Universidade de Aveiro 2019
Departamento de Engenharia de Materiais e Cerâmica
Viviana dos Santos
Gomes
MATERIAIS
HÍBRIDOS
COM
PROPRIEDADES
ANTIBACTERIANAS
PARA
ENGENHARIA
DE
TECIDOS
HYBRID
MATERIALS
WITH
ANTIBACTERIAL
Universidade de Aveiro 2019
Departamento de Engenharia de Materiais e Cerâmica
Viviana dos Santos
Gomes
MATERIAIS
HÍBRIDOS
COM
PROPRIEDADES
ANTIBACTERIANAS
PARA
ENGENHARIA
DE
TECIDOS
HYBRID
MATERIALS
WITH
ANTIBACTERIAL
PROPERTIES FOR TISSUE ENGEENIRING
Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Mestrado em Materiais e Dispositivos Biomédicos, realizada sob a orientação científica da Doutora Maria Helena Figueira Vaz Fernandes, Professora associada do Departamento de Engenharia de Materiais e Cerâmica da Universidade de Aveiro, e do Doutor José Carlos Martins de Almeida, Investigador do mesmo departamento.
Dedico este trabalho à minha mãe, ao meu pai e à minha irmã.
“Success is the sum of small efforts, repeated day-in and day-out”
o júri
Presidente Prof. Doutora Maria Margarida Tavares Lopes de Almeida
Professora Auxiliar, Universidade de Aveiro
Doutora Bárbara Joana Martins Leite Ferreira
Investigadora Doutorada (nível 1), Universidade de Aveiro
Doutor José Carlos Martins de Almeida
Acknowledgment Initially I would like to thank my main supervisor, Professor Maria Helena Fernandes, for all the support she has provided over the months. To Doctor José Carlos, my co-supervisor, I owe my most sincere gratitude for all the accompaniment, guidance and dedication in sharing with me teachings from various areas. Although my area of academic training is not the same as the work I did, the knowledge shared by both of them and all their trust in me and my work made it possible to develop all the work.
I would also like to thank Professor Isabel Salvado for all the knowledge shared with me in the sol-gel area, as well as for all the advice shared with me.
To Dr.ª Claudia Oliveira, Professor of the Department of Biology and researcher at CESAM, University of Aveiro, my most sincere thanks for all the follow-up and for all the help during the antibacterial assays.
I also want to thank the technical team of the Department of Materials and Ceramic Engineering (DEMAC), in particular Eng. Artur and Eng. Ana, as well as Celeste Azevedo, technician of the Department of Chemistry.
I would also like to give special thanks to the people with whom I shared the laboratory, especially Dr.ª Marisa Costa for all the advice and help at various times throughout the work. To Inês Rodrigues, my colleague and laboratory friend, thank you for all the friendship, all the smiles and all the positive thoughts we share. I also want to thank my old friends Adriana Azevedo, Carmen Nunes and Filipa Rodrigues for sharing with me one more stage of my academic life and my personal achievements.
To my boyfriend, Pedro Brás, I am very grateful for all the trust you place in me every day. Thank you for all the affection and encouragement.
I also want to thank my grandparents, Armanda Gomes and António Gomes, for all their support and affection. Part of all that I got is due to them.
Finally, I want to thank my parents, especially my mother, for having done everything in her power to follow my dreams. Thanks to my mother and sister, Bruna Gomes, for all the conversations, for all the encouragement and patience, and for all the love over all these years, never doubting my abilities.
To all who were left without mention but who directly, or indirectly, contributed to the development and implementation of this work, thank you.
palavras-chave Materiais híbridos, propriedades antibacterianas, engenharia de tecidos, método sol-gel, zinco, cobre, tetraetilortosilicato (TEOS), polidimetilsiloxano (PDMS).
Resumo O aumento da esperança média de vida e a necessidade crescente de melhorar as técnicas terapêuticas já existentes, levaram ao desenvolvimento de novas abordagens para a engenharia de tecidos. A utilização de vidros bioativos como biomaterial para a recuperação de tecidos é uma prática muito comum. Recentemente têm sido desenvolvidos de modo a permitir a libertação controlada de iões clinicamente benéficos, melhorando assim os seus efeitos terapêuticos. Uma das desvantagens destes materiais é o facto de, geralmente, possuírem propriedades muito diferentes das dos tecidos que substituem. Nesse sentido surgiram os materiais híbridos orgânico-inorgânicos, que são formados por uma componente inorgânica, tetraetilortosilicato (TEOS), à qual é adicionada uma componente orgânica, polidimetilsiloxano (PDMS). Este trabalho surge na continuação de um trabalho anterior, intitulado de “Materiais híbridos orgânico-inorgânicos de borossilicato obtidos por sol-gel para aplicações em engenharia de tecido ósseo”. A novidade deste trabalho consistiu no desenvolvimento de materiais do sistema híbrido PDMS-SiO2-B2O3 com propriedades antibacterianas,
através da adição de zinco ou de cobre. Dessa forma, foram preparadas três composições distintas para cada um dos sistemas, alterando as quantidades de zinco, ou cobre, adicionadas mas mantendo constantes os restantes reagentes. Para cada um dos sistemas híbridos foi utilizado um protocolo distinto, sendo todos os materiais foram obtidos através do método sol-gel. Os materiais resultantes da adição de zinco ao sistema híbrido PDMS-SiO2-B2O3 apresentam-se na forma de monólito branco, poroso
e opaco, enquanto que as amostras resultantes da adição de cobre apresentam-se na forma de um monólito sem porosidade. A análise estrutural dos diferentes materiais foi realizada por difração de raios-X (DRX), espectroscopia de infravermelhos por transformada de Fourier (FTIR) e por ressonância magnética nuclear (NMR), que permitiu identificar a presença de ligações híbridas, devido à existência de ligações entre a fase orgânica e a fase inorgânica (D-Q), assim como ligações características de borosiloxano (B-O-Si). Os resultados de RMN 1H, juntamente com a análise de DRX, permitiram concluir que as amostras com adição de zinco possuíam cadeias de PDMS mais curtas, enquanto que nas amostras com adição de cobre estavam presentes cadeias de PDMS mais longas. A análise da RMN 11B permitiu concluir que nas amostras com adição de cobre apenas existe boro na forma trigonal (BO3), mas nas
amostras com adição de zinco, por se tratar de uma espécie intermediária que pode desempenhar a função de ião modificador de rede, ocorre a alteração na coordenação do boro de trigonal (BO3) para tetraédrico (BO4). A análise microestrutural foi realizada
através da determinação da área superficial por adsorção de azoto com recurso à isotérmica de Brunauer, Emmett, Teller (BET) e microscopia eletrónica de varrimento (SEM), que permitiu concluir que as amostras com adição de zinco apresentam uma estrutura porosa, com uma área superficial especifica (SSA) superior à da apresentada pelas amostras com adição de cobre. O teste de degradação foi realizado de acordo com a norma ISO 10993-13 para diferentes tempos de imersão em água desionizada: 30 minutos, 1 hora e 5 horas. Posteriormente, a análise de espectrometria de emissão ótica por plasma acoplado indutivamente (ICP-OES), revelou a capacidade do material em libertar os iões adicionados, zinco ou cobre. Por fim, os ensaios antibacterianos, realizados de acordo com a norma ASTM E2149, revelaram a capacidade dos materiais desenvolvidos em reduzirem a atividade antibacteriana para as estirpes bacterianas
keywords Hybrid materials, antibacterial properties, tissue engineering, sol-gel method, zinc, copper, tetraethyl orthosilicate (TEOS), polydimethylsiloxane (PDMS).
abstract The increasing average life expectancy and the increasing need to improve existing therapeutic techniques have led to the development of new approaches to tissue engineering. The use of bioactive glasses as biomaterials for tissue recovery is a very common practice. However, they have recently been developed to allow the controlled release of clinically beneficial ions, thereby improving their therapeutic effects. One of the disadvantages of these materials is the fact that they generally have properties very different from those of the tissues in which they are implanted. In this sense new materials arose, including the organic-inorganic hybrid materials, which are formed by an inorganic component, such as tetraethyl orthosilicate (TEOS), to which an organic component, polydimethylsiloxane (PDMS) is added.
