UNIVERSITÀ DI PISA
DOCTORAL SCHOOL IN CHEMISTRY AND
MATERIALS SCIENCE
Bionanocomposites Based on
Biodegradable Polyesters and
Cellulose Nanowhiskers for Tissue
Engineering Applications
PhD Thesis
iii To Steven Bradbury
Who taught me that in life it is important to keep trying and keep standing
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Index
1. Introduction...1
1.1 Tissue engineering ... 1 1.2 Scaffolds ... 5 1.2.1 Scaffold manufacture ... 7 1.3 Biodegradable polymers ... 121.3.1 Synthesis of PGA, PLA and PCL: Ring Opening Polymerization .. 17
1.4 Bionanocomposite ... 20
1.4.1 Cellulosic nanofillers ... 22
1.4.2 Cellulose Nanowhiskers ... 23
1.4.3 Polymer Grafting on CNWs ... 27
1.5 Aim of the work ... 31
2. Results and Discussion...32
2.1 Statistical P(LLA-CL) Copolymers ... 32
2.1.1 Comonomers relative concentration ... 32
2.1.2 Molecular weight ... 34
2.1.3 Randomness factor ... 35
2.2 Synthesis and characterization of P(LLA-CL) copolymers ... 39
2.2.1 FT-IR Analysis ... 41
2.2.2 1H-NMR Analysis ... 42
2.2.3 Thermal analysis ... 49
2.2.4 Differential Scanning Calorimetry (DSC) ... 52
2.2.5 Gel Permeation Chromatography ... 54
2.2.6 Final Considerations ... 56
2.3 Synthesis of P(LLA-CL) copolymers with 4-Dimethyl-aminopyridine 58 2.3.1 1H-NMR Analysis ... 60
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2.3.3 DSC Analysis ...62
2.3.4 GPC Analysis ...63
2.3.5 Final considerations ...65
2.4 CNWs modification with P(LLA-CL) copolymers ...67
2.4.1 CNWs Treatment ...69
2.4.2 FT-IR characterization of organophilic CNWs ...71
2.4.3 Thermogravimetric analysis of organophilic CNWs ...72
2.4.4 CNWs functionalization ...73
2.4.5 CNWs functionalization with BiSS obtained copolymer ...77
2.4.6 Soxlet extraction ...77
2.4.7 FT-IR Analysis ...78
2.4.8 TGA ...79
2.4.9 Reduction of the molecular weight of the P(LLA-CL) ...80
2.4.10 CNWs functionalization with DMAP obtained copolymer ...83
2.4.11 Soxlet extraction ...83
2.4.12 FT-IR Analysis ...84
2.4.13 TGA ...86
2.4.14 Functionalization degree of CNWs...88
2.4.15 Enhancing of the functionalization yield...89
2.4.16 Final considerations ...91
2.5 Synthesis of P(LLA-CL)-CNWs nanocomposites ...92
2.5.1 Solution casting synthesis of nanocomposites ...92
2.5.2 TGA ...93 2.5.3 DSC ...96 2.5.4 Degradative analysis ...98 2.5.5 Final considerations ...100 2.6 Scaffold manufacture ...101 2.6.1 Additive manufacturing ...101
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2.6.2 Computer aided wet spinning ... 103
2.6.3 Scaffold fabrication by CAWS ... 104
2.6.4 Synthesis of the PLLA homopolymer ... 105
2.6.5 FT-IR Analysis ... 106
2.6.6 1H-NMR Analysis ... 106
2.6.7 TGA ... 107
2.6.8 DSC ... 108
2.6.9 GPC ... 109
2.6.10 Functionalization of CNWs with PLLA ... 109
2.6.11 Scaffold manufacture ... 111
2.6.12 SEM analysis ... 113
2.6.13 Final considerations ... 117
3. Conclusive remarks...118
4. Materials and Methods...121
4.1 Materials ... 121
4.1.1 Monomers and polymer ... 121
4.1.2 Catalysts ... 121
4.1.3 Solvents ... 122
4.2 Analysis methodologies and instrumentation ... 122
4.2.1 1H-NMR Spectroscopy ... 122
4.2.2 FT-IR Spectroscopy ... 122
4.2.3 Gel Permeation Cromatography... 122
4.2.4 Thermogravimetric Analysis (TGA) ... 123
4.2.5 Differential Scanning Calorimetry ... 123
4.2.6 CAWS Instrumentation ... 123
4.3 Experimental procedures ... 124
4.3.1 Synthesis of P(LLA-CL) copolymers ... 124
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4.3.3 Functionalization of CNWs with P(LLA-CL) copolymers ...128
4.3.4 Solution Casting ...129
4.3.5 Hydrolysis degradation ...130
4.3.6 Scaffold manufacture ...130
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List of abbreviation
LLA: L-Lactic Acid PLLA: poly-L-Lactic Acid CL: ε-Caprolactone
PCL: poly-ε-caprolactone CNWs: Cellulose nanowhiskers
P(LLA-CL): statistical copolymer of L-Lactic Acid and ε-Caprolactone R: Randomness character
SnOct2: Tin (II) Ethylhexanoate BiSS: Bismuth Subsalycilate
DMAP: 4-Dymethylamminopyridine BzOH: Benzyl Alcohol
