: Main reactions between diisocyanate groups and different compounds.

Reaction Product

Diisocyanate + Alcohol = Urethane

Diisocyanate + Amine = Urea

Diisocyanate + Water = Carbon dioxide + amine

Diisocyanate + Urethane = Allophanate

Diisocyanate + Urea = Biuret

Table 3.2: Relative reaction rate related to the active hydrogen compound.

Active hydrogen compound Relative reaction rate at 25 oC

Primary aliphatic amine 100,000

Secondary aliphatic amine 20,000-50,000

Primary aromatic amine 200-300

Primary hydroxyl 100

Carboxylic acid 40

Secondary hydroxyl 30

Tertiary hydroxyl 0.5

Urethane 0.3

3.2.1 Isocyanates process description

Isocyanate was first synthesized by Wurtz in 1848 by reacting diethyl

sulfate and potassium cyanide (Reaction 3.1).

R2SO4 + KNCO RNCO

Reaction 3.1: Reaction of Wurtz.

The discovery of polyurethanes in 1937 by Otto Bayer and co-workers

became one of the most produced chemical in the world. Otto Bayer discovered the

polyurethane by the reaction from polyester diol and diisocyanate. From this

discovery, several new routes for the production of isocyanates have emerged.

In 1884, Hentschel mentioned the most important industrial synthesis of

isocyanates by phosgenation of primary amines (Figure 3.2). This reaction leads to


the formation of hydrochloric acid as a by-product, and the electric energy

consumption is high. It is also possible to add alternative compounds as diphosgene

and triphosgene. Phosgene is a highly toxic and flammable gas, which causes

environmental hazards. It is common to add alternative compounds such as

nitrobenzene, which is highly explosive.

R-NH2 + COCl2 = R-NCO + 2 HCl

Reaction 3.2: Phosgenation of amines to produce isocyanates.

The classic process for MDI production is by the nitration of benzene,

obtaining nitrobenzene. This is hydrogenated to form aniline. The next step is the

reaction of aniline and formaldehyde in presence of hydrochloric acid as a catalyst, to

produce a mixture of polyamines. The mixture of polyamines obtained by

condensation of aniline has 4,4 and 2,4-diamino di phenyl methane are treated with

phosgene to form MDI. The process is also complex, nitrobenzene is a highly

explosive compound, and the presence of phosgene also occurs (Reaction 3.3).

Reaction 3.3: Classic process for MDI production (Vilar, 2004).


The classic process for obtaining TDI is divided into following steps. The

first step is the nitration of toluene to obtain 2,4 and 2,6-dinitrotoluene. The isomers

are separated by fractional distillation. The next step is the

reduction of dinitrotoluene to toluene diamine by catalytic hydrogenation. Then, the

toluene diamine isomers are dissolved in chlorobenzene or xylene and added

constinuously with excess phosgene liquid. Residual phosgene and hydrogen

chloride are removed and then concentrated to give pure products. Distillation of

toluene diisocyanate is the last phase as dilute TDI is distilled using evaporator. The

classic process is shown in Reaction 3.4.

Reaction 3.4: Classic process of TDI production (Vilar, 2004).

Another possibility for isocyanate synthesis is starting from carbamates,

which are esters of carbamic acid. Therefore, carbamates are obtained from amines

and alcohols with the addition of phosgene or derivatives (Kreye et al., 2013). Most of

the amines are toxic and their addition should be avoided in the synthesis of

biomaterials.

Another strategy is to obtain isocyanates via the oxidation of isonitriles

(reaction 3.5). Isonitriles are characterized by an atom of Nitrogen and Carbon, linked

by triple bond and connected to a radical. As described by Le & Ganem (2011),

isocyanides was oxidized to isocyanate in the presence of dimethyl sulfoxide and

trifluoroacetic anhydride, as a catalyst. The disadvantage of this method is certainly

the absence of a sustainable route to isonitriles.

Reaction 3.5: Oxidation of isonitriles to isocyanates.


Figure 3.5 shows the chemical structures of phosgene and nitrobenzene

presents in the diisocyanate production.

Figure 3.5: Chemical structure of phosgene and nitrobenzene, respectively.

Alternative routes have emerged in order to replace the isocyanate in the

PU synthesis, to suit environmental and safety standards, and as far as medical

application is concerned an extra care has to be taken. In the PU chemistry, having

the isocyanate as one of the monomers is clearly the versatility of urethane and urea

linkages in addition to the higher reactivity of the isocyanate. Due to the high toxicity

of isocyanates, several studies have emerged in order to replace the isocyanate

monomer in the polyurethane synthesis reaction to suit the environmental and safety

standards.

A recent strategy is the transcarbamylase, aiming the synthesis of

polyurethane by the reaction of the polyol monomer and a urethane diol. The

technology of blocked isocyanates is another strategy that enables the blocking of

the isocyanate function in order to remove them from the formulation and then the

isocyanate function is regenerated by heating to high temperature. This presents

advantages like marked reduction of moisture and water sensitivity but due to this

process, the reactivity of isocyanate groups is reduced. Another strategy is to use the

called polyurethane NIPUs, which produce polyurethane by reaction between cyclic

carbonates, which are non-toxics and biodegradables, and an amine or polyamine. It

is necessary to add a catalyst to enhance the reaction rate and selectivity (Guan et

al., 2011; Delebecq et al., 2013).

Currently, Brazil has only one MDI production plant, with an insufficient

capacity to satisfy the demand for the product. In April 2012 was announced the end

of the only plant for the production of TDI (Industrial Plastics, 2012).


3.2.2 Isocyanates applied In biomedical polyurethanes

In the medical field, PUs based on aromatic isocyanates are considered

less biocompatible than PUs based on aliphatic isocyanates. This happens because

the degradation products of aromatic isocyanates are toxic to the human body, such

as aromatic carcinogenic amines from the rigid segments of polyurethanes. So

aliphatic diisocyanates are replacing the aromatic diisocyanates, promoting suitable

mechanical properties, better oxidative and ultraviolet stabilities (Yildirimer et al.,

2015).

The process of photodegradation can be applied as biomaterials for the

immobilization of cells and controlled release of drugs. UV irradiation can be applied

for the process of situ polymerization, producing materials with desired elasticity and

texture. Furthermore UV radiation is the most effective sterilization method for

biomedical devices, but there are some restrictions, for example some sterilization

techniques can react with functional groups of the polymer. Cherng et al. (2013)

studied PU based on MDI in vivo and his results concluded that the aromatic amine

diphenylmethane dianilines (MDA) was considered toxic due to the degradation of

the hard segments of PU.

The most common aliphatic diisocyanates applied in TE are HDI and

dicyclohexylmethane diisocyanate (HMDI), as they have been reported to degrade

into nontoxic decomposition products (Barrioni et al., 2015), such as nontoxic amines

(Oprea, 2010; Alishiri et al., 2014), and the degradation products can be metabolized

by the Krebs cycle.

Table 3.3 illustrates different types of aromatic and aliphatic diisocyanates

that are currently being applied as monomers to biomedical polyurethanes, and their

authors. As previously mentioned, most applications use aromatic diisocyanates.


Table 3.3: Some isocyanates applied as monomers to biomedical polyurethanes.

Diisocyanate Reference

1,4-Butane diisocyanate (BDI)

4,4-Dibenzyl diisocyanate (DBDI)

de Mulder et al. (2013)

Prisacariu and Scortanu (2010)

1,6-Hexamethylene

diisocyanate (HDI)

4,4-Dicyclohexylmethane

diisocyanate (HMDI)

4,4-Diphenylmethane

diisocyanate (MDI)

Toluene diisocyanate (TDI)

4,4-Methylene dicyclohexyl

diisocyanate (H12MDI)

Asefnejad et al. (2011)

Caracciolo et al. (2013)

Peponi et al. (2013)

Kalajahi et al. (2016)

Jamadi et al. (2016)

Nakhoda & Dahman (2016)

Ganji et al. (2016)

Barrioni et al. (2015)

Dulinska-Molak et al. (2013)

Yildirimer et al. (2015)

Hernándes-Cordova et al. (2016)

González-Paz et al. (2013)

Hu et al. (2012)

Kiran et al. (2012)

Kuranska et al. (2013)

Macocinschi et al. (2013)

Mândru et al. (2013)

Prisacariu and Scortanu (2010)

Das et al. (2013a)

Das et al. (2013b)

Filip et al. (2016)

From Table 3.3 it is possible to create the Figure 3.6, which shows the

isocyanate and the corresponding percentage that it is being employed. The highest

percentage (34.8%) represents the aliphatic diisocyanate HDI, followed by the

aromatic isocyanate MDI (30.4%). HMDI is also representative (13%).


Figure 3.6: Graph created from Table 3.3 showing the percentage that some

isocyanates have been applied in TE.

3.3 POLYOLS

The soft segment domain of PUs is based on the long chain linear diol

applied (polyol or macrodiol) and affected the chain flexibility. They can be classified

in polyether, polyesthers, polycaprolactone (PCL) and polycarbonate. The most

common polyols applied in PU synthesis are hydroxylated polyethers obtained by

anionic polymerization of a propylene oxide or by the block copolymerization of

propylene and ethylene oxides, such as 1,4-butanediol (Choi et al., 2010),

poly(ethylene glycol) (PEG) (Niu et al., 2014), PCL (Baheiraei et al., 2014), poly

(tetramethylene glycol) (De Ilarduya et al., 2010), ethylene glycol (Hevus et al.,

2010). The main polyols applied in medical area is PCL and PEG.

3.4 CONCLUSION

The high toxicity of the diisocyanates is a determinant fator in the PU

synthesis. The difference in the structure, aliphatic or aromatic, can also affect the

final product. The degradation products derived from aromatic diisocyanates can be

toxic to the human body, so this is a determinant factor. Alternative production

methods are required. The existent techniques, as transcarbamylase, blocked

isocyanates, and non-isocyanate polyurethanes are alternative routes, but these

routes are still superficial and may produce several secondary reactions and


unwanted sub-products, and can affect the chemical and mechanical properties of

polyurethanes.