This work follows on from a previous work entitled "Hybrid organic-inorganic borosilicate materials by sol-gel for bone tissue engineering", The novelty of this work consisted of the development of materials in the PDMS-SiO2-B2O3 hybrid system with antibacterial
properties through the addition of zinc and copper. Thus, three distinct compositions were developed for each of the systems, changing the amounts of added zinc, or copper, but keeping the remaining reagents constant. For each of the hybrid systems a different protocol was used, but all materials were obtained by the sol-gel method. The materials resulting from the addition of zinc to the PDMS-SiO2-B2O3 hybrid system are
porous and white opaque monoliths, while the samples resulting from the addition of copper are monolithic samples with no porosity. The structural analysis of the different materials was performed by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and magnetic nuclear resonance (NMR), which allowed to verify the presence of hybrid bonds, due to the existence of the bonds between the organic phase and the inorganic phase (D-Q), as well as characteristic bonds of borosiloxane (B-O-Si). The 1H NMR results, together with the XRD analysis, led to the conclusion that zinc-containing samples had shorter PDMS chains, while in copper-containing samples had longer PDMS chains were present. The 11B NMR analysis allowed to conclude that in copper-containing samples boron is present only in the trigonal form (BO3), However,
in the zinc-containing samples, as zinc is an intermediate species that can behave as a network modifier, the change of boron coordination from trigonal (BO3) to tetrahedral
(BO4) occurs. The microstructural analysis was performed by determining the specific
surface area by Brunauer-Emmett-Teller (BET) method and scanning electron microscopy (SEM), which led to the conclusion that, unlike copper-containing samples, zinc-containing samples have a porous structure with a much larger specific surface area (SSA). The degradation test was performed according to ISO 10993-13 for different immersion times in deionized water: 30 minutes, 1 hour and 5 hours. Subsequently, analysis by inductively coupled plasma optical emission spectrometry (ICP-OES) revealed the ability of the material to release the added ions, zinc and copper. Finally, antibacterial assays performed according to the ASTM E2149 method revealed the ability of the developed materials to reduce bacterial activity for Escherichia coli ATCC 25922 and Staphylococcus aureus NCTC 6871 bacterial strains.
Table of Contents
List of Figures ... v
List of Acronyms and Abbreviations... ix
Chapter 1 ... 1
Introduction ... 1
1.1 Background ... 1
1.2 Objectives ... 3
1.3 Structure of the thesis ... 3
Chapter 2 ... 5
State of the art ... 5
2.1 Tissue engineering... 5
2.2 Bioactive glasses ... 6
2.3 Sol-gel method ... 8
2.4 Hybrid materials in the PDMS- SiO2 system ... 10
2.5 Zinc, copper and boron in tissue engineering ... 12
2.5.1 Zinc... 12 2.5.2 Copper ... 12 2.5.3 Boron ... 13 2.6 Polydimethylsiloxane ... 13 Chapter 3 ... 17 Experimental Procedure ... 17 3.1 Materials ... 17 3.2 Samples preparation ... 18 3.3 Characterization Technics ... 23
3.3.1 X-ray diffraction (XRD) ... 23
3.3.2 Fourier transform infrared (FT-IR) spectroscopy ... 24
3.3.3 Nuclear magnetic resonance (NMR) ... 24
3.3.4 Specific surface area by Brunauer-Emmett-Teller (BET) method ... 25
3.3.5 Scanning electron microscopy (SEM) ... 27
3.4 Antibacterial tests ... 27
3.4.1 Sieving technique ... 27
3.4.2 Inductively coupled plasma optical emission spectroscopy (ICP-OES) ... 28
3.4.3 Antibacterial assays ... 28
Chapter 4 ... 31
Results and Discussion ... 31
4.1 Hybrids structural analysis ... 31
4.1.1 XRD analysis ... 31
4.1.2 FT-IR analysis ... 34
4.1.3 NMR analysis ... 37
4.2 Hybrids Microstructural analysis ... 41
4.2.1 BET analysis ... 41
4.2.2 SEM analysis ... 42
4.3 Hybrids antibacterial analysis ... 44
4.3.1 ICP-OES analysis ... 44
4.3.2 Antibacterial assays ... 46
Chapter 5 ... 51
5.2 Future works ... 53
References ... 55
Annex ... 65
Annex I ... 65
I. Results obtained by replicating a previous procedure ... 65
III. Results obtained with addition of nitric acid in the experimental procedure of copper-containing samples... 67
Annex II... 69
List of Figures
Figure 1: Tissue engineering triad: scaffolds, cells, and growth factors 22. ... 5
Figure 2: Tissue Regeneration, through the implementation of growth factor and mechanical stimulus 25. ... 6
Figure 3: Effects of ions released from incorporated ions on bioactive glasses 5. ... 8
Figure 4: Scheme of preparation of materials by sol-gel process. Adapted by 33. ... 10
Figure 5: Formation of an Organically Modified Silicate (ORMOSIL) 12. ... 11
Figure 6: Experimental procedure used to prepare the zinc-containing samples. ... 20
Figure 7: Zinc-containing samples after stirring, 2 days at room temperature and monolithic samples. ... 20
Figure 8: Experimental procedure used to prepare the copper-containing samples. ... 22
Figure 9: Copper-containing samples after stirring, 2 days at room temperature and monolithic samples. ... 22
Figure 10: Different types of gas adsorption isotherms, according to the IUPAC classification. ... 26
Figure 11: Different types of hysteresis, according to the IUPAC classification. ... 27
Figure 12: XRD spectra of the four zinc–containing samples after dried. ... 32
Figure 13: XRD spectra of the four copper-containing samples after dried. ... 33
Figure 14: XRD spectra of samples B0Zn0 and B0Cu0 after dried. ... 33
Figure 15: FT-IR spectra for the zinc-containing samples in the interval of 700-1300 cm-1 (resolution of 2 cm-1 and 250 scans). ... 35
Figure 16: FT-IR spectra for the copper-containing samples in the interval of 700-1300 cm -1 (resolution of 2 cm-1 and 250 scans). ... 36
Figure 17: FT-IR spectra for the zinc-containing samples, B10Zn0 and B10Zn10, and copper-containing samples, B10Cu0 and B10Cu10, in the interval of 350-1500 cm-1 (resolution of 4 cm-1 and 128 scans). ... 37
Figure 18: 1H NMR spectra of the samples B10Zn0 and B10Zn10. ... 38
Figure 20: 1H NMR spectra of the samples B10Cu0 and B10Zn0. ... 39
Figure 21: 11B NMR spectra of the samples B10Zn0 and B10Zn10. ... 40
Figure 22: 11B NMR spectra of the samples B10Cu0 and B10Cu10. ... 40
Figure 23: Isotherms of B10Zn2.5, B10Zn5, B10Zn10 e B10Zn0 by BET method. ... 42
Figure 24: SEM micrographs of (a) B10Zn0, (b) B10Zn2.5, (c) B10Zn5 and (d) B10Zn10, xerogel fracture surfaces. ... 43
Figure 25: SEM micrographs of (a) B10Cu0, (b) B10Cu2.5, (c) B10Cu5 and (d) B10Cu10, xerogel fracture surfaces. ... 43
Figure 26: Ionic concentration of (a) boron, (b) zinc, (c) silicium in deionized water at different soaking times (30, 60 and 300 minutes), and released from the zinc-containing samples. ... 45
