Biomaterials Based On Synthetica And Natural Polimers
Contents
CHAPTER 1. TISSUE ENGINEERING
1.1. INTRODUCTION
1.2. NATURAL AND SYNTHETIC POLYMERS
1.2.1. Chitosan
1.2.2. Poly(vinyl-alcohol) (PVA)
1.2.3. Polymer blending
1.2.4. Alginate
1.3. GRAPHENE/ GRAPHENE OXIDE (GO) MATERIALS
1.3.1. Graphene
1.3.2. Graphene Oxide (GO)
1.3.3. Graphene oxide properties
1.3.4. Synthesis of Graphene Oxide (GO)
1.3.5. Applications
1.3.6. Graphene oxide-polymer composites
CHAPTER 2. DRUG DELIVERY
2.1. INTRODUCTION
2.2. MESOPOROUS MATERIALS
2.3. MESOPOROUS SILICA NANOPARTICLES (MSN)
2.3.1. Synthesis
2.3.2. Structure of MSN
2.3.2. Properties
2.3.4. Applications
CHAPTER 3. OBJECTIVES AND ORIGINALITY
CHAPTER 4. CHARACTERIZATION METODS
CHAPTER 5. MOLECULAR MODELING OF MECHANICAL PROPERTIES OF THE CHITOSAN BASED GRAPHENE COMPOSITES
5.1. SIMULATION METHODOLOGY
5.2. EQUILIBRATION OF THE BULK MODEL
5.3. CHARACTERIZATION
5.4. CONCLUSIONS
CHAPTER 6. NANOCOMPOSITES BASED ON BIOPOLYMERS AND GRAPHENE OXIDE
6.1. EXPERIMENTAL STUDY OBJECTIVES
6.2. RAW MATERIALS
6.2.1. Characterization of GO
6.3. NANOCOMPOSITE FILMS BASED ON CHITOSAN AND GRAPHENE OXIDE
6.3.1. Preparation of graphene oxide-based Chitosan composite films
6.3.2. Structural and morphological characterization
6.3.3. Thermal and mechanical characterization
6.3.4. Biocompatibility assessment
6.3.5. Conclusions
6.4. NANOCOMPOSITE FILMS BASED ON CHITOSAN- POLYVINYL ALCOHOL AND GRAPHENE OXIDE
6.4.1. Preparation of PVA-Chitosan/graphene oxide composite films
6.4.2. Structural and morphological characterization
6.4.3. Thermal and mechanical characterization
6.4.4. Biocompatibility assessment
6.4.5. Conclusions
6.5. NANOCOMPOSITES BASED ON ALGINATE AND GRAPHENE OXIDE
6.5.1. Preparation of graphene oxide-based sodium alginate composite films
6.5.2. Characterization
6.5.3. Conclusions
6.6. NANOCOMPOSITE 3D SCAFFOLD BASED ON CHITOSAN AND GRAPHENE OXIDE
6.6.1. Preparation of graphene oxide-based Chitosan composite 3D scaffold
6.6.2. Characterization
6.6.3. Mineralization assay
6.6.4. Conclusions
6.7. NANOCOMPOSITE 3D SCAFFOLDS BASED ON CHT-PVA AND GO
6.7.1. Preparation of graphene oxide-based Chitosan composite 3D scaffold
6.7.2. Characterization
6.7.3. Mineralization assay
6.7.3. Conclusions
CHAPTER 7. HYBRID MATERIALS BASED ON MESOPOROUS SILICA NANOPARTICLES AND DIFFERENT INORGANIC GUEST FOR DRUG DELIVERY
7.1. EXPERIMENTAL STUDY OBJECTIVES
7.2. RAW MATERIALS
7.3. ADSORPTION OF THE DRUG ONTO MESOPOROUS SILICA PORE OR SURFACE
7.3.1. Experimental protocol
7.3.2. Characterization of MSN modified with BZC
7.3.3. The influence of reaction parameters on the interaction between drug and MSN
7.4. SYNTHESIS OF POLYMER-BZC AND POLYMER-MSN-BZC HYBRID MATERIALS
7.4.2. In vitro drug release
7.5. CONCLUSIONS
CHAPTER 8. GENERAL CONCLUSIONS AND FUTURE PERSPECTIVES
CHAPTER 9. BIBLIOGRAPHY
Publication list
Figure 1-1 Properties and characteristics to consider for the design of optimal scaffolds for bone tissue engineering 16
Figure 1-2 Chemical crosslinking of chitosan with a) dicarboxylic acid, b) glutaraldehyde, c) epichlorohydrin (21) 18
Figure 1-3. Physico-chemical and biological properties of chitosan 19
Figure 1-4 Chitosan chemical structure (20) 20
Figure 1-5 Structure of poly(vinyl-alcohol) (44) 23
Figure 1-6 Effect of molecular weight and hydrolysis level on the physical properties of PVA (46) 24
Figure 1-7 Formation of hydrogen bonds between PVA chains and between PVA and CS chains 26
Figure 1-8 Structural of alginate acid (58) 27
Figure 1-9 Possible junction point in alginate: a) GG/GG junction; b) MG/MG junction; c) GG/MG junction (58) 28
Figure 1-10 The mechanism of the acid catalyzed hydrolysis of methyl-glycopyranosides (69) 29
Figure 1-11 The proposed structure of graphene oxide (96) 33
Figure 1-12 Oxidation of graphite to graphene oxide(93) 34
Figure 1-13 Biomedical applications of graphene and graphene oxide (107) 35
Figure 2-1 Comparison between classical mechanisms (a) and controlled release systems (b)(134) 40
Figure 2-2 Mesophase structures of M41S: a) MCM-41, b) MCM-48 and c) MCM-50 (42) 42
Figure 2-3 Schematic illustration of silanol group 45
Figure 2-4 Mesoporous silica agglomeration 45
Figure 2-5 Controlled release mechanisms (154) 46
Figure 5-1 The computational bulk model of A) graphene/CHT (7.67 wt. % graphene) and B) graphene/CHT (14.28 wt. % graphene) after equilibration process 59
Figure 6-1 FT-IR spectrum of GO 62
Figure 6-2 XPS Spectrum of GO 63
Figure 6-3 Raman Spectum of GO 64
Figure 6-4 The XRD Spectrum of GO 64
Figure 6-5 TEM image of GO 65
Figure 6-6 Experimental protocol 66
Figure 6-7 (A) SEM image of the Chitosan film surface, (B) Chitosan/GO (2.5 wt%) film surface, (C) Chitosan fracture surface, (D) Chitosan/GO (2.5 wt%) fracture surface 68
Figure 6-8 High-resolution TEM micrograph of the chitosan/GO (2.5 wt%). 68
Figure 6-9 FT-IR spectra of the (A) GO, (B) Chitosan and (C) Chitosan/GO biocomposite film 2.5 wt%. 69
Figure 6-10 XRD patterns of (A) GO, (B) Chitosan, (C) Chitosan/GO 0.5 wt%, and (D) Chitosan/GO 2.5 wt% films 70
Figure 6-11 TG analysis curves of CHT and CHT/GO composites 72
Figure 6-12 Tensile stress versus strain curves of neat chitosan and chitosan/GO composite films 73
Figure 6-13 Fluorescence microscopy evaluation of living (green-labelled) and dead (red-labelled) murine osteoblasts at 2, 4 and 7 days post seeding on 2D control, Chitosan and Chitosan/GO composite materials 75
Figure 6-14 (A) Quantification of murine osteoblasts proliferation rate on Chitosan and Chitosan/GO films as revealed by MTT assay, (B) cytotoxic potential of the Chitosan and Chitosan/GO films in contact with osteoblasts as revealed by LDH assay 77
Figure 6-15 Experimental protocol 80
Figure 6-16 FT-IR spectra of the A) GO, B) CHT-PVA, and C) CHT-PVA/GO 2.5 wt. % 80
Figure 6-17 Raman spectra of A) GO, B) CHT-PVA/GO composite film with 1 wt.% GO content and C) CS-PVA/GO composite film with 6 wt. % GO content 81
Figure 6-18 XRD patterns of A) GO, B) CHT-PVA, C) CHT-PVA/GO 2.5 wt.% and D) CHT-PVA/GO 6 wt.% 82
Figure 6-19 (A) SEM image of the CHT-PVA film surface, (B) CHT-PVA film fracture surface, (C) CHT-PVA/GO 2.5 wt. % film surface, (D) CHT-PVA/GO 2.5 wt. % film fracture surface 83
Figure 6-20 High-resolution TEM micrograph of the CHT-PVA/GO 2.5 wt. % 84
Figure 6-21 The TGA curves of CHT-PVA and CHT-PVA / GO nanocomposites 85
Figure 6-22 Tensile strain versus tensile stress for CHT-PVA and CHT-PVA / GO films 87
Figure 6-23 The cytotoxic potential of the CHT-PVA/GO films in contact with osteoblasts at 2, 4 and 7 days of culture, as revealed by LDH assay 89
Figure 6-24 The quantification of cell proliferation rate on CHT-PVA/GO films as revealed by MTT test at 2, 4 and 7 days of culture 89
Figure 6-25 Fluorescence microscopy evaluation of living (green-labeled) and dead (red-labeled) murine osteoblasts at 2, 4 and 7 days post-seeding on CHT-PVA and CHT-PVA/GO films. 90
Figure 6-26 FTIR spectra of A) Al, B) GO, and C) Al/GO composite film with 2.5 wt.% 93
Figure 6-27 XRD patterns of A) GO, B) Al and Al/GO composites with different GO content: C) 1% and D) 2.5 % 94
Figure 6-28 TEM images of Al/GO 2.5 wt % composite films 95
Figure 6-29 SEM film surface images (A-B) and fracture surface images (C-D) of (A, C) Al, (B, D) Al/GO 1 wt %. 96
Figure 6-30 Schematic representation Al/GO composite films structure 97
Figure 6-31 TGA curves of neat Al and Al/GO composite films. Inset: Magnification of first stage of degradation. 98
Figure 6-32 Mechanical behavior of Al and Al / GO films 99
Figure 6-33 Experimental protocol 101
Figure 6-34 Raman Spectra of A) GO; B) composite CHT/GO 0.5 wt.%; C) CHT/GO 1 wt.%; D) CHT/GO 2 wt.% and E) CHT/GO 3 wt.% 102
Figure 6-35. SEM Images for A) CHT; B) CHT/GO (0.5 wt.%); C) CHT/GO (1 wt.%); D) CHT/GO (2 wt.%); E) CHT/GO (3 wt.%) 104
Figure 6-36 Swelling degree in water versus time for CHT/GO scaffolds and CHT control after 4 h 105
Figure 6-37 Maximum liquid uptakes for the investigated porous CHT/GO composite materials 106
Figure 6-38 In vitro degradation behavior for the designed scaffolds. The degradation was studied at 37°C in the presence of lysozyme 107
Figure 6-39 Experimental protocol 109
Figure 6-40 SEM images and EDAX spectra of the morphology of mineral deposits onto the surface for A) CHT; B) CHT/GO 1 wt.%; C) CHT/GO 3 wt.% 110
Figure 6-41 XRD-HA 111
Figure 6-42 SEM Images for A) CHT_PVA; B) CHT-PVA/GO (0.5 wt.%); C) CHT-PVA/GO (1 wt.%); D) CHT-PVA/GO (2 wt.%); E) CHT-PVA/GO (3 wt.%) 114
Figure 6-43 XRD curves of CHT-PVA and composites after mineralization 115
Figure 6-44 SEM images of CHT-PVA A) and CHT-PVA with 1 wt.% GO B), 2 wt.% GO C) and 3 wt.% GO D) after mineralization 116
Figure 7-1 Adsorption of the drug onto mesoporous silica surface or pore 119
Figure 7-2 The FT-IR spectra for MSN, BZC and MSN modified with BZC 120
Figure 7-3 The XPS survey spectra for MSN and modified MSN with BZC 121
Figure 7-4 TGA curves for MSN, BZC and MSN modified with BZC 122
Figure 7-5 The influence of the contact time of BZC 123
Figure 7-6 Intra-particle diffusion model for BZC adsorption onto MSN 125
Figure 7-7 Adsorption of BZC at different temperature 126
Figure 7-8 Adsorption of BZC at different pH values 127
Figure 7-9 The influence of drug concentration 128
Figure 7-10 Synthesis of hybrid materials based on CHT/Al-MSN-BZC 128
Figure 7-11 UV-VIS spectra recorded for solution BZC 130
Figure 7-12 The calibration curve for determining the amount of BZC 130
Figure 7-13 The percent of drug release from CHT and CHT-MSN hybrid materials 131
Figure 7-14 The percent of drug release from Al and Al-MSN hybrid materials 132
List of table
Table 2-1: Porous materials classification 41
Table 5-1 The characteristics of the CS and the CS-graphene computational models 57
Table 5-2 Step of equilibration protocol 58
Table 5-3 Elastic module for the CS and CS-graphene computational bulk models 60
Table 6-1 Temperature at which the mass loss is 3%, tensile modulus and tensile strength of neat Chitosan and Chitosan/GO composite films 72
Table 6-2 The ID/IG of GO and the CS-PVA/GO composite films 81
Table 6-3 Td3% and tensile modulus of CHT-PVA blend and CHT-PVA/GO nanocomposites 86
Table 6-4 XRD data of Al and Al /GO composite films 95
Table 6-5 Decomposition temperature and mechanical properties of neat Al and Al/GO composite films 98
Table 6-6 The ID/IG of GO and the CS/GO composite 3D scaffold 103
Table 7-1 FT-IR spectra assigned of MSN and BZC 121
Table 7-2 Kinetic parameters for BZC adsorption onto MSN 124
Table 7-3 Thermodynamic parameters for BZC adsorption onto MSN 126
Table 7-4 Absorbance of solutions obtained by diluting the standard solution measured at 262 nm 129
ACKNOWLEDGEMENTS
Even if the thesis has one author it would not have been achieved without the support of many people who I am grateful.
First, I would like to thank to my adviser Prof. dr. ing. Horia Iovu who gave me the opportunity to develop this research topic and for his support and guidance offered throughout this thesis.
I would like to address special thanks to Dr. Mariana Ionita and Dr. Sorina Alexandra Garea for theirs guidance, encouragement and support offered during the thesis.
I gratefully acknowledge to a special person who had an important role in my research, Corina Andronescu. She gave me a lot of advices in thesis writing and she is friend in daily life. She's the reason I came to be part of this group.
Thanks to Dr. Celina Damian which made me a great pleasure to work with when I joined this group. Thanks to Dr. Adi Ghebaur for the help and the explanations offered in the drug release field. Also to Dr. Catalin Zaharia and Dr. Adriana Lungu for helping me with mineralization assay and enzymatic degradation studied.
Also thanks to Dr. Eugeniu Vasile, Roxana TMSN with BZC 121
Figure 7-4 TGA curves for MSN, BZC and MSN modified with BZC 122
Figure 7-5 The influence of the contact time of BZC 123
Figure 7-6 Intra-particle diffusion model for BZC adsorption onto MSN 125
Figure 7-7 Adsorption of BZC at different temperature 126
Figure 7-8 Adsorption of BZC at different pH values 127
Figure 7-9 The influence of drug concentration 128
Figure 7-10 Synthesis of hybrid materials based on CHT/Al-MSN-BZC 128
Figure 7-11 UV-VIS spectra recorded for solution BZC 130
Figure 7-12 The calibration curve for determining the amount of BZC 130
Figure 7-13 The percent of drug release from CHT and CHT-MSN hybrid materials 131
Figure 7-14 The percent of drug release from Al and Al-MSN hybrid materials 132
List of table
Table 2-1: Porous materials classification 41
Table 5-1 The characteristics of the CS and the CS-graphene computational models 57
Table 5-2 Step of equilibration protocol 58
Table 5-3 Elastic module for the CS and CS-graphene computational bulk models 60
Table 6-1 Temperature at which the mass loss is 3%, tensile modulus and tensile strength of neat Chitosan and Chitosan/GO composite films 72
Table 6-2 The ID/IG of GO and the CS-PVA/GO composite films 81
Table 6-3 Td3% and tensile modulus of CHT-PVA blend and CHT-PVA/GO nanocomposites 86
Table 6-4 XRD data of Al and Al /GO composite films 95
Table 6-5 Decomposition temperature and mechanical properties of neat Al and Al/GO composite films 98
Table 6-6 The ID/IG of GO and the CS/GO composite 3D scaffold 103
Table 7-1 FT-IR spectra assigned of MSN and BZC 121
Table 7-2 Kinetic parameters for BZC adsorption onto MSN 124
Table 7-3 Thermodynamic parameters for BZC adsorption onto MSN 126
Table 7-4 Absorbance of solutions obtained by diluting the standard solution measured at 262 nm 129
ACKNOWLEDGEMENTS
Even if the thesis has one author it would not have been achieved without the support of many people who I am grateful.
First, I would like to thank to my adviser Prof. dr. ing. Horia Iovu who gave me the opportunity to develop this research topic and for his support and guidance offered throughout this thesis.
I would like to address special thanks to Dr. Mariana Ionita and Dr. Sorina Alexandra Garea for theirs guidance, encouragement and support offered during the thesis.
I gratefully acknowledge to a special person who had an important role in my research, Corina Andronescu. She gave me a lot of advices in thesis writing and she is friend in daily life. She's the reason I came to be part of this group.
Thanks to Dr. Celina Damian which made me a great pleasure to work with when I joined this group. Thanks to Dr. Adi Ghebaur for the help and the explanations offered in the drug release field. Also to Dr. Catalin Zaharia and Dr. Adriana Lungu for helping me with mineralization assay and enzymatic degradation studied.
Also thanks to Dr. Eugeniu Vasile, Roxana Trusca and Dr. Getuta Voicu for morphological analyzes performed in this research.
I thank my colleagues Dr. Paul Stanescu, Dr. Izabela Cristina Stancu, Brindusa Balanuca, Claudiu Ciobotaru, Dr. Nicoleta Florea, Dr. Florentina Constantin, Livia Crica, Anda Mihai, Ileana Duta for the support and help. I would like to thank to my high school friend, Simona Sanda-Mir who does not guide me scientifically but she helped me each time I have an English problem.
Finally to my family and friends who were patient with me and support me during all this time. Thanks to my baby who was very silent and allowed me to continue my doctoral work after childbirth returning to work when he was only a month.
The work has been funded by the Sectoral Operational Programme Human Resources Development 2007-2013 of the Romanian Ministry of Labour, Family and Social Protection through the Financial Agreement POSDRU/107/1.5/S/76903 and by the National Research Grant PN-II-PCCA-140, CNCS-UEFISCDI.
State of the art
TISSUE ENGINEERING
1.1. INTRODUCTION
Life quality improvement is one of the most important objectives of global research efforts. Tissue engineering known as regenerative medicine is an interdisciplinary field found in constant development, which combines the knowledge and skills of several disciplines including engineering, biology and medicine. By restoring, maintaining or enhancing tissue and organ function, tissue engineering is based on the discovery of ways to improve the health and the quality of life for millions of people around the world. The main aim of the tissue engineering field is to replace and regenerate the diseased or damaged tissue. The repairmen is generally achieved by using complex artificial architectures constructs combining three-dimensionally polymeric scaffold material (3D scaffold) with cells and bioactive molecules (1). The scaffold, which is an artificial extracellular matrix, should mimic the behavior of natural extracellular matrix in terms of physical and chemical processes favoring cell adhesion, migration, proliferation and new tissue formations (2).
Bone grafting was first established in the 1800s. Since then, development of scaffolds has been one of the main topics of this domain research. By introducing a biocompatible matrix into a bone defect, the bone healing will be enhanced. After the failed use of empty scaffolds, researchers have been trying to improve the efficacy of the scaffold by using it as a delivery system or a carrier for cells and/or growth factors. A good carrier device is often needed to deliver the cells, drugs, and extracellular matrix material to a localized injured bone. In order to support the osteoinduction, osteogenesis and osteoconduction, the scaffold should be biocompatible and osteocompatible (Figure 1-1). Moreover, the scaffold has to show mechanical strength, elasticity, and its residual products must not initiate any inflammatory reaction. Additionally, the material should be able to be easily processed in different irregular shapes taking into account that defects vary from patient to patient (3). The cell spread onto the scaffold surface has an import role in bone growth/regeneration. The factors which influence the design of tissue engineering scaffolds are presented in Figure 1-1.
The bone tissue engineering field critically depends on scaffold key functionality i.e. biocompatibility, controlled biodegradation to non toxic products, processability in different irregular shapes, with appropriate porosity to favor the growth, differentiation and proliferation of cells. The mechanical strength of the scaffold should be maintained during the regeneration process (4). Almost all biocompatible materials known until now for scaffold fabrication for bone reconstruction have most of those properties, but there are still some challenges for which no breakthrough solution s have been found and that many times are essential for the proper functioning of the new tissue and the patient’s safety. A few characteristic examples are:
optimum degradation behavior for scaffold materials;
optimum biocompatibility of materials in contact with cells and blood;
to offer anchorage points that allow cells adhesion and outgrowth;
permissive – allows cell migration and tissue remodeling;
similar mechanical properties with the tissue (bone) that enclosed the biomaterial;
allowing diffusion of nutrient metabolites (5, 6)
Figure 1-1 Properties and characteristics to consider for the design of optimal scaffolds for bone tissue engineering
Materials that combine most of these features can be placed into two main categories: natural polymers such as chitosan, alginate, collagen, gelatin and hyaluronan and synthetic polymers such as polyacrylates, polyesters, polyaminoamides and their blends and copolymers. The most widely used natural polymers are chitosan and alginate.