The chemistry of polyurethanes making use of isocyanates is old but it is

the most promising technique and proposes several challenges in the areas of

chemistry and materials science. The strategies mentioned may be alternatives, but

occurs several other secondary reactions and unwanted by-products.

CHAPTER 4 – Antibacterial properties of the polyurethane nanocomposites 45

CHAPTER 4

ANTIBACTERIAL PROPERTIES OF THE POLYURETHANE

NANOCOMPOSITES

Over the several last decades, bioactive materials have attracted special

attention in order to reduce the number of hospital-acquired infections.

Nanomaterials (or materials with at least one dimension less than 1 nm) have

demonstrated significant excitement towards the reduction of bacteria adhesion and

growth without the use of antibiotics due to their ability to easily control surface

energy and manipulate the bacteria membrane to keep bacteria from attaching.

However, there have been few studies creating nanofeatures on currently implanted

biomaterials for such purposes to allow for their quick introduction into the market.

Keywords: Polyurethane; Antibacterial activity; Bacterial growth; Nanoparticles;

Silver; Hydroxyapatite; and Zinc oxide.

4.1 INTRODUCTION

The hospital environment is highly susceptible to different types of

infections due to the presence of pathogens and multi-drug resistant micro-

organisms. The risk of micro-organism presence on hospital surfaces and

implantable medical devices is high and cannot be avoided no matter how much they

are cleaned. The statistics show significant patient morbidity and mortality due to this

unavoidable presence of bacteria in hospitals (Hogan et al., 2015). Specifically, the

most common bacteria found in the hospital environment are Gram-negative

Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli as well as Gram-

positive Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus

(Paul et al., 2013). In order to reduce hospital contamination, environmental cleaning

and decontamination are important measures that need to be taken (Livshiz-Riven et

al., 2015), but it is also necessary to use bioactive materials to reduce microorganism

growth, the transmission of different infections, and destroy pathogens and multi-drug

resistant organisms. One strategy is to modify the surface of polymer films using

nanoparticles that present antibacterial properties, in order to obtain bioactive


nanocomposites. Nanotechnology can be used to inhibit bacteria adhesion and

growth since their surface energies can be easily controlled to repel bacteria or the

adsorption of proteins bacteria adhere to, enhance surface area exposure of

antibacterial chemistries, and if created properly, nanofeatures can minimize bacteria

membrane curvature to keep them from attaching. An approach that should be

definitely avoided is the use of antibiotics for which bacteria easily develop a

resistance towards, rendering such approaches meaningless.

Along these lines, inorganic/organic polymer nanocomposites have gained

attention due to their synergic and hybrid properties (Wang et al., 2011). Moreover,

inorganic metal oxides as zinc oxide (ZnO), magnesium oxide, titanium dioxide, and

silicon dioxide, have attracted great interest because they present strong

antibacterial behavior at low concentrations (Ozkan et al., 2015). In recent years,

ZnO nanoparticles have been widely included in commonly used products, including

cosmetic products, and as sunscreens, food packaging (Lin et al., 2014),

semiconductors (Puay et al., 2015), solar panel devices, paints (Brun et al., 2014),

and even as medical materials. ZnO significantly inhibits the growth of a wide range

of bacteria (Ozkan et al., 2015).

Polymer-silver combinations have also been widely studied for different

anti-bacterial applications. Ag nanoparticles are strong bactericidal agents, and in

this way, many studies have focused on their evaluation for bactericidal properties in

plastics, textiles, the paint industry and medicine (Paul et al., 2013).

Calcium phosphate-based ceramics and their associated composites are

some of the most used chemistries for bioactive material applications, such as

orthopedics due to mechanical strength. Of this family, hydroxyapatite

(Ca10(PO4)6(OH)2) (HA) has been the most applied towards numerous biomaterial

applications due in part since it is the majority mineral phase of bone with

osteocondutivity (Hickey et al., 2015) and osteoinductive properties.

On the organic side, recently, polyurethanes (PUs) have been considered

excellent materials for biomedical applications due to their various controllable

properties (Hood et al., 2010) and high biocompatibility properties due to their

microphase separated structure composed of hard and soft segment domains

(Mishra et al., 2014a). Even though they are biocompatible, a wide range of


nanoparticles have been added to PU to promote select properties (such as

mechanical, etc.).

4.2 OBJECTIVE

Bearing all this in mind the objective of the present in vivo study was to

incorporate nanofeatures onto materials currently used as medical devices and

determine bacteria growth on such materials. In the present research,

nanocomposites of polyurethane fims (PU; an FDA approved biomaterial) doped with

the addition of either zinc oxide (ZnO), silver (Ag) and hydroxyapatite (HA)

nanoparticles in order to impart antibacterial properties without resorting to the use of

antibiotics were developed. The nanoparticles were evaluated by transmission

electron microscopy. Bacteria morphology and the distribution of nanoparticles in the

polymer were characterized by scanning electron microscopy and energy-dispersive

spectroscopy, and antibacterial properties were evaluated using well-established

bacterial assays against the Gram-positive bacterium S. epidermidis and the Gram-

negative bacterium P. aeruginosa, the most common microbial pathogens

encountered in the hospital environment.

4.3 MATERIALS AND METHODS

4.3.1 Materials

All chemicals were of analytical grade and were used without further

purification. PU in pellets form (medical grade SG85A) was kindly provided by

Lubrizol Advanced Materials. Nanoparticles of zinc oxide (#721077), silver dispersion

(#730807) and hydroxyapatite nanopowder (#677418), as well as chloroform,

dicloromethane and dimethylformamide were purchased from Sigma Aldrich.


4.3.2 Methods

4.3.2.1 PU nanocomposite film preparation

It was prepared a PU stock solution in a final concentration of 0,8%

PU/chloroform (weight/volume) during sonication (Sonicator 3000; Misonix,

Farmingdale, NY, USA) for 2 hours. The nanoparticles stock solutions were prepared

to a final concentration of 10% weight/volume (wt/v) of NPs/solvent, during sonication

for 30 minutes. ZnO was dispersed in dichloromethane, Ag and HA stock solutions

were dispersed in dimethylformamide. A range of solutions were mixed to provide the

PU/ZnO, PU/Ag and PU/HA composites at 75:25 weight ratios which were then

sonicated for 2 hours. After that, solutions were then pipetted into petri dish culture

glass (60 x 15 mm, Fisher Scientific, Pittsburgh, PA) and were dried in a vacuum

oven (Vacuum Oven 3500, Shel Lab, Cornelius, OR, USA) for 48 hours at 50 oC in a

vacuum of 10 mmHg to obtain the films. Then, the films were cut into 1 x 1 cm disks.

4.3.2.2 Material characterization

4.3.2.2.1 Nanoparticle characterization

Transmission electron microscopy (TEM) (JEOL JEM-101) was used to

characterize the average size and shape of the ZnO, Ag and HA nanoparticles.

4.3.2.2.2 Nanocomposite film characterization

Scanning electron microscopy (SEM) (Hitachi S-4800; Hitachi Ltd., Tokyo,

Japan) was performed to evaluate the morphology and distribution of nanoparticles

and energy-dispersive spectroscopy (EDS) was applied to indicate the nanoparticle

chemistry. Samples were coated with palladium using a Sputter coater (Cresington

208 HR). The optical density was measured using a SpectraMax M3 microplate

reader (Molecular Devices).


4.3.2.2.3 Cell culture:

Human dermal fibroblast (HDF, Lonza, Basel, Switzerland) were cultured

in Dulbecco's Modified Eagle Medium (DMEM, Sigma Aldrich) supplemented with

10% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin/streptomycin (P/S,

Sigma Aldrich) in a 37°C, humidified, 5% CO2/95% air environment.

4.3.2.2.4 In vivo cell viability assays:

MTS assay (Promega, Fitchburg, WI) was used to determine the cell

activity of the films. Before cell seeding, the scaffolds were sterilized with 70%

ethanol (Sigma-Aldrich) with subsequent exposure to UV light overnight. HDFs were

cultured to ~90% confluence, rinsed with Dulbecco's phosphate-buffered saline

(DPBS, Sigma Aldrich) without calcium chloride and magnesium chloride, and

detached from the tissue culture plate by using 0.25% trypsin-EDTA (Sigma-Aldrich).

Detached cells were then centrifuged at 2000 r.p.m. and resuspended at a density of

50,000 cells/mL before seeding onto the silk scaffolds in a 96 well-plate at 100 μl in

each well (5000 cells/well). The HDFs incubated for 1, 3, and 5 days. Afterwards, the

medium was removed from the sample and 100 μl solution of 1:5 MTS dye with

DMEM medium (v/v) were added to each well. Samples were placed back into the

incubator for 2.5 h. to allow the MTS to react with the metabolic products of the

adherent cells before reading in a SpectraMax M3 microplate reader (Molecular

Devices, Sunnyvale, CA) at an absorbance wavelength of 490 nm.

The data are presented as mean ± S.E.M. Statistical analysis of the data

was carried out using ANOVA, followed by Tukey's HSD post hoc test (equal

variances). Differences were considered statistically significant when the P-value was

less than 0.05.

4.3.2.2.5 Antibacterial activity of PU nanocomposites films

Samples were cut into disks, washed in ethanol for 5 minutes and then

sterilized under UV light overnight. S. epidermidis was used as a Gram positive

bacteria and P. aeruginosa was used as a Gram negative bacteria. Both were


hydrated and streaked for isolation on a trypic soy agar plate. Following bacteria

growth, a single isolated colony was selected and added to inoculate 5 mL of tryptic

soy broth (TSB) (Fluka Analytical #22092) and it was incubated for growth on a

shaking incubator for 16 hours at 37 oC. After that, the bacterial solution was diluted

to a concentration of 107 bacteria/mL, which was confirmed by measuring the optical

density of the bacterial solution using an absorbance of the bacterial solution in each

well at a wavelength of 600 nm. After that, 500 µL of Dulbecco’s phosphate buffered

saline (DPBS) (Sigma Aldrich #D8537) were immediately pipetted into each well, with

complete submersion of the samples for 5 minutes. Then, all the PBS (phosphate-

buffered saline) was completely removed. This procedure was repeated three times.