Figure 27: Ionic concentration of (a) boron, (b) copper, (c) silicium in deionized water at different soaking times (30, 60 and 300 minutes), and released from the copper-containing samples. ... 46
Figure 28: Effect of zinc matrix (B10Zn0) and copper matrix (B10Cu0) on the amount of (a) E. coli ATCC 25922 and (b) S. aureus NCTC 6871 cell colony forming units (CFU). 47 Figure 29: Influence of zinc and copper addition on E. coli ATCC 25922 cell reduction. . 48
Figure 30: Influence of zinc and copper addition on S. aureus NCTC 6871cell reduction. 48 Figure 31: Zinc-containing samples after stirring and monolithic samples obtained by replicating the previous experimental procedure... 65
Figure 32: Copper-containing samples after stirring and monolithic samples obtained by replicating the previous experimental procedure... 65
Figure 33: Copper-containing samples after stirring, 2 days at room temperature and monolithic samples. ... 66
Figure 34: XRD spectra of the copper-containing samples after dried. ... 66
Figure 35: Copper-containing samples after stirring, 2 days at room temperature and monolithic samples. ... 67
List of Tables
Table 1: Reagents, chemical formula, purity and brand. ... 17
Table 2: Composition (in molar ratio) of zinc-containing samples. ... 18
Table 3: Composition (in molar ratio) of copper-containing samples... 18
Table 4: Values of the maximum angle and distance between organic centers of the four zinc-containing samples. ... 32
Table 5: Values of the maximum angle and distance between organic centers of the four copper-containing samples. ... 33
Table 6: Values of surface specific area calculated from the nitrogen adsorption isotherms, for zinc-containing samples. ... 42
Table 7: Values of surface specific area calculated from the nitrogen adsorption isotherms, for copper-containing samples... 42
Table 8: Zinc-containing samples analytical results... 45
Table 9: Copper-containing samples analytical results. ... 46
Table 10: Band assignments for the four zinc-containing samples. ... 69
List of Acronyms and Abbreviations
a.u. Arbitrary Units
ca. Circa
ORMOSIL Organically Modified Silicate XRD X-Ray Diffraction
FT-IR Fourier Transform Infrared Spectroscopy NMR Nuclear Magnetic Resonance
BET Brunauer-Emmett-Teller method SEM Scanning Electron Microscopy
ICP-OES Inductively coupled plasma optical emission spectroscopy
ISO International Standard Organization IUPAC International Union of Pure and Applied
Chemistry
MAS Magical Angle Spinning ppm Parts-per-million
IPA Isopropanol
PDMS Polydimethylsiloxane TEOS Tetraethyl Orthosilicate TMB Trimethylborate
rpm Rotation per minute SSA Specific Surface Area
LB Liquid Broth
PCA Plate Count Agar
CFU Colony Forming Units ANOVA Analysis of Variance
List of Symbols
δ Chemical shift (NMR)
⁰C Celsius degree
Ɵ Angle of incident radiation (XRD) wt.% Weight percent
δa Bending (asymmetric) vibrational mode
δs Bending (symmetric) vibrational mode
νa Stretching (asymmetric) vibrational mode
Chapter 1 - Introduction
Chapter 1
Introduction
1.1 Background
Situations of disease, injury and trauma can often lead to irreversible damage to various tissues of the human body. Conventional treatment involves replacing the damaged tissue with the patient's own tissue (autograft) or tissue from another donor of the same species (allograft) or of another species (xenograft). However, these techniques are associated with a number of risks to the patient himself, including tissue unavailability, rejection, or disease transmission. In this sense, tissue engineering emerged as a way to overcome this problem by using materials, either synthetic or natural, capable of contributing to the restoration of functions lost by the affected tissues.
Tissue engineering is an interdisciplinary area focused on regeneration or repair of damaged tissue 1,2. Contrarily to the common approach based on the implantation of new materials (biomaterials) into the body, tissue engineering aims to induce tissue formation and regeneration 1. In one of the most explored strategies, tissue engineering depends on the use of porous supports, designated scaffolds, that allow cell infiltration, fixation and growth 2,3.
For a successful tissue engineering approach, choosing of the scaffold material plays an essential role 4. Scaffolds are three-dimensional porous structures with a controlled and interconnected porosity 5. Although the mechanical properties of this material depend on both the manufacturing technique and its future use, there are some general properties that are indispensable. Thus, among other characteristics, scaffold materials must be biocompatible, bioresorbable and possess mechanical properties similar to those of the tissue where they will be implanted 6,7.
During the last years the use of bioactive glasses as biomaterial for tissue recovery, in particular bone tissue, has been largely increasing 5. Some of the major advantages of using this material include its bioactive nature and its osteoconductive and osteoinductive properties, leading to a controllable degradation rate 4,5. This is mostly due to the fact that bioactive glasses have the ability to induce the formation of a calcium phosphate layer on their surface, thus promoting a strong bond with the surrounding tissue 8.
The first bioactive glass, better known as 45S5 Bioglass, was synthesized in 1960 by Hench and his group, with a composition of 45 SiO2 - 24.5 Na2O - 24.5 CaO - 6 P2O5
(wt.%) 9. Since then, numerous studies have been conducted using this material. Recently, investigations in the field of bioactive glasses have been directed to the exploitation of
Chapter 1 - Introduction their angiogenic effects 5,9. Bioactive glasses can be developed to allow controlled release of clinically beneficial ions, thereby improving their therapeutic effects, in particular allowing local release of antibacterial agents 5,6. From a mechanical point of view, these glasses generally exhibit very different properties from those of the tissues they replace. The development of new materials that present improved mechanical properties has received special attention, justifying for example the appearance of the organic-inorganic hybrid materials, or ORMOSIL's (Organically Modified Silicates). These materials contain an inorganic component, such as tetraethyl orthosilicate (TEOS), to which an organic component, polydimethylsiloxane (PDMS) is added 10–13. The preparation of organic-inorganic hybrid materials is carried out through the sol-gel process by the alkoxide method, which includes hydrolysis and condensation reactions of metal alkoxides. The organic component used is PDMS, as it provides silica bonds that will facilitate and promote a stronger bond (covalent bonds) between the organic component and the inorganic component 12,14.