Biopolymers can be classified based on their monomer units in three main categories:
long nucleoside polymers (13 or more nucleoside monomers);
short polypeptides (amino acid monomers);
polysaccharides (carbohydrate monomers).
Polysaccharides such as starch, chitosan, dextran or alginates have been shown to be promising materials for applications such as hemodialysis, vehicle or modulator of bioactive substances, tissue engineering, carriers for the drugs, etc.
The recent researches in nanotechnology and polymer chemistry have led to the development of many novel and ingenious scaffold materials. In the last years, new bio and synthetic polymers and composite materials have been developed and tested for scaffolds.
Biopolymers such as chitosan, collage or fibrin offer a number of advantages over other materials for the developing of scaffolds due to their excellent properties, such as biocompatibility, biodegradability, bioadhesivity, antibacterial activity, etc. (7-10). Osathanon et al. recently described the use of a nanofibrous fibrin scaffold for bone regeneration in a mouse calvarias defect model (8). Some of the disadvantages of these biopolymers are their poor processability, mechanical properties and high cost.
1.2. NATURAL AND SYNTHETIC POLYMERS
1.2.1. Chitosan
Chitosan (CHT) is a natural polysaccharide extracted from the exoskeleton of crustaceans and cell wall of fungi. Industrially, it is derived from chitin by de-acetylation of N-acetyl-glucosamine residue under alkaline conditions or by enzymatic hydrolysis.
Biopolymers such as chitosan are widely investigated for biomedical applications because they offer a number of advantages over other materials for the biomaterials development (11). CHT can be processed in different forms including films (12), hydrogels (13), 3D porous scaffolds (14), fibers (15), tablets (16), capsules (17), and mico/nanoparticles (18).
Chitosan can be crosslinked by several methods: chemical, thermal and by radiation exposure. By chemical crosslinking, covalent or ionic crosslinked networks can be achieved. The covalent crosslinked network is obtained when dicarboxylic acid, glutaraldehyde and epichlorhydrine reacts with CHT (19) (see Figure 1-2). Using sodium sulfate, tripolyphosphate (TPP) or other multiple-charged anion molecules, ionical interactions are formed with the amino group from CHT structure, leading to an ionical crosslinked structure (20). These reactions are simple and carried out under mild conditions.
Figure 1-2 Chemical crosslinking of chitosan with a) dicarboxylic acid, b) glutaraldehyde, c) epichlorohydrin (21)
1.2.1.1. Structure and properties
Having good physico-chemical (many reactive NH2 and OH groups on its surface) and biological (biocompatible, biodegradable) properties, chitosan is an attractive material for various biological applications (Figure 1-3). These properties give the ability to bind to various materials including fats, metal ions, tumor cells, cholesterols and proteins. The physico-chemical properties (degree of deacetylation, molecular weight, polydispersity, crystallinity or the pattern of acetylation) of chitosan solution might be manipulated by many factor such as temperature, pH, ionic strength, concentration, solvent (22).
Figure 1-3. Physico-chemical and biological properties of chitosan
The chitosan is mainly processed in solution since its glass transition temperature (Tg) and melting point are higher than its degradation temperature, this being a general problem for polysaccharides with extensive hydrogen bonding (23). Both chitosan and chitin are insoluble in water (pH = 7) or common organic solvent. They are soluble in aqueous solutions of some acids (acetic, formic and lactic acid), hexafluoro-2-propanol, N,N-dimethylacetamide or hexafluoroacetate (6). The solubility in classical organic solvents has been improved, by tailoring its physical properties through chemical reactions (24).
The properties of chitosan are strongly influenced by two parameters: degree of deacetylation (DDA) and molecular weight (MW). Processing conditions are another important parameter because they influence the deacetylation degree and thus the properties of chitosan.
a. Influence of DDA
Due to the synthesis procedure which consists in the deacetylation of chitin, the chitosan structure contains also residual chitin unit structures (N-acetylglucoseamine unit) which were not fully hydrolyzed (Figure 1-4). The DDA represents the ratio between the average number of D-glucoseamine units and the sum of D-glucoseamine and N-acetylclucaosemine units (25). By changing the DDA and the distribution mode of the chitosan units, one could modify the properties of the final product (26).
Figure 1-4 Chitosan chemical structure (20)
The DDA controls the viscosity and solubility of the polymer through the pKa values (about 6.5) which is strongly influenced by the degree of N-acetylation. Chitosan is slightly soluble in dilute acidic solutions below pH = 6. The amount of free amino groups in the polymer chain is also controlled by the degree of deacetylation. At pH lower than 6, the amine groups get protonated to NH3+ and give chitosan a positive charge density that makes it a water-soluble cationic polyelectrolyte. As the pH increases over 6, the amino groups become deprotonated and the polymer becomes insoluble losing its charge (27).
Due to its positive charge, it could electrostatic interact with many negatively charged molecules. The two reactive groups, amino and hydroxyl groups, allow it to be a very reactive polymer (28).
The degradation kinetics according to Inmaculada Aranaz et al. appears to be inversely to the degree of crystallinity, a parameter also controlled by the DDA (29). The DDA can be increased from 80% to higher values by a second deacetylation reaction of chitosan using NaOH concentrate solution at 100 °C temperatures. However, it has been found that this process leads to degradation of the polymer and therefore a decrease of molecular weight. To avoid this phenomenon enzymatic deacetylation method was used most commonly (26). A low DDA causes an inflammatory response while a higher DDA causes a minimal response due to a lower degradation rate (30).
b. Influence of MW
The MW also influences the solubility of the polymer. The ionic strength is directly influenced by MW. This favours the condensation of counter ions and polymer chains aggregation. Also, an increase in MW implies an increase in polymer solution viscosity and thus, it is difficult to obtain a chitosan solution with a concentration higher than 2-3 wt.%. Therefore, by decreasing the MW of the polymer one could improve its solubility. Furthermore, various researche's reported that the MW affects the rate of degradation and biocompatibility (31).
In the case of chitosan hydrogels, according to H.Y. Zhou et. all (32), the aspect of the hydrogels becomes more compact and regular with increase of chitosan MW. The release rate of the drug loaded is lower when the high MW chitosan is used.
1.2.1.2. Processing techniques for chitosan
In order to obtain a structure that mimics the tissue structure exactly, several techniques were performed such as electrospinning (33), lyophilization (34) or the solvent evaporation method (35).
Electrospinning method is an efficient method in synthesis of 3D scaffold materials for tissue engineering. This method consists in using electrostatic force to form fiber from polymer solution. (36) Through this advanced method, nanofibers that are infinitely long, continuous, with a diameter from a few microns to 100 nm, forming 3D cell culture media similar to extracellular matrix, are obtained.
Lyophilization, also known as freeze-drying process or cryodesiccation, is the process of removing water from a compound by sublimation and desorption. This process is most often performed in a device called Lyophilizator and is a technique for obtaining the 3D scaffolds with high porosity and controlled pore size (37).
The solvent evaporation method is the process of drying, removal of solvent from a compound, in normal environmental conditions. This is a simple method which allows synthesis of a 2D scaffolds (films) with low porosity and low pores size (the pores’ size strongly depends on the nature of the solvent used and the rate of solvent evaporation) (38).
1.2.1.3. Applications
Chitosan has a large area of applications including water engineering, paper industry, textile industry, agriculture, photography, chromatography separations, light-emitting diode (LED) and biomedical and pharmaceutical industry such as ophthalmology and artificial skin (39).
Since it can be obtained in a large variety of forms (porous scaffolds, films and beads) and it is biocompatible, biodegradable and non-toxic, chitosan has a wide range of potential applications in tissue engineering and drug delivery systems as well.
Using tissue engineering technology, artificial tissues that can mimic the performance of the natural ones by combining various type of scaffolding materials with adjusted cells have been elaborated. Because of its polycationic nature (positive charge), chitosan has been found to have an accelerating effect on in vitro seeding and proliferation of relevant cells. It has also been found that the DDA of chitosan influences the degree of cell attachment (40).
In the past years, drug delivery technology has rapidly developed and become one of the most important fields in medical applications (41). Biopolymers such as chitosan offer various advantages as medical devices. To become a perfect drug delivery system a polymer has to be biocompatible, inert or biodegradable, able to accomplish high drug loading, mechanically strong, comfortable for the patient, easy to administer and remove, and simple to fabricate and sterilize (42). The release of drug from the chitosan depends on several factors including the physicochemical properties of the drug, morphology, size, density, degree of cross-linking of the particular system, the hydrophilicity and hydrophobicity of the polymer, the capacity of gel forming of the polymer, swelling capacity, bioadhesive ability, etc.
1.2.2. Poly(vinyl-alcohol) (PVA)
PVA is a non-toxic, synthetic, water-soluble polymer, which has good physical and chemical properties (43) (Figure 1-5). It is obtained by polymerization of vinylacetate to polyvinylacetate (PVAC), followed by the hydrolysis of PVAC into PVA. In practice, polyvinyl alcohol is obtained by the transesterification reaction with various alcohols. The reaction is named alcoholysis.
Figure 1-5 Structure of poly(vinyl-alcohol) (44)
1.2.2.1. Properties of PVA
The degree of crystallinity and the physical properties of PVA depend on various factors such as: the technology used, the degree of hydrolysis, the molecular weight. The degree of hydrolysis represents the degree of conversion of PVAC to PVA. Based on the total number of acetate groups in the polymer structure, PVA can be completely hydrolyzed (98-99 %) having a Tg of 85 °C and a Tm (melting temperature) of 230 °C or partially hydrolyzed (86-89 %) with a Tg of 58 °C and a Tm of 180 °C. PVA is stable at normal temperatures and has very good physical properties (45). Also, the solubility in water of PVA is strongly influenced by the hydrolysis degree, lower degrees increasing the water solubility. Thus, PVA with a low hydrolysis degree can be processed by solution casting method in order to obtain high performance PVA films. The way in which the properties of PVA-based materials vary depends on the degree of hydrolysis and molecular weight and is presented in Figure 1-6.
Figure 1-6 Effect of molecular weight and hydrolysis level on the physical properties of PVA (46)
It is a polymer resistant to oil, grease and solvent, odorless and nontoxic. It has good oxygen and aroma barrier properties and high mechanical properties (tensile strength and flexibility). PVA can absorb water, its properties depending on the humidity. Water acts as a plasticizer and decreases the tensile strength, but in the same time enhances its elongation at break. A disadvantage of PVA is that it has a high energy cost of water evaporation which limits its applications.
PVA can be subject to crosslinking reactions by several methods: freeze-thaw inducement of crystallisation, heat treatment, acid catalysed deshydratation, irradiation, radical production, chemical using formaldehyde, glutaraldehyde or other aldehyde (44).
Since the early 1930s, PVA has been considered a biodegradable synthetic polymer (47). However, the rate of degradation of the PVA is relatively small when compared to other aliphatic polyesters such as the polylactic acid. A similar trend was observed for starch-PVA based composites (46, 48). Another disadvantage is its high cost compared with other polymer such as polyethylene and polypropylene. Blending PVA with a biodegradable and cheap polymer might be a solution to lower the overall cost and increase its biodegradation rate.
1.2.2.2. Applications of PVA
PVA presents excellent film forming and adhesive properties being used in the industrial field such as water soluble packaging films, paper adhesives, textile sizing agent and paper coating.
Its chemical versatility and physical properties combined with good biocompatibility determined the use of polyvinyl alcohol in the biomedical field (49). PVA have been extensively studied as biomaterial for artificial kidney and pancreas, glucose sensors, immune-isolation membranes, artificial cartilage and muscle, contact lenses and drug delivery systems (50). Also, it is used in the manufacture of composite implants (PVA – bioactive glass) and hydrogels for cartilage replacement.
In recent years, it has drawn a lot of attention in tissue engineering field for repairing or regeneration different tissues or organs having properties similar to natural tissues and a low cost (51). PVA can provide porous and hydrophilic interface with body tissue.
1.2.3. Polymer blending
Synthetic polymers possess a great number of advantages involving excellent processing characteristics, biocompatible and biodegradable properties, which are important for future applications. Additionally, synthetic polymers have foreseeable and consistent mechanical and physical properties (e.g. tensile strength, elastic modulus, and degradation rate) and can be manufactured with great precision (10). Conversely, a large number of such polymers have some shortcomings implying incompatibility with host tissue, can give inflammatory reactions and are easily eroded (52).
The drawbacks associated with use of synthetic polymers were expected to be averted by combining them with biopolymers. In the last years, polymeric blends have been widely used especially in bioapplication such as biomaterials, summarized in numerous publications (53) (54). Synthetic and natural polymeric blends are a new class of materials, whose final ability combine the specific properties of both polymeric components. The properties of the final material depend on the composition and the miscibility of the two polymer constituents (55).
Because chitosan is a polymer soluble in acetic acid aqueous solution, it permits the possibility of mixing with water soluble synthetic polymers such as poly(vinyl-alcohol) (PVA). Chitosan and PVA exhibit a huge amount of hydroxyl functional groups on their molecular chain, which lead to the formation of strong hydrogen bonds (Figure 1-7). The intermolecular interactions between the polymers and the formation of an homogeneous blend give improved mechanical properties compared with the pure polymers (56). Besides the enhancement of the mechanical properties, the synthesis of the polymeric blends also aims to improve properties such as processability and overcome the difficulties related to biocompatibility associated with the use of synthetic polymers (57). An important parameter that influences the properties and the preparation of the blend is the bulk and surface hydrophilicity of the synthetic polymer which also affect the biological behavior of the blend.
Figure 1-7 Formation of hydrogen bonds between PVA chains and between PVA and CHT chains
The crosslinking process is the formation of bonds between polymer chains leading to a three-dimensional structure. The process can be achieved by using crosslinking agents or thermal and radiation treatment. A common method for curing these polymeric blends is thermal treatment for several hours. As the exposure temperature increases, the number of hydrogen bonds formed is greater and therefore properties such as the degree of swelling and permeability are reduced because the hydrogen bonds formed prevent penetration of water molecules.
Until now, several applications of the CHT/PVA blends have been published including tissue engineering, drug delivery systems, medical devices, cosmetics, food packaging, etc.
1.2.4. Alginate
Alginate (Al) is a natural water-soluble polymer extracted from brown algae such as Laminaria hyperborean, Ascophyllum nodosumand Macrocystis. Alginate has a linear structure composed of block copolymers of 1-4 linked β-D-mannuronate acid (M) and α-L-guluronate acid (G). The structural units are placed consecutively in GG and MM blocks, as well as MG blocks (Figure 1-8). The sequence of M and G blocks in the copolymer and the molecular weights depend on the source and species that produce the alginate (58).
Due to the fact that alginate has a highly hydrophilic nature, the cell could be seeding into the scaffold rapidly and simply (59).
Figure 1-8 Structural of alginate acid (58)
Alginate is a pH-sensitive material, shrinking at low pH and with increasing pH, the swelling degree increases up to 200% which facilitates its disintegration.
Alginate can be processed in various forms due to its reversible solubility including beads (60), films (61), hydrogels (62), fibers (63), porous scaffold (64), microspheres (65), beads (66).
1.2.4.1. Physical and chemical properties
a. Physical properties
Solubility
The alginate water solubility depends on 3 parameters: 1) pH of the solution; 2) ionic strength of the medium and 3) the presence of the gelling ions in the solvent. Alginate solubility is influenced by the state of the carboxylic acid groups. If the carboxylic acid groups are found in protonated form, the alginate is not entirely soluble in any solvent, including water. Na-alginate was not fully soluble in any solvent but it could be dissolved in water (58) Thus, in order to solubilise the alginate it is very important to maintain the pH of the solution above a certain critical values in order to deprotonate the carboxylic acid groups from the alginate backbone. By changing the ionic strength, the polymer conformation, chain extension, viscosity and the solubility of the polymer could be affected.
Ionic crosslinking
Despite its unique properties, alginate presents a major disadvantage such as low mechanical strength. The properties of the alginate can be improved by crosslinking with divalent cations in aqueous solution (e.g. Ca2+, Sr 2+ and Ba2+), through ionic interactions (67). The crosslinking process is fast and the affinity of alginates towards divalent ions increases in the following order: Mn < Zn, Ni, Co < Ca < Sr < Ba < Cd < Cu < Pb. The most commonly used cation is Ca 2+ , which can form 3 possible junction types (Figure 1-9) (68).
Figure 1-9 Possible junction point in alginate: a) GG/GG junction; b) MG/MG junction; c) GG/MG junction (58)
The crosslinking of alginate using Ca2+ can be done by 2 methods. The first one called the "diffusion" method consists in the diffusion of the crosslinking ions into the polymer solution from an outside reservoir. The second method is named "internal setting". In this last method, the ions source is within the alginate solution and a trigger controlled release (pH or ion source solubility) of the ions into the solution leads to gel formation.
b. Chemical properties
Figure 1-10 The mechanism of the acid catalyzed hydrolysis of methyl-glycopyranosides (69)
Under acidic conditions, polysaccharides undergo hydrolytic cleavage. The reaction implies 3 steps: 1) protonation of the glycosidic oxygen (I); 2) heterolysis of the formed conjugate acid (II) , which forms non-reducing end groups and a carbonium-oxonium ion (III) which can be in equilibrium with a carbonium-oxonium ion with a reducing end group (IV); 3) rapid addition of water to the carbonium-oxonium ion with reducing end group forming free sugar (glucose) (Figure 1-10).
The degradation of the alginate can be performed in an enzymatic manner, by using lyase or by immersing it into strong alkaline environment. The rate of degradation is higher above pH of 10 (from the β-elimination mechanism) and below pH 5 (due to the acid catalyzed hydrolysis). Alginate degradation could be realized even at neutral pH value in the presence of reducing compounds such as hydroquinone, sodium sulfite, sodium hydrogen sulfide, cysteine ascorbic acid, hydrazine sulfate and leuco-methylene blue (58).
Blending and spinning with non-toxic polymers such as gelatin (70), chitosan (71) or polyethylene oxide (72) are other methods used to improve the properties of alginate.
1.2.4.2. Applications
Alginate is a natural polymer used in several medical applications showing durability in biological performances and biocompatibility characteristics. It is commonly used in tissue enginneering and drug delivery applications due to its low cost, biocompatibility, low toxicity, biodegradability, immunogenecity, potential bioactivity etc. (67). Alginate is currently used for the treatment of acute and chronic wounds as a wound dressing materials (73).
The polymers’ properties such as mechanical strength and thermal conductivity can be also enhanced by reinforcing with inorganic materials such as carbon nanotube (74, 75), graphene oxide (76, 77), clays (78), silica nanoparticles (79), bioactive glass ceramic (80) etc.
1.3. GRAPHENE/ GRAPHENE OXIDE (GO) MATERIALS
1.3.1. Graphene
Graphene is a planar monolayer of carbon atoms arranged into two dimensional sheets of sp2 hybridized carbon atoms, which attracted much interest in the recent years because of its excellent thermal, mechanical, electric and barrier properties. Graphene is in fact a carbon allotrope formed (carbon nanotube, fullerene, diamond) by benzene rings joined to give a structure in which the carbon atoms are covalently bonded to form sp2 hybridized co-planar atoms with zigzag or armchair terminated edges (81).
In 2010, Andre Geim and Konstantin Novoselov (University of Manchester, UK) received the Nobel prize in physics for their successfully isolation of a single layer graphene using a micromechanical exfoliation done in 2004. Since the study published by Dai et al in 2008 (82) on the use of graphene in biomedicine, a number of publications have expanded particularly in drug/gene delivery (83), tissue engineering, antibacterial materials, biological sensing and imaging. Until now, there are only few studies as regards the toxicity of graphene and GO, at in vitro and in vivo level (84).
1.3.1.1. Graphene synthesis
Graphene can be obtained by a sequence of processes starting with graphite, which is a very widespread mineral in the world. It was prepared for the first time unambiguously in 2004 by peeling a single layer of graphene using sticky tape and a pencil (85).
Prevention of aggregation of graphene is very important, the unique properties of this material existing just for the individual sheets. Agglomeration can be hindered by one of the following methods: exfoliation (mechanical exfoliation in solution, intercalation of small molecules by mechanical exfoliation), chemical vapor deposition (CVD) (thermal CVD, plasma enhanced CVD and thermal decomposition on SiC and other substrates) and chemically derived graphene (synthesis of graphene oxide and reduction).
The most common approach is first to oxidize graphite, then to obtain monolayer graphene oxide from graphite oxide and finally to perform a reduction of the graphene oxide to graphene by chemical, thermal or electrochemical treatment (86). The reduction of graphene oxide to graphene is a challenge considering the problems encountered by each method. Thus, reduction by hydrazine monohydrate gives finally N-covalent blended in the chain, which reduces conductivity (87). An alternative is to use sodium borohydride which is unfortunately slowly hydrolyzed by water (87). Another approach is thermal reduction of GO, which uses heat to eliminate the functional groups from the graphene oxide surface (88).
1.3.1.2. Graphene properties
The thickness of a graphene sheet is 0.33 nm and the surface area is 2600 m2/g (89). Therefore, a stack of graphene layers separated by 0.33 nm forms the structure of graphite with three possible graphene stacking sequence. Moreover, graphene exhibits impressive mechanical, thermal, electronic and optical properties. It has a high Young’s modulus (~1100 GPa) (90), a very high thermal conductivity (~5000 W/m·K) (91), good electrical conductivity (high electron mobility at room temperature, 250,000 cm2/V*s), non-toxicity, low cost, intrinsic biocompatibility (92) and facile biological/chemical functionalisation of graphene oxide (GO).