The bacterial solution was then added to the samples. Immediately after this

procedure, the bacterial solution was added to the 48 well plates with the samples

and the plate was incubated for 16 hours at 37 oC. After that time period, the

bacterial solution was removed and samples were diluted with 1 mL of PBS and

vortexed for 5 minutes. This procedure was repeated two times. The samples were

spread on agar plates, and the bacteria colonies were counted after 16 hours of

incubation.

4.3.2.2.6 Statistical analysis

Bacterial tests were conducted in triplicate. The data were analyzed using

Student’s t-tests between populations of interest, with p<0.05 considered statistically

significant.


4.4 RESULTS AND DISCUSSION

4.4.1 Nanoparticles characterization

ZnO (Fig. 4.1a), Ag (Fig. 4.1b) and HA (Fig. 4.1c) nanoparticles were

characterized using TEM and the images are shown in the Figure 1. As expected, all

particles possessed nanoscale sizes. The TEM images revealed that the ZnO

nanoparticles were dispersed in as a rod-shaped particle with an average diameter of

~33.4 ± 4.6 nm. The Ag nanoparticles were circular with an average diameter of

~43.1 ± 3.2 nm. The TEM of HA nanoparticles showed a circular shape with an

average particle diameter of ~65.9 ± 8.4 nm.

Figure 4.1: TEM images of the nanoparticles: a) ZnO, b) Ag, and c) HA. Scale bars

represent 100 nm.

4.4.2 PUs nanocomposite characterization

Surface morphology of the nanocomposites was evaluated using SEM.

Figure 4.2 shows the micrograph of plain polyurethane and Figures 4.3, 4.4 and 4.5

show EDS measurements and micrographs of the PU/ZnO, PU/Ag and PU/HA

nanocomposites, respectively.


Figure 4.2: SEM of Plain PU. Scale bar represents 500 nm.

Figure 4.3: EDS and SEM of PU/ZnO nanocomposite. Scale bar represents 500 nm.

Figure 4.4: EDS and SEM of PU/Ag nanocomposite. Scale bar represents 50 µm.

Figure 4.5: EDS and SEM of PU/HA nanocomposite. Scale bar represents 5 µm.


For the plain PU, there were no nanoparticles observed, showing that the

polyurethane was successfully dispersed in the solvent.

Figure 4.3 shows the EDS measurements in which was verified the

presence of ZnO in the nanocomposite and SEM showed that the ZnO nanoparticles

were well distributed in the PU matrix completely covering the surface. EDS

measurements of the PU/Ag nanocomposites revealed the presence of Ag in the

nanocomposite and SEM showed smaller as well as well distributed nanoparticles in

the nanocomposite (Figure 4.4). As can be seen from Figure 4.5, EDS

measurements conducted in the PU/HA nanocomposite verified the presence of

phosphorus and calcium, indicating the presence of HA nanoparticles and SEM

showed the nanoparticles were well dispersed in the PU matrix, showing a circular

shape.

4.4.3 In vivo cell viability of nanocomposites

Citotoxicity test was performed to study the cytotoxicity of the

nanocomposites. Human dermal fibroblast (HDF) cells were seeded directly onto PU

nanocomposites during 1, 3 and 5 days and the colorimetric results are depicted in

Figure 4.6.

Figure 4.6: In vivo cell viability assay after 1, 3 and 5 days of incubation with control,

PU/ZnO, PU/Ag and PU/HA.

According to the two-way ANOVA, a statistically significant (p<0.05)

difference was found between the nanocomposites and the control when cell viability

was considered.


It was verified that the control showed high levels of optical density.

Unfortunatelly results obtained after 1, 3 and 5 days showed that a statistical

differences were observed in the nanocomposites with respect to the control, the

nanocomposites induced toxicity to the HDF cell line for the nanocomposites,

showing cell death.

4.4.4 Antibacterial activity of PU nanocomposites

As mentioned, bacteria growth on the nanocomposites was evaluated by

counting the number of viable cells (i.e., colony forming units) after 16 h of

incubation. The antibacterial activity of the following nanocomposites were assessed

against S. epidermidis and P. aeruginosa: PU/ZnO, PU/Ag and PU/HA. Figure 4.7

shows the CFU ratio for nanocomposites against S. epidermidis (Fig. 4.7a) and P.

aeruginosa (Fig. 4.7b).

Figure 4.7: (a) S. epidermidis and (b) P. aeruginosa bacteria growth after 16 hours

on the nanocomposites of interest to the present study. Asterisks (*) indicate

significant differences at p < 0.05 between testing conditions. Data = mean +/- SEM;

n = 3.

According to the obtained results, the PU/ZnO nanocomposites completely

inhibit growth of S. epidermidis and P. aeruginosa. The PU/Ag nanocomposites

showed better inhibition of S. epidermidis, compared to the PU/HA nanocomposite.

No significant difference in P. aeruginosa was observed between PU/Ag and PU/HÁ

nanocomposites.


4.5 DISCUSSION

Impressively, the present research provides the first data that PU/ZnO

nanocomposites can inhibit bacteria growth of both bacteria types under the

conditions used without the use of antibiotics. Figure 4.7a demonstrates the greatest

inhibition of PU/Ag toward Gram-positive bacteria, compared to the PU/HA

nanocomposite. There was no significant difference in bacteria between PU/Ag and

PU/HA.

The exact mechanism of the antibacterial action of PU/ZnO

nanocomposite still needs to be determined, but certainly can be attributed to the

bactericidal phenomenon of zinc toxicity toward the bacteria whereas nano ZnO

particles with increased surface area possess greater antibacterial properties

(Raghupathi et al., 2011). One possible mechanism of the antibacterial action of ZnO

nanoparticles is the increased levels of reactive oxygen species (ROS) and

accumulation of nanoparticles either in the bacteria cytoplasm or in the periplasmic

region causing disruption of bacteria function and/or disruption and disorganization of

their membranes (Brayner at al., 2006; Zhang et al., 2007). However, it is puzzling

why complete inhibition of bacteria was not observed on PU/Ag nanocomposites due

to the documented increased bacteria inhibition on ZnO compared to Ag, especially

when ZnO and Ag were added at the same weight ratio. Certainly, different amounts

of ZnO and Ag present on the PU surface may be the answer (as supported by AFM

images) but requires more investigation.

An additional possible reason for this difference in bacteria behavior

between PU/ZnO and PU/Ag may be related to changes in polymer properties when

Ag is added. Bacterial adhesion depends on the polymer hydrophobicity, the

presence of functional groups on the polymer, surface roughness, charge, and

flexibility. It is known that Ag nanoparticles influence the distribution of hard and soft

segments domains in the PU matrix (Hsu et al., 2010; Rai et al., 2012). The

difference in bacteria wall structure between the S. epidermidis and P. aeruginosa

can help to further explain these results as each bacteria presumably depends on

polymer composite properties differently. More specifically, one of the hypotheses for

the antibacterial properties of Ag is that Ag nanoparticles attack Gram-negative

bacteria by penetrating the cell wall, causing uncontrolled transport across the


cytoplasmic membrane (Morones et al., 2005; Sondi & Salopek-Sondi, 2004). As

expected, the smaller Ag nanoparticle sizes found in the TEM images were silver

nanoparticles and provided better antibacterial activity compared with HA

nanoparticles. The ZnO nanoparticles presented an even a smaller nanoparticle size

to potentially penetrate bacteria cell walls in greater numbers.

Lastly, it is important to comment on the antibacterial properties of the

PU/HA nanocomposites as there are few literature reports on the role HA

nanoparticles may play to reduce bacteria growth. This study provided evidence of

decreased P. aeruginosa growth on PU/HA compared to PU/Ag nanocomposites.

Since Ag is already a well establish antibacterial chemistry, these results may

indicate a promising future for HA nanoparticles to reduce bacteria growth for

numerous medical applications (including orthopedic) without using antibiotics

previously undiscovered.

4.6 CONCLUSION

This work successfully prepared nanocomposites of zinc oxide, silver and

hydroxyapatite in polyurethane by a simple, quick, and efficient method. Results

showed for the first time in the literature complete inhibition of the growth of both

bacteria after 16 hours of culture on the PU nanocomposites with ZnO nanoparticle

this effect will need to be elucidated in future work to exploit this surprising finding.

Moreover, the presence of silver nanoparticles in the nanocomposites

showed better antibacterial activity against S. epidermidis compared to the

hydroxyapatite nanocomposite whereas the hydroxyapatite nanocomposites showed

better antibacterial activity against P. aeruginosa than silver nanocomposites. In

summary, this research provides the first evidence of complete inhibition of bacteria

growth when using nanoparticles of ZnO embedded in PU without resorting to the

use of antibiotics, for which bacteria are developing a resistance towards anyway

(and, thus, should be avoided).

CHAPTER 5 – The influence of Hydroxyapatite Nanoparticles on the structure, termal and biological behavior of bio-based polyurethane

composites 57

CHAPTER 5

THE INFLUENCE OF HYDROXYAPATITE NANOPARTICLES ON THE

STRUCTURE, THERMAL AND BIOLOGICAL BEHAVIOR OF BIO-

BASED POLYURETHANE COMPOSITES

In this work, thermoset polyurethane composites were prepared by the

addition of hydroxyapatite nanoparticles using the reactants polyol polyether and an

aliphatic diisocyanate. The polyol employed in this study was extracted from the

Euterpe oleracea Mart. seeds (açaí berry) from the Amazon Region of Brazil. The

influence of hydroxyapatite nanoparticles on the structure and morphology of the

composites was studied using scanning electron microscopy (SEM) and energy

dispersive spectroscopy (EDS), the structure was evaluated by Fourier transform

infrared spectroscopy (FTIR), thermal properties were analyzed by thermogravimetry

analysis (TGA), and biological properties were studied by in vivo and in vivo studies.