TDI: 2, 4-Toluene Diisocyanate AM: Additive Manufacturing
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Summary
The aim of this work is the synthesis and characterization of polymeric bionanocomposites, based on cellulose nanowhiskers and copolyesters of lactic acid and ε-caprolactone, usable for tissue engineering application. The first part of the work was focused on the synthesis of copolymers at different concentrations of the two comonomers, using three different catalysts. The properties of the copolymers were studied by FT-IR, TGA and GPC analysis.
In the second part of the work the obtained copolymers were used for the surface modification of cellulose nanowhiskers by an innovative approach that takes the name of grafting to. The modified filler was then dispersed at different concentration in a copolymer matrix by solution casting. The effect of copolymers structure on the modify reaction, together with the thermal and degradative properties of the obtained nanocomposites were studied.
The third part of the work was focused on the synthesis of bionanocomposites based on poly-lactic acid an modified cellulose nanowhiskers. The nanocomposites were used for the realization of tissue engineering scaffolds by computer assisted wet spinning, whose morphology was studied by SEM analysis.
1. Introduction
1.1 Tissue engineering
The regeneration and substitution of damaged human tissues and organs are, since the beginnings of the human race, the greatest challenge for medical sciences. The first attempt of repairing damaged human skin through an autologous transplant is reported in Sanskrit texts of India, which goes up to 3000 b.C.[1].
Starting from 18th century, several attempts of substitution of skin were performed by doctors and researchers throughout all Europe, using either autologous or heterologous sources, like animals or corpses [2], leading in 1908 to the first successful skin allograft-transplantation. In 1940s were discovered several methods to refrigerate [3] or cryopreserve [4] skin cells, thus allowing to preserve implantable tissue in safe and sterile conditions and using it when necessary. Following the road opened by these pioneers, since the middle of the last century the procedures for the transplant of several tissues or whole organs were fine-tuned.
In all these cases, however, we are talking about implanting an already formed tissue or organ from a living (or dead) donor to a patient. The idea of replacing a human tissue with a substitute built up from zero began to take a step at the early beginnings of 1960. It was in fact in 1962 that William M. Chardack et al. developed the first artificial substitute of skin tissue [5]. Intuiting that human autologous cells have the possibility of migrate in a porous media, they obtained good results in burns healing applying on the wounds a polyvinyl alcohol (PVA, a biodegradable polymer) sponge. They obtained by this way a substitute of the extra-cellular matrix that could help the regeneration of the keratinocytes (the most abundant cells of the epidermis), and successfully implanted it in pig (fig. 1.1).
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Figure 1.1: PVA implanted on pig skin [5]
The idea of providing a synthetic biodegradable support for the regeneration of the cells was then resumed by the researchers of the Massachussets Institute of Technology (MIT), who developed a porous matrix cross-linking bovine type collagen I and shark chondroithin 6-sulfate [6]. The material was approved by United States Food&Drug Administration (FDA), and is still commercialized with the name of Dermal Regeneration Template. I
Thirteen years after the study of Chardack, the first artificial skin was created by Howard Green et al. in Harvard Medical School, proliferating in vitro heterologous keratinocytes obtained by a biopsy, thus obtaining sheets of epithelium with a diameter of few centimetres [7]. These study led to the realization and commercialization of the first types of artificial epithelium (EPICEL [7]).