Recent publications display the fact that graphene was used as inexpensive substitute for carbon nanotubes in nanocomposites materials due to its excellent mechanical, structural, thermal and electrical properties. These important properties are applicable at nanoscale level and the performances of the manufactured nanocomposite depend on the degree of exfoliation of the graphite into a single graphene sheet in the polymer (93). The main challenge still remains to obtain a full and homogeneous dispersion of individual graphene layers in various solvents. Without proper separation, graphene sheets tend to agglomerate or even rearrange to form graphite through Van der Waals interactions.
1.3.1.3. Applications
The 2-D network of graphene has gained more interest in a wide range of applications including nanoelectronics, biosensors, drug delivery, supercapacitors, flue cells, hydrogen storage, transistors and polymer nanocomposities.
Soo-Ryoon Ryoo and coworkers report the behavior of NIH-3T3 fibroblast cells grown on support thin film of graphene and carbon nanotubes. The study suggests that these compounds present a high potential for medical application due to its high biocompatibility especially as surface coating for implants (94). Lim et al. describe the synthesis and characterization of graphene hydrogel and its applications in tissue engineering and demonstrate the compatibility of graphene for bioapplications (95).
However, in most of the cases, using pure graphene has had some disadvantages such as tendency of aggregation and difficulty in processing which limit its applications. To overcome these drawbacks, graphene oxide, which is a graphene derivate, can be used.
1.3.2. Graphene Oxide (GO)
Compared with graphene, GO exhibits additional functional groups such as epoxy groups and hydroxyl groups linked to the sp3 hybridized carbon on the base plane and carboxyl and carbonyl groups on the sheet edges linked to the sp2 hybridized carbon (Figure 1-11). These functional groups gives GO a highly hydrophilic character and can be easily exfoliated in water. Moreover, they keep individual sheets separated from each other improving dispersion in water.
There is still some confusion between graphite oxide and graphene oxide. Chemically, these two are identical but structurally, graphite oxide is different from graphene oxide, the latter being exfoliated into monolayers or few-layered stacks (96).
Figure 1-11 The proposed structure of graphene oxide (96)
The presence of the reactive groups (carboxyl, hydroxyl and epoxy gropus) on GO surface enables its interaction with different polymeric systems (97), biomolecules (98), protein, magnetic nanoparticles (99), DNA (100) etc. All these functional groups are fundamental for further chemical functionalization of graphene oxide to produce chemically modified graphene, which is important in the graphene – polymer nanocomposites synthesis.
1.3.3. Graphene oxide properties
Having a 2D crystal structure, graphene oxide has similar mechanical properties as carbon nanotubes. Additionally, GO possesses superior electrical and thermal properties and even a higher aspect ratio and large surface area (2620 m2/g) than other reinforcing compound. Due to its 2D structure, graphene exhibits a thermal conductivity of about 3000 W/m*K, making it an excellent candidate for enhancing polymer composites thermal stability. Compared with graphene, GO is electrically insulating. It partially restores its electrical conductivity by eliminating the oxygen functional groups using thermal reduction.
1.3.4. Synthesis of Graphene Oxide (GO)
Synthesis of graphite oxide involves the oxidation of graphite to various levels using oxidants. Thus Brodie and Standenmaier (101) used a combination of potassium chlorate (KClO3) and nitric acid (HNO3) to oxidize graphite. The most commonly used method for synthesis of GO is the Hummers method (102) which involves the treatment of graphite with potassium permanganate (KMnO4) and sulfuric acid.
Graphene oxide may be produced from graphite oxide by sonication in water (Figure 1-12). However, this process encountered some disadvantages coming from the damage of the graphene oxide platelets, which may decrease the dimensions from several µm per size to several hundred nm per size (86).
Figure 1-12 Oxidation of graphite to graphene oxide (93)
1.3.5. Applications
GO can be successfully used in the same applications as graphene (section). Besides this, the use of graphene oxide in biomedical area is quite new, showing great development potential (103). GO was used for the first time in 2008 by Dai et al. (104) as an efficient drug nanocarrier, opening thus a new research domain, where a large number of works have been carried out using GO in biomedical field ranging from drug delivery, antibacterial materials, cancer therapy, biosensing and bioimaging to biocompatible scaffold for cell cultures, tissue engineering and component of implant device (Figure 1-13) (105).
Another important application of graphene oxide is the fabrication of biosensors to diagnose dseases (106). Due to the presence of reactive COOH on GO surface, GO facilitates conjugation with biomolecules, DNA, protein, quantum dots and various hydrophilic and hydrophobic drugs.
Figure 1-13 Biomedical applications of graphene and graphene oxide (107)
1.3.6. Graphene oxide-polymer composites
From the materials science point of view, a single material type does not usually supply the necessary mechanical and/or chemical properties required (Figure 1-1); hence, the properties of two or more materials can be combined in a composite material. Polymers and ceramics (and glass) are often used for hybrid scaffolds due to their ability to degrade in vivo concomitant with the new tissue formation. Massive release of acidic degradation from polymers was observed to cause inflammatory reactions (108). The research of graphene-biomacromolecules composite materials is limited.
Biopolymer-graphene composites possess good electric conductivity, thermal conductivity, and mechanical stiffness established as one of the most fascinating materials. By comparing the mechanical properties of graphene/epoxy with those of carbon nanotube/epoxy nanocomposites, it was shown that the graphene platelets have enhanced reinforcing capacity when compared to carbon nanotubes (109). Such unique reinforcing behaviour of graphene sheets combined with their biocompatibility reported by Fun at al (110) attracted a lot of interest since these properties are highly desirable for scaffolds in bone tissue engineering (111). Fan et al. reported a significant increase of the elastic modulus of chitosan when a small amount of graphene (0.1-0.3 wt %) was added. Also, enhanced proliferation of L929 cells was observed in the same study for composite materials compared with neat chitosan, showing thus that graphene increases the material biocompatibility (110). However, additional studies regarding graphene-reinforced natural biomacromolecules (chitosan, collagen) should be carried on in order to consider these materials for a certain application. In tissue engineering, the scaffold should provide good mechanical properties and favour good cells (osteoblas) attachment and growing. Pure biopolymers do not meet these requirements. (112). Enhanced biological and mechanical performance of biopolymer-graphene composites can be realized by obtaining good chemical and/or physical bonding between the biopolymer and graphene. Scientific studies have shown that chemical factors (e.g., ECM proteins and certain peptide sequences, topographical features of biomaterials,and substrate stiffness) play critical roles in cells adhesion, growth, and tissue remodelling (113).
1.3.6.1. Graphene oxide-polymer composites synthesis
Due to the excellent properties of graphene oxide, different polymers composites have been synthesized. Polycarbonate (114), polyurethane, polyester (115), epoxy (116), polystyrene (117), polymethylmethacrylate (118), polyvinyl alcohol (119), polypropylene (120), chitosan (121), alginate (66), etc were used for the synthesis of graphene-polymer composites. Similar to other fillers with multilayer structure, the dispersion of GO layers within the polymer matrix is crucial for achieving improvement of the mechanical properties of the composite material. GO can be easily agglomerated into flakes due to strong hydrophobicity and van der Waals attraction and the aspect ratio of the reinforcement is dramatically reduced. During drying, GO sheets restack easily and are strongly bonded by hydrogen bonding. In order to achieve a good dispersion, restacking of GO sheets must be avoided. Dispersion of GO in individual sheet can be achieved in water, which acts as a spacer and forms a stable dispersion after ultrasonication.
Thus, obtaining highly-exfoliated platelets, which are well dispersed within the polymer mass, is a desideratum (88, 118). For polymer composites synthesis, solution blending, melt mixing and in situ polymerization are usually employed (93).
a. Solution blending
This method implies the solubilization of the polymer in a proper solvent followed by its mixing with the solution of the previous dispersed GO suspension. To obtain a homogenous suspension, the high power ultrasonication can be used. Considering that a long exposure to the high power of ultrasonicater can cause defects in GO layers, which may subsequently affect the composites properties, the sonication process should be carefully performed. Mixing graphene oxide sheets in viscous polymer matrix is very difficult even after ultrasonication for a long time especially for higher GO amount. During the mixing, the polymer chains coat the individual layer surface and afterwards, the solvent evaporation interconnects each sheet. If the solvent evaporation is too slow, the GO sheets tend to form aggregates, which leads to an inhomogeneous distribution of layers into polymer matrices.
b. Melt mixing
This technique involves dispersion of GO into polymer matrices using high temperatures and shear forces. In this method, the use of toxic solvents is avoided. High temperatures lead to the polymer melt and the dispersion or intercalation of GO within the polymer chain can be easily realised. This method is less efficient than the previous one due to the higher viscosity of the polymer.
c. In situ polymerization
In situ polymerization technique means the dispersion of GO in monomer followed by the polymerization of the monomer in the presence of GO layers.
1.3.6.2. Graphene oxide-polymer composites properties
A good dispersion of nanomaterials into polymer matrix is a key factor in determining the final properties of the materials (122). GO – polymer nanocomposites showed a significant increase of elastic modulus, tensile strength, thermal stability and electrical conductivity even at low loading of GO filler due to the large interfacial area (118, 123).
a. Mechanical properties
GO exhibits high mechanical properties such as elastic modulus and tensile strength. The polymer composites reinforced with GO show a significant improvement of mechanical properties, having an elastic modulus of about ̴ 1TPa and an intrinsic strength of 125 GPa. The improvement of mechanical properties depends on a good dispersion of the filler within polymer matrices, interface bonding and reinforcement phase concentration (124).
b. Electrical properties
One of the most important properties of graphene is its excellent electrical conductivity. By mixing GO with an insulator polymer the electrical conductivity of the composite material can be greatly improved. Electrical conductivity of the composite depends on several factors such as agglomeration of the filler, concentration, aspect ratio, inter-sheet junction, filler dispersion within polymer matrices, processing methods, etc. (125)
c. Thermal properties
The thermal properties of the composites are affected by several factors similar to those which also influence the electrical and mechanical properties (aspect ratio, orientation and dispersion of GO sheets, etc.) (126).
1.3.6.3. Graphene oxide-polymer composites applications
GO-polymer nanocomposites show great potential for a wide range of application such as electric conductive composites (116), supercapacitors (127), sensors (128), batteries (129), ultrafast laser mode-locker (130) and thermally stable and mechanically reinforced materials.
The unique properties of GO have drawn the attention of a large number of researchers who have started to use it as reinforcing agent in biocompatible composites materials in order to obtain materials with improved mechanical, thermal and electrical properties (131). GO-based composite materials experienced a broad attention in applications such as biomedical engineering and biotechnology. The presence of reactive groups on GO surface is important for improving the interaction and compatibility with a particular polymer but may reduce the thermal stability of the composite material (132).
DRUG DELIVERY
2.1. INTRODUCTION
Drug delivery system (DDS) is an efficient method to control the loading and the release of the therapeutic agent and to improve their bioavailability.
During the last three decades, drug delivery technology has developed rapidly and has become one of the most important fields in modern medicine. It presents some advantages when compared to traditional dosage forms such as low side effects, low toxicity, improved dosing efficacy and reduced patient complications (Figure 2-1). Nevertheless, the conventional nano-formulation has some drawbacks that motivate scientist to look for new nano-carriers with optimal performances. Firstly, it is hard to find a system to include sufficient active agent. Secondly, the toxicity of the carrier is still a pendant problem. Thirdly, the cost of the product should be considered. Finally, the nano-carrier delivery to a desire location is still difficult to achieve (133). Inorganic materials meet these requirements and among them mesoporous silica seems to cover a large number of applications in drug delivery systems.
Figure 2-1 Comparison between classical mechanisms (a) and controlled release systems (b) (134)
An ideal drug delivery system has as a main goal the release of the drug to a particular site, in a specific time and release pattern. Most of the drugs fail in achieving these objectives because they do not have the ability to reach the target site of the action. A significant amount of the drug is delivered to the normal organ or tissue causing severe side effects damaging the healthy cells. To overcome this problem, scientists have to obtain a controlled release system that delivers the drug at the desired location hence the therapeutic efficacy of the active substance being increased and the patient complications during drug administration decreased (21). Despite the fact that a lot of attention has been paid to the idea of developing a controlled drug release system in the desired location in the last years, little progress has been made in this field.
The drug delivery system consists in three main components: an active substance (the drug), a target moiety (a receptor) and a carrier system. Choosing the right carrier is very important because it affects the pharmacokinetics and pharmacodynamics of the drug. Several materials have been employed as drug delivery carrier including natural and synthetic polymers (135), mesoporous nanoparticles (136), lipids (137), surfactants and dendrimers (138), anionic (139) and cationic clays (140), etc. The drug can be loaded into the carrier by physical adsorption (141) or chemical conjugation (142).
Drug delivery systems should be inert or biodegradable, biocompatible, mechanically strong, comfortable for the patient, capable of achieving high drug loading, simple to administer and remove, and easy to fabricate and sterilize (42).
2.2. MESOPOROUS MATERIALS
Mesoporous materials are materials with pores in the range 2-50 nm. According to IUPAC classification, the pores are divided into three main classes presented in Table 2-1.
Table 2-1: Porous materials classification
The pore shapes can be seen in Figure 2-2. The pores may be arranged in different structures and can take various forms such as a a) hexagonal phase (MCM-41), b) cylindrical or spherical (MCM-48) and c) a lamellar phase (MCM-50) (143).
Different oxides such as SiO2, TiO2, ZnO2, Fe2O3 or combination of metal oxide can be used for the synthesis of mesoporous materials. Among them, mesoporous SiO2 is the most commonly used as controlled drug delivery systems.
Figure 2-2 Mesophase structures of M41S: a) MCM-41, b) MCM-48 and c) MCM-50 (42)
2.3. MESOPOROUS SILICA NANOPARTICLES (MSN)
Mesoporous silica has been discovered for the first time by a Japan researcher named Yanagisawa and his coworker in 1990 (144). In 1992, it was produced by Mobil Corporation Laboratories (145) under the acronym of M41S. The most widely known silica from the M41S family are MCM-41 (hexagonal), MCM-48 (cubic), MCM-50 (lamellar). The differences between them arise from the synthesis method and lead to different applications. The most studied compound of this family is MCM-41 due to its hexagonal structure which favours the loading of the drug.
In 2001, Vallet-Regí, used for the first time MSN as drug delivery system for an antimflamatory drug which was load into their pores. The system showed high drug loading and high drug release capacity (146).
Mesoporous silica nanoparticles possess tremendous advantages over traditional nano-based formulations and were found to be one of the greatest carrier materials for hydrophobic or hydrophilic drugs (147). The mesoporous carrier is selected based on the characteristics of the guest molecule and the targeted application. Therefore, different guest molecules have been successfully confined into mesoporous silica. Various categories of drugs such as anti-inflammatory agent, hormones, antibiotics, antifungal, antiseptic, steroids and vaccines were tested as guests (148).
Pore size, geometry and connectivity, the reaction between the matrix and the surrounding media (dissolution properties), adsorption properties (interactions between drug molecules and matrix), are just several factors that one should consider when designing a controlled drug delivery system.
The pore size of mesoporous materials limits the size of the molecule that can be absorbed into the mesopores. The adsorption of the molecules in the mesoporous matrix is governed by size selectivity. Commonly, pore diameters slightly larger than the drug molecule dimensions (pore/drug size ratio>1) are enough to allow the adsorption of drug inside the pores (149). The increase in drug loading depends on pore size and pore surface chemistry.
Silica is accepted as having a low toxicity and a good biocompatibility at nano-scale. In 1986, at the European Society for Biomaterials conference, the word biocompatibility was assigned as "the ability of a material to perform with an appropriate host response in a specific application” (150). Also, for the mesoporous silica some disadvantages were reported. The agglomeration tendency of the particles has a strong influence on the drug-loading capacity. This phenomenon has a negative effect on drug encapsulation by decreasing the amount of drug loaded due to the steric hindrance. Some biocompatibility studies showed that MSN particles with diameters ranging from 150 nm have significant toxicity at high concentrations in vitro, and cause severe systemic toxicity in vivo after intraperitoneal and intravenous injections (151).
On the other hand, mesoporous polymer/silica composites are very interesting carrier materials because they can encapsulate large amounts of guest molecules and subsequently release them at later stages in an optimal way (42).
2.3.1. Synthesis
MCM-41 is one of the most used type of MSN for biomedical applications. In the synthesis of MCM-41, cetyltrimethylammonium bromide (CTAB) is used as surfactant, tetraethyl orthosilicate (TEOS) or sodium metasilicate (Na2SiO3) as the silica precursor, and alkali as catalyst. By using Na2SiO3 as precursor, MSN with larger pores and higher specific surface area is obtained compared with the MSN synthesized from TEOS.
The alkoxide precursor Si(OR)4 is hydrolyzed and condensed in order to form siloxan bonds as is outlined below:
The surfactant can be removed by two methods: calcination and solvent evaporation. During the calcination process, the surfactant is decomposed into CO2, NOx and steam. After calcination (400-550 °C), the unreacted silanol groups condense and thus more functional groups are lost. The extraction process, where no high temperatures are used, has the advantage of decreasing the loss of silanol group. This method is performed by using mixtures of acid/alcohol for cationic surfactants or alcohol for neutral surfactants.
Depending on the type of surfactant used, synthesized silica has different properties. Resulted mesoporous silica can have interconnected pores, which are energetically unfavorable, or an amorphous arrangement of the pores walls. The synthesized MCM-41 is an agglomerated white powder with low mechanical stability.
For the medical field it is very important to control the pore size, geometry, shape and particular size. The orientation and the size of the pore depend on the carbon chain length of the surfactant templates. The morphology and the size of the particle is tailored by the ratio between the silica precursor and the surfactant, by the pH of the reaction medium, the solvent used for swelling and the organoalkoxysilane precursor used in the co-condensation reaction. Hydrothermal treatment of mesoporous materials is another method which leads to the synthesis of stable and enlarged mesopores.
2.3.2. Structure of MSN
Mesoporous silica presents a three-dimensional network structure. The surface of MSN is dominated by silanol and siloxan groups leading to the hydrophilic nature of the particles. The silanol groups may be classified as: single or isolated, hydrogen bonded or vicinal and geminal (Figure 2-3.)
Figure 2-3 Schematic illustration of silanol group
The silanol groups from the MSN surface form hydrogen bonds which lead to agglomeration (
Figure 2-4). This drawback might be solved by chemical functionalization. The silanol groups from the silica surface and pore walls are susceptible for chemical modification with different organic groups. Depending on the functional groups from the drug molecules to be adsorbed, different functional groups can be introduce on silica surface in order to be able to create a physical/ chemical interaction, the amount of the drug loaded being influenced by the silica pore/surface nature.
Figure 2-4 Mesoporous silica agglomeration
Under ambient humidity level, the drug molecules are adsorbed from MSN pores by hydrogen bonding or chemical interactions. The drug molecules are adsorbed on MSN surface or pores by direct impregnation methods. The silica powder is immersed directly into the drug solution with a given concentration. For a good drug loading, several factors might be considered such as drug solubility and polarity, solution pH, reaction temperature and drug chemical nature. Usually the adsorption of drugs by porous materials takes place in three stages. In the first one, the instantaneous adsorption of the drug on silica surface occurs. In the second stage, gradual adsorption occurs, the adsorption rate being limited by the diffusion of the drug into the silica pores. In the last stage, a low rate adsorption is observed due to the low concentration of the drug left in the solvent (152).
Monitoring the drug release is achieved by insertion of silica drug into a simulated body fluid (SBF) solution until the equilibrium is reached. Chemical composition of mesoporous silica surface, pore size and pore connectivity are just a few factors that one might considered for release kinetics of the drug. Usually the release of drugs takes place by three different mechanisms: a) erosion; b) diffusion and; c) release from the surface of the particles (Figure 2-5). In most cases, the drug release follows more than one type of mechanism. In the first case, the adsorbed drug is release upon contact with the release medium. This could be diminished by use of a crosslinking agent such as glutaraldehyde, formaldehyde, genipin etc. or by washing the microparticles with a proper solvent (153).
Figure 2-5 Controlled release mechanisms (154)
2.3.2. Properties
Mesoporous silica has many advantageous properties that recommend it as drug carrier and catalyst support. It exhibits pores with uniform size and shape, large surface area and high thermal and hydrothermal stability. The size and the surface chemistry of the pore could be easily controlled and changed. Depending on the drug which should be encapsulated, the chemistry of the surface and the dimensions of the pores can be controlled in order to obtain the proper loading and release of the drug.
Another important advantage of MSN for the medical field is its degradable behavior in
aqueous solution, which can avoid further problems related to the removal of the material after usage.
Unlike other drug carrier, in addition to the mentioned properties, MSN has a higher resistance to heat and pH variation, mechanical stress and hydrolysis-induced degradations, showing thus a stable and rigid framework. Moreover, MSN present two functional surfaces, an internal surface caused by the presence of cylindrical pores and the external particle surface. Thus both internal and external surface can be functionalized with various reactive groups (155).
MSN proved to have a higher versatility in drug delivery systems compared with other systems like polymer nanoparticles, liposomes, etc.
2.3.4. Applications
The controllable morphology, porosity and chemical stability of MSN make it an attractive material in applications like adsorption, catalysis, sensing and separation, medical usage, ecology and nanotechnology.
Since 2001, when MCM-41 was proposed as controlled delivery system for the first time (156), many reports have been devoted to tailoring the chemical properties of mesoporous carriers to the nanometer scale to achieve a better control over loading and release of molecules. Furthermore, in early 2006, silica-based ordered mesoporous materials were also found to act as implantable bioceramics with bone regeneration capability (157).