PDMS is one of the most widely used polymeric materials in tissue engineering due to its biocompatibility, flexibility, low toxicity and high thermal and oxidative stability. However, it has some disadvantages such as its extreme hydrophobicity and lack of antimicrobial activity. Thus, after numerous studies, it was suggested that the addition of zinc or copper nanoparticles to the surface of PDMS, allows the modification of its hydrophobicity and surface roughness, improving its anti-inflammatory and antibacterial activity 15.
Zinc is an essential element in the body, in particular in stimulating bone regeneration. According to the literature doping of bioactive glasses with zinc contributes to the emergence of an anti-inflammatory activity 9,17. The addition of zinc further contributes to the increased acellular formation of the calcium phosphate layer on the surface of bioactive glass, thereby promoting a better bond between material and bone 9. Also copper corresponds to a chemical element relevant to the human body being responsible for the formation of blood cells. The addition of copper to bioactive glasses induces the formation of capillary networks, promoting vascularization in tissue engineering structures 16.
This work follows on from an earlier work entitled “Hybrid organic-inorganic borosilicate materials by sol-gel for bone tissue engineering” in which the PDMS-SiO2-B2O3 hybrid
system was studied 18. The addition of copper and zinc ions, proposed in this thesis work, is a novelty. The way these ions, added separately to the PDMS-SiO2-B2O3 hybrid system,
affect the structure, microstructure and antibacterial properties of the final product will be studied in detail.
Chapter 1 - Introduction
1.2 Objectives
The main objective of this work is the production, through the sol-gel process, of PDMS-SiO2-B2O3 hybrid materials, with antibacterial properties, for future application in tissue
engineering. In order to obtain such properties, zinc and copper ions will be added. Analysis of the structure and microstructure of the obtained materials will be performed and the release kinetics of the copper and zinc ions will be determined. The evaluation of antibacterial activity will focus on materials with selected formulations and will take place in research units of UA within already established joint research partnerships.
At the end of this work it is expected to have acquired a better understanding of the potential application of hybrid materials in tissue engineering. The methodology for preparing these sol-gel hybrids will be properly designed and it is expected to establish the correlation between process parameters, structural and microstructural characteristics, and materials behaviour in contact with living entities, such as bacteria.
1.3 Structure of the thesis
The thesis is divided into five chapters. Chapter 1, corresponding to the introduction, describes the motivation behind this thesis, as well as its purpose and structure. Chapter 2 refers to the state of the art, which approaches topics related to the work developed, namely, tissue engineering, bioactive glass, sol-gel method, method used for the processing of the prepared hybrid materials, and the use of the added ions in tissue engineering. The experimental procedure was detailed in chapter 3, as well as the characterization techniques used to analyze the developed materials. The results obtained by the characterization techniques performed at structural, microstructural and antibacterial assays were discussed in chapter 4. And finally, Chapter 5 indicates the conclusions obtained from the work done, as well as indications and/or guidelines of possible future work.
Chapter 2 – State of the art
Chapter 2
State of the art
2.1 Tissue engineering
The first definition of tissue engineering emerged in 1987 from the National Science Foundation and is described as "an interdisciplinary field that applies engineering and life sciences principles to the development of biological substitutes that restore, maintain or improve tissue function" 19. Currently, there is a growing need for tissues, particularly for the skeletal system, due essentially to increased average life expectancy. However, although tissue or organ transplantation is the most common alternative, it has limited availability and may be associated with a number of risks, including the need for immune suppressing drugs as a means of avoiding graft rejection 20. Within this context, combining the innumerable knowledge gained in various fields such as biology, medicine, chemistry and physics, tissue engineering has emerged as an innovative alternative for replacing or repairing injured tissues or organs.
The development of biocompatible materials can be carried out in two different ways, one of which is, based on the in vitro production of tissue to be later implanted, and the other depends on the building of a structure that can trigger cell growth after its implantation in the organism 21. Figure 1 shows the three essential components of tissue engineering, namely scaffolds, cells and growth factors 22. Typically, scaffolds incorporate cells and growth factors, which upon implantation into the organism will allow cell proliferation and differentiation by promoting repair or regeneration of damaged tissue 22.
Chapter 2 – State of the art The basic principle of tissue engineering is to provide a biomimetic environment that supports cell adhesion, proliferation and differentiation in order to provide tissue regeneration (figure 2) 23. The great success of tissue engineering also comes from the fact that three-dimensional structures, called scaffolds, can be used that allow controlled release of drugs, or support the addition of growth factors to provide vascular and tissue growth
20,24
. Due to the relation between cost and effectiveness, some inorganic elements, such as boron, tend to be increasingly used as angiogenic agents, responsible for stimulating the formation of vascular structures, compared to common growth factors.
Figure 2: Tissue Regeneration, through the implementation of growth factor and mechanical stimulus 25.
2.2 Bioactive glasses
The first definition of biomaterials, referred to in 1976 by the European Society of Biomaterials, was limited to a material intended to interact with the body without influencing biological processes. However, biomaterials are currently defined as materials intended to interact with biological systems in order to evaluate, treat and / or replace any damaged tissue or organ 25. The regeneration of damaged tissues requires the use of biomaterials with suitable characteristics for stimulating the tissue renewal process. Bioactive glasses have gained special attention for tissue engineering applications due to their osteoconductivity, biocompatibility and ability to bind to tissues 5.
A material is considered bioactive when it induces a specific biological response at the interface, resulting in the formation of a bond between tissues and materials. In the particular case of bioglasses this bond is related to the formation of a hydroxyapatite
Chapter 2 – State of the art composition (wt. %) of 45 SiO2 - 24.5 Na2O - 24.5 CaO - 6 P2O5 9. Due to its
biocompatibility and high bioactivity, translated into its ability to promote tissue regeneration, this bioactive glass and its derivatives have been widely used clinically 5,6,9. Bioactive glasses are produced using network former oxides, such as SiO2, B2O3 and P2O5,
which allows its identification as silicate, borosilicate, borate and phosphate-based glasses
5
. The bioactivity of these glasses can be tailored by the addition of network modifier oxides such as Na2O, CaO, MgO, K2O, or intermediate oxides such as Al2O3, ZnO, ZrO2
and TiO226.
Bioactive glasses have an osteoinductive behavior, that is, they have the ability to induce an undifferentiated mesenchymal cell to turn into an osteoblast (bone forming cell), helping the formation of a hydroxyapatite layer. This hydroxyapatite layer is considered responsible for the strong bond between bioactive glasses and human bone 9. Bioactive glasses can be used in the manufacture of various clinical products (e.g. cast monoliths, micrometric particles, porous granules and injectable mass) 27.
Recently, extensive work has been done in the field of bioactive glasses, mainly related to the evaluation of the angiogenic activity of these biomaterials. Increasing evidence in the literature shows that ionic dissolution products released by bioactive glasses can stimulate not only osteogenesis, but also angiogenesis, promoting tissue regeneration 5,9. Angiogenesis is related to the formation of new blood vessels, which ensure the delivery of nutrients, growth factors and oxygen, as well as allowing stem cells to reach the injured site 27.
Bioactive glasses can be developed to release clinically beneficial ions such as copper (Cu+ and Cu2+) or zinc (Zn2+) 5,6. The release of copper and zinc may induce antibacterial mechanism. Additionally zinc is also referred to as inducing anti-inflammatory mechanisms 5,6.