From the above-mentioned studies is possible to notice that the regeneration of the skin tissue (but also of every other type of human tissue) can be achieved using two different approaches:
It’s possible to grow up in vitro the new tissue starting from a cell culture and then graft it on the damaged tissue. The main problem inherent in this method is the impossibility to obtain large portion
3 of tissue in just one production step. Moreover the grafting of the artificial tissue could be problematic in case of severe wounds, where the internal layer of skin are damaged [8].
It’s possible to provide a synthetic support where new cells can easily adhere and proliferate. The cells can migrate in the porous media from the healthy tissue secreting new extracellular matrix and progressively degrading the support [10]. Despite this type of procedure has the great advantage that can be used to heal really extensive wounds, the migration of autologous cells in the synthetic matrix can take a really long time.
However, there is a third possibility to promote the regeneration of a human tissue: the combination of the two approaches. The first functioning, artificial skin was in fact obtained by seeding dermal fibroblasts (the most common cells of the connective tissue) in a collagen lattice, and covering it with a suspension of epidermal cells [10]. When grafted on a living tissue, the result was a well differentiate, vascularised functioning tissue. Basing on this research work, the first artificial skin (APLIGRAF) was later developed. It in figure 1.2 is possible to observe the presence in APPLIGRAF of the two different families of cells, the keratinocytes and the fibroblasts, that makes APLLIGRAF extremely similar to human skin.
4 For the first time in history the two techniques for tissue regeneration were joined in a single one; even if they did not know at that time, Bell and his colleagues applied the principles of a science that would have been defined only in the successive decade: tissue engineering.
While the studies on the regenerative methods for human tissue date back to the beginnings of 1960s, the concept of tissue engineering as a science in itself was proposed to the academic world for the first time in 1987 by professor Y.C. Fung during a scientific symposium. A clear definition of this new field of research was although given only six years later by Langer and Vacanti. They defined tissue engineering as
“...an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function” [11], [12]
Langer and Vacanti, in the same work, defined the three strategies through which tissue engineering can fulfil its goal:
Use of isolated cells
Use of tissue inducing substances
Use of cells placed in a matrix
Starting from 1990s private companies and research groups invested money and resources in the implementation of this new research field. In the past twenty years, different types of skin, cartilage and vascular synthetic tissues have been produced and approved by FDA [8]. The excellent results in the production of skin, cartilage and vascular engineered tissues are mainly due to their relative simplicity. These are in fact two-dimensional tissues with a low grade of differentiation. For the reconstruction of these simple tissues, the first two methodologies seen before showed an high efficiency. Nowadays however, the real challenge of tissue engineering is the production of more complex systems like bones, part of organs or even whole organs. These systems contain different kinds of tissues, an high cellular differentiation and complex three dimensional geometries. The key to achieve this goal could be represented by the third methodology seen before, that is the growth of cells on a three-dimensional support.
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1.2 Scaffolds
As said before, in tissue engineering we can find to critical points. The first one is represented by the cells: living cells can be obtained from a donor, which can be a problem if there is the necessity of an high quantity of cells, or can be derived by stem or progenitor cells. The second method is the most profitable due to the high speed of proliferation of stem cells and their pluripotency, that is their ability to differentiate in different families of cells.
The second critical point is represented by the support: living cells need a suitable environment for their differentiation, proliferation and natural functions. This environment takes the name of extra-cellular matrix (ECM). When implanted in a damaged tissue, cells need time to secrete new ECM, so there is the necessity to provide them with a surrogate. In tissue engineering this surrogate takes the name of scaffold.
Scaffolds are three-dimensional supports that assists proliferation, differentiation, and biosynthesis of cells. Scaffolds allow to reconstruct human tissue through a process which is illustrated in figure 1.3.
Figure 1.3: Scheme of realization of a reconstructed tissue
After its realization the scaffold is seeded with the human cells in a bioreactor to allow their proliferation, then it is applied on the damaged tissue.
To be usable in tissue engineering, a scaffold must meet certain requirements [9] [13]. First of all it must have a structure with
6 interconnected micropores, to allow the seeding and migration of an high number of cells. Too large pores in fact may interfere with the vascularisation of the tissue, while too small pores may influence the good diffusion of nutrients, waste products and oxygen. It has been seen how the optimal average diameter of the micropores is between 100 and 500 μm [14].
A second important requirement