According to Fangqiong Tang review (133), MSN should no longer be considered a simple drug nanocarrier for medical application since the chemical and physical properties of mesoporous silica also have an exceptional influence on its biocompatibility.
Original contributions
OBJECTIVES AND ORIGINALITY
The bone tissue engineering field depends critically on scaffold key functionality i.e. biocompatibility, controlled biodegradation to non toxic products within the time frame required for the application, processability to complicated shapes with appropriate porosity, ability to support cell differentiation, growth, and proliferation, and appropriate mechanical properties as well as maintaining mechanical strength during the largest part of the tissue regeneration process. Almost all biocompatible materials known until now for scaffold fabrication for bone reconstruction have most of those properties, but they miss others that, many times, are essential for the good functioning of the new tissue and patient safety.
Based on data from literature (110, 158-161), we believed that combining biopolymer such as CHT, PVA, Al or blends and graphene or GO, new materials including the characteristics of the nanofiller, such as good mechanical properties, and the specific properties of biodegradability and compatibility against cells associated with biopolymers could be obtained.
The first research direction consists in fabrication of biomaterial biopolymer-graphene oxide with optimal properties to be used for the fabrication of scaffold for bone reconstruction having improved performance. We tried to couple efforts using complex computational tools of materials modelling and advanced experimental assays for the understanding and knowledge-based design of an ideal biomaterial with end-use as scaffold for bone.
At international level, only a very limited number of studies reported molecular modelling strategies, dedicated to polymer-graphene materials characterization and to the best of our knowledge, no study has attempted to capture the behavior of more complex structures such as graphene/biopolymer. Thus, a reliable protocol at multiscale in order to design and characterize materials based on chitosan and graphene was developed. Results show that graphene has a high agglomeration capacity, making its dispersion in chitosan quite difficult. Thus, graphene oxide, which has functional groups on its surface (section 1.3.2.), was used for experimental assays instead of graphene because it has higher hydrophilicity and it can be easily exfoliated in water.
Several outstanding papers reported numerous protocols for polymers, protein or graphene investigation approaches. Our approach is to take advantages of complementary properties of the three materials, biocompatibility associated with CHT and Al, processability and versatility associated with PVA and physical properties of graphene oxide. Therefore, simple and low cost methods for the preparation of chitosan/GO, alginate/GO and chitosan-polyvinyl alcohol/GO biocomposite films and 3D scaffolds by incorporation of GO into polymers matrix using water as solvent were involved.
Recently, chitosan/GO composites have been obtained by casting method (100). We obtained chitosan/ GO by a new approach which involves the thermal treatment of the composites in order to diminish its solubility and increase mechanical properties. A detailed structural and morphological characterization of the composites was realized. Biocompatibility studies were performed for the first time on this type of materials.
Despite its high biocompatibility, chitosan shows low processability and mechanical properties. An increase of chitosan/GO mechanical properties was achieved by blending it with PAV (20/80 v/v ratio), this being presented in a recent published paper (162). As far as we know, only a structural and mechanical characterization of these materials was performed (162). We considered the increase of the CHT/PVA ratio in biopolymer/GO composite materials a solution to increase the biocompatibility of the materials. After the advanced material characterization, biocompatibility studies were carried out; this type of studies has never been reported before for chitosan/PVA/GO composite materials.
In chapter 6.6 , new 3D composite scaffolds chitosane/GO were obtained. This approach was used for the first time for these composites and it was envisaged to increase the biocompatibility since a high porosity network should be obtained compared with film structure. Chitosan/GO composite films have sufficient mechanical support but do not have osteoconductive properties. SEM was used to study the morphology of the scaffolds. Enzymatic degradation and mineralization studies were performed.
The results obtained were the subject of the following papers:
Pandele A.M., Ionita M., Iovu H., Molecular modeling of mechanical properties of the chitosan based graphene composites, U.P.B. Scientific Bulletin, Accepted
Pandele A.M., Dinescu S.,Costache M., Vasile E., Obreja C., Iovu H., Ionita M. Preparation and in vitro, bulk, and surface investigation of Chitosan/graphene oxide composite films, Polymer Composite 34 (12), 2116-2124, 2013
Pandele A.M., Ionita M., Crica L., Dinescu S.,Costache M., Iovu H.,
Synthesis, characterization, and in vitro studies of graphene oxide/chitosan-polyvinyl alcohol films, Carbohydrate Polymers, 102, 813-820, 2014
Another biopolymer, alginate was used for the first time in the synthesis of films biopolymer/GO. Alginate is the most used biopolymer in medical applications due to its water solubility, which favours cell adhesion (18). Structural, morphological and mechanical tests were conducted, all this being for the first time reported in the literature.
Ionita M., Pandele A.M., Iovu H., Sodium Alginate/graphene oxide composite films with enhanced thermal and mechanical properties, Carbohydrate Polymers, 94 (1), 339-344, 2013
The second direction of the thesis consists in synthesis of hybrid materials based on biopolymers and MSN functionalized with an anti-inflammatory drug such as benzalkonium chloride (BZC). The BZC was loaded into the MSN pore or surface in order to achieve reduced and controlled drug release. Drug adsorption was monitored, both on the surface and in the pores of mesoporous silica nanoparticles by varying various parameters of the reaction: time, pH, temperature and initial drug concentration. Films based on two biopolymers (chitosan and alginate) and MSN modified with BZC were synthesized and the influence of drug concentration on the process of drug release from hybrid materials was followed.
Due to its great adsorption capacity, MSN shows a higher tendency of agglomeration leading to prevention of drug release. We believed that incorporation of MSN and the anti-inflammatory drug into the polymer will lead to a better dispersion of MSN and, consequently, will increase the amount of drug released. The concept of MSN/BZC hybrid materials is entirely new, no data being reported in the literature.
CHARACTERIZATION METODS
FOURIER TRANSMISSION INFRARED (FT-IR)
FTIR spectra were recorded on a Fourier transform infrared spectrometer – Bruker Vertex 70. The FTIR spectra were recorded in 600 ÷ 4000 cm-1 range with 4 cm-1 resolution. The samples were analyzed from ATR for polymer-Graphene oxide composites and KBr pallets for MSN-BZC hybrid materials. All ATR-FTIR spectra were recorded on a FTIR spectrometer with germanium ATR crystal cell at room temperature. The pallets were obtained from a mixture of intimate mixing of the sample (1 mg) and anhydrous KBr (200 mg). The resulting mixture was pressed in the form of a very thin, virtually transparent, disc with a hydraulic press to compress about 109 N/m2, removing the air from the sample simultaneously with a vacuum pump.
RAMAN SPECTROMETRY
Raman spectra were performed on a DXR Raman Microscope from Thermo Scientific using a 633 nm laser line and a number of 10 scans. The laser beam was focused with the 10x objective of the Raman microscope. It was used a 633 nm filter and a holographic diffraction grating of 900 lines/nm. The aperture used was 5 μm pinhole and the exposure time was 10 s. The samples were set on a microscope slide and the laser beam was focused on sample surface.
SCANNING ELECTRON MICROSCOPY (SEM)
The morphology of the films was investigated on a QUANTA INSPECT F scanning electron microscope (SEM) equipped with field emission gun (1.2 nm resolution) and with an energy dispersive X-ray spectrometer with a resolution of 133 eV to MnKα.
TRANSMISION ELECTRON MICROSCOPY (TEM).
The composite films’ structure was further investigated by transmission electron microscopy (TEM), the images being recorded on a TECNAI F30 G2 S-TWIN equipment provided with 300 kV emission gun. To investigate the polymer-GO composites by transmission electron microscopy (TEM), small pieces of the films were embedded in epoxy resin, cut on microtome, transferred to a copper grid covered with a thin amorphous carbon films with holes.
THERMOGRAVIMETRICAL ANALYSIS (TGA)
Thermogravimetrical analysis (TGA) curves were registered on a Q500 TA Instruments equipment, using nitrogen atmosphere from room temperature to 600 °C and a heating rate of 10 °C/min.
X-RAY DIFFRACTION ANALYSIS (XRD)
The X-Ray Diffraction analysis (XRD) was done on a Panalytical X’Pert Pro MPD instrument with CuKα radiation (λ=1.5418Å). X-Ray diffraction (XRD) spectra were registered in the range 2 θ = 1 – 40.
MECHANICAL TESTS
A mechanical tests were performed by using an universal mechanical tester (Instron, Model 3382, USA) at a speed of 2 mm/min at room temperature and relative humidity was 45–50%. The size of the test specimens was 10 cm in length and 1 cm in width. A minimum of ten specimens were tested for each sample and the average values were reported.
ULTRAVIOLET-VISIBLE SPECTROSCOPY (UV-VIS)
To study the samples in the UV-VIS range, a UV-3600 SHIMADZU spectrophotometer was used. It is a double beam spectrophotometer with two double networks monochromatic diffraction, fully controlled by microprocessor. This allows the system to operate in four diffraction gratings according to the selected wavelength. The device is equipped with a light source tungsten halogen lamp for the visible and NIR (295-3600 nm) and deuterium lamps for the UV (185-364 nm) two quartz cuvettes having a path length of 10 mm are employed. The samples were analyzed in transmittance in the range 200-400 nm. UV absorption and release of BZC was performed on a UV 3600 Shimadzu equipment provided with a quartz cell having a light path of 10 mm and was measured at λ=262 nm.
X-RAY PHOTOELECTRON SPECTROSCOPY (XPS)
XPS spectra were recorded on a Thermo Scientific K-Alpha integrated equipment fully fitted with a monochromatic aluminum anode. Preparation of the sample prior to loading on the support of the analysis included the remove of the dust by spraying compressed air support. The sample was loaded into the vacuum chamber analysis. After the vacuum reached a value exceeding 10-8 atm,, the parameters of the experiment were established: X-ray source is turned on and blast load compensation, the positions of the points that will be analyzing the sample are set, respectively ray spot size X and the height of focusing on the sample surface. Surface analysis by XPS was recorded for each sample in three different points, for each point being registred the primary spectra over the range 0-1200 eV with a X-Ray spot of 200 μm and na auto focus area. The primary XPS spectra (0-1200 eV) were recorded with the purpose of identification of constituents.
BIOCOMPATIBILITY STUDIES
All biocompatibility studies were performed on the murine osteoblastic cell line MC3T3-E1 (ECACC, code 99072810). The cells were cultivated in Minimum Essential Medium Eagle (MEM) alpha culture medium, supplemented with 2 mM Glutamine and 10 % Fetal Bovine Serum (FBS). Murine osteoblasts from MC3T3-E1 cell line in the 10th passage were seeded on the surface of the materials with an initial density of 2.5 x 104 cells/cm2. The resulted culture systems were incubated for 7 days under standard culture conditions: 37 oC, 5 % CO2 and adequate humidity. Cell proliferation and viability within the cell-material culture systems were evaluated by fluorescence microscopy using Live/Dead Kit (Invitrogen, Life Technologies, Foster City, CA) (165). Briefly, at 2, 4 and 7 days post seeding, the chitosan/GO films (preseeded with murine osteoblasts) were exposed to a staining solution, calcein AM and ethidium bromide. After 15 minutes, the stained cultures were analysed by fluorescence microscopy using an Olympus IX71 inverted microscope and images were captured with Cell F Imaging Software (Olympus). MC3T3-E1 osteoblasts capacity to proliferate on the surface of the chitosan and chitosan/GO films was quantitatively assessed by MTT test at 2, 4 and 7 days post seeding. Cell-material culture systems were incubated in 1 mg/ml MTT solution for 4h (166).The formazan crystals were solubilized in isopropanol and the concentration of the resulted solution was spectrophotometrically quantified at 550 nm (Appliskan Thermo Scientific). Eventually, the cytotoxic potential of the composite films on the osteoblasts was evaluated using in vitro toxicology assay kit lactate dehydrogenase based (Sigma Aldrich Co., Steinheim, Germany) (167). The culture media were harvested at 2, 4 and 7 days post seeding and mixed with test solution. After 20 minutes of incubation, the reaction was stopped with 1N hydrochloric acid (HCl) and the spectrophotometric detection of lactate dehydrogenase (LDH) concentration was determined by measuring the optic density of the resulting solution at 490 nm (Appliskan Thermo Scientific).
MOLECULAR MODELING OF MECHANICAL PROPERTIES OF THE CHITOSAN BASED GRAPHENE COMPOSITES
Graphene – polymer nanocomposites showed a significant increase in elastic modulus, tensile strength, thermal stability and electrical conductivity even at low loading of graphene filler (163). The main challenge in manufacture of such composite is to ensure a homogeneous dispersion of graphene nanosheets in polymer hosts. On the other hand, controlling the uniformity of dispersion and the degree of alignment of graphene sheets is difficult by experimental means, but computational modelling can ensure some essential vision. However, performing such simulation is not a trivial task due to the complex nature of composite system (polymer and nanofiller).
The aim of this study is to use molecular modeling at atomistic scale in order to predict the mechanical properties of the CHT-graphene biomaterial by varying the graphene composition.
The results presented in this subchapter have been accepted for published in the follow paper: Pandele A.M., Ionita M., Iovu H., Molecular modeling of mechanical properties of the chitosan based graphene composites, U.P.B. Scientific Bulletin.
5.1. SIMULATION METHODOLOGY
Atomistic simulations using Material Studio 5.5 (Accelrys, Inc) software were performed in order to construct and equilibrate the bulk models of the CHT and CHT-graphene composite. The force field applied in the simulation for both polymer and the composites was COMPASS (164). Material Studio 5.5 software provides a versatile sketching tool „Build Polymers/Homopolymer”, which allows constructing the monomer of chitosan manually. The CHT chain models were constructed by linking randomly 100 structural repeat units. In order to obtain a stable starting structure the energy of polymer chain was minimized. A polymer conformation was achieved by packing the polymer chain models into a cubic cell using Amorphous Cell tool. The density of computational cubic cell was decrease by two/three orders of magnitude (0.01/0.001 g/cm3) in order to avoid the ring catenation phenomenon (164). The number of the atoms within the computational bulk model of CHT biopolymer and CS-graphene composites was chosen according to Hofmann and co-workers suggestions (165): the cell has to be sufficiently large so that the interaction between the atoms is avoided; the number of the polymeric chain allows random orientation; the computational costs are low.
The graphene sheet was manually constructed, further charge groups were assigned and energy was minimized in order to obtain a stable starting structure. The CHT and graphene atomic model has been well equilibrated and reaches an energy derivative of 7.94×10-4 and 9.3×10-4 respectively.
Computational bulk model of graphene/CHT with various amount of graphene (4, 7.67, 14.28 wt. %) was constructed using Amorphous Cell module. Computational models were implemented with different contents of graphene in order to determine the influence of graphene amount on the mechanical behavior of the composite systems. Each bulk model includes about 9000 atoms.
5.2. EQUILIBRATION OF THE BULK MODEL
All the models were subject to a complex equilibration protocol. First, molecular dynamics simulation (MD) was performed. The simulations were carried out with constant number of atoms, volume and temperature (NVT) ensemble at a temperature of 300 K for 10 ps by using a time step of 1 fs. The temperature was controlled during simulations by using the Anderson thermostat. Table 5-1 describes the main features of the models.
Table 5-1 The characteristics of the CHT and the CHT-graphene computational models
*Graphene density was considered to be 2.2 g/cm3 (166).
Each MD simulation was followed by molecular mechanic calculations (MM) of about 300.000 steps. The energy minimization was achieved using Conjugate Gradient- Fletcher Reeves algorithm (164).
Afterwards, the computational bulk models were subject of a short MD-NPT simulation (constant number of atoms, pressure and temperature) at p = 0.5 GPa and 300 K for about 5000 steps. The aim of this cycle was to compress the models in order to reach a density closer to that of the real material. The pressure control during simulation was done using Anderson barostat.
After the compression stage the simulated packing models were further equilibrated. For the equilibration of the computational bulk models a complex approach which combined various steps of MM and MS calculations with scaling factors (SF) was used (164). A sequence of 7 MM and MD simulations in which both bond interaction and non-bond interaction were scaled down using SF from 0.001 to 1 were performed. In more details our equilibration routine is described in Table 5-2.
Table 5-2 Step of equilibration protocol
5.3. CHARACTERIZATION
After the equilibration procedure, the density of the CHT and graphene/CHT composite systems was in accordance with that of the real material. Moreover, the computational systems were energetically stable and present an uniform and random distribution (Figure 5-1A) of polymer chain and graphene sheets.
For the composite systems containing 14.28 wt. % graphene sheets (Figure 5-1B) an agglomeration of the inorganic filler within the polymer matrix was observed. Nevertheless, several configurations have been generated after the equilibration proceedure, graphene nanosheets agglomeration was observed in all the cases. The total potential energy of the composite systems after equilibration varies between 2.92×10-4 Kcal/mol for CHT and 5.84×10-4 Kcal/mol in the case of 14.28 wt. % graphene contents.
Figure 5-1 The computational bulk model of A) graphene/CHT (7.67 wt. % graphene) and B) graphene/CHT (14.28 wt. % graphene) after equilibration process
Further, mechanical behavior of the composite systems was evaluated. In order to test the mechanical properties we used the Elastic Properties Analysis tool implemented in the MS software. A procedure described elsewhere was applied for the calculation of the Young’s moduli (167) and the results are summarized in table 5-3. The Young’s moduli outcame from the MD simulations at 300 K increase from 8.55 GPa in the case of pure CHT to 11.55 GPa in the case of graphene/CHT with 14.28 wt. % graphene. It was noticed that by adding 4 wt. % or 7.67 wt. % of the graphene to the CHT matrix an increase of about 6.76 % and 23.25 % respectively was obtained. By future increasing the graphene content to 14.28 wt. % just a marginal effect was observed. This lower increase of the Young’s moduli displayed by the computational system with highest amount of graphene might be due to the graphene agglomeration. The improvement of the mechanical properties of CHT might be assigned to the electrostatic interaction between the inorganic reinforcing agent and the polymer matrices.
Table 5-3 Elastic module for the CHT and CHT-graphene computational bulk models
5.4. CONCLUSIONS
The elastic moduli constants for the pure CHT and graphene/CHT composite systems have been calculated by using molecular modeling at atomistic scale. A reinforcement of CHT was observed with the addition of the graphene. The largest increase (~ 23%) was noticed in the case of the composite with 7.67 wt. % graphene in their structure, by further increasing the graphene amount this tends to agglomerate and produced just a marginal effect.
NANOCOMPOSITES BASED ON BIOPOLYMERS AND GRAPHENE OXIDE
Biopolymers such as chitosan and alginate offer a number of advantages over other materials for developing biomaterials due to theirs excellent properties, such as biocompatibility, biodegradability, bioadhesivity, antibacterial activity, etc. Although, theirs poor processability and mechanical properties which limit the number of applications, a combination of biopolymer and graphene oxide may have a beneficial effect on the characteristics of the composites.
Graphene oxide–biopolymer composites showed a significant increase of elastic modulus, tensile strength, thermal stability and electrical conductivity even at low loading of graphene filler which compensate with the fact that they are non-biodegradable.
6.1. EXPERIMENTAL STUDY OBJECTIVES
In the present study we aimed to synthesize and characterize new nanocomposites based on biopolymers and graphene oxide. To achieve this goal the following secondary objectives were performed:
Preliminary tests are necessary to determine the optimum synthesis and optimization of biomaterial properties such as influence of solvent type; solvent removement (evaporation or freeze-drying);
Synthesis and characterization of new nanocomposites based on biopolymers and graphene oxide;
The structure, morphology and mechanical properties of the films were investigated by FT-IR and Raman spectrometry, TEM, TGA, XRD and mechanical test.
Biodegradability assessment for various polymer-based composite graphene
6.2. RAW MATERIALS
Graphene oxide was purchased from National Institute for Research and Development in Microtechnologies (Romania) and prepared according to Hummers method. Acetic acid (≥ 99.7%), Chitosan from crab shells, Alginate, Poly (vinyl alcohol), Thiazolyl blue tetrazolium bromide (MTT) and in vitro toxicology assay kit lactate dehydrogenase were supplied from Sigma Aldrich. All materials were used without future purification and the water used in this work was double distillate water.
6.2.1. Characterization of GO
FT-IR Spectrum
Figure 6-1 FT-IR spectrum of GO
The FT-IR spectrum of GO is shown in Figure 6-1. The GO functional groups are confirmed by the presence of the following signals: the one around 3400 cm-1, assigned to O-H stretching vibration, 1736 cm-1 attributed to C=O stretching vibration from carboxylic groups, 1618 cm-1 attributed to the stretching vibration of the sp2 network, 1166 cm-1 due to the C-OH stretching vibrations, 1050 cm-1 assigned to C-O stretching vibrations and 879 cm-1 due to the C-O-C stretching vibration from oxirane rings.
XPS Spectrum
According to XPS spectrum (Figure 6-2) on the GO surface it can be identified the following chemical species: C1s (284 eV) and O 1s (530 eV).
Figure 6-2 XPS Spectrum of GO
Raman Analysis
The Raman Spectrum of GO (Figure 6-3) displays four peaks. The peak around 1604 cm-1, named G band, is assigned to the ordered sp2 bonded carbon and the peak at 1343 cm-1 (D band) is assigned to the disorder in the sp2–hybridized carbon atoms. The G peak is due to the bond stretching of all pairs of sp2 atoms in both rings and chains and to the breathing modes of sp2 atoms in rings (168). In the Raman spectrum of graphene oxide the G band is broadened and the D band becomes more preominent in the spectrum to indicate the creation of sp3 domains due to the extensive oxidation.