Figure 3 illustrates the effects produced by some metal ions released from bioactive glasses. The release of these ions improves the therapeutic effects of bioactive glasses, including osteogenesis, angiogenesis, antibacterial and anti-inflammatory mechanisms 5.
Chapter 2 – State of the art
Figure 3: Effects of ions released from incorporated ions on bioactive glasses 5.
In addition to the osteogenic and angiogenic stimulation provided by the use of metal ion-doped bioactive glasses, the local release of antibacterial agents is another advantage of these biomaterials, overcoming the problem of bacterial resistance caused by the use of antibiotics. However, some toxicity may be associated with metal ion doped bioactive glasses, which requires consideration of the levels of metal ions used 5,9.
2.3 Sol-gel method
Bioactive glasses can be produced by two different methods, the traditional method, where the materials are melted at high temperatures (approximately 1400 °C) followed by a sudden cooling to solidification, without crystallization, or the sol-gel method. However, unlike the second method, the traditional method has demonstrated some limitations associated with the high melting temperatures involved, or the need of glass milling which may produce bioactive glasses with no homogeneous particle size distribution 28.
The sol-gel process involves two different methods of preparation: the colloidal and the alkoxide. While the colloidal method consists in the preparation of colloidal fine particle suspensions, the alkoxide method includes hydrolysis and condensation reactions of metal alkoxides. In this study, we will use the alkoxide method. This is a technique that over the years has been an alternative in the preparation of these materials as it allows their compositional and microstructural control at low processing temperatures 28–31. Metal alkoxides belong to the family of organometallic compounds, compounds containing at least one carbon-metal bond, such as tetraethyl orthosilicate (TEOS) 31.
Chapter 2 – State of the art Sol-gel synthesis allows the preparation of materials in a wide variety of shapes such as porous structures, fine fibers, dense powders and thin films 30,32.
The sol-gel process occurs in several steps, including hydrolysis and condensation, gelation and aging, each of which is described in more detail below 32.
Hydrolysis
In the hydrolysis step, which may come under acidic or basic conditions, nucleophilic attack occurs by OHZ- groups to the MZ+ metal cations, promoting increased coordination of the metal atom 31. Subsequently, the proton (H+) is transfered through an alkoxide group and the resulting R (OH) molecule is released. Equation 1 describes the hydrolysis process, where formation of reactive hydroxyl groups (M-OH) occurs, where R is an alkyl group, ROH an alcohol and M a metal 30,32.
M(OR)n + nH2o M(OR)n + nR(OH) (1)
The chemical reactivity of the metal alkoxides depends essentially on the "strength" of the nucleophilic agent, as well as on the electrophilic character of the metal atom, that is, its electronegativity. Silicon alkoxides, since silicon has a low electrophilicity value, exhibit relatively low reactivity. However, by the addition of catalysts the hydrolysis and condensation reactions can be accelerated.
The species formed must be soluble so that the polymerization and gelation processes can be carried out, to prevent precipitation of powders 30.
Condensation
During the condensation process three-dimensional polymer networks are formed due to the various condensation reactions. Within condensation steps the formation of units M-O-M occur with the loss of water molecules, equation 2 31. Condensation, lead to the formation of a larger molecule. Initially, the polymers formed during this polymerization process combine so as to occupy the entire volume occupied by the sol. M(OR)n + (OH)nM nM(O)M + R(OH)n (2)
Condensation is a dynamic process that can be improved by adjusting a number of parameters such as precursor type, alkoxide-water (RW) ratio, solvent and catalyst type, pH and order of addition of compounds 28,32.
When several alkoxides are used, hydrolysis of the most stable alkoxide is performed first, and then the one that reacts most rapidly. That is, since silicon alkoxides slowly hydrolyze compared to other alkoxides, they are first hydrolyzed and silanol groups are formed. These react quickly with other alkoxides to form new groups 28.
Chapter 2 – State of the art
Starting solution Sol Gel Dried monolithic gel
Gelation
Due to the condensation reactions, the formed aggregates collide and then bonds are formed between them. Repetition of this process causes the formation of a larger aggregate, that is, the gel. Thus, the gel is formed as soon as the last link between aggregates is established, thus forming a continuous solid network.
As the sol aggregates there is an increase in viscosity, and at when the sol-gel transition (gelling point) occurs, verifies that a sharp increase in viscosity 32. The gelation temperature is one of the factors that directly influences the product obtained, that is, with the increase of the gelation temperature there is an increase of the porosity of the gels 8.
Aging
Aging time refers to the amount of time a gel remains at the gelling temperature before drying. As the viscosity increases, the solvent remains within the gel as mentioned above 8. Condensation reactions continue to occur even after the gelation point is reached, together with the expulsion of liquid from the pores during aging, causing bonds to form with a consequent reduction in gel volume 8,32.
In the figure 4 it is possible to visualize the materials preparation scheme through the sol-gel process.
Figure 4: Scheme of preparation of materials by sol-gel process. Adapted by 33.
2.4 Hybrid materials in the PDMS- SiO
2system
In general, bioactive glasses exhibit little advantageous mechanical properties. In order to be able to improve these properties, hybrid materials emerged, as a combination of organic and inorganic materials.
During the last decade the synthesis of organic-inorganic hybrid materials has received special attention 10,11. This is because these materials have attracted a great interest in a variety of applications, notably anti-corrosion coatings or scaffold production 14. These
Chapter 2 – State of the art is incorporated into an inorganic network 10–14. Then, the polymer (the organic component) is added and inorganic chains are formed around the polymer molecules, as shown in Figure 5 12. One of the polymers most commonly used in the preparation of hybrid materials is polydimethylsiloxane, PDMS 12.
Figure 5: Formation of an Organically Modified Silicate (ORMOSIL) 12.
The characteristics of the formed ORMOSIL depend on the properties of the organic and inorganic components, as well as the conditions under which the process is performed 34. The materials formed from the reaction of tetraethyl orthosilicate, TEOS (inorganic component), with the PDMS have been the most studied. In this work, TEOS and PDMS will also be used as precursors. The advantage of using these two precursors is that it is possible to produce more or less flexible ORMOSIL's, depending on the amount of PDMS and TEOS that is used. That is, since PDMS has some flexibility, materials with a higher amount of TEOS will become less flexible. According to some studies, namely by Chen et
al.35, this combination also allows to obtain potentially bioactive ORMOSL's 10.
ORMOSILs can be divided into two classes, class I and class II, depending on the interactions between their inorganic and organic components 12,14,36.
Class I corresponds to the incorporation of the organic component into the inorganic one during condensation. Bonding between both components is accomplished through weak bonds between both phases, i.e. hydrogen bonds and / or Van der Waals forces 14,36. On the other hand, in class II, organic and inorganic components are bonded by strong bonds, i.e. covalent bonds 36. For this to happen, a coupling agent must be used to allow the strong bond between the silica and the polymer, or a polymer that already contains the silane bonds, such as PDMS 14.
Chapter 2 – State of the art
2.5 Zinc, copper and boron in tissue engineering
The addition of some elements, such as copper, zinc or boron, in bioactive glasses has been performed as a way to improve their properties when implanted in the body. This is because, when released into the body, these elements can contribute to antibacterial activity, or facilitate angiogenesis 5.