It was found that the addition of HA nanoparticles promoted fibroblast adhesion, and

may further the biodegradation of the composite. In addition, in vivo investigations

with histological studies confirmed that the composites promoted connective tissue

adherence and did not induce inflammation. Hence, this study supports the further

investigation of bio-based, polyurethane/hydroxyapatite composites as biocompatible

scaffolds for numerous orthopedic applications.

Keywords: Polyurethane, Hydroxyapatite, Tissue Engineering, Biodegradation, and

Nanotechnology.

5.1 INTRODUCTION

Hydroxyapatite (HA) has attracted a great deal of attention since it is the

major component of the mineral phase of the bone and has been consequently

widely applied to numerous tissue engineering (TE) applications (Ramier et al., 2014;

Sun et al., 2014; Rajzer et al., 2014). These nanoparticles can be obtained by

different techniques, such as sol-gel (Tredwin et al., 2014; Jia et al., 2014),

precipitation (Hao et al., 2014; Cox et al. 2014), and microwave processes (Zhao et


composites 58

al., 2014; Mishra et al., 2014a). It has already been confirmed that HA is conductive

and osteoinductive, showing that it promotes fibroblastic cell (a model cell line) and

osteoblastic cell (bone forming cell) adhesion, growth, differentiation and bone repair

(Kim et al., 2015). In order to improve its osteocondutivity properties, HA

nanoparticles have been used to produce different polymer composites (Mi et al.,

2014a; Pan et al., 2014) with polyurethanes (Mi et al., 2014b; Cetina-Diaz et al.,

2014) for various TE applications. Dulinska-Molak et al. (2013) produced the

composite PU-calcium carbonate and showed that the nanoparticles improved the

biocompatibility of the composite and also improved the osteoinduction process.

Polyurethanes (PUs) based on polyols from renewable resources have

been extensively studied (Dubé & Salehpour, 2014; Septevani et al., 2015;

Zieleniewska et al., 2014; Zhang et al., 2014) and are promising for TE applications

due to the possibility to present some desirable properties for biomaterials, such as

anti-inflammatory and anti-oxidant activities.

However, the major disadvantage in the synthesis of PU nowadays is the

dependence on petroleum-based products; therefore, in order to contribute to global

sustainability, the use of renewable biomass as raw materials was recognized as

viable (Dubé and Salehpour, 2014; Miao et al., 2014). Besides it is worthwhile to

evaluate the consequences of oil-based products on the human body. In this way,

different types of vegetable oils have been applied, such as sunflower oil, soybean oil

(Heinen et al., 2014), peanut and linseed oil (Garrison et al., 2014).

Euterpe oleracea Mart., popularly known as açaí, is a palm tree widely

distributed in the northern part of South America, especially in the Brazil Amazon

Region. The açaí fruits are spherical dark purple berries with a high content of

flavonoids. Each berry is covered by a thin pulp layer, covered by a shell and also

has one large seed covered with a layer of fibers. Biochemical studies have shown

that the açaí seeds contain cellulose and hemicellulose (Wycoff et al., 2015), present

antioxidant and anti-inflammatory characteristics due to compounds such as

flavonoids and polyphenolic molecules, such as catechin and epicatechin (Bonomo

et al., 2014; Schauss et al., 2006., Kang et al., 2010) (Figure 5.1).


composites 59

Figure 5.1: Polyphenolic compounds found in açaí seed.

Moreover, the synthesis of PU-HA nanocomposites have been widely

investigated in the TE field (Schwinté et al., 2015). It was already confirmed (Du et

al., 2014) that composites synthesized from renewable polyol, showed high affinity to

bone forming cells, and had good prospects for bone repair. It was also

demonstrated (Mi et al., 2014b) that PU-HA nanocomposites induced in vivo

proliferation of human mesenchymal stem cells in comparison to scaffolds without

HA.

5.2 OBJECTIVE

In all of the aforementioned PU materials, renewable, or environmentally

friendly sources of PU, were not used. The purpose of this study was to obtain a new

polyurethane based on a renewable polyol and an aliphatic isocyanate (TolonateTM

HDB 75 BX) via prepolymer method. The produced materials were studied through

chemical and characterizations in order to evaluate the structure, morphology, and

thermal properties. It was also proposed to study the addition of HA nanoparticles on

the structure and in the biological response of the composite. Figure 5.2 depicts the

research proposed in this chapter.


composites 60

Figure 5.2: Abstract graph of the proposed research.

Importantly, the polyol employed in this study was extracted from the

Euterpe oleracea Mart. seeds (acai berry) from the Amazon Region of Brazil. It is a

polyether polyol containing a nanofiller cellulose, a natural catalyst, which possesses

anti-oxidant and anti-inflammatory properties. Thus, this unique study emphasizes a

bio-based, green chemistry polyurethane for TE applications.


5.3.1 Materials

Tolonate® HDB 75 BX, derived from hexamethylene diisocyanate was

purchased by Perstorp. Acaí berry polyol powder was kindly provided by the

Laboratory of Eco-composites from the Federal University of Pará, Brazil. The

extraction method was previously described (Barreira, 2009).


composites 61

5.3.2 Methods

5.3.2.1 Polyurethane preparation

PU was prepared by a two-step procedure in a nitrogen atmosphere. In

the first step, the synthesis was carried out in a batch reactor by a mixture of polyol

and HDB at a heating rate of 10 oC/min to 75 oC, by stirring at 100 rpm and 4 kgf/cm2

of pressure in order to form an NCO-terminated prepolymer. At 75 oC, a sample of

prepolymer was taken out of the reactor and placed in a high density polyethylene

bottle for cooling. In the second step, the resulting mixture in the reactor was heated

from 75oC to 120 oC, in the same conditions, to obtain complete polymerization of

PU.

5.3.2.2 Hydroxyapatite nanoparticle preparation

HA was prepared as previously described using a water-based sol-gel

method (Rodrigues et al., 2012). The reagents used were phosphoric acid in a 85%

aqueous solution (LAFAN), calcium nitrate tetrahydrate (99% purity, Synth) and

water (purified by reverse osmosis). The molar ratio of Ca/P was 1.67.

5.3.2.3 PU-HA composite preparation

The prepared HA nanoparticles were mixed at a 20 wt% in a soluble PU

solution using ultrasound for 30 minutes. The resulting mixture was polymerized in a

batch reactor at 120 oC for 30 minutes.

5.3.3 Material Characterization

5.3.3.1 Morphological and structural characterization

Morphology analysis was performed using a scanning electron microscope

(SEM, model 430i Leo, Zeiss). The voltage used was 10 kV and the current was 100

pA. To analyze the distribution of the HA through the composites, EDS (model 6070,


composites 62

Leo) was applied. A metallic SC7620 Sputter Coater was used for coating the sample

with gold.

5.3.3.2 Structural characterization

FTIR studies were conducted at room temperature using a thermo

scientific Nicolet IR100 FTIR spectrophotometer (Nicolet Instrument Inc.,Madison,

WI, USA). FTIR spectra were collected after casting a film on KBr disks, from 128

scans, within a range of 675–4000 cm-1 using a resolution of 4 cm-1.

5.3.3.3 Thermal characterization

The investigation of the thermal composition of the PU was carried out

using TGA, model TGA-50 (Shimadzu Instruments Kyoto, Japan). A sample mass of

about 8 mg was heated under a flowing nitrogen atmosphere (50 mL/min) from 30 to

600 oC, at a heating rate of 10 oC/min.

5.3.3.4 Cell culture

Human normal lung fibroblasts (a model cell line, MRC-5) were obtained

from ATCC (Manassas, VA, USA). Cells were cultured in Dulbecco’s Modified Eagle

Medium (DMEM) with 10% fetal bovine serum (FBS, Gibco). Growth media contained

100 units/mL penicillin and 50 μg/mL streptomycin and cells were cultured in a 37 oC,

humidified, 5% CO2/95% air environment. Cells were used at population numbers

below 5.

5.3.3.5 In vivo cell viability assays

To determine the toxicity of the polyurethane and the nanocomposite, cells

were seeded onto 96-well culture plates at 0.5×105 cells/mL and were incubated for

24 hours. After that, cells were incubated with 100 μL of the aforementioned cell

culture medium for 24 hours. The amount of formazan crystals formed was measured

after 2 hours of exposure to the (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-


composites 63

tetrazolium bromide) (MTT) solution in dimethyl sulfoxide (DMSO) and absorbance

values were measured at 570 nm by a scanning multi well spectrophotometer plate

reader (ELISA reader). Cytotoxicity experiments were performed in triplicate and

results were presented as the mean ± standard deviation. The data are presented as

mean ± S.E.M. Statistical analysis of the data was carried out using ANOVA,

followed by Tukey’s HSD post hoc test (equal variances). Differences were

considered statistically significant when the P-value was less than 0.05.

5.3.3.6 In vivo study

For our experiments, we used 6 to 8-week-old male Swiss mice with a

body weight of 15–20 g. The mice were housed individually and provided with chow

pellets and water ad libitum. The present study was approved by the Ethics

Committee in Animal Experimentation of Federal University of Pará, Brazil. The

Ethics Committee in Animal Experimentation obtained (CEPAE 86-2015) is

presented in Appendix 1.