Figure 6-3 Raman Spectum of GO
XRD Analysis
The XRD pattern of GO exhibits a characteristic peak at 2θ = 10.92 º and the d spacing obtained from Bragg equations as 8.06 Å (Figure 6-4 A), which are close to that of GO previously reported (169).
Figure 6-4 The XRD Spectrum of GO
TEM Image
Figure 6-5 TEM image of GO
Figure 6-5 clearly illustrates flake-like shapes of few hundred nanometer sized staked together of GO.
6.3. NANOCOMPOSITE FILMS BASED ON CHITOSAN AND GRAPHENE OXIDE
For better understanding and better use of graphene or its derivatives based materials in the biomedical field, until now; there are just few studies on how different kinds of cells respond to such materials. This lack of studies has inspired us to explore biological effects associated with chitosan/GO materials on murine osteoblastic cell and determine their potential for applications within the bone regeneration field.
In this Subchapter we report a simple and low cost method for the preparation of chitosan/GO biocomposite films by incorporation of GO into chitosan matrix using water as solvent, the most common solvent used in biomedical applications. Further, this Subchapter investigated relationship between the GO content and the structure, morphologies, thermal stability, and mechanical properties of the composites. The biological activity of the biocomposite films, the status of the cell viability, proliferation, and the quantification of the cytotoxic potential, were carefully assessed.
The results presented in this subchapter have been published in Polymer Composites [Pandele, A.M., Dinescu, S., Costache, M., Vasile, E., Obreja, C., Iovu, H., Ionita, M., Preparation and in vitro, bulk, and surface investigation of chitosan/graphene oxide composite films, 34 (12), 2013, DOI: 10.1002/pc.22620]. Section 6.3.1., 6.3.2., 6.3.3., 6.3.4 and 6.3.5 are directly cited from the article with a few modifications. Published data include: Figure 6-7 (A, B, C and D), Figure 6-8, Figure 6-9 (A,B and C), Figure 6-10 (A, B, C and D), Figure 6-11, Figure 6-12, Figure 6-13 and Figure 6-14 (A and B).
6.3.1. Preparation of graphene oxide-based Chitosan composite films
Figure 6-6 Experimental protocol
The preparation of the samples was made following a procedure described by Han and coworkers (175): 10% (by weight) solution acetic acid in water was prepared. 10 g chitosan was added and stirred continuously at ~ 50° C in 100 ml acetic acid solution to form a uniform viscous solution. The GO solution (1 mg/ml) was gradually added to the chitosan solution and sonicated for 1h at room temperature (Figure 6-6). A series of chitosan/GO solutions with different GO content, 0.5, 1, 2.5 and 6 wt. % were prepared. Finally, homogeneous Chitosan/GO solutions were casted onto transparent glass Petri dish and left undisturbed for 72 h at ambient temperature allowing forming thin films.
The method described in literature (175) was modified in the final stages by using a thermal protocol to form some physical interactions between CHT and GO. The films were peeled off from the mould and thermal treated in vacuum according to the following procedure: 30 min 50 °C, 30 min 70 °C and 4 h at 90 °C.
6.3.2. Structural and morphological characterization
SEM Analysis
Motivated by the success of using GO as a reinforcing phase in biocomposites, Chitosan/GO films were obtained.
Figure 6-7 (A) SEM image of the Chitosan film surface, (B) Chitosan/GO (2.5 wt%) film surface, (C) Chitosan fracture surface, (D) Chitosan/GO (2.5 wt%) fracture surface
The chitosan/ GO films look and feel smooth and have thickness of 50 ± 10 µm. Even at high magnification the surface of chitosan film displays a homogeneous, smooth morphology (Figure 6-7 A). However, under SEM observation, composite films surface appear rough as shown in Figure 6-7 B which displays the surface of chitosan/GO composite with 2.5 wt% GO content. The GO nanosheets can be faintly seen on the surface of the composite films. Conversely, the cross-section of the Chitosan (Figure 6-7 C) and chitosan/GO films become bristlier, particularly when GO is added (Figure 6-7 D). The GO nanosheets appear uniformly distributed and embedded into the chitosan matrix, seldom GO nanosheets staked together were observed. In addition, the GO nanosheets observed in the cross section seem to be unidirectional distributed parallel to the biocomposite film surface. The orientation of GO nanosheets is probably assisted by gravitational attraction and the method used for the biocomposite preparation; i.e. solution casting.
TEM Analysis
Figure 6-8 High-resolution TEM micrograph of the chitosan/GO (2.5 wt%).
The TEM results also confirmed the presence of GO into the chitosan matrix. Figure 6-8 shows typical TEM picture indicating the well dispersed status of the GO, in addition, clear stratification of GO parallel to the surface of the film is observed. The apparent alignment of GO sheets is exactly in agreement with SEM assessment. The same observations were reported in the literature for similar materials (i.e. chitosan/GO and chitosan/reduced graphene oxide) fabricated by the same method (i.e. solution casting). Pan et al reported that GO sheets tend to lie down inside the chitosan solution due to their 2D structure and gravitational attraction (170).
FT-IR Analysis
FT-IR experiments were carried out to investigate the interaction between chitosan and GO. The FT-IR spectra of chitosan, GO, and chitosan/GO (2.5 wt%) biocomposite film are shown in Figure 6-6. In the spectrum of GO, there are two characteristics bands at 1736 cm-1 and 1618 cm-1 assigned to C=O stretching vibration of the carboxylic group and C=C stretching mode of the sp2 network (171). For Chitosan FT-IR spectrum, a strong peak at 1554 cm-1 corresponding to N-H bending of –NH2 and an absorption peak at 1379 cm-1 attributed to C-CH3 deformations in amide III groups are observed (172). As shown in Figure 6-6 C, the spectrum of chitosan/GO composite film is roughly similar to that of pristine chitosan. However, the C=O stretching vibration of the –COOH group has diminished to disappear, besides that intensity of the N-H banding of –NH2 decreases; this indicates that some amine groups from chitosan chain reacted with –COOH groups on GO surfaces and changed in –NHCO- groups and therefore, the intensity of the peak at 1379 cm-1 assigned to C-CH3 deformation in amide group has increased.
Figure 6-9 FT-IR spectra of the (A) GO, (B) Chitosan and (C) Chitosan/GO biocomposite film 2.5 wt%.
XRD Analysis
The structure of the biocomposite films was further studied by XRD. Figure 6-10 depicts the X-ray diffractograms of GO, chitosan and chitosan/GO biocomposite films with 0.5 and 2.5 wt% GO measured at 25 ºC. The XRD pattern of GO was presented in figure 6-5 (Section 6.3.). XRD pattern of chitosan shows three diffraction peaks at 8.8 º, 11.4 º and 19.48 º (Figure 6-10 B). The weak diffraction peaks at 8.8 º, 11.4 º correspond to the hydrated crystalline structure, whereas the broad peak at 2θ = 19.48 º, indicates the presence of an amorphous structure (173). XRD pattern (Figure 6-10 C) of the biocomposite film with low GO content (0.5 wt%) is similar to that of chitosan, presents two diffraction peaks at 2θ ~ 8 and 18 º, by further increasing the GO content to 2.5 wt% the peak at 18 º diminish to disappear (Figure 6-10 D).
Figure 6-10 XRD patterns of (A) GO, (B) Chitosan, (C) Chitosan/GO 0.5 wt%, and (D) Chitosan/GO 2.5 wt% films
The diffraction peak characteristic for GO disappeared or is weak and overlapped by the broad diffraction peak of chitosan at 2θ ~ 8 º, indicating that GO is efficiently dispersed within chitosan matrix. Conversely, even if in biocomposite some GO are still staked together, as indicating by SEM and TEM, these are rarely. The addition of GO seems to affect the crystalline structure of the chitosan. It is noticed that the diffraction peak at about 8 º broadened indicating that GO is hindering the relatively ordered arrangement of the chitosan. Previous studies included reports of decreases and increases in the crystallinity of the polymer matrix with increases in nanofiller loading. Yang et. all have introduced GO in chitosan matrix. An increase in crystallinity of chitosan was observed which is attributed to the electrostatic interaction and hydrogen bonding which contribute to a relatively ordered arrangement of the chitosan chains (173).
The composites chitosan/GO prepared by Wang et. al exhibit almost similar crystallinity as the chitosan itself, even if they observed good contact and electrostatic interactions between the GO and chitosan . On the other hand, Jose et. all have argued that the incorporation of MWNTs into polypropylene matrix hinders the recrystallization of the matrix (174). The dissimilarity between those reports may be attributed to differences in filler–matrix interactions. The XRD analysis for our chitosan/GO biocomposites indicate that the incorporation of the GO within the chitosan matrix lead to a decrease of the chitosan crystallinity probably due to the covalent bonding, evidenced by FT-IT investigation, of the polymer chain to rigid GO sheets which limits chitosan chain mobility and hinder its relatively ordered arrangement.
6.3.3. Thermal and mechanical characterization
TG Analysis
The thermal stability of the biocomposites chitosan/GO was assessed by thermogravimetric analysis. From the TGA profiles (Figure 6-11) of the samples, it was shown that the biocomposites chitosan/GO exhibited similar thermal degradation steps as that of neat matrix. Figure 6-11 and Table 6-1 showed that the temperature at which the mass loss is 3% (Td3%) for neat chitosan degradation was about 65°C. The Td3% increased by about 32 ºC in the case of the composite with the highest amount of GO (6 wt. %) added to the pure biopolymer. Conversely, the residual weight loss of the composites is higher than that of neat chitosan, which could be ascribed to the excellent thermal stability of GO which generally shows a decomposition temperature around 200 ºC. The improved thermal stability could be contributed also by the strong interaction between chitosan and GO at the interface, which suppresses the mobility of the polymer chains. Similar trends concerning enhancement of thermal stability was observed for the chitosan/GO composite by Fan et al. (110).
Figure 6-11 TG analysis curves of CHT and CHT/GO composites
Table 6-1 Temperature at which the mass loss is 3%, tensile modulus and tensile strength of neat chitosan and chitosan/GO composite films
Mechanical Tests
The interest in graphene-based composites stems also from their potential high mechanical properties. The observed homogeneous dispersion and alignment of GO nanosheets within the chitosan matrix evidenced by SEM, TEM, and XRD combined with the strong interfacial adhesion due to specific interactions between chitosan amino groups and GO carboxyl groups indicated by FT-IR is expected to significantly enhanced the mechanical properties of the chitosan/GO composite films. The mechanical behaviour of the chitosan and chitosan/GO composite films was investigated by tensile tests. The representative tensile stress versus strain curves are presented in Figure 6-12 for comparison. The key tensile properties are listed in Table 6-1. The data are the average results measured for 7-10 samples.
Figure 6-12 Tensile stress versus strain curves of neat chitosan and chitosan/GO composite films
The results show that the incorporation of GO can improve the chitosan Young’s modulus and tensile strength. Even at low GO loading, the composite films chitosan/GO exhibit an enhancement of the mechanical performance. For example, with a GO loading of only 0.5 wt%, the tensile strength and Young’s modulus increased by 37% (from 73.46 to 107.25 MPa) and 16% (from 2.78 to 3.22 GPa), respectively, as shown in Table 6-1. Further addition of GO (1 and 2.5 wt%) increased the Young’s modulus slightly from 3.22 GPa to 3.29 and 3.70 GPa respectively without any pronounced changes. However, these improvements are not as significant as expected. The incorporation of 6 wt% GO leads to the best overall reinforcing effect in modulus by about 61%. The main reason for the modulus improvements is probably because of the large aspect ratio of the GO sheets, the overall good dispersion of GO sheets within the chitosan matrix, and the strong interfacial adhesion due to covalent-bonding between graphene and chitosan. Similar observations were reported in the literature for chitosan associated with GO. Han et al. reported that chitosan/GO blended films show improved elastic properties at relatively low weight fraction of the GO (175).
6.3.4. Biocompatibility assessment
Murine osteoblasts were qualitatively tested for viability and proliferation potential at 2, 4 and 7 days post seeding. As shown in Figure 6-13, the ratio between the living (green labelled) and the cells that lost membrane integrity (red labelled) was highly positive, suggesting high cell viability in contact with the chitosan/GO composite materials. Fluorescence microscopy images revealed that cells progressively proliferated moreover, after 7 days of culture reached confluent monolayers on all chitosan/GO biocomposite films. The cellular density was found to be higher on the composite materials with 2.5 and 6 wt% GO content than that on the chitosan/GO composite films with lower GO content or 2D control. Moreover, cell distribution was observed to be different on the surface of the biocomposite films than on the control. According to Figure 6-13, the cell distribution on the surface of the biocomposite films with low GO content (0.5 and 1 wt%) is similar to that of the chitosan and control. Conversely, a particular cell distribution was observed for the biocomposites containing higher GO content (2.5 and 6 wt%). A possible explanation could be that the content of GO influence cell behaviour and distribution, as well as cell proliferation potential.
Murine osteoblasts proliferation potential on chitosan/GO biocomposites was quantitatively assessed by MTT test. Viability and cell proliferation profiles generally described ascending trends up to 7 days of culture in standard conditions (Figure 6-14 A). Murine osteoblasts were able to proliferate on the surface of the tested materials, generating cell monolayers on all biocomposites. The proliferation potential was proved to be higher for cells cultivated on the surface of chitosan and chitosan/GO films, as compared to the classical bidimensional 2D control (Figure 6-14 A). These observations are in total accordance with the conclusions of the Live/Dead assay.
Figure 6-13 Fluorescence microscopy evaluation of living (green-labelled) and dead (red-labelled) murine osteoblasts at 2, 4 and 7 days post seeding on 2D control, Chitosan and Chitosan/GO composite materials
When comparing cell viability at 2 days post seeding, no important differences were registered between the materials with low GO content (0.5 and 1 wt%) and the controls which suggests that cellular behaviour on these composites is similar with that of the chitosan and control. Conversely, significant higher levels of cell proliferation were observed for chitosan/GO biocomposites with 2.5 and 6 wt.% content. This could be an indication that the GO amount exhibits a significant impact on cell behaviour and proliferation since the first days of culture. The results revealed by MTT test at 4 and 7 days of culture is similar to the one registered at 2 days post seeding; the films with 2.5 and 6 wt% GO content highly exceeded cell proliferation of the films low GO loading, chitosan or 2D control, particularly after 7 days of culture. Consequently, chitosan/GO 6 wt% composite film proved to condition the highest cell proliferation rates and viability during one week of culture in standards conditions, as compared to the controls and the other tested composites.
Cytotoxic potential of the chitosan/GO biocomposite films was tested on the murine osteoblasts during one week. The results revealed higher levels of LDH activity in the culture media harvested from the chitosan and chitosan/GO biocomposite than LDH activity level corresponding to the 2D control (Figure 6-14 B). No significant differences between the biocomposite and chitosan in terms of LDH levels released in the culture medium after 2 days of culture were observed. After 4 days of culture in standard conditions, the cytotoxic potential of chitosan/GO biocomposites with low content of GO (0.5, 1 and 2.5 wt%) was comparable with the cytotoxic potential displayed by the chitosan, while lower level of the LDH activity was registered for chitosan/GO biocomposite with 6 wt% GO content. A similar trend was observed after 7 days post seeding; the LDH activity registered for chitosan / GO 6 wt.% biocomposite was significantly lower than the LDH activity revealed for the chitosan and the lowest among the tested biocomposite. These data are in accordance with MTT test results, which indicated the highest cell proliferation potential for the biocomposite chitosan/GO 6 wt% after 7 days of culture. One can say that the addition of GO within chitosan matrix has beneficial effect on biocompatibility features. Furthermore, high GO content influences cell proliferation potential, in contrast with low content GO which apparently does not interfere with cellular behaviour.
Figure 6-14 (A) Quantification of murine osteoblasts proliferation rate on Chitosan and Chitosan/GO films as revealed by MTT assay, (B) cytotoxic potential of the Chitosan and Chitosan/GO films in contact with osteoblasts as revealed by LDH assay
6.3.5. Conclusions
The nanocomposites based on exfoliated GO nanosheets and chitosan were prepared successfully using a simple solution blending method. GO sheets containing carboxylic and hydroxyl groups may form strong interactions with the chitosan matrix as indicated by means of FT-IR spectrometry. The SEM, TEM, and XRD results confirm overall good dispersion of GO within the chitosan matrix. The thermal stability of the composites films was higher than that of pristine chitosan. The TG analysis indicates that the incorporation of 6 wt% GO within the chitosan matrix leads to an increase of Td3% with about 32 ºC. The tensile modulus of the composite containing 6 wt% GO was up to 4.47 GPa, whereas that of pure chitosan was only 2.78 GPa; i.e., a 61% increase in tensile modulus. The viability and proliferation data together with the quantification of the cytotoxic potential suggested that the murine osteoblasts tend to adapt faster and proliferate more in contact with the chitosan/GO biocomposites with a higher content of GO. The biocomposite chitosan/GO 6 wt% proved to be biocompatible and displayed the most equilibrated ratio between the pro-proliferative and cytotoxic potential. The excellent biocompatibility, good mechanical properties and thermal stability place chitosan/GO composites, as materials with remarkable potential in biomedical applications such as bone repair or bone augmentation.
6.4. NANOCOMPOSITE FILMS BASED ON CHITOSAN- POLYVINYL ALCOHOL AND GRAPHENE OXIDE
Chitosan (CHT) is widely investigated for biomedical applications because of its good biocompatibility, biodegradability, bioadhesivity and antibacterial properties. However, some major drawbacks such as low mechanical strength and hard processability restrict its use in tissue scaffolds field. By combining biopolymer such as CHT and PVA some of the CHT drawbacks mentioned above are overcome.
Our approach in the present Subchapter is to take advantage of complementary properties of the three materials, biocompatibility associated with CHT, processability and versatility associated with PVA and exceptional physical properties of GO in order to obtain a composite material which merges the above-mentioned properties. Incorporation of GO within the polymer is an active approach for improving the physical and chemical properties of the blend. Currently, to the best of our knowledge, there have been no reports on biological effects associated with CHT-PVA/GO materials on murine osteoblastic cell.
In this Subchapter we report a simple and effective method for the fabrication of thin films based on incorporation of GO in CHT–PVA blends.
The results presented in this subchapter have been published on Carbohydrate Polymer [Andreea Madalina Pandele, Mariana Ionita, Livia Crica, Sorina Dinescu, Marieta Cistache, Horia Iovu, Synthesis, characterization, and in vitro studies of grapheneoxide/chitosan–polyvinyl alcohol films, Carbohzdrate Polymer, 102, 813– 820, 2014]. Section 6.4.1., 6.4.2., 6.4.3., 6.4.4 and 6.4.5 are directly cited from the article with a few modifications. Published data include: Figure 6-16 (A, B and C), Figure 6-17 (A,B and C), Figure 6-18 (A, B, C and D), Figure 6-19 (A, B, C and D), Figure 6-20, Figure 6-21, Figure 6-22, Figure 6-23, Figure 6-24 and Figure 6-25.
6.4.1. Preparation of PVA-Chitosan/graphene oxide composite films
For the preparation of the samples we used the method described by Jun Ma et al (162) using different CHT:PVA ratio.
Chitosan was mixed with an acetic acid solution (10 % by weight in water) at ~ 50 °C in order to form 1 wt. % homogenous viscous solution. Separately, 1 wt.% PVA aqueous solution was obtained by dissolving PVA powder in distilled water at 120 °C in an autoclave. Blends were prepared by mixing the two polymer solutions (50:50 v/v ratio). The composite mixtures with different GO amount (0.5, 1, 2.5, 6 wt.%) were prepared by gradually adding the GO aqueous solution (1 mg/ml) to the CHT-PVA solution and sonicating for 1 hour at room temperature. The homogeneous CHT-PVA/GO solutions were poured onto transparent Petri glass dish and left undisturbed at room temperature for 72 hours in order to evaporate the solvent and form the film (Figure 6-15).
The method described in literature (162) was modified in the final stages by using a thermal protocol. After that the films were peeled off form the mould and thermal treated in vacuum according to the following procedure: 30 minute at 50 °C, 30 minute at 70 °C and over the night at 120 °C.
Figure 6-15 Experimental protocol
6.4.2. Structural and morphological characterization
FT-IR Analysis
The FT-IR experiments were performed to investigate the interaction between the CHT-PVA and GO. The FT-IR spectra of CHT-PVA, GO, and CHT-PVA /GO (2.5 wt. %) biocomposite films are shown in Figure 6-16. GO spectrum was already discussed in section 6.3., Figure 6-1. CHT-PVA blend exhibits FT-IR absorption bands around 3291 cm-1 and 2914 cm-1, concerned -OH stretching and -CH2 asymmetric stretching from PVA. The peaks at 1647 cm-1, 1563 cm-1, and 1327 cm-1 are assigned to amide I and III of C=O stretching vibration, N-H bending of –NH2 and -CH2 wagging coupled with OH group from CS. The peak at 1411 cm-1 corresponds to oscillations of -OH and -CH groups (53). In comparison with CHT-PVA blend spectrum, CHT-PVA/GO composite spectrum (Figure 6-16 C) shows new peak at 1743 cm-1 assigned to carboxyl groups from GO surface which indicates the presence of GO within the CHT-PVA blend. Furthermore, the peak at 3291 cm-1 (O-H stretching vibration) was shifted to lower values (3241 cm-1) and becomes more broadened which could be attributed to the hydrogen-bonding interaction of GO with CHT-PVA blend.