Zinc is related to bone formation, having anti-inflammatory effects and antimicrobial activity 5,9,37. The incorporation of copper in bioactive glasses leads to improvements in vitro and in vivo angiogenesis, further inducing cell differentiation 5,9. On the other hand, boron allows the stimulation of messenger ribonucleic acid (mRNA) synthesis, responsible for the transfer of information from DNA to the cytoplasm, promoting an angiogenic action 9,38.
A revision on the biochemical behaviour and therapeutic role of these ions will be presented in the following sections.
2.5.1 Zinc
Zinc is an essential chemical element for the human body, as it stimulates the activity of various enzymes, intervening in protein metabolism and DNA replication. Zinc is also known to play an important role in bone metabolism, stimulating bone formation in vitro by activating protein synthesis in osteoblastic cells, and having an anti-inflammatory effect 9,26. For this reason, the addition of this element to bioactive glasses has attracted some attention from the scientific community due to the combination of its non-toxicity, low cost and its ability to promote an anti-inflammatory and antibacterial effect, thus contributing to an improved biocompatibility of these materials 9,26.
Although the addition of zinc is beneficial as it has stimulatory effects on bone formation, it should be performed in a controlled manner as otherwise it may result in cytotoxicity. Generally, the dissolution of zinc-doped bioglasses in adequate amounts allows for controlled release of critical metal ion concentrations, further revealing beneficial anti-inflammatory effects 9,17,39.
2.5.2 Copper
Copper is an element widely distributed in nature, and it is considered essential in the body acting in numerous physiological and biochemical functions, being constituent of several metalloproteins and metalloenzymes 40. The presence of copper in bioglasses has the ability to increase cell proliferation, stimulate angiogenesis, and improve its mechanical properties (increases its microhardness and bending strength) 9. According
Chapter 2 – State of the art presence of copper, the authors reported a satisfactory association between angiogenesis growth factor, FGF-2 (Fibroblast Growth Factor-2), and copper.
Besides stimulating endothelial cell proliferation copper ions also contribute for the formation of capillary networks and play a role in extracellular matrix degradation and remodeling, by regulating gene expression and protein levels 16.
Copper is referred by many authors as a non-cytotoxic element until a certain limit, but it can occur cell destruction if the copper ion concentration is exceeded. Different authors found different copper cytotoxicity values for different cell cultures. Yinan Lin
et al.41 carried out a study in which they analyzed the release of copper ions from bioactive glass structures. The scaffolds included different copper concentrations (0 to 2 wt. % CuO) and after soaking in SBF the authors concluded that doped bioactive glasses with 0.8 wt. % copper concentration did not alter their biocompatibility. However for 2 wt. %, copper has already demonstrated a cytotoxic effect on MC3T3-E1 pre-osteoblastic cells, being detrimental to bone regeneration. A similar study was conducted by Hui Wang et al.42, varying copper concentrations between 0 and 3 wt. %, and using human bone marrow derived stem cells. The authors concluded that the 3 wt.% copper concentration showed no cytotoxicity to bone marrow derived human stem cells, representing an improvement in bone regeneration as well as an increase in angiogenesis capability when compared to undoped scaffolds 43,44.
2.5.3 Boron
Boron is an element with an extremely important role in various processes that take place in the body, including bone growth and maintenance, immune function and stimulation of estrogen production. The use of estrogen for the treatment of bone loss caused by menopause has proved to be one of the most effective methods. In this sense, the study of the addition of boron in bioactive materials for bone recovery has received special interest 45,46.
According to the literature, boron allows stimulation of mRNA translation, which in turn, allows stimulation of growth factors involved in angiogenesis 9,47.
Once the use of boron improves the osteogenic and angiogenic properties of biomaterials, it seems obvious that the controlled release of boron ions from bioactive glass is a promising alternative for tissue engineering 48,49.
2.6 Polydimethylsiloxane
Polydimethylsiloxane, or PDMS, is one of the most widely used polymeric materials in tissue engineering. Its applications include, among others, scaffolds biomaterials due to their durability and low reactivity when immersed in aqueous solutions 50–53. Other features that make this polymer so special are its high biocompatibility, flexibility, low toxicity and
Chapter 2 – State of the art high thermal and oxidative stability 50,51,54. Despite these advantages, the high utilization rate of PDMS is still achieved due to its low cost 50. However, there is usually a need for additional modifications to the surface properties of PDMS due to its extreme hydrophobicity, in some cases corresponding to a water contact angle greater than 150⁰, and ease of non-specific protein adsorption 50,52–57.
In last years, PDMS has been associated with inorganic materials, such as silica, thus producing an ORMOSIL (PDMS-SiO2). The combination of the two materials has shown
promising results for biomedical applications, since ORMOSIL´s PDMS-based have revealed high bioactivity, leading to the formation of an apatitic layer when soaked in SBF
53,58
. Compoint et al.58 shows that the association of PDMS with in a silica network allows adjusting the elastic properties of the material.
Although PDMS is an excellent implant material for various tissue engineering applications, it often fails due to lack of antibacterial activity 15. Thus, the incorporation of nanoparticles on the surface of PDMS has proved to be the most viable alternative, affecting bacterial populations and thus improving their antibacterial properties. 59. Both the contact angle of the material and the hydrophobicity of the cell surface directly influence the extent of bacterial fixation. The authors observes a change in hydrophobicity and surface roughness of the material, resulting in a reduction in initial bacterial adhesion, that is, an increase in PDMS antibacterial activity.
Due to the anti-inflammatory properties of zinc, ZnO nanoparticle has been investigated in order to understand its action when in ZnO-PDMS systems. Similar to other studies performed 15, also Ammar et al. 60, wanted to check which would be the improvement relatively to corrosion as well as surface properties of PDMS, with the addition of ZnO. This led to the conclusion that there was an improvement in the hydrophobicity as well as the roughness.
The coating of CuO nanocomposites on the PDMS surface, as mentioned above, promotes significant antibacterial behavior as well as appropriate biocompatibility to enable its use in tissue engineering. Tavakoli et al.59 verified, recently, been that in fact, the incorporation of CuO nanoparticles into PDMS-SiO2 coatings has proved to be a promising technique, because it has improved the material hydrophobic properties and roughness, leading to a significant increase in the antibacterial rate.
None of the aforementioned articles evaluated antibacterial activity in function of the release of zinc or copper ions. In the articles cited above, only the increase of antibacterial characteristics was observed by changing the hydrophobic / hydrophilic character and the surface roughness of the materials.
Chapter 2 – State of the art these materials, copper and zinc ions, referred in the literature as stimulatory of bactericidal effects, have been added to the base system. The addition of copper and zinc ions to this hybrid is a new approach, as it is reported in the literature that these ions have only been used as a PDMS coating to form a composite. Thus, it is expected that this new approach will provide hybrid materials with unique characteristics, high potential for use as scaffolds in biomedical applications and the possibility of other applications in the form of films or coatings.
Chapter 3 – Experimental Procedure
Chapter 3
Experimental Procedure
Throughout chapter 3, the experimental procedures and characterization techniques used will be specified. Initially, will be mentioned the various reagents used for the synthesis of formulations in the PDMS-SiO2-B2O3-Zn and PDMS-SiO2-B2O3-Cu systems, as well as a
detailed description of both procedures using the sol-gel process.