The biocompatibility properties of the presently fabricated substrates were

evaluated for an inflammatory host tissue response through in vivo tests. For this,

mice were anesthetized by intraperitoneal injection of a mixture of ketamine (50

mg/kg) and xylazine (5 mg/kg) and the present samples were implanted into the

subcutaneous dorsal area of individual Swiss mice and controls were used (without

material) (n=4) for 7 days. At the end of the in vivo experiments, the animals were

killed with an overdose of the anaesthetic and the speciments were excised for

histological examinations. For light microscopy, specimens were embedded in

paraffin at day 14 after scaffold implantation. Sections of 5 µm thick were cut and

stained using the classical haematoxylin-eosin standard procedures. Tissues

sections were observed in a light microscope (Carl Zeiss, Oberkachen, Germany).


composites 64


5.4.1 Structure and morphology

Rigid, green chemistry polyurethane foams and composites were obtained

following the methods described above. The composites were low density and of

high mechanical resistance due to the hard PU segments and physical crosslinks.

The chemical reaction for the formation of PU is presented in Figure 5.3.

Figure 5.3: Reaction formation of the natural, green chemistry, polyurethane.

At the end of the process, thermoset PU and PU-HA foams were obtained.

In order to investigate the structure and the morphological properties of the the

composite, EDS and SEM were used. The results of the composite characterization

are shown in Figures 5.4 and 5.5.

Figure 5.4: EDS of the composite. Figure 5.5: SEM image of the composite. Scale bar represents 500 µm.


composites 65

The Figures (5.4-5.5) depicted that the materials contained high porosity,

interconnected and well-distributed pores throughout the scaffold. It is possible to

observe in Figure 5 that the nanoparticles of HA were dispersed homogeneously in

PU. EDS results indicated HA formation and incorporation. These results confirmed

that the HA nanoparticles deposited on the porous polyurethane composite,

confirming that the composites contained HA in the form of calcium and phosphorous

atoms.

5.4.2 Microphase-separated structure

The influence of HA and the polymerization reaction were investigated by

infrared spectroscopy measurements on the polyurethane and composite after 7

days of implantation in vivo. The main functional groups that confirm the formation of

polyurethane are shown in Table 5.1.

Table 5.1: Functional groups and its absorption peaks (cm-1) that confirm the

formation of urethane.

Functional group Absorption peak (cm-1)

-NH 3280

-C=O 1740

C-O-C 1160

The microphase-separated structure is generated by the rigid hard

segments of the urethane group which originated from diisocyanate, and by the

flexible soft segments of the hydroxyl groups which originated from the polyol (Fig.

5.2). According to the reaction between the polyurethane reactants, when an active

hydrogen originated from polyol attacks the nitrogen atom of isocyanate, the –NH

group is a proton donor, so hydrogen bonding is formed. The oxygen of carbonyl is a

proton acceptor, so it is possible to study this formation through these bands in the

FTIR spectra. The FTIR spectra of polyurethane and the composites are shown in

Figure 5.6.


composites 66

Figure 5.6: FTIR spectra of polyurethane and composites.

The band present at 3280 cm-1 can be assigned to the –N–H stretching

vibration of urethane groups, this new band confirms the presence of urethane

groups. The peaks at 2920 and 2850 cm-1 are the asymmetric and symmetric

vibrations of the -CH2 groups and mostly represent the hard and soft segments,

respectively. The band at 2350 cm-1 corresponds to the stretching vibration of the

isocyanate group, –N=C=O. Carbonyl stretching bands (-C=O) of urethane bonds

appear at 1740 cm-1. The absorption peaks at 1540 cm-1 corresponds to the

deformation vibration of (-NH-) bonds in the urethane (-NHCOO-) groups from amide

II. The stretching of the ether group C-O-C is associated with the absorption at 1160

cm-1, which indicates the soft segments. In addition, the presence of HA in the

composite was verified by peaks at 1070 cm-1 (PO43-) and at 1420 cm-1 (CO3

2-).


composites 67

5.4.3 The thermal degradation of polyurethane

The thermal degradation of PU and composite are shown in Figure 5.7.

Figure 5.7: Thermal degradation of PU and PU-HA by TGA/DTG.

The TGA curves allow to verify three steps of mass loss. The first step

was attributed to the decomposition of the oligo-polyssacarides present in polyol. The

second step is attributed to the decomposition of the rigid segments of PU, and the

third step is attributed to the decomposition of flexible segments of PU. It is possible

to note that the addition of HA improves the thermal stability of PU. This behavior can

be attributed to the formation of hydrogen bonding interactions between hydroxyl

groups of HA and the amine groups of PU. The PU developed in this work is a

thermoset polymer and the covalent bonds are responsible for crosslinking between

chains formed in the healing process. This polymer can be sterilized by using

different process, including autoclave. These connections are only broken by the

introduction of high amounts of energy and cause the degradation of the polymer.

Thermoset polymers are not easily reprocessed through pressure and temperature

due to the type of connection between the polymer chains.


composites 68

5.4.4 In vivo analysis

In vivo studies are necessary to evaluate the cytotoxicity of the samples.

For this purpose, a control, the polyurethane and the composites were tested for 24

hours in the presence of a model cell line, fibroblasts (Fig. 5.8).

Figure 5.8: MRC-5 viability of the PU and composite after 24 hours. Data = mean +/-

SEM. No statistical difference was observed with respect to the controls.

Colorimetric assays for testing the biocompatibility were used in this study.

After 24 hours, PU had 98% ± 12 MRC-5 viability and the composite had 97% ± 13

MRC-5 viability. According to the two-way ANOVA, no statistically significant

difference (p<0.05) was found between the samples and the control. In summary, the

results obtained in this study indicated that the PU and the composite did not induce

any toxicity to the MRC-5 cell line for the polyurethane and the composites, and

showed potential to be applied in TE.

5.4.5 In vivo analysis

The In vivo tests further showed positive interaction between the material

and the animal’s body without a large scale inflammatory response for the control,

polyurethane and composites after 7 days (Figures 5.9, 5.10 and 5.11, respectively).


composites 69

Figure 5.9: Negative control. Figure 5.10: PU after 7 days. Figure 5.11: Composite after 7 days.

It is already well-known that the initiation of foreign body responses

involves recruitment of macrophages, lymphocytes, and neutrophils to the site of

implantation. None of the samples showed a significant inflammatory response.

Figure 5.12 shows the hematoxylin-eosin histological images of the

subcutaneous dorsal area of Swiss mice after 7 days after implantation.


composites 70

a) a)

b)

c)

d)

e)

f)

g)

h)

i)

Figure 5.12: Representative histological sections of tissue after 7 days of

implantation for control (a-c), for PU (d-f) and for the composites (g-i). Scale bars in

a, d and g represent 100 µm, scale bars in b, e and h represent 50 µm, and scale

bars in c, f and i represent 25 µm.

In the control group (Figures 5.12 a-c), intact tissue was observed. Figures

5.12 (d-f) present the histological results for the PU. The histological images of the

composite are presented in Figures 5.12 (g-i). No adverse inflammatory responses

can be noticed after 7 days. The yellow square in the Figure 5.12 (e-f) show the

migration of cells to the site of PU implantation.


composites 71

The black indication in the Figure 5.12 (g-h) showed the composite, the

white indication in the Figure 5.12 (h) presented the subcutaneous tissue, and the

red square in the Figure 5.12 (i) indicated the material-tissue interaction. It was

noticed that HA nanoparticles may provide cellular binding sites and the scaffold can

provide a suitable structure for cell ingrowth, demonstrating that these composites

should be further studied for orthopedic applications since they show no cytotoxicity

or biocompatibility problems.

5.5 DISCUSSION

This study highlights a critical need in the next generation of nanomedicine

research: green nanomedicine. While other science and engineering fields

continuously strive to find environmentally-friendly, green chemistry, approaches to

new material design, the field of biomaterials has not (to a large extent). Synthetic

materials, such as Ti, Co, Cr, Mo, and other metals are commonly used across all

paths of biomaterials. Since the polyurethane (PU) used in this study was obtained

through a natural process, it demonstrates a new green chemistry approach to

biomaterials and nanomedicine.

With the addition of hydroxyapatite (HA) nanocrystals to the polyurethane,

this study was able to maintain functions of fibroblasts (shown in vivo) and show

promise for a non-inflammatory response for connective tissue growth in vivo. It has

been well demonstrated to date that nanomaterials can alter the surface energy of

materials to control initial protein adsorption and confirmation to inhibit inflammatory

cell functions and promote tissue forming cell functions. To the best of our

knowledge, this is the first time this was accomplished for the naturally-derived PU as

described in this study. Although certainly more studies are required (such as

optimizing HA nanocrystal distribution and weight percentage in PU composites,

targeted in vivo studies aimed as assessing connective tissue formation, and

determining a mechanism of action), the present first-time results are encouraging for

the further emphasis of green nanomedicine across all of biomaterial synthesis.


composites 72

5.6 CONCLUSION

A natural, green chemistry, composite was successfully obtained in this

work by the addition of hydroxyapatite HA nanoparticles in polyurethane. The

structure and morphological studies showed a high porosity of the present materials

and the nanoparticles of HA were dispersed homogeneously in PU. FTIR showed a

microphase-separated structure of PU, which can be modified to improve blood

compatibility. The TGA results showed a high temperature of degradation, ideal for

processes such as sterilization, and its application as a biomaterial. The biological

results demonstrated that the natural, green chemistry, composite formulated

possessed safe biocompatibility in vivo and in vivo characteristics which could be

promising for numerous tissue engineering applications.

CHAPTER 6 – Synthesis and characterization of electrospun polyurethane membranes for Tissue Engineering applications 73

CHAPTER 6

SYNTHESIS AND CHARACTERIZATION OF ELECTROSPUN

POLYURETHANE MEMBRANES FOR TISSUE ENGINEERING

APPLICATIONS

In this Chapter, polyurethane was prepared using an electrospinning

apparatus in order to obtain membranes for different applications in Tissue

Engineering (TE), such as ephitelial, drug delivery or cardiac applications.