Figure 6-16 FT-IR spectra of the A) GO, B) CHT-PVA, and C) CHT-PVA/GO 2.5 wt. %
Raman Analysis
Figure 6-17 Raman spectra of A) GO, B) CHT-PVA/GO composite film with 1 wt.% GO content and C) CS-PVA/GO composite film with 6 wt. % GO content
Figure 6-17 shows the Raman spectra of pristine GO and CHT-PVA/GO composite films. The Raman spectrum of GO is presented in Figure 6-3. From Figure 6-17 B and C it can be observed that CHT-PVA/GO composites exhibit a similar spectrum with the pristine GO, with a slight shifting of D and G band to ~1340 and 1602 cm1 respectively. Furthermore, by adding the GO into the polymer blends the intensity ratio of D and G bands (ID/IG) increases and thus the degree of disorder increase (Table 6-2).
Table 6-2 The ID/IG of GO and the CHT-PVA/GO composite films
*ID intensity of D band, IG intensity of G band, ID/IG intensity ratio of D band to G band
At 1 wt.% GO, the intensity of ID/IG is higher than for the GO powder meaning that the filler is well dispersed into the polymer. However, increasing the GO amount (2.5wt.% and 6 wt.%), the ID/IG ratio decrease due to the aggregation of the reinforcing agent. By agglomeration the GO crystallite size increase forming a more orderly structure. The intensity ratio of D and G band (ID/IG) is used to measure the distance between the defects in the GO structure (176). Cancado et al. noted that the ratio of the D to G band intensities varied inversely with the crystallite size (177).
XRD Analysis
The crystallinity of the CHT-PVA/GO composites was further examined by XRD investigations. The GO spectrum is presented in Figure 6-4 (section 6.3.). The XRD pattern of CHT-PVA blend shows two weak diffraction peaks located at 2θ = 9.03° and 11.9° assigned to CS indicating an almost amorphous structure for this compound.
Figure 6-18 XRD patterns of A) GO, B) CHT-PVA, C) CHT-PVA/GO 2.5 wt.% and D) CHT-PVA/GO 6 wt.%
The peak appearing at 2θ=19.82° belongs to PVA crystallites (178). In the CHT-PVA/GO composite films spectra (Figure 6-18 C, D) it is observed that the incorporation of the GO obviously increase the intensity and sharpness of the characteristic peaks of CHT (2θ = 9.45 º and 2θ = 11.87 º). Moreover, there is an increase in intensity of the peak centered at 2θ=19.82° corresponding to PVA as the GO content increases in the polymer blend. The absence of the characteristic peak of GO in CHT-PVA/GO films is indicating that most GO sheets were dispersed within polymer matrix, or the peak is weak (just limited number of GO aggregates are formed) and overlapped by the peak of CHT at 2θ= 9.45°. X-ray diffraction pattern clearly illustrates that the incorporation of GO affect the crystalline structure of CHT-PVA. Similar behavior was found for other nanocomposites polymer/carbon nanofiller and is believed to be a result induced by the carbon nanofiller (173, 179). In our particular case of CHT-PVS/GO composite films, the increase of the polymer blend crystallinity could be attributed to the intermolecular hydrogen bonding, confirmed by FT-IR, between CHT-PVA blend and GO which could give a relatively ordered adjustment of the polymer chains along the GO nanosheets.
SEM Analysis
Figure 6-19 (A) SEM image of the CHT-PVA film surface, (B) CHT-PVA film fracture surface, (C) CHT-PVA/GO 2.5 wt. % film surface, (D) CHT-PVA/GO 2.5 wt. % film fracture surface
The morphology of the composite films was characterized by SEM and TEM techniques. Figure 6-19 C and D exhibit a typical SEM image of surface and the cross-section of the CHT-PVA/GO composite films. Unlike the pure polymer (Figure 6-19 A and B), whose structure is smooth, the morphology of the nanocomposite changes, becomes rough and exhibits little wrinkles assigned to the GO nanosheets presence.
TEM Analysis
The TEM investigation confirms the homogenous dispersion of GO sheets within polymer matrix with hardly any aggregations for lower GO contents (0.5, 1 wt. %). Conversely, it seems that the GO content increase to 2.5 and 6 wt. % leads to formation of isolated aggregation of the nanofiller (Figure 6-20). The orientation of the GO nanosheets seems to be almost parallel distributed to the biocomposite film surface.
Figure 6-20 High-resolution TEM micrograph of the CHT-PVA/GO 2.5 wt. %
6.4.3. Thermal and mechanical characterization
The interest in CHT-PVA reinforced composites is based on their potential applications in biomedical field such as bone repair or bone augmentation, utilization which requires good thermal and mechanical properties together with excellent biological activity. The observed good dispersion of GO within CHT-PVA matrix showed by SEM, TEM and XRD combined with hydrogen bonds interaction between GO and CHT-PVA polymer chains, evidenced by FT-IR spectrometry, is anticipated to produce an enhancement of mechanical and thermal properties of the materials. Moreover, the presence of the GO produced modification of materials surface which can facilitate the cells attachment. On the other hand GO nanosheets from the film surface could act as anchors for cells.
TG Analysis
The thermogravimetrical analyses were used to explain the CHT-PVA blends thermal behavior with GO loading. The TGA curves of pure CHT-PVA blend and the CHT-PVA/GO composite films with various contents of GO were outlined in Figure 6-21. As can be seen, both pure CHT-PVA films and the composite present three decomposition steps. The first step between 70 °C and 120 °C is due to the moisture inside the film. The second degradation step starts at 250 °C and ends at 410 °C and is assigned to the decomposition of the polymer structure. The final stage (̴ 420 °C) is attributed to the further decomposition of the residue (180). If GO is loaded into the polymer matrix the degradation stages were shifted toward higher values of temperature.
Figure 6-21 The TGA curves of CHT-PVA and CHT-PVA / GO nanocomposites
The Td3% (temperature at which the mass loss is 3%) values obtained for the pure CHT-PVA and the composites are summarized in Table 6-3 which shows an increase with 7-12 °C in the case of the composite with lower (0.5 and 1 wt. %) amount of GO due to the good dispersion of the filler into CHT-PVA matrix and the presence of hydrogen interaction between CHT-PVA chains and GO which suppress the polymer chain mobility. However, as the GO loading further increases, the tendency for Td3% is to decrease and even reach a lower value than for the neat reference (CHT-PVA) probably due to the aggregation of GO within the polymer blend.
Table 6-3 Td3% and tensile modulus of CHT-PVA blend and CHT-PVA/GO nanocomposites
Mechanical Tests
The mechanical behavior of the CHT-PVA blend and CHT-PVA/GO composite materials with different content of GO was further characterized by tensile tests. The representative tensile stress versus strain curves are reported in Figure 6-22 and the average (measured for 6-8 samples) tensile modulus are listed in Table 6-3. An improvement of the mechanical performance of the nanocomposite compared to pure CHT-PVA matrices was observed. It can be noticed that the addition of GO improves the mechanical response of CHT-PVA matrix. By adding a small amount of GO within the CHT-PVA matrix just a marginal effect was observed, the Young's modulus increased from 1.83 GPa to 1.96 GPa. By further increasing the GO content in polymer matrix a significant increase of the Young’s modulus was noticed, by loading 6 wt. % GO within CHT-PVA matrix an increase by about 200 % of the Young’s modulus was observed. This trend is however normal and ascribed to the interaction between GO and CHT-PVA matrix and the change in the crystallinity, which are both important factors for the enhancement of tensile properties of a polymer. These findings could be also correlated with the largely good dispersion of GO within the polymer matrix (173). Clearly here the crystallinity plays an important role in the increase of the mechanical properties for CHT-PVA/GO composites. The incorporation of GO affects the crystallinity of CHT-PVA as indicated by XRD and Raman spectroscopy pattern. The addition of 2.5 and 6 wt. % of GO produced and important change of the crystalline structure of the CHT-PVA and significant increased the Young’s modulus of composites. The incorporation of less than 1 wt% GO only slight affect the crystalline structure of CHT and produced insignificant increase of the Young’s modulus of the composites. Similar findings have been revealed for carbon nanotubes (181) and GO based composites (173) whereas the largest improvement of tensile modulus was observed for the composites in which a great increase of crystallinity of the polymer was noticed.
Figure 6-22 Tensile strain versus tensile stress for CHT-PVA and CHT-PVA / GO films
6.4.4. Biocompatibility assessment
All materials were tested for cytotoxicity in contact with murine osteoblasts during one week of culture. Although all samples displayed low cytotoxicity at 2, 4 and 7 days, the CHT-PVA/GO composites with 2.5 wt.% and 6 wt.% GO content showed a significantly lower cytotoxic potential than CHT-PVA/GO composites with 0.5 wt% and 1 wt%. The samples with 0.5 wt.% and 1 wt.% GO content registered the same levels of LDH released in the culture medium as the control and CHT-PVA blend (Figure 6-23). Thus, the addition of 0.5 wt.% and 1 wt.% graphene oxide in the composition of CHT-PVA film apparently has no significant impact on the material’s cytotoxicity. Conversely, the samples with 2.5 and 6 wt.% GO revealed a similar cytotoxic potential at 2 days of culture as compared to the control. However, at 4 and 7 days of culture, LDH levels measured for composite films with 2.5 and 6 wt.% GO were found to be lower than the levels registered for control. Consequently, CHT-PVA/GO 6 wt.% displayed the lowest cytotoxic potential among the studied compositions, which may suggest that the addition of graphene oxide in the structure of chitosan films enhances material biocompatibility and ensures a favorable microenvironment for cell viability and proliferation.
Quantitative MTT test revealed cell viability and proliferation on all studied samples (Figure 6-24). An increasing cell proliferation potential was generally observed during 7 days of culture in standard conditions. Murine osteoblasts were able to proliferate on the surface of the tested materials, generating cell monolayers on all composites. However, the proliferation potential was proved to be higher for cells cultivated on the surface of the CHT-PVA and CHT-PVA/GO films, as compared to the classical bidimensional (2D) culture. At 2 days post-seeding, cell viability for CHT-PVA/GO 0.5 wt.% and 1 wt.% composite films and the controls was similar. Significant differences were observed for CHT-PVA/GO composite film with 6 wt.% GO content, for which the highest cell viability at 2 days of culture was registered. Furthermore, the quantification performed after 4 and 7 days of culture revealed the same cell viability and proliferation pattern, with a higher proliferation rate for osteoblasts cultivated on CHT-PVA/GO composite films with 2.5 wt. % and 6 wt. % GO content, as compared to the materials with lower GO content. In terms of cell viability and proliferation, the best sample was found to be CHT-PVA/GO 6 wt.%, suggesting that a higher percent of GO within the film composition is a "must" for better biocompatibility and cell bioactivity than in the absence of GO.
Cell behaviour (viability, morphology, distribution) was qualitatively assessed after 2, 4 and 7 days of culture by fluorescence microscopy, based on the simultaneous staining of live and dead cells (Figure 6-25). The results obtained by Live/Dead assay confirmed the quantitative determinations performed during MTT and LDH tests, concluding that CHT-PVA/GO composite films with higher percentage of GO in their composition display higher biocompatibility that the ones with low GO content.
Figure 6-23 The cytotoxic potential of the CHT-PVA/GO films in contact with osteoblasts at 2, 4 and 7 days of culture, as revealed by LDH assay
Figure 6-24 The quantification of cell proliferation rate on CHT-PVA/GO films as revealed by MTT test at 2, 4 and 7 days of culture
Figure 6-25 Fluorescence microscopy evaluation of living (green-labeled) and dead (red-labeled) murine osteoblasts at 2, 4 and 7 days post-seeding on CHT-PVA and CHT-PVA/GO films.
Briefly, it was observed that cell behaviour varies depending on the GO content. For example, cells displayed a particular morphology and distribution in lines on the surface of CHT-PVA/GO 6 wt.%, as compared to the other samples and to the controls. In terms of cell proliferation, it was observed a constant increasing cell number on all samples, suggesting a significant proliferation potential which cells displayed in contact with the tested materials. However, a lower cell number was found on the samples with 0.5 and 1wt.% GO content at 2, 4 and 7 days, as compared to those with 2.5 and 6 wt.% GO content. These results were correlated with the higher percent of dead (red-labeled) cells which was observed on the low-GO-containing materials, as compared to the rest of tested materials. Additionally, different cell morphology was revealed on the GO-containing materials than on the controls. Cell grouping tendency was revealed for the materials with higher GO content (2.5 wt.% and 6 wt.%), as observed in Figure 6-25.
6.4.5. Conclusions
Mechanically strong with excellent biocompatibility CHT-PVA/GO composite films have been successfully synthesized by solution blending method. It was observed by SEM, TEM and X-ray diffraction that graphene oxide is largely dispersed on molecular scale in the CHT-PVA matrix. FTIR investigation indicated the occurrence of some interaction between graphene oxide nanosheets and CHT-PVA blend. The composite film containing 6 wt.% GO showed highest tensile modulus of 5.78 GPa, which is about twice higher than for the neat matrix (CHT-PVA). The composite films show a higher thermal stability if lower (0.5 and 1 wt.%) amount of GO was incorporated within CHT-PVA.
A higher content of GO within the composite film led to a significant increase of the cell proliferation rate. The CHT-PVA/GO films with high mechanical strength, good thermal stability and excellent biological activity are competitive candidates for various biological application, such as bone repair and tissue engineering.
6.5. NANOCOMPOSITES BASED ON ALGINATE AND GRAPHENE OXIDE
Alginates as in the case of CHT displays some unsatisfactory properties such as poor mechanical strength and loss of structural integrity which limits its applications. In the present thesis many systems that seemed promising were studied in order to select the most suitable material for bone repair. To the best of our knowledge there is no report in the literature on alginate (Al) embedded with graphene or GO to obtain composite films.
In the present study, we employed casting techniques for the preparation of novel Al / GO nanocomposite films and then structural, morphological, thermal and mechanical properties of the films were explored.
The results presented in this subchapter have been published on Carbohydrate Polymer [Mariana Ionita, Andreea Madalina Pandele, Horia Iovu, Sodium alginate/graphene oxide composite films with enhanced thermal and mechanical properties, Carbohydrate Polymers, 94 (1), 339-344, 2013]. Section 6.5.1., 6.5.2., 6.5.3., 6.5.4 and 6.5.5 are directly cited from the article with a few modifications. Published data include: Figure 6-26 (A, B and C), Figure 6-27 (A,B, C and D), Figure 6-28, Figure 6-29 (A, B, C and D), Figure 6-30, Figure 6-31 and Figure 6-32.
6.5.1. Preparation of graphene oxide-based sodium alginate composite films
Graphene oxide aqueous suspensions were prepared by ultrasonic treatment using double distillate (DD) water. Briefly, 0.1 g of GO was mixed in 100 ml of DD water. The mixture was sonicated for 1 h to get a homogenous dispersion. The alginate solution was obtained by dissolving the sodium alginate in DD water as 1 % (w/v) solution. Composite films with different GO contents (0, 0.5, 1, 2.5, and 6 wt %) were produced by dropwise GO suspension to the sodium alginate solution. The resulting mixture was constantly stirred for about 30 min using a magnetic stirrer. Each solution was cast onto transparent glass Petri dish and left undisturbed for 72 h at ambient temperature allowing formation of thin films. The films were peeled off from the mould and then thermal treated in vacuo according to the following procedure: 30 min 50 °C, 30 min 70 °C and 4 h at 120 °C.
6.5.2. Characterization
By employing casting technique well-dispersed Al / GO nanocomposite films were fabricated with thickness of 70 ± 10 µm.
FT-IR Analysis
Figure 6-26 FTIR spectra of A) Al, B) GO, and C) Al/GO composite film with 2.5 wt.%
FT-IR experiments were carried out to investigate the interaction between GO and Al. Figure 6-26 shows absorption bands from FT-IR spectrum of Al, GO and Al / GO composite films. The FT-IR spectra of GO and Al were similar to those previously presented in literature (102) (182). In the Al spectrum the bands at 1600 cm-1 and 1414 cm-1 correspond to symmetric and asymmetric COO- stretching vibration of carboxylate salt group, and the peak at 1030 cm-1 is assigned to the stretching vibration of C-O-C group. For the GO spectra, dominant peaks appear at 1735 cm-1, 1624 cm-1, 1043 cm-1 and are assigned to C=O stretching vibration of the carboxylic group, C=C stretching mode of the sp2 network and C-O stretching vibration respectively (102), demonstrating the presence of the reactive groups in the GO product.
From FT-IR spectrum of the composite films, a shoulder peak at 1735 cm-1 due to the carboxyl groups from the GO surface can be observed in addition to Al spectrum which shows the presence of GO in composite films.
XRD Studies
Figure 6-27 XRD patterns of A) GO, B) Al and Al/GO composites with different GO content: C) 1% and D) 2.5 %
Figure 6-27 depicts the X-ray diffractograms of Al, GO, and Al/GO composite films with 1 and 2.5 wt % GO measured at 25 ºC. The GO spectrum is previously reported in section 6.3. (Figure 6-4) the values being within the range of values that has been previously reported (169). In the XRD pattern of Al film the weak, broad diffraction peak at 13.32 º, with average intermolecular distance 6.63 Å indicates a rather amorphous structure for this compound (183). All of the XRD patterns of the composite films show two diffraction peaks at 2θ ~ 5 and 13 º associated with GO and Al respectively. The diffraction angle characteristic for GO is shifted toward lower angle values (Table 6-4) and the GO interlayer spacing increases from 8.06 Å for pristine GO to ~ 17.19 Å for Al / GO composite film with 2.5 wt % GO, indicating formation of intercalated structures for which the Al chains managed to penetrate between the GO sheets. As a consequence if the GO content increases the GO interlayer space decreases as a less amount of Al is available to intercalate within GO sheets. It is noticed that the incorporation of GO within Al matrix slightly broadened the diffraction peak at about 13 º indicating that GO is hindering the relatively ordered arrangement of the Al (184).
Table 6-4 XRD data of Al and Al /GO composite films
TEM and SEM Studies
Figure 6-28 shows typical TEM micrographs of the nanocomposite films AL /GO indicating that GO are indeed present and partially exfoliated into individual sheets within Al matrix. Apparently, GO sheets were unidirectionally distributed in the Al matrix.
Figure 6-28 TEM images of Al/GO 2.5 wt % composite films
Figure 6-29 SEM film surface images (A-B) and fracture surface images (C-D) of (A, C) Al, (B, D) Al/GO 1 wt %.
To further determine the quality of the GO dispersion and the composite films morphology SEM was employed. Figure 6-29 illustrates images of Al and Al/GO composite films with 2.5 % GO loading. From the films surface micrographs (Figure 6-29 A, B) noticeable difference between pure Al and Al / GO composite films can be observed. The surface of Al film displays a homogeneous, smooth morphology (Figure 6-29 A). Changes in surface morphology were observed for the composite films Al / GO, these revealed a rather rough morphology with some salient edged structures which might be attributed to the GO sheets embedded into Al, there were also few GO sheets dragged out (pointed by black arrows in Figure 6-29 B). The cross section of the Al/GO composite films is totally different from that of the pristine Al. As shown in Figure 6-29 D the films became coarser when GO is added with grooves which might be generated by the different stiffness between Al and GO when the films are snapped (175). Figure 6-29 D also indicates the well dispersed status of the GO in the Al matrix without any aggregation, in addition. The alignment of GO sheets is in agreement with TEM assessment.
Figure 6-30 Schematic representation Al/GO composite films structure
The same observations were reported in the literature for similar materials (i.e. chitosan-GO) prepared by the same method (i.e. solution casting). Pan et al., 2011, reported that GO sheets tend to lie down inside the chitosan solution due to their 2D structure and gravitational attraction (170). On the basis of XRD, TEM and SEM results and similar behaviour found in the preparation of similar materials (170) (184) we propose a similar structure of the Al / GO composites. Figure 6-30 depicts penetration of the Al chains within the ordered GO structure as already revealed by XRD. FT-IR results suggested the possible formation of hydrogen bonds between Al and GO oxygen functional group as schematically shown in Figure 6-30, but additional work is needed to investigate this feature. The homogeneous uniform dispersion and certain degree of alignment of GO within Al matrix coupled with good interfacial adhesion between GO and Al should lead to significant improvement in thermal and mechanical properties of Al/GO nanocomposite films.
TG Analysis
The TG analysis of Al / GO composite films shows higher starting decomposition temperature around 100 ºC and less weight loss than of pure Al films (Figure 6-31 and Table 6-5 at 620 °C). Td3% increased by about 30 degrees in the case of the composites with the highest amount of GO added to the pure biopolymer. Conversely, the residual weight loss of the composites is higher than that of neat Al, which can be attributed to the excellent thermal stability of GO which generally presents a decomposition temperature around 200 ºC.
Figure 6-31 TGA curves of neat Al and Al/GO composite films. Inset: Magnification of first stage of degradation.
Table 6-5 Decomposition temperature and mechanical properties of neat Al and Al/GO composite films
*the temperature at which the mass loss is 3%
Mechanical Tests
The mechanical behavior of the films, pure Al and Al/GO composites was investigated by tensile tests. Figure 6-32 shows typical stress–strain curves of neat Al and its composites with GO. The tensile strength and modulus as a function of GO content are summarized in Table 6-5. The data are the average results measured for ten samples. Generally a reinforcement of the Al was observed with the addition of the GO. The reinforcement was related to the GO amount, it is evident that even a small amount of GO significantly increases the tensile modulus of Al biopolymer. Moreover, higher values of GO loading from 2.5 to 6 wt% cause a significant increase of tensile strength and Young’s modulus from 82 to 113 MPa and from 1.39 to 4.18 GPa respectively. The improvement of the mechanical properties of the composites is mainly attributed to alignment, compatibility and specific interactions of Al hydroxyl groups with GO functional groups (hydroxyl, epoxide) rather than to Al crystallinity modification.