The structural and microstructural analysis techniques to characterize the obtained samples will be described, as well as evaluation of copper and zinc ion release kinetics by deionized water immersion tests.
The analysis of the antibacterial activity of the samples will be performed through antibacterial assays and the experimental protocol will be detailed.
3.1 Materials
The reagents used in both experimental procedures are presented in table 1, as well as their chemical formula, purity and brand. For the preparation of the zinc-containing samples hydrochloric acid was used, while in the copper-containing samples formic acid was used.
Table 1: Reagents, chemical formula, purity and brand.
Reagents Chemical Formula Purity (%) Brand
Tetraethyl orthosilicate (TEOS) C8H20O4Si > 99.0 %
Sigma-Aldrich #BCBV4115
Propanol anhydrous (IPA) C3H8O > 99.5 %
Sigma-Aldrich #STBG2375V Poly(dimethylsiloxane)silanol
terminated (550 g/mol average molecular weight) (PDMS)
(C2H6OSi) n - Sigma-Aldrich
#MKBS9904V0
Hydrochloric acid (HCl) HCl - Panreac
0000441340
Formic acid (CH2O2) CH2O2 99-100%
Analar NORMAPUR 20318.320
Lote: 14k100501
Trimethyl borate (TMB) B(OCH3)3 > 99.0 %
Fluka 2044689 Zinc acetate dihydrate (ZnAc) C4H6O4Zn·2H2O > 98 % Sigma-Aldrich
#MKBW6784V Copper (II) acetate monohydrate (CuAc) Cu(OOCCH3)·H2O > 98 % Alfa aesar
Chapter 3 – Experimental Procedure
3.2 Samples preparation
Hybrid materials of the PDMS-SiO2-B2O3 system were prepared by the sol-gel process at
room temperature. The antibacterial properties were studied thought the addition of zinc or copper ions. To comply these objectives six different compositions based on PDMS-SiO2
-B2O3 system have been prepared, three different compositions for zinc-containing samples
and three different compositions for copper-containing samples.
The composition of the samples, in molar ratio, and respective notation, are presented in table 2, for zinc-containing samples, and in table 3, for copper-containing samples. The hybrid materials arise from the combination of organic and inorganic materials, so, in this work, the inorganic part of these hybrid materials is provided by tetraethyl orthosilicate (TEOS), trimethyl borate (TMB) and copper (II) acetate monohydrate (CuAc) or zinc acetate dihydrate (ZnAc), while the organic part is provided by polydimethylsiloxane silanol terminated (550 g/mol average molecular weight) (PDMS). The materials were prepared using a fixed molar ratio composition, so, for zinc-containing samples preparation the following molar ratio was used: IPA:TEOS = 7.5:1, HCl:TEOS = 0.68:1, TMB:TEOS = 0.10:1 and PDMS:TEOS = 0.18:1. For the preparation of copper-containing samples other molar ratios were used: IPA:TEOS = 19:1, CH2O2:TEOS = 0.08:1, TMB:TEOS =
0.10:1 and PDMS:TEOS = 0.18:1.
Table 2: Composition (in molar ratio) of zinc-containing samples. Notation Composition (in molar ratio)
TMB/TEOS ZnAc/TEOS
B10Zn2.5 0.10 0.025
B10Zn5 0.10 0.05
B10Zn10 0.10 0.10
Table 3: Composition (in molar ratio) of copper-containing samples. Notation Composition (in molar ratio)
TMB/TEOS CuAc/TEOS
B10Cu2.5 0.10 0.025
B10Cu5 0.10 0.05
Chapter 3 – Experimental Procedure Initially, a previously sol-gel process developed in a early work from the PDMS-SiO2
-B2O3-CaO system was replicated 18, however a phase separation was observed, probably
due to the presence of water and / or acetone (Annex I). Knowing that line acetate dissolves in isopropanol a new approach was developed in which we initially removed water and acetone. Although this new methodology worked for the procedure involving the addition of zinc acetate, this did not occurs when copper acetate was added. In this case a precipitation occurred, forming copper chloride dihydrate (Annex I).
Figure 6 shows the experimental protocol used to produce the PDMS-SiO2-B2O3-Zn
system hybrid materials. IPA and ZnAc were initially added, always under continuous stirring, using a magnetic stirrer, and at room temperature. After addition of ZnAc, the medium was acidified by the addition of HCl, that was used as a catalyst to the hydrolysis / condensation process. The solution remained under the magnetic stirrer to which after 20 minutes TMB was added. Subsequently, the solution continued to be stirred continuously for 45 minutes, TEOS was then added and it was kept on the magnetic stirrer for further 45 minutes. Finally, PDMS was added and then stirred for 1 hour at room temperature.
After stirring, the final suspension was divided into three equal petri dishes, which were kept at room temperature for 2 days for aging, remaining liquid and transparent. Later, to gelify the samples, they were placed in the oven at 60 ⁰C for 3 days. Finally, in order to remove all solvent from the samples, they were placed for 1 hour in an oven at 150 ⁰C. Figure 7 shows the zinc-containing samples after stirring, 2 days at room temperature as well as the monolithic samples obtained after the drying step. All zinc-containing samples, after dried at 150 ⁰C during 1 hour, resulted in white opaque and homogeneous monolithic samples.
Chapter 3 – Experimental Procedure
Chapter 3 – Experimental Procedure As noted before, replication of the procedure used with zinc did not lead to satisfactory results with copper, once there was the formation of a precipitate, copper chloride dihydrate. Thus, in a first attempt the HCl was replaced by nitric acid (HNO3), but another
precipitate, copper nitrate hydroxide, did form (Annex I).
After a number of experimental failures it was decided to discard the above acids and use a completely organic acid: formic acid (CH2O2). In this case, two different copper acetates,
monohydrate and dihydrate, were used in separate procedures, in order to understand which would lead to more satisfactory results. Copper (II) acetate monohydrate showed better results, with a lower amount of CH2O2 added. Thus it was used in all further
preparations with copper.
The experimental procedure used to produce the hybrid materials of the PDMS-SiO2-B2O3
-Cusystem, is shown in figure 8. The materials have been prepared by adding IPA, ZnAc, using the molar ratios previously described. The prepared solutions were then stirred for 45 minutes at room temperature. After that time, TEOS and TMB were added, as copper is difficult to dissolve, it was necessary to bring the solution to the ultrasonic processor for 3 minutes at 70 %. The ultrasonic processor used was the VCX130 model, vibrating cell, sound, with the following characteristics, power = 130 W and frequency = 20 KHz. Subsequently, PDMS was added and the medium was acidified with CH2O2. The final
mixture was dissolved again with the aid of the ultrasonic processor, during 3 minutes at 70%, and then stirred, on the magnetic stirrer, for 30 minutes at room temperature and divided equally by three petri dishes.
Mixtures were aged, after stirring, for 2 days at room temperature and posteriorly placed in an oven at 60 ºC during 3 days to gellify. Afterwards, the samples were dried at 150 ºC during1 hour. Finally, all copper-containing samples resulted in blue translucent monolithic samples with homogeneous appearance, as shown in figure 9.