The influence of fibers on the structure and morphology of the scaffold

were studied using scanning electron microscopy (SEM), the structure was evaluated

by Fourier transform infrared spectroscopy (FTIR), and thermal properties were

analyzed by thermogravimetry analysis (TGA). In vivo cells attachment and

proliferation was studied by SEM, MTT and Live/Dead® assays. The degradation

behavior of the membranes was investigated by in vivo degradation studies.

In summary, the results showed that the membrane present an

homogeneous morphology, the first step of mass loss started at 254 oC, SEM results

showed that the membrane presents high porosity, high surface area:volume ratio, it

was observed a random fiber network. In vivo evaluation of fibroblasts cells showed

that fibroblasts spread over the membrane surface after 24, 48 and 72 hours of

culture, and in vivo degradation study showed that the developed membrane can be

considered for long-term applications. This study supports the investigation of

electrospun polyurethane membranes as biocompatible wound healing as long-term

devices for tissue engineering applications.

Keywords: Electrospinning, fibroblast proliferation, implants, in vivo degradation and

polyurethane.

6.1 INTRODUCTION

The skin wounds market has been considered the most successful market

in the TE area (Teoh et al., 2015). This is a large market due to offer products to heal

damage skin, severe burns and diabetic and pressure ulcers. Skin wounds may

appear as a result of accidents, but also as a result of surgical incisions. Pressure


ulcers may be caused by immobility, and cause infections or more severe

complications. Patients with chronic diseases such as diabetes and obesity are also

subject to more difficult wound healing. In the United States, it is estimated that,

about 6.5 million cases per year of skin ulcer (Sen et al., 2009), about 285 million

people had diabetes mellitus in 2010 and it is estimated more than 360 million people

by 2030 will have diabetes mellitus (Shaw et al., 2010; Whiting et al., 2011).

The use of polymeric membranes in the medical area has grown

considerably. From the membranes, it is possible to obtain artificial skin, bandages,

angioplasty balloons, intra-gastric balloons, scaffolds and neural connections.

Several polymers have been used for these applications.

The implantation of synthetic polymers in the body and their duration as

medical devices can be divided in two groups: (1) biodegradable devices (2)

biostable devices. Biodegradable devices should provide an initial substrate to cell

adhesion, proliferation and differentiation and maintain the mechanical properties

while it degrades until the newly tissue should be regenerating, releasing non-toxic

products to the human body (Bártolo et al., 2011). Biostable devices should maintain

the architecture and mechanical properties over time in vivo. In addition, they will not

release degradation products in the human body (Major et al., 2016).

Recently, different synthetic polymers have been applied as implants in

TE, including polymers derived from poly(α-hydroxyacids) such as, poly(lactic acid)

(PLA), poly(glycolic acid) (PGA), and their copolymer poly(lactic-co glycolic acid)

(PLGA), as well as polymers derived from polyesters class, such as

polyhydroxybutyrate (PHB) and polyhydroxyalkanoate (PHA).

Scaffolds derived from poly(α-hydroxyacids) may undergo mechanical

deformation, which can promote a significant size reduction of the scaffold pores

(Laschke et al., 2009).

Among the synthetic polymers, polyurethanes (PUs) are versatile

polymers due to their segmented structure, composed of two thermodynamic

incompatible phases (Yilgör et al., 2015; Hiob et al., 2016), then by modification of

the structure, PUs can be biostable or biodegradable as well as rigid or flexible.

The degradation rate of PUs can be easier achieved by introducing

hydrolysable chain extenders in the hard segment, such as butanediol (BDO), 1,2-

ethanediol and 1,2-ethanediamine and glycerol (Barrioni et al., 2015). Diamines are

https://en.wikipedia.org/wiki/Polyester

https://en.wikipedia.org/wiki/Polyhydroxyalkanoates


more reactive than diols or triols. The hydrophilic character of the PU can be easily

controlled in this way.

The soft segment has a significant influence on the degradation rate of

PUs. In general, the use of polyols with high functionality produces a crosslinked

structure and reduces the hydrolytic degradation capacity due to the difficult to the

water reach the hydrolytic segments (ester and ether groups) of the PUs (Gamerith

et al., 2016).

Polyether PUs are recognized as hydrolytically stable at neutral and basic

pH and have been applied for long-term applications. However, polyether PU in a

combination with metal parts, have been subjected to metal ion oxidation (Cooper et

al., 2016). Polyester PUs can suffer hydrolytic degradation and are no longer used in

devices designed for long-term implantation.

PUs based on polycaprolactone (PCL) are used as long-term implantation

and can be hydrolyzed and presents non-toxic degradation products and have also

been applied for long-term implants (2-4 years) due to degradation rate slower than

PLA, PGA and PLGA. Polycarbonate PUs are used in long term implantation and not

undergo hydrolytic degradation.

The soft segment of PUs also affected the flexibility of the chain polymer.

Flexible PUs have the advantage that they present easy and almost frictionless

integration into the host tissue. Table 6.1 illustrates different types of polyols,

diisocyanates, chain extenders, the PU application and the degradation character

that are currently being applied in TE.


Table 6.1: Currently monomers and their applications in TE.

Polyol Diisocyanate Chain extender Application Degradation

character

Author

PCL Aliphatic Putrescine

(diamine)

Cardiac TE

Biodegradable

Hernándes-

Cordova et al.

(2016)

PCL Apliphatic Putrescine

(diamine)

Shape

memory

polymers

Biodegradable

Kalajahi et al.

(2016)

PCL Aliphatic Butanediamine Cardiac TE Biodegradable

Jamadi et al.

(2016)

PEG Aliphatic PPG TE

applications

Biodegradable

Filip et al. (2016)

PCL Aliphatic BDO TE

applications

Biodegradable

Nakhoda &

Dahman (2016)

BDO Aliphatic Ethylenediamine

/diethylamine

TE

applications

Biodegradable

Yildirimer et al.

(2015)

Castor oil Aliphatic PEG Cardiac TE Biodegradable

Ganji et al.

(2016)

PCL Aliphatic POSS diol TE

applications

Biodegradable

McMullin et al.

(2016)

PCL Aromatic BDO Drug delivery Long-term Claeys et al.

(2015)

PTMG Aliphatic BDO Drug delivery Long-term Claeys et al.

(2015)

PCL-triol Aliphatic Glycerol TE

applications

Biodegradable

Barrioni et al.

(2015)

From the Table 6.1 it is possible to see that the main polyol applied in TE

is the PCL. PCL is a biocompatible polymer with aliphatic ester linkage that is

susceptible to be hydrolyzed. Poly(ethylene glycol) (PEG) is another polyol

commonly applied in TE. PEG is biocompatible and presents non-toxic degradation

products. The main diisocyanate applied is the HDI, due to the aliphatic segment and

degrade in non-toxic products; furthermore the addition of BDO to the hard segment

is frequently used. Polyhedral oligomeric silsesquioxane (POSS) diol it was also

founded as chain extender this is a silicon-oxigen caged structure, it is biocompatible,

and presents thermal and oxidative stability (McMullin et al., 2016).


The primary mechanism of PUs degradation is the hydrolysis of ester and

urethane groups. The cleavage of the ester bonds occurs via simple hydrolysis

generating free carboxylic acids and hydroxyl groups, and may cause the pH

decrease (Martin et al., 2014). The degradation rate depends on the crystallinity,

molecular weight, copolymer composition and morphological structure. The cleavage

of urethane groups generates amine and hydroxyl groups, and may cause the pH

increase. PUs can also be degradated by oxidation of the ether segments in a

presence of enzymes or calcification.

This versatile polymer has enormous potential applications as degradable

and non-degradable implants. Along these lines, PUs have gained attention in

different applications. In the cardiovascular (Hernández-Córdova et al., 2016; Ganji

et al., 2016) area mostly of the implants are stable, as intraortic balloons, cardiac

valves, vascular prostheses and grafts (Han et al., 2013; Hou et al., 2014). In drug

delivery applications, PUs have been applied as long-term intravaginal rings

presenting potential to prevent HIV disease, or containing a microbicide (Hiob et al.,

2016; Malcolm et al., 2016). PUs can also been applied as a capsule for oral dosage

forms (Claeys et al., 2015). Bioactive PUs have also been developed by adding

nanoparticles or antibiotics in order to promote antibacterial and antimicrobial

properties (Sommer et al., 2010). Another approach in the PU applications is related

to the epithelial applications, as wound dressings devices (Rottmar et al., 2015),

artificial skin and bandages. PU can also be applied on the prevention of biofilm

formation in devices.

Electrospinning was first observed by Rayleigh in 1897, is a unique

technique, an important tool for synthesizing one-dimensional nanostructures

(Bhattarai et al., 2014). Focusing on the TE applications the electospinning have

gained attention due to the possibility of creating mimicking scaffolds that present

properties of native tissues due to aligned fiber matrix, prepared from polymers

(Kucinska-Lipka et al., 2015).

This technique presents advantages, such as, high surface

area:volume ratio (Selvakumar et al., 2016), high porosity, small more size (Budun et

al., 2016; Wenguo et al., 2010), extraordinary length, highly porous and

interconnected structure for cell attachment and oxygen and nutrient transport (Zou

et al., 2012). However, this technique presents limitations based on scarce cell


infiltration and ingrowth, inadequate mechanical strength, possible toxicity due to the

residual solvent, and slow production rate. The optimum pore sizes for tissues are in

the range 100-500 micrometers (Wright et al., 2011).

It is an attractive technique, but many factors determine the success of the

fiber production, from the solution parameters, such as, concentration, conductivity

and viscosity, the controlled variables, such as, the flow rate, the electrical field

strength, the distance between the tip and the collector, and the laboratory

environment parameters, such as temperature, humidity and air velocity.