Figure 6-32 Mechanical behavior of Al and Al / GO films
As shown in XRD patterns in Figure 6-27, the addition of GO affects only slightly the crystalline structure of Al therefore here the crystallinity plays a less important role. The phenomenon may be explained by the fact that macroscopic properties such as tensile strength are relatively insensitive to small changes in the crystallinity of the composites. The same observations were reported in the literature for chitosan associated with GO. Yang et al., reported that the addition of 0.5 wt% GO in the composite films, slightly affects their crystallinity and significant increases the strain modulus, Young’s modulus and elongation at break. In this case, the improved tensile properties are ascribed to GO and Al hydrogen bonding and preferential alignment of GO shown by SEM and TEM investigation. As schematically shown in Figure 6-30, the brick and mortar structure of the Al/GO composites leads to more uniform stress distribution and minimizes the presence of stress concentration center, thus significantly increase the mechanical properties of the nanocomposite films
6.5.3. Conclusions
This work confirms that high performance Al /GO nanocomposite films could be successfully prepared using a simple solution casting method. All results evidenced that GO sheets, containing abundant functional groups on the surface, well interact with the Al matrix. In addition, uniform distribution and preferential aligned GO sheets were suggested. The incorporation of GO sheets within Al matrix exhibits a beneficial effect on mechanical integrity and thermal properties of nanocomposites. Among the studied nanocomposite films, Al / GO with 6 wt % GO exhibited the highest increase of the tensile strength and Young’s modulus up to 113 MPa and 4.18 GPa respectively. Meanwhile, the TG analysis indicates that the incorporation of 6 wt % GO in the Al matrix leads to an increase of Td3% with about 25 ºC. The results reported below are intended for publication.
6.6. NANOCOMPOSITE 3D SCAFFOLD BASED ON CHITOSAN AND GRAPHENE OXIDE
The idea of developing an ideal biomaterial, suited for bone repair applications, which have both osteoconductive and osteoinductive properties is still a question of basic research for which no breakthrough solution has been found yet. Polymer composites films possess sufficient mechanical support and biocompatibility with the cell but do not have osteoconductive properties. This means that these materials do not have sufficient porosity to allow cell attachment and proliferation inside the material.
In this Subchapter porous chitosan/GO scaffold were synthesized, using a freeze drying method. Chitosan/GO 3D scaffolds are favorable materials that meet all the requirements of an osteoconductive material. Such materials facilitate cell attachment, allow diffusion of nutrients and metabolites, are permissive (allow cell migration, proliferation and tissue remodelling) and may have similar mechanical properties with the tissue (bone) that enclosed the biomaterial.
This approach was used for the first time for these composites and it was envisaged in order to increase the biocompatibility. SEM was used to study the morphology of the scaffolds. Studies such as swelling, enzymatic biodegradation and mineralization assay of the polymer composite were conducted to see the effect of the network structure.
6.6.1. Preparation of graphene oxide-based Chitosan composite 3D scaffold
Figure 6-33 Experimental protocol
2.5 g of chitosan were mixed with 250 ml acetic acid solution (10% by weight in water) at ~ 50 °C in order to form an homogenous viscous solution. Briefly, different contents of graphene oxide (0; 0.5; 1; 2 and 3 wt.%) were added into the chitosan solution and ultrasonicated for 1h at room temperature. The homogenous solutions were casted onto transparent glass Petri dish, then frozen over night an -70 °C and freeze-dried for 2 days at -50 °C (0.040 mbar). After sublimation of ice crystals by freeze-dryer the polymer structure were obtained porous (Figure 6-33). The 3D dried scaffolds were thermal treated in vacuum according to the following procedure: 30 min at 50 °C, 30 min at 70 °C and overnight at 90 °C.
6.6.2. Characterization
Raman Spectroscopy
Figure 6-34 shows Raman spectra of GO and CHT/GO composites scaffolds.
Figure 6-34 Raman Spectra of A) GO; B) composite CHT/GO 0.5 wt.%; C) CHT/GO 1 wt.%; D) CHT/GO 2 wt.% and E) CHT/GO 3 wt.%
All spectra display two intense bands at 1333 and 1604 cm-1 associated to G and D modes respectively. The intensity ratio of D and G band (ID/IG) is used to measure the distance between the defects in the GO structure according to Eigler and coworkers (Table 6-6). Furthermore, J. Jiu et al. suggest that the ID/IG is proportional with the average size of sp2 domain. By adding GO into the CHT matrix the ID/IG increases significantly from 0.81 (GO) to 1.51 (CHT/GO 0.5 wt.%) meaning that the nanofiller is well dispersed into the polymer matrix. However, ID/IG decreases slowly with increasing the GO content reaching a value of 1.27 at 3 wt.% GO perhaps due to the agglomeration tendency of GO at higher concentration, this value being higher than for the GO powder.
Table 6-6 The ID/IG of GO and the CHT/GO composite 3D scaffold
SEM Studies
Figure 6-35 shows the SEM micrograph of the CHT/GO nanocomposites 3D scaffolds. Under SEM observation, all samples present a porous structure with open and well interconnected pores. However, differences between pure CHT and CHT / GO composite films can be observed. The surface of pure CHT displays a rough morphology with undefined pores (Figure 6-35 A). Conversely, even for small GO amount added, the pores begin to define forming an heterogeneous pores arrangements (Figure 6-35 B and C). Moreover, by further increasing the GO content the porosity of the samples increases too. In addition, for the composite 3D scaffold with 3% GO, SEM images reveal a highly interconnected porosity and well defined pores (Figure 6-35 E). The highly porosity structure is advantageous for cell attachment and proliferation allowing ingrowth of cells.
The presence of COOH groups linked to the edges of the basal plane of GO makes it suitable to promote interfacial interaction with other cationic molecules. The individual dispersion of the GO into the polymer matrices is randomly.
From the pattern of SEM results we can see that the addition of GO into the polymer matrix leads to a decrease of interconnected pore size. The pore size of the CHT ranged between 22 μm and 55 μm while for the CHT/GO nanocomposite scaffolds varied from 4 μm to 30 μm.
Swelling measurements
Further, for an advanced characterization the water uptake capacity and the enzyme-mediated degradation behavior were also investigated in order to better understand and to correlate the material composition with the corresponding properties.
To determinate the water retention, an important property for scaffolds when is used in biomedical filed, the swelling behavior and equilibrium water content of different concentrations of GO (0%, 0.5%, 1%, 2% and 3%) embedded in the chitosan scaffolds were measured. The swelling behavior was determined by immersing samples in double-distilled water (ddw) at room temperature (RT). Hence swelling studies were performed to equilibrium in ddw. A conventional gravimetric procedure was used to determine the swelling degree of the investigated scaffolds at pre-determined time intervals. The dry weight (drymass) of various chitosan composite materials was measured after drying, and the fully swollen films weight (wetmass) was taken after submerging the composite films in water removing excessive surface water with filter paper. The swelling ratio (%) was calculated using the well-known equation:
All data points are the mean ± standard deviation of two separate measurements. The equilibrium swelling degree (MSD) was estimated as the maximum value of the swelling degree.
Figure 6-36 Swelling degree in water versus time for CHT/GO scaffolds and CHT control after 4 h
Figure 6-37 Maximum liquid uptakes for the investigated porous CHT/GO composite materials
Figure 6-36 and Figure 6-37 reveal the swelling ratios and the MSD for all the chitosan/GO composites. The water uptake was monitored for 4h till equilibrium was achieved and it was found to be significantly affected by compositional factor. Within the first hour, all the samples reach the equilibrium very quickly. This behavior is mainly due to the 3D porous structure of the materials which favor the water penetration within the polymer scaffold.The presence of GO in the materials tends to decrease the swelling behavior when compared with the control chitosan scaffold.
A decrease of the MSR with increasing the GO content into the polymer is also noticed. It could be seen that the swelling degree was inversely proportional to the GO content in the synthesized composite materials: i.e., 3 wt. % GO nanosheets in chitosan had a much lower swelling ratio (1400 %) than those of pristine chitosan (2500 %). On one hand this is due to less elastic chitosan in scaffold with higher ratios of GO which is a dense structure and unfavorable for water penetration. On the other hand, more uniform packing of CHT chain in the presence of GO seems to hindrance water penetration. Thus, tissue engineering scaffolds of chitosan/GO are expected to maintain their integrity for longer periods of time in comparison with pristine chitosan.
Moreover, the obtained outcomes are absolutely consistent with the enzymatic degradability studies presented below.
In Vitro biodegradability studies
The enzymatic degradation of the chitosan scaffolds was investigated in vitro using the ferricyanide method described in literature (185, 186). Briefly, freeze-dried samples (40 mg, 0.5 × 0.5 cm) were incubated in phosphate buffer (pH 7.4; 0.1 M; 25 ml) with lysozyme (30 mg) in a shaking water bath at 37oC. At pre-defined time intervals filtered aliquots (1 ml) were cooled on ice for 5 min in order to stop the degradation, made up to 25 ml with buffer, mixed with an alkaline solution (4 ml) of potassium ferricyanide (prepared from 0.25 g potassium ferricyanide complex salt dissolved in sodium carbonate; 0.5 M, 500 ml) and quenched in boiling water (100oC; 15 min). Following rapid cooling to 25oC (within 5 min; ice bath), the optical absorbance was recorded at 420 nm, using phosphate buffer as reference. The amount of chitosan that was released into solution was assessed using a N-acetyl-D-glucosamine calibration curve.
Figure 6-38 In vitro degradation behavior for the designed scaffolds. The degradation was studied at 37°C in the presence of lysozyme
Enzymatic hydrolysis was performed in vitro in the presence of lysozyme at a lysozyme to substrate ratio of 0.6:1 (Figure 6-38). Ferricyanide method was used for determination of the available number of reducing end groups of chitosan chains. These reduction groups are formed as a result of the enzymatic degradation (185, 186). Yellow ferricyanide become colourless ferrocyanide by reduction in the presence of N-acetyl-D-glucosamine and hot:
The quantification of the enzymatic degradation can be done using the variation of the ferricyanide aborbance with time which is inversely with the available number of reducing chain ends. Under the used experimental protocol (pH 7.2; 37oC; lysozyme concentration 1.2 mg/ml), it can be easily observed the effect of different GO content on the enzymatic degradation of the scaffolds. As we expected the amount of degraded chitosan is lower when GO content is increased probably due to the GO sheets that hinder the penetration of the enzyme inside the polymer matrix. No significant difference was noticed between the samples with GO up to 20h of enzymatic hydrolysis. The in vitro degradation reached at equilibrium after approximately 50h of incubation in the solution enzyme. Scaffolds reinforced with 1 or 2 % GO respectively exhibit insignificant differences concerning the in vitro enzymatic degradation profile. The samples with higher GO (3%) exhibit limited susceptibility to biodegradation in comparison to the unreinforced specimens.
These results indicate a strong composition/ enzyme degradability dependency and thus the in vitro degradation with lysozyme can be controlled by the composition of the materials: GO-free material is degraded more readily, whereas loading of GO leads to more resistant scaffolds to lysosyme attack.
6.6.3. Mineralization assay
For mineralization assay (Figure 6-39), CHT and CHT/GO composite scaffolds were immersed in a medium with the following composition: 0.6 g tris(hydroxy-methyl) aminomethane (Tris), 2.9 g CaCl2 and water at pH 7.4, adjusted with HCl 1N. The samples were incubated for 24 h at 37 °C. After 24h, the samples were rinsed with distilled water for three times to remove any traces of salts from the surface or pores and incubated in Na2HPO4 solution in water (1.7g at 100 ml water) for another 24h at 37 °C. The method was repeated. After washing with distilled water, the samples were dried at 40 °C for 24h.
Figure 6-39 Experimental protocol for mineralization procedure
6.6.3.1. Characterization after mineralization
The presence of mineral crystals onto CHT and CHT/GO composite scaffolds surface or pores was evaluated by SEM and XRD. The Ca/P molar ratio was investigated by EDAX spectroscopy.
SEM Studies
SEM investigations were used to assess the formation of HA layers onto CHT and their composites surface. As representative samples for the composite scaffolds the composites with 1 and 3 wt.% GO were selected. According to Figure 6-40 all samples are covered with a mineral phase made of microgoblule needle type. The presence of the reactive OH and NH2 groups onto chitosan surface favors the formation of the HA crystals which are homogenous distributed over the surface of the material. Based on SEM images the morphology of the crystal layers are not influenced by the addition of GO.
Figure 6-40 SEM images and EDAX spectra of the morphology of mineral deposits onto the surface for A) CHT; B) CHT/GO 1 wt.%; C) CHT/GO 3 wt.%
The presence of Ca and K elements was proved using EDAX as a complementary analysis to SEM, this being another evidence of the HA formation on CHT and CHT/GO composites after the immersion of the samples in saline solution.
XRD Studies
More information's about the development of the HA-like crystals onto CHT and CHT/GO composites scaffolds are observed from XRD. The XRD patterns of CHT and its composites after the immersion exhibit the characteristic diffraction peaks of the HA. Figure 6-36 displays two peaks at 2θ ~ 26° (002) and at 2θ ~ 32°. The peak from 2θ ~ 26° is sharp and strong which means that a higher degree of formed HA crystallinity was formed. Considering this peak one may see that the formation of HA depends on GO, as GO amount increases the intensity of the peak decreases and thus the size of HA decreases to nanolevel.
Figure 6-41 XRD curves of CHT composites after mineralization
6.6.4. Conclusions
Chitosan and graphene oxide are used to fabricate porous scaffolds by a freeze drying method. Raman spectra display a well dispersion of GO within the biopolymer matrix. SEM investigations exhibit a porous structure with well interconnected pores for all the obtained samples after the sublimation of ice crystals by freeze-dryer. The morphology of the pure CHT changes with the addition of GO. The pores become more defined and the porosity increases. Swelling measurements reveal a decrease of water retention and MSR with increasing the GO amount because the CHT structure becomes more dense and unfavorable for water penetration. These results are consistent with those obtained from enzymatic degradation
A mineralization procedure was used to enhance CHT and CHT/GO composites biocompatibility and bioactivity. According the SEM, EDAX and XRD analysis these materials have been proved to be satisfactory for the mineralization. The presence of GO does not influence the amount of HA formation, but only the size of the crystal layers.
6.7. NANOCOMPOSITE 3D SCAFFOLDS BASED ON CHT-PVA AND GO
3D polymeric scaffolds are suitable materials for cell attachment, proliferation and allowing ingrowths of the cells. As it was mentioned in the previous section they meet all the requirements to be used as an osteoconductive material since unlike composite films facilitate the formation of links at the interface.
The aim of this study consists in the synthesis of a material with improved mechanical and osteoconductive properties which permits a good growing and proliferation of the cells. In this Subchapter CHT-PVA/GO 3D scaffolds were obtained, using a simple freeze drying method. This approach was used for the first time for these composites and it was envisaged in order to increase the biocompatibility of the material with the cells. SEM was used to study the morphology of the scaffolds. Studies such as mineralization assay of the polymer composite were conducted to increase the biocompatibility and bioactivity of the polymeric blends and also to see if these kind of materials represent a suitable support to form hydroxyapatite (HA). The results reported in this subchapter will be future intended for publication.
6.7.1. Preparation of graphene oxide-based Chitosan composite 3D scaffold
1 wt.% chitosan solution was obtained by dissolving CHT powder in 10 wt.% acetic acid aqueous solution. Separately, 1 wt.% PVA aqueous solution was obtained after solubilization of PVA powder in distilled water at 120 °C in an autoclave. Blends were prepared by mixing the two polymer solutions (50:50 v/v ratio). Briefly, different concentrations of graphene oxide (0, 0.5, 1, 2 and 3 wt.%) were added into the blend solution and ultrasonicated for 1 h at room temperature. The homogenous solutions were casted onto transparent glass Petri dish, then frozen over night an -70 °C and freeze-dried for 2 days at -50 °C (0.040mbar). The 3D dried scaffolds were thermal treated in vacuum according to the following procedure: 30 min at 50 °C, 30 min at 70 °C and overnight at 120 °C.
6.7.2. Characterization
SEM Images
Figure 6-42 shows the surface structure of CHT-PVA and the composites with different concentration of GO for which a porous structure with open and interconnected pores was observed. This structure is advantageous for cell attachment and proliferation allowing ingrowth of cells.
CHT-PVA exhibit heterogeneous microstructure with a non alignment arrangement of the pores (Figure 6-42 A). The presence of GO within the polymer, even in small amount, leads to significant changes in the material morphology. When GO is loaded, the porosity of the material increases and entangles pores uniformly distributed in the material are produced. This type of pores induces a decrease of the network density which disturbs the 3D polymer structure. The porosity of the material, however, is not influenced by the amount of graphene oxide introduced within polymer network. We can conclude that the introduction of GO within the polymer bend leads to an increase of the porosity.
6.7.3. Mineralization assay
The mineralization assay was performed following the experimental protocol extensively detailed in section 6.6.3.
XRD studies
Figure 6-43 XRD curves of CHT-PVA and composites after mineralization
Figure 6-43 displays the formation of HA on the scaffold surface. The XRD patterns of the mineralized surface show sharp diffraction peaks at 2θ ~ 26° (002) and at 2θ ~ 32° (002) assigned to HA-like crystals. Considering the sharpness of the characteristic peak at 2θ ~ 26 ° one may say that HA formation depends on GO amount, as the GO quantity increases a decrease of HA nanocrystallites size being observed. Similar trend was observed by a SEM investigation subsequently presented.
SEM analysis
The ability of composite materials to form HA has been proved also by SEM analysis. As shown in Figure 6-44 a large number of microglobules with a needle-like shape emerged uniformly covering the entire surface of all samples. Regarding the XRD and SEM measurements it can be stated that the presence of GO influences only the size of the HA-like crystals growth on the scaffold.
Figure 6-44 SEM images of CHT-PVA A) and CHT-PVA with 1 wt.% GO B), 2 wt.% GO C) and 3 wt.% GO D) after mineralization
6.7.3. Conclusions
CHT-PVA/GO 3D composite scaffolds were successfully obtained using a freeze drying method. Their properties could be easily manipulated by varying the GO content. By increasing the GO amount the morphology of all materials undergoes a significant change, entangle and uniformly distributed pores being produced. According to SEM and XRD results all scaffolds present a good capability to formed HA-like crystals under biomimetic conditions. The composites surface is covered by a mineral layer whose uniformity dispersion and morphology is influenced by the GO amount.
HYBRID MATERIALS BASED ON MESOPOROUS SILICA NANOPARTICLES AND DIFFERENT INORGANIC GUEST FOR DRUG DELIVERY
MSN is an attractive material for drug delivery applications due to its remarkable properties such as high surface area and controlled pore size which favors drug adsorption, biocompatibility and ability to degraded in simulated body fluid (SBF) (187).
Benzalkonium chloride (BZC), a quaternary ammonium salt with the general formula [C6H5CH2N(CH3)2R]Cl where R=n-C8H17 to n-C19H39 is a bacteriostatic agent used as a preservative and disinfectant in the pharmaceutical industry.
In the present thesis we discussed the BZC absorption into the pore channel of the MSN and its drug release. The influence of the contact time, pH and temperature were considered. The absorption mechanism of the drug has been determinated by using the equilibrium data. Drug adsorption was monitored both on the surface and in the pores of MSN by varying various parameters of the reaction: time, pH, temperature and initial drug concentration. Films based on two biopolymers (chitosan and alginate) and MSN modified with BZC were synthesized. The influence of drug concentration on the process of drug release from hybrid materials was followed.
7.1. EXPERIMENTAL STUDY OBJECTIVES
This objective is based on three main stages:
The synthesis of hybrid materials based on mesoporous silica and the drug.
The characterization of the hybrid materials based on MSN and the drug by FT-IR, TGA, XPS, UV-VIS;
Monitoring the drug release from hybrid material using UV-VIS spectroscopy in SBF media (7.4).
7.2. RAW MATERIALS
Benzalkonium chloride (BZC), Chitosan with avarage molecular weight, alginate, glutaraldehyde (GA), calcium chloride used as crosslinking agents and mesoporous silica nanoparticles (MSN) with a pore size of about 2.1-2.7 nm, 0.98 cm3/g pore volume and a specific surface area ~1000 m2/g were purchased from Sigma Aldrich.
Sodium hydroxide, potassium phosphate monobasic, hydrochloric acid, potassium chloride were received from Sigma Aldrich.
7.3. ADSORPTION OF THE DRUG ONTO MESOPOROUS SILICA PORE OR SURFACE
7.3.1. Experimental protocol
Figure 7-1 Adsorption of the drug onto mesoporous silica surface or pore
0.015g benzalkonium chloride was dissolved in pH 5 solution and then 0.1 g of mesoporous silica was added under magnetic stirring. The stirring was maintained for 2h at RT. The obtained suspension was centrifuged and dried at 35 °C for 24 h in a vacuum oven.
7.3.2. Characterization of MSN modified with BZC
FT-IR Analysis
Figure 7-2 shows the MSN, BZC, and MSN-BZC FT-IR spectra. The FT-IR absorption bands are given in Table 7-1.