Chapter 3 – Experimental Procedure
Chapter 3 – Experimental Procedure
3.3 Characterization Technics
Each zinc and copper monolith samples obtained by the methods described above were characterized at structural and microstructural level. Then, we will be briefly describes the characterization techniques that were performed in the materials. The samples were first pulverized in agate mortar in order to create thin powders with a specific particle size.
3.3.1 X-ray diffraction (XRD)
X-ray diffraction analysis (XRD) is one of the structural analysis methods used for the identification and quantification of crystalline phases in the material. This technique consists of directing a monochromatic X-ray beam to the material to be analyzed. Thus, the cathode ray tube generated the X-rays which are filtered to produce monochromatic radiation, then, the X-rays are collimated to concentrate and directed towards the sample. This interaction, between the incident rays and the sample, generates a constructive interference (and a diffracted ray) when the conditions satisfy the Bragg Law (n λ = 2dsinθ) 61
. This law relates the wavelength of electromagnetic radiation to the diffraction angle and the lattice spacing in a crystalline sample. According to Bragg Law, when the X-ray is incident on a crystal surface, its angle of incidence, Ɵ, will reflect back with the same scattering angle, Ɵ. When the path difference, d, is equal to an integer, n, wavelength, λ, constructive interference occurs 62. When sweeping the sample over a range of 2θ angles, all possible directions of diffraction of the net must be obtained due to the random orientation of the powder material. The phase identification is made by comparison with pure phase diffractograms. While the position of the peaks depends on the crystalline phases of the sample, their intensity is related to the abundance of each phase in the sample. On the other hand, the width of the peaks is related to the size of the lens, possible network defects and diffractometer optics.
In this work, XRD analysis was important because it allowed us to verify the amorphous character of the hybrid material. A Pan Analytical-X'pert-PRO diffractometer equipped with a copper anode (Cu), which operates with a current of 40 mA and a voltage of 45 kV, emitting a Kα1 radiation of λ = 1.540598 Å and Kα2 of λ = 1.544426 Å was used. The
spectra were recorded through a continuous scan from the smallest possible angle, which was 2θ = 3 ⁰, to 2θ = 60 ⁰, with a pitch size of 0.0263 ⁰ and a pitch time of 150 seconds, at room temperature. The identification of the crystallographic phases was done using the data of the powder diffraction file (PDF) provided by JCPDS / ICDD (Joint Committee of Diffraction Patterns of Dust / International Center for Diffraction Data, ICDD).
Chapter 3 – Experimental Procedure
3.3.2 Fourier transform infrared (FT-IR) spectroscopy
Fourier transform infrared (FT-IR) spectroscopy has been a successfully analytical technique to study the chemical bonds or molecular structure of different materials. The FT-IR analysis method uses infrared light to scan test samples and observe chemical properties. When the sample molecules are exposed to infrared radiation, usually around 10,000 to 100 cm-1, they selectively absorb the radiation of specific wavelengths, which in turn, cause the dipole moment change of the sample molecules. Consequently, the vibrational energy levels of the sample molecules transfer from the ground state to the excited state. The emitted wavefront beam (analog spectral output) hits the detector and generates an electrical signal as a response. These processing of data, to convert the collected data to the desired result are done using the Fourier transform. The resulting signal in the detector is presented as a spectrum, typically between 4000 and 400 cm-1, thereby representing the molecular fingerprint of the sample. This method of structural characterization is a fast non-destructive technique that allows the determination of the functional groups present in the sample, since the intensity and position of each peak corresponds to the vibrational mode of a specific chemical bond 63.
In order to analyze the different samples, two distinct regions recorded in a Bruker Tensor model 27 were used. One analysis corresponded to the region of 350 - 1500 cm-1 with a resolution of 4 cm-1 and 128 scans, while the other occurred in the range of 700 - 1300 cm-1 with a resolution of 2 cm-1 using 250 scans. The preparation of the material to be analyzed consisted of mixing the powdered material with a small amount of potassium bromide (KBr), which was then pressed so as to produce fine grains. Subsequently, the spectra were obtained by subtraction with the pure KBr spectrum and were reported in transmittance mode.
3.3.3 Nuclear magnetic resonance (NMR)
Nuclear magnetic resonance (NMR) spectroscopy is a technique that allows the observation of local magnetic fields around atomic nuclei. The principle of this technique is based on the fact that many nuclei have spin and all nuclei are electronically charged. Thus, the molecules are placed on the action of a strong magnetic field, generating energy levels for atoms with nonzero angular momentum of spin. In turn, radiofrequency absorption will cause excitation of some nuclei, and the frequency and intensity of radiation absorbed by the various atoms is very accurately measured. The spectrometers can then be adjusted to multiple cores, as is the case with 1H and 11B 64.
In this work, samples were characterized by solid-state 1H NMR at 12 KHz sample rotation speed, using a probe of 4 mm, with a pulse width of 3 μs, and a delay time of 5 sand 11B HAHN-ECHO NMR, using a Bruker Avance III HD 700MHz (16.4 T), with a pulse length
Chapter 3 – Experimental Procedure In the present work it is important to determine the 1H NMR and 11B NMR spectra, once on the one hand the first technique will be useful because it allows to solve the physisorbed water peaks, hydrogen-linked water, hydrogen-linked silanol and isolated silanol with different hydrogen bonding forces. On the other hand, the second technique will be important in determining the coordination of boron in the samples. Spectra were simulated using DMFIT software and analyzed according to literature data.
3.3.4 Specific surface area by Brunauer-Emmett-Teller (BET) method
The physical nitrogen adsorption method developed by Brunauer, Emmett and Teller (BET) can, essentially, considered a mathematical way of analyzing the adsorption isotherm. In this work, this technique was used to determine the specific surface area (SSA) of the hybrid materials produced, which is defined as the surface area of a solid particle per unit mass, usually expressed in m2/g. Previously, the samples were degassed at 150 °C for 12 hours and cooled to room temperature. This analysis was then performed on Micromeritics - Germini 2370V5 equipment, where at least 30 points were acquired with a 5-second equilibrium time.The principle of this technique is to place known amounts of an inert gas, usually nitrogen, into the sample in which the particles adsorb a molecular layer of nitrogen. Pressures lower than atmospheric pressure is obtained by creating partial vacuum conditions. When the saturation pressure is reached, adsorption no longer occurs, regardless of any increase in pressure. After formation of the adsorption layers, the sample is removed from the nitrogen atmosphere and heated so that the adsorbed nitrogen is released from the material and quantified. Finally, the data collected is displayed as a BET isotherm representing the amount of adsorbed gas as a function of relative pressure (p / p0), where p0 is the saturation vapor pressure of the adsorbed substance at the temperature at which assay is performed. The amount of adsorbed gas will depend on the exposed surface as well as the temperature, gas pressure and interaction force between gas and solid.
In the context of physiology, depending on pore size, the materials can be classified into macroporous, mesoporous and microporous. Thus, pores with widths greater than about 50 nm (0.05 μm) are referred to as macropores, widths between 2 nm and 50 nm correspond to mesopores and pores with smaller than 2 nm micropores. There are 6 types of possible isotherms obtainable, according to the International Union of Pure and Applied Chemistry (IUPAC). Figure 10 shows the different graphs corresponding to each type of isotherms, as well as the description of each one 65.