The synthesis of electrospun polyurethanes is relatively new. Moreover,

there have been few studies proving that this is a viable and promising technique for

the fabrication of medical devices. For fibroblasts culture, the optimal pore size was

found to be 20 micrometers (Whang et al., 1995), at the sime time, a large pore size

of 500 micrometers was found to be excellent for fibroblast vascular tissue ingrowth

(Wake et al., 1994). However, few in vivo studies of electrospun PU scaffold were

done (Kucinska-Lipka et al., 2015). Electrospun polyurethane have been applied as

artificial skin (Wang et al., 2016), bandages (Amina et al., 2013), stents (Aguilar et

al., 2015), grafts (Bergmeister et al., 2013), and scaffolds (Tetteh et al., 2014).

The purpose of this study was to prepare PU membranes using the

electrospinning apparatus and to investigate the produced membrane through SEM,

FTIR, TGA, in vivo cell adhesion and viability and in vivo degradation test. The PU

applied in this work is a medical grade commercial elastomer (Tecoflex SG-85A), an

aliphatic poly(ether-urethane) prepared from poly(tetramethylene glycol) (PTMG),

HMDI and BDO. PTMG is a polyol polyether that exhibits good flexibility properties

and it is biocompatible. Their structure and reactants are presented in Figure 6.1.


Figure 6.1: Structure of the polyurethane applied in this work.

6.2 OBJECTIVE

The purpose of this research was to obtain a polyurethane membrane via

electrospinning apparatus. The electrospun polyurethane was studied through

morphological, structural and thermal properties, the biological response was studies

through in vivo cell viability and adhesion assays and the biodegradation character

was studied through in vivo degradation test.


6.3.1 Materials:

All chemicals were of analytical grade and were used without further

purification. PUs in pellets form (medical grade SG85A) was kindly provided by

Lubrizol Advanced Materials. Chloroform was purchased by Sigma Aldrich.


6.3.2 Methods

6.3.2.1 PU electrospinning solution preparation:

PU stock solution was prepared by dissolving 7.0 g of PU in 80 mL

chloroform during sonication (Ultrasonic clear, Unique, São Paulo, Brazil) for 2 hours.

6.3.2.2 Preparation of the electrospun membranes:

The electrospinning apparatus (Figure 6.2) employed in this research was

designed and it is located in the National Institute of Biofabrication (INCT-Biofabris),

consisted of a syringe pump, a high-voltage direct-current power supplier (Testtech)

generating a positive dc voltage up to 30 kV, and a grounded collector that was

covered with aluminum foil. The solution was loaded into a syringe, and a positive

electrode was clipped onto the syringe needle. The feeding rate of the polymer

solution was controlled by a syringe pump, and the solutions were electrospun onto

the collector. The syringe pump was set at a volume flow rate of 7 mL/h, the applied

voltage was 18 kV, the tip-to-collector distance was 10 cm, and all solution

preparations and electrospinning were carried out at room temperature.

Electrospinning apparatus Electrospun fiber

Figure 6.2: Electrospinning apparatus and electrospun fiber, respectively.


6.3.3 Material characterization

6.3.3.1 Morphological and structural characterization

SEM analysis was performed in a scanning electron microscope (model

LEO 440i; Leo Electron Microscopy; Cambridge, England). The voltage used was 20

kV and the current was 100 pA. A metallic SC7620 Sputter Coater was used for

coating the sample with gold.

6.3.3.2 Structural characterization

FTIR studies were conducted at room temperature using a thermo

scientific Nicolet 6700 FTIR spectrophotometer (Nicolet Instrument Inc.,Madison, WI,

USA), connected with an ATR accessory. Spectra were collected from 40 scans,

within a range of 675–4000 cm-1 using a resolution of 4 cm-1. All spectra were ATR

corrected using Smart Omni sampler (Thermo Scientific, Madison, WI).

6.3.3.3 Thermal characterization

The investigation of the thermal composition of the PU was carried out

using TGA, model TGA-50 (Shimadzu Instruments Kyoto, Japan). A sample mass of

about 15 mg was heated under a flowing nitrogen atmosphere (50 mL/min) from 30

to 600 oC, at a heating rate of 10 oC/min.

6.3.3.4. Human cardiomyocytes cell adhesion

Human cardiomyocytes derived from induced pluripotent stem cells (iPS)

were provided from Pluricell Biotech (São Paulo, Brazil). Cells were cultured in

Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum

(FBS, Gibco). Growth media contained 100 units/mL penicillin and streptomycin and

cells were cultured in a 37 oC, humidified, 5% CO2/95% air environment. Cells were

used at population numbers below 5.


To determine the cardiomyocyte adhesion in the polyurethane, cells were

seeded onto 96-well culture plates at 1×106 cells/mL and were incubated for 24

hours. After that, cells were incubated with 100 μL of the aforementioned cell culture

medium for 24 hours. The cell adhesion after 24 hours was verified by fluorescent

microscopy (model Evos FL, Thermo Fischer Scientific).

6.3.3.4. Fibroblasts cell culture

VERO fibroblasts cells (African green monkey kidney fibroblasts) were

provided from Adolfo Lutz Institute (São Paulo, Brazil). Cells were cultured in

Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum

(FBS, Gibco). Growth media contained 100 units/mL penicillin and streptomycin and

cells were cultured in a 37 oC, humidified, 5% CO2/95% air environment. Cells were

used at population numbers below 5. The DMEM-LG with 10 % of phenol it was used

as positive control of toxicity (PCT) and the polystyrene extract was used as negative

control of toxicity (NCT).

6.3.3.5 Evaluation of fibroblasts cells using SEM

VERO cells were seeded on the PU surface with a final density of 3 x106

cells/mL. After growth time of 24, 48 and 72 hours, the samples were fixed in

glutaraldehyde 2.5% in 0.1 M sodium cacodilate buffer for about 2 h. The samples

were washed in PBS and then in water for 15 minutes. Afterwards, they were

dehydrated in a graded series of ethanol (50%, 70%, 95% and 100%), critical point

dried with CO2 (Balzers, CTD-030), and sputter coated with gold in a SC7620 Sputter

Coater apparatus. The cell-seeded PU scaffolds were characterized by Scanning

Electron Microscope (SEM, model 440i Leo, Zeiss) operated at 20 kV and 100 pA.

6.3.3.6 In vivo cell viability assays using MTT

To determine the toxicity of the polyurethane and the nanocomposite, cells

were seeded onto 96-well culture plates at 1×106 cells/mL and were incubated for 24

hours. After that, cells were incubated with 100 μL of the aforementioned cell culture


medium for 24, 48 and 72 hours. The amount of formazan crystals formed was

measured after 4 hours of exposure to the (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-

2H-tetrazolium bromide) (MTT) solution in dimethyl sulfoxide (DMSO) and

absorbance values were measured at 595 nm by a scanning multi well

spectrophotometer plate reader (Microplate Reader F5, Molecular Probes).

The data are presented as mean ± S.E.M. Statistical analysis of the data

was carried out using ANOVA, followed by Tukey's HSD post hoc test (equal

variances). Differences were considered statistically significant when the P-value was

less than 0.05.

6.3.3.7 Cytotoxicity assay using Live/Dead

A viability study on PU membranes was performed with a Live–dead

assay kit (Kit Live/Dead® Viability Cytotoxicity Molecular Probes TM. The cells were

seeded (1 x 103 cells/mL) into a 96-well plate and incubated with DMEM-LG

containing 10% of FBS at 37 oC for 24 hours. After this period, the membranes were

incubated with the cells for 24, 48 and 72 hours. A Live/Dead fluorescence assay kit

was applied to qualify the cells viability. After the specific times, the cells were rinsed

with 200 µL of PBS and stained with calcein/ethidium homodimer, according to the

manufactures’ instructions. The cells were incubated at 37 oC for 30 minutes and

then rinsed and maintained in PBS. After that, the membranes were visualized using

fluorescence microscopy (Nikon E800) with a specific program (Image Pro-Plus

software).

6.3.3.8 In vivo degradation test

The degradation experiments were performed following ASTM F1635-11.

The dried electrospun polyurethane samples were cut into 2 x 2 cm2 pieces. All the

cut specimens had weight of 13.2 ± 0.01 mg and then were placed in a test tube

containing 20 mL of pH 7.4 PBS (phosphate-buffered saline) at physiologic

temperature (37 oC) to simulate the hydrolytic environment. The tubes were placed in

an electronically controlled thermostat and kept in these conditions for 90 days. At

regular time intervals, (30, 60 and 90 days), the polyurethanes were taken out from


the degradation media and weighted. The samples were washed with distilled water

and then vacuum-dried at 37 oC to constant weight. The mass loss was calculated

according to the following equation:

( )

Where: wi and wd represent the initial weight and dry weight of the samples,

respectively.


6.4.1 Microphase-separated structure

The FTIR-ATR spectrum of PU is shown in Figure 6.3.

Figure 6.3: FTIR-ATR spectra of polyurethane.

The band present at 3322 cm-1 can be assigned to the –N–H stretching

vibration of urethane groups, this new band confirms the presence of urethane

groups. The peaks at 2940 and 2858 cm-1 are the asymmetric and symmetric

vibrations of the -CH2 groups and mostly represent the hard and soft segments,


respectively. The band at 2350 cm-1 corresponds to the stretching vibration of the

isocyanate group, –N=C=O. Carbonyl stretching bands (-C=O) of urethane bonds

appear at 1713 cm-1. The absorption peaks at 1529 cm-1 corresponds to the

deformation vibration of (-NH-) bonds in the urethane (-NHCOO-) groups from amide

II. The bands at 1447 and 1370 cm-1 corresponds to C-N stretching. The stretching of

the ether group C-O-C is associated with the absorption at 1109 cm-1, which

indicates the soft segments. It is possible confirm that there is no residual solvent in

the polyurethane.

6.4.2 The thermal degradation of polyurethane

The thermal degradation of PU is depicted in Figure 6.4.

Figure 6.4: TGA/DTG of polyurethane.