Figure 7-2 The FT-IR spectra for MSN, BZC and MSN modified with BZC
From FT-IR spectrum of modified MSN some additional peak can be observed which evidences the presence of BZC. The bands at 2961 cm-1, 2926 cm-1 and 2858 cm-1 are attributed to symmetric and asymmetric stretching vibration of C-H bond of BZC tail. The peaks from 700 cm-1 corresponds to the C-H bending vibration from aromatic ring while 1458 cm-1 and 1488 cm-1 are assigned to C-H bending from methyl (-CH3) and methylene (-CH2) groups (188).
Table 7-1 FT-IR spectra assigned of MSN and BZC
XPS Analysis
Figure 7-3 The XPS survey spectra for MSN and modified MSN with BZC
From the XPS survey spectra (Figure 7-3) of the modified MSN, the presence of C 1s and N 1s signal at 282 eV and 402 eV respectively can be clearly observed which is another proof of a true interaction between MSN and BZC.
TG Analysis
Figure 7-4 TGA curves for MSN, BZC and MSN modified with BZC
The absorption of BZC onto MSN was also confirmed from TGA curves (Figure 7-4). The MSN modified with BZC shows two thermal decomposition steps. The first step, around 200 °C, is attributed to the thermal degradation of the drug and the second step occurs at a higher temperature, around 300 °C, and is assigned to thermal degradation of inorganic fraction.
The MSN modified with BZC exhibits also an increase of weight loss from 4.4 % for unmodified MSN to 17.64 % for MSN-BZC. This increase is due to the thermal degradation of the organic compound grafted onto MSN surface or/and within MSN pores.
7.3.3. The influence of reaction parameters on the interaction between drug and MSN
The influence of contact time was performed by mixing 0.015 g BZC and 0.1 g MSN at pH 5 for 10, 30, 60, 120, 240, 360 min. The same quantities of drug and MSN were mixed to determine the thermodynamic parameters. The reaction was kept for 1 h, at room temperature, 40 °C, 60 °C and 80°C. pH effect was studied by maintaining the reactions 1 h at 80 °C in solutions of different pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. Another important parameter that was studied was the initial drug concentration: 3 g/L, 6 g/L, 11 g/L, 18 g/L, 24 g/L, 36 g/L, 47 g/L, 200 g/L.
The unabsorbed drug concentration was determined by UV adsorption at 262 nm, after the centrifugation of MSN-BZC suspension. The amount of drug adsorbed at time t (Qt, mg/g) and at equilibrium (Qe, mg/g) were calculated with the following equations:
(1)
(2)
Where C0, Ct, Ce (mg/L) are the initial, t time and equilibrium concentrations of BZC solution; V (L) is the volume of BZC solution, W (g) is the mass of MSN used.
Contact time influence
The effect of the contact time for the adsorption of BZC onto MSN is presented in Figure 7- 4. MSN has a good adsorption capacity, within 60 min the equilibrium was achieved. There was no significant change from 1 h to 6 h.
Figure 7-5 The influence of the contact time of BZC
In order to determine the adsorption process type and to predict the adsorption rate, the kinetic parameters were determined. The adsorption of BZC onto MSN was calculated using the pseudo-second order equation:
2 (3)
where k2 is the rate constant of second-order adsorption in (g mg−1 min−1).
Eq. 3 can be integrated using boundary conditions t=0 to t=t and q=0 to q=q and gives:
(4)
Eq. 4 can be linear written as:
(5)
The straight-line plots of (t/q) versus t have been drawn to obtain rate parameters, k2 and qe. (189).
The high correlation coefficient (r2 = 1) suggests that the adsorption process of BZC onto MSN follows the pseudo-second-order kinetic model. Also, the calculated qe has almost the same value as the qe determined experimental (190).
The pseudo-second-order kinetic model can not determine if the transport of the drug onto MSN takes place by diffusion. This possibility was explored by using intra-particle diffusion model:
(6)
where qt (mg/g) is the amount of BZC adsorbed at time t (min), kp (mg/g min) is the intra-particle diffusion rate constant and C is the intercept.
Table 7-2 Kinetic parameters for BZC adsorption onto MSN
Figure 7-6 Intra-particle diffusion model for BZC adsorption onto MSN
Usually the adsorption of drugs by porous materials takes place in three stages: a) first, the surface or instantaneous adsorption occurs; b) a gradual adsorption occurs where intra-particle diffusion is rate limiting; c) the intra-particle diffusion takes place with a low rate due to the low drug concentration left. This is the equilibrium stage (152). The first part (Figure 7-6.) of the process the BZC molecules enter into MSN particles by intra-particle diffusion. The second stage of the figure is attributed to the equilibrium step
The temperature influence
The temperature effect on drug adsorption onto MSN was studied at 298, 313, 333, 353 K. As can be observed in Figure 7-7, the highest amount of drug, 144.9 mg/g, was adsorbed at 298 K. At higher temperature values BZC starts to degradate, according to TG analysis (Figure 7-4).
Also, the thermodynamic parameters were calculated. The free energy (G0), enthalpy (H0) and entropy (S0) were calculated using the following equations:
G0 = -RT ln (Kc) (6)
G0 = H0 – TS0 (7)
Kc = Qe/Ce (8)
(9)
where Kc (L/g) is the adsorbed capacity to retain the active substance, R (8.314 J/mol K) is the universal gas constant and T (K) is the temperature.
Figure 7-7 Adsorption of BZC at different temperatures
The values of ΔH0 and ΔS0 are calculated from the slope and intercept of the plot of ln (Kc) versus 1/T. ΔG0 is determined from equation 6 and 7. The results are summarized in table 2. The negative values of free energy indicate a spontaneous and physic adsorption process. This is confirmed by ΔH0 values, which are smaller than 25kJ/mol (191). The positive values of S0 indicate a higher disorder of MSN particles with increasing the adsorbed drug amount onto its surface. Because ΔG0 is negative and S0 positive, the adsorption process is spontaneous with high affinity for BZC (192).
Table 7-3 Thermodynamic parameters for BZC adsorption onto MSN
The pH influence
Figure 7-8 Adsorption of BZC at different pH values
The pH solution is one of the most important parameter in adsorption of pharmaceutics onto mesoporous silica surface or pores. The pH solutions range between 2 to 11, pH values were adjusted with HCl 0.1 N and NaOH 0.1 N solutions. Figure 7-8 shows the pH dependence on adsorption of benzalkonium chloride at a concentration of 6g/L onto MCM-41 surface or pores. The amount of the BZC adsorbed increases to pH 5, slowly decrease to pH 6 and furthermore an abrupt decrease at higher pH values was observed.
The influence of initial drug concentration
Figure 7-9 illustrates the effect of initial BZC concentration. It was noticed an increase of the equilibrium values for adsorbed amount of drug onto MSN from 18.56 to 352 mg/g for an increase of initial drug concentration from 3000 to 200000 mg/l. The higher the drug concentration is the mass transfer resistance between liquid phase and solid phase is reduced (193).
Figure 7-9 The influence of drug concentration
7.4. SYNTHESIS OF POLYMER-BZC AND POLYMER-MSN-BZC HYBRID MATERIALS
Due to its great adsorption capacity, MSN shows a higher tendency of agglomeration leading to prevention of drug release. We believed that incorporation of MSN and the antinflamatory drug into the polymer will lead to a better dispersion of MSN and as a consequence it will increase the amount of drug released. The concept of MSN/BZC hybrid materials is entirely new, no data being reported in the literature.
Figure 7-10 Synthesis of hybrid materials based on CHT/Al-MSN-BZC
50 mg of CHT/Al was dissolved in 50 ml acetic acid aqueous solution for 24 h at RT to form a uniform viscous solution. The CHT/Al-BZC and CHT/Al-MSN-BZC suspension was obtained by mixing the two respectively three compounds and mechanical stirring at RT for 2h. Then 0.0225 ml aqueous solution of GA or CaCl2 was added and the stirring continued for 1h. CHT/Al-BZC and CHT/Al-MSN-BZC was cast onto transparent Petri dish and left undisturbed for 72 h at RT (Figure 7-10).
7.4.2. In vitro drug release
Experimental protocol
The drug release was fulfilled into a thermostatic shaking bath by immersing a dialysis membrane bag with certain amount of BZC, BZC-MSN and CHT/Al-BZC or CHT/Al-MSN-BZC hybrid materials and 4 ml buffer solution of pH 7.4 (simulated intestinal fluid, SIF) prepared as described by A. Ghebaur and coworkers (194), in 50 ml of the same buffer solution. The temperature was kept constant at 37 °C and the rotation speed was 100 rpm. At various time intervals, 3 ml of dissolution medium were extracted and the BZC concentration was calculated by UV adsorption at 262 nm. The analyzed solution was put back to maintain the same volume.
Results
In order to determine both the concentration of the drug adsorbed onto MSN surface or within MSN pores and the concentration of drug release from the MSN using UV-VIS Spectroscopy it is required to run a calibration curve. For this purpose we prepared a standard solution of BZC 10-3 mol / L concentration in water, and then by successive dilutions solution of different concentrations were made (Table 7-4).
Table 7-4 Absorbance of solutions obtained by diluting the standard solution measured at 262 nm
Figure 7-11 UV-VIS spectra recorded for solution BZC
In Figure 7-11 it is observed the UV-VIS curve registered for BZC solution. The calibration curve shown in Figure 7-12 presents linear dependence between sample concentration and absorbance of the solution at 262 nm.
Figure 7-12 The calibration curve for determining the amount of BZC
In vitro release of BZC was investigated in SIF (pH 7.4). In Figure 7-13 and Figure 7-14 it was observed the percentage of BZC released from MSN and the two biopolymers. At pH 7.4 the amount of the drug release from the CHT was 100% (Figure 7-13) and about 42 % from Al (Figure 7-14). The rapid release from CHT matrices within 24 h might be due to a good swelling behavior of the biopolymer in SIF which favors the diffusion of the drug molecules. The lower release rate of BZC from Al matrix was attributed to a higher crosslinking density of the polymer which slows down considerably the diffusion of the drug and inhibits the crossing of water molecules through the polymer chains. Xiujuan Huang and coworkers reports that the delivery of the drug might be determinate by varying the amount of sodium alginate and CaCl2 (195). Also, the release profile reveals that the percentage of BZC released from MSN-BZC systems is lower than the amount of drug released from the CHT-MSN-BZC and Al-MSN-BZC hybrid materials. These results were attributed to a good dispersion of MSN in polymer matrices which reduces nanoparticles agglomeration and enables a better drug diffusion. On the other hand, the percentage of the drug release from Al-MSN-BZC hybrid materials was even higher than that from the pure polymer. This could be assigned to a lower crosslinking density of the hybrid materials than the pure polymer after adding the inorganic guest.
Figure 7-13 The percent of drug release from CHT and CHT-MSN hybrid materials
Figure 7-14 The percent of drug release from Al and Al-MSN hybrid materials
7.5. CONCLUSIONS
The MSNs were modified with different amount of BZC. The modification process was proved by FT-IR spectra which exhibit distinctive bonds assigned to BZC. The presence of the drug within the MSN and surface was confirmed also by the XPS spectra.
The study shows that there are several factors which display a strong influence on the adsorption of the BZC onto MSN such as pH values of the medium, the contact time and the reaction temperature. Therefore, MSN presents a good adsorption capacity at 60 min, at room temperature using a pH loading medium of about 5.
The polymers are intended to increase the amount of the drug released by improving the dispersion of the MSN which allows a better diffusion of the drug. This is evident from the drug release curves of the MSN-BZC hybrids materials with and without the polymer. There is also a difference between the percent of the drug released from the pure polymer and the Al-MSN system. The drug released from the Al-MSN-BZC hybrid materials is higher than that from the pure polymer due to the difference between the crosslinking density. It seems that the presence of the inorganic guest decreases the crosslinking density of the system which favors the diffusion of the drug and thus the increase of the drug released.
GENERAL CONCLUSIONS AND FUTURE PERSPECTIVES
Biopolymers have proved to be good candidates for tissue engineering and drug delivery systems due to their excellent properties including biocompatibility and biodegradability to non-toxic products. The foremost drawbacks of these polymers are low mechanical strength and low processability. The first direction of the study uses chitosan, alginate as biopolymers and a mixture of chitosan and polyvinyl alcohol in order to select the material that is most promising for tissue engineering. The biopolymers were processing both as films and as 3D porous materials.
An appealing approach to design a practical material consists in the dispersion of graphene in the biopolymer matrix. Graphene has generated a high interest owing to remarkable mechanical, thermal and superior electronically properties. One main challenge of using graphene for the improvement of the physico-chemical properties of the nanocomposite materials is the distribution of graphene sheets in the polymer matrix (particularly at high loading of graphene), as well as the interfacial bonding between the graphene sheets and the polymer chain.
The molecular modeling results reported in Chapter 5 show that graphene has a big agglomeration capacity making its dispersion in chitosan quite difficult. The lack of good adhesion between CHT and graphene matrix generates just a marginal effect on the mechanical properties of the composite materials. Considering the observation presented in this chapter, in Chapter 6, we used a graphene derivative named graphene oxide which presents hydroxyl, epoxy and carboxyl functional group on its surface. The experimental results show that GO is significantly more compatible with the polymers. When appropriately incorporated into polymer matrix, it can significantly improve physical properties of the polymer host, even at low nanofiller concentration.
For the CHT/GO composite films, GO was successfully embedded in the polymeric scaffold. SEM, TEM and XRD results confirm a good dispersion of GO within the CHT matrix. GO presence within the CHT reveals higher thermal stability of the composite material compared to the pure polymer as it was observed from TG Analysis. The results of mechanical test display an improvement of about 61% compared with the pure biopolymer. Murine osteoblasts tend to adapt faster and proliferate more in contact with the chitosan/GO biocomposites with a higher content of GO. These observations were suggested based on the viability and proliferation data together with the quantification of the cytotoxic potential.
For the CHT-PVA/GO composite films the ability of GO to enhance the CHT-PVA properties was confirmed in section 6.4. From XRD, SEM and TEM analysis it was shown a largely dispersion of GO within the polymeric blends. According to FT-IR analysis some non-bond interaction between CHT-PVA matrix and GO were observed. The mechanical properties were significantly improved, the obtained values being nearly twice compared with those of the pure polymer. The composite films show a higher thermal stability even at lower amount of GO incorporated within CHT-PVA matrix. In contrast to thermal analysis, a higher content of GO in the composite film leads to a significant increase of cell proliferation rate.
For the Al/GO composite films, the results show that GO nanosheets are uniformly distributed within the polymer matrix. These results were confirmed by SEM, XRD and TEM analysis. The loading of GO sheets within Al matrix exhibits a significant effect on mechanical integrity and thermal stability of the composite, the Young's modulus and tensile strength being highly increased.
For the CHT/GO 3D scaffolds, the presence of GO within the biopolymer changes the morphology of the material. SEM images show an increase of the porosity of the sample by increasing the GO amount. Further, for an advanced characterization the water uptake capacity and the enzyme degradation behavior were also investigated. Due to the 3D porous structure of the materials it was observed that all the sample reach very quickly the equilibrium within the first hour. The GO loaded in the polymer tends to decrease the swelling behavior and the MSR compared with the pure CHT. The results are consistent with those obtained from enzymatic degradation of the materials.
CHT/GO 3D scaffold materials have been also subjected to a mineralization treatment. XRD and SEM / EDAX analysis highlight that the mineralization was successful achieved.
For the CHT-PVA/GO 3D scaffold composites a change in the morphology of pure material was also evidenced by SEM. These materials are also submitted to a mineralization treatment revealed by XRD and SEM analysis.
. The second direction of the thesis consists in synthesis of hybrid materials based on biopolymers and MSN functionalized with an anti-imflammatory drug such as benzalkonium chloride (BZC). In this Chapter chitosan and alginate were used as biopolymers. The modification process of MSN with BZC was successfully achieved and it is confirmed by FT-IR and XPS analysis. Also in this section it was demonstrated that BZC adsorption within MSN surface depends on a number of factors such as pH values of the reaction medium, contact time and temperature. After several tests it was observed that the best adsorption conditions for the BZC on MSN are: room temperature, 1 h reaction time and pH value of 5.
Since MSN shows a high agglomeration capacity, the polymer was used in order to improve the dispersion of silica which allows the drug release in a larger quantity. These results were highlighted in the release profiles of BZC from MSN and biopolymer-MSN.
Since CHT/GO and CHT-PVA/GO 3D scaffolds offer promising properties more detailed studies such as mechanical testing and in vitro and in vivo analysis to investigate the cell and host response to the 3D scaffold materials need to be done in the future.
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Publication list
ISI Papers
Andreea Madalina Pandele, Mariana Ionita, Livia Crica, Sorina Dinescu, Marieta Costache, Horia Iovu, Synthesis, characterization, and in vitro studies of graphene oxide/chitosan–polyvinyl alcohol films, Carbohydrate Polymers, 102, 813-820, 2014 FI=3.47
Andreea Madalina Pandele, Sorina Dinescu, Marieta Costache, Eugeniu Vasile, Cosmin Obreja, Mariana Ionita, Preparation and in vitro, bulk, and surface investigation of chitosan/graphene oxide composite films, Polymer composite, 34 (12), 2116-2124, 2013, FI=1.48
Mariana Ionita, Madalina Andreea Pandele, Horia Iovu, Sodium alginate/graphene oxide composite films with enhanced thermal and mechanical properties, Carbohydrate Polymers, 94, 339-344, 2014, FI=3.47
BDI Papers
Andreea Madalina Pandele, Mariana Ionita, Horia Iovu, Molecular modeling of mechanical properties of the chitosan based graphene composites, U.P.B. Scientific Bulletine, Series B, 76,1, 2014
Oral presentations
Andreea Madalina Pandele, Mariana Ionita, Livia Crica, Preparation and characterization of sodium alginate/graphene oxide composite films, 21st Annual International Conference on Composites or Nano Engineering, Tenerife, Spain, 21-27:07:2013.
Andreea Madalina Pandele, Livia Crica, Mariana Ionita, Horia Iovu, The effect of graphene oxide on thermal and mechanical properties of chitosan-poly(vinyl alcohol) blend, 18Th Romanian International Conference on Chemistry and Chemical Engineering, Sinaia, Romania, 01-07:09:2013.
Poster presentations
Andreea Madalina Pandele, Mariana Ionita, Horia Iovu, Well-dispersed alginate/graphene oxide nanocomposites. Thierd International Conference on Multifunctional, Hybrid and Nanomaterials, Sorrento, Italy, 3-7 Marth 2013.
Mariana Ionita, Madalina Andreea Pandele, Horia Iovu, Molecular modelling of the mechanical behavior of the collagen embedded with graphene and graphene oxide. Fibrous Protein Nanocomposites for Tailored Hybrid Biostructures and Devices, Greece 08-13 Octomber 2012.
Andreea Madalina Pandele, Horia Iovu, Mariana Ionita, Well-Dispersed Alginate / Graphene Oxide Nanocomposites With Enhanced Mechanical Strength For Biomedical Applications, Biomateriale Inginerie Tisulara si Dispozitive Medicale, Constanta, Romania, 28 August-1 September 2012.
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Publication list
ISI Papers
Andreea Madalina Pandele, Mariana Ionita, Livia Crica, Sorina Dinescu, Marieta Costache, Horia Iovu, Synthesis, characterization, and in vitro studies of graphene oxide/chitosan–polyvinyl alcohol films, Carbohydrate Polymers, 102, 813-820, 2014 FI=3.47
Andreea Madalina Pandele, Sorina Dinescu, Marieta Costache, Eugeniu Vasile, Cosmin Obreja, Mariana Ionita, Preparation and in vitro, bulk, and surface investigation of chitosan/graphene oxide composite films, Polymer composite, 34 (12), 2116-2124, 2013, FI=1.48
Mariana Ionita, Madalina Andreea Pandele, Horia Iovu, Sodium alginate/graphene oxide composite films with enhanced thermal and mechanical properties, Carbohydrate Polymers, 94, 339-344, 2014, FI=3.47
BDI Papers
Andreea Madalina Pandele, Mariana Ionita, Horia Iovu, Molecular modeling of mechanical properties of the chitosan based graphene composites, U.P.B. Scientific Bulletine, Series B, 76,1, 2014
Oral presentations
Andreea Madalina Pandele, Mariana Ionita, Livia Crica, Preparation and characterization of sodium alginate/graphene oxide composite films, 21st Annual International Conference on Composites or Nano Engineering, Tenerife, Spain, 21-27:07:2013.
Andreea Madalina Pandele, Livia Crica, Mariana Ionita, Horia Iovu, The effect of graphene oxide on thermal and mechanical properties of chitosan-poly(vinyl alcohol) blend, 18Th Romanian International Conference on Chemistry and Chemical Engineering, Sinaia, Romania, 01-07:09:2013.
Poster presentations
Andreea Madalina Pandele, Mariana Ionita, Horia Iovu, Well-dispersed alginate/graphene oxide nanocomposites. Thierd International Conference on Multifunctional, Hybrid and Nanomaterials, Sorrento, Italy, 3-7 Marth 2013.
Mariana Ionita, Madalina Andreea Pandele, Horia Iovu, Molecular modelling of the mechanical behavior of the collagen embedded with graphene and graphene oxide. Fibrous Protein Nanocomposites for Tailored Hybrid Biostructures and Devices, Greece 08-13 Octomber 2012.
Andreea Madalina Pandele, Horia Iovu, Mariana Ionita, Well-Dispersed Alginate / Graphene Oxide Nanocomposites With Enhanced Mechanical Strength For Biomedical Applications, Biomateriale Inginerie Tisulara si Dispozitive Medicale, Constanta, Romania, 28 August-1 September 2012.
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