The TGA/DTG curves show two steps of mass loss. The first step of mass

loss occurred from 254 to 403 oC is attributed to the decomposition of the rigid

segments of PU, and the second step occurs between 403 and 487 oC due to the

decomposition of flexible segments of PU.


6.4.3 Human cardiomyocytes cell adhesion by fluorescent microscopy

After 24 hours of culture, the resulting morphology of the PU surfaces

was investigated using fluorescent microscopy. Figure 6.5 shows the samples (a-c)

and the cells (d) seeded onto 96-well culture plate.

a)

b)

c)

d)

Figure 6.5: Cell adhesion of human cardiomyocytes monitored by fluorescent

microscopy. Scale bar in a represent 1000 µm, and in b, c and e represent 400 µm.

Figures 6.5 (a-c) present the samples and Figure 6.5 (d) present the cells

seeded onto 96-well culture plate. The experiment showed that human

cardiomyocytes cells did not adhere in the PU surface. Unfortunately, the cells did

not migrate through pores of a membrane and, in fact, the cells just migrated to the

walls and to the bottom of the 96-well culture plate.


6.4.4 Evaluation of the fibroblasts cells adhesion of the electrospun fibers

using SEM

The resulting morphology of the electrospun surfaces were investigated

using SEM. Figure 6.6 shows the control sample (Plain PU) and the samples after

24, 48 and 72 hours of fibroblasts culture.

1000 x 3000 x

Plain

PU

PU 24

hours

PU 48

hours


PU 72

hours

Figure 6.6: SEM images of the plain PU and the fibroblasts after 24, 48 and 72 hours

of culture.

Electrospun PUs showed relatively uniform fiber diameter distributions,

exhibiting fiber diameter of approximately 20 micrometers, it was also observed a

random fiber networks, with pore size between 50-150 micrometers. The

experiments show that fibroblasts adhered and spread over the surface of the

polyurethane, following the fiber morphology. The results also showed that cells

migrated and proliferated after 24 hours. Specifically after 72 hours, the sample

showed that cell proliferation was enhanced, generating an extensive network of

fibroblasts cells.

6.4.5 In vivo fibroblasts cell viability assays

Cytotoxicity test was performed to study the polyurethane biocompatibility.

Fibroblasts cells were seeded directly onto PU membranes during 24, 48 and 72

hours and the amount of formazan crystals formed was measured after 4 hours of

exposure to the MTT. The results obtained after 24, 48 and 72 hours to PU, positive

control of toxicity (PCT) and negative control of toxicity (NCT) are presented in Figure

6.7.


Figure 6.7: Cell viability monitored by MTT assay following 24, 48 and 72 hours

exposure of fibroblasts cells.

Figure 6.7 represents the number of viable fibroblasts cells measured by

the MTT assay. According to the two-way ANOVA, a statistically significant (p<0.001)

difference was found between the groups when cell viability was considered. Results

obtained after 24 hours showed no toxic effect of polyurethane compared to the NCT

control group (polystyrene extract). Cytotoxic effect was observed after 48 hours

(p<0,005) and after 72 hours (p<0,001). The cytotoxic effect was also verified

comparing the PU to the PCT control group (phenol solution) for 24 hours (p<0,014),

48 hours (p<0,012) and 72 hours (p<0,001).

6.4.6 Live/Dead

Cell viability as a function of culture time (24, 48 and 72 hours) was

determined by using a Live–dead assay kit. The viable fibroblasts cells (stained

green) and dead fibroblasts cells (stained red) were visualized by fluorescence

microscopy and are presented in Figures 6.8 and 6.9.


24 HOURS 48 HOURS 72 HOURS

NCT

PU

Figure 6.8: Live-dead assay showing fibroblasts incorporated in PU membranes,

after 24, 48 and 72 hours. Amplification: 10x.

24 HOURS 48 HOURS 72 HOURS

NCT

PU

Figure 6.9: Live-dead assay showing fibroblasts incorporated in PU membranes,

after 24, 48 and 72 hours. Amplification: 20x.

Figures 6.8 and 6.9 shows that at all culture times, predominantly living

cells were found within the PU membranes. Fibroblasts cells adhered through the

membrane and have a good cytocompatibility. These findings were further confirmed

with the MTT assay (Figure 6.6) and SEM images (Figures 6.7). The viable


fibroblasts cells which stained green were observed throughout the membrane,

showing a uniform cell distribution. Specifically at 24 and 48 hours, the results

strongly suggest that the membrane did not compromise cell viability and the

transport of nutrients and oxygen to the cells. After 72 hours, it was showed the

decrease of stained green cells, resulting in an increase of toxic effect of

polyurethane compared to the NCT.

6.4.7 In vivo degradation study

The in vivo degradation measurements of the electrospun membranes

were performed during 30, 60 and 90 days. Figure 6.10 depicts the mass loss of the

membranes as a function of the degradation time.

Figure 6.10: Mass loss during in vivo degradation study.

No changes were observed in the surface morphologies of the

membranes during the experiment. The surfaces of the samples and the flexibility

remained very smooth, with no evidence of any degradation. After the incubation

period, the PU membranes exhibited a very slow weight loss rate. Specifically after

90 days on in vivo incubation, about 0,7% mass loss was observed. Thus, this

polyurethane is suitable for long-term applications due to their stability in vivo.


6.5 DISCUSSION

This study highlights the synthesis and characterization of polymeric

membranes for numerous TE applications.

This study was able to maintain function of fibroblasts, due to

interconnected structure obtained, adequate for cell attachment, migration and

proliferation, oxygen and nutrient transport, and physical support for cell seeding.

The obtained membranes showed the success of fiber production,

indicating homogeneous morphology, appropriate concentration, temperature,

electrical field strength, and more important, that all residual solvent was consumed

during the electrospinning process.

Although certainly more studies are required, such as mechanical

resistance, the present results are encouraging for further emphasis of

electrospinning technique as an attractive technique for membranes synthesis.

This fascinating area of research demonstrate just how multidisciplinary

field of TE is vast, the opportunities for improving human health are immense. Many

challenges are still limited to this multidisciplinary area and the complexity of

biological systems involved in the repair of different tissues. Significant progress has

been made to achieve the TE goals. While simple polymeric scaffolds that were

being developed in the past were not providing adequate interactions between cells

and the ECM, future advances in TE are depending on new systems that will actively

modulate the behavior of cells to a functional TE. The success of TE depends on the

researchers efforts to understand the results of science and clinical applications. The

integration of knowledge of life sciences areas along with materials science,

medicine, chemistry, biology and engineering is crucial in the scaffolds design, and it

will also generate new technologies for application in regenerative medicine.


6.6 CONCLUSION

This work successfully prepared electrospun polyurethane membranes by

a simple and efficient method. The structure and morphological studies showed

random fibers, exhibiting fiber diameter of approximately 20 micrometers. All the

chloroform was consumed during the electrospinning process. The TGA results

showed the temperature of degradation starting at 254 oC, ideal for processes such

as sterilization. Cell viability results indicated fibroblast adhesion, generating an

extensive network of fibroblasts. The in vivo degradation investigations showed that

this polyurethane shoud be applied for long-term applications. In summary, the

electrospun membrane developed in this research is very promising for different

applications in TE, such as ephitelial, drug delivery or cardiac applications.

CHAPTER 7 – Conclusions and suggestions for future work 94

CHAPTER 7

CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK

This chapter highlights the conclusions of this research work. It also

outlines a number of suggestions for future work.

7.1 CONCLUSIONS

The main conclusions that can be drawn for this work are summarized below:

It was successfully prepared polyurethane films doped with zinc oxide, silver

and hydroxyapatite in order to impart antibacterial properties. The

morphology, structure and antibacterial properties of the nanocomposites were

tested. For the first time, it was observed the complete inhibition of bacteria

growth after 16 hours of culture on the PU-ZnO nanocomposite. The PU-Ag

nanocomposite showed better antibacterial activity against S. epidermidis and

the PU-HA nanocomposite better antibacterial activity against P. aeruginosa.

This study provides evidence of bacteria inhibition of materials current applied

as medical devices due to the possibility to modify the surface of medical

devices using nanocomposite films doped with nanoparticles.

A green chemistry composite scaffold was prepared and the influence of

hydroxyapatite nanoparticles was tested on the structure, thermal and

biological properties. The results showed that the hydroxyapatite promoted the

fibroblast adhesion, and in vivo studies confirmed that the developed medical

device promoted tissue adherence, presented biocompatibility, and could be

applied for numerous orthopedic applications.

Polyurethane membranes were prepared by a simple method. The

electrospun fibers were studied through morphological, structural, thermal and

biological properties. In summary, the membrane presented homogeneous

morphology, exhibited fiber diameter of approximately 20 micrometers. In vivo

experiments showed fibroblast adhesion and proliferation. This study supports

the investigation of electrospun membranes as biocompatible wound healing.

CHAPTER 7 – Conclusions and suggestions for future work 95

7.2 SUGGESTIONS FOR FUTURE WORK

Based on the understanding gained in this PhD, the suggestions for

future work are:

To modify the surface hydrophobicity of the polyurethane scaffolds in order to

govern cell response to promote cell adhesion and to obtain biodegradable

scaffolds;

The next step would be to include investigations of the PU biodegradation

using enzymes;

To test the adherence of different cells in the polyurethane membrane, such

as liver and lung cells;

To test the mechanical resistance of the developed films, foams and

membranes;

To develop different bioactive films using different nanoparticles and test the

cell proliferation, in order to understand the mechanism of the bacteria action;

To produce membranes with different pores size, ranging to nanopores to

micropores, to evaluate the pore size in the cell attachment.

To produce three-dimensional scaffolds using additive manufacturing and test

cell proliferation using different geometries.

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APPENDIX 1 110

This appendix presents the Ethics Committee in Animal Experimentation

obtained to in vivo studies presented in Chapter 5.

development of medical devices based on...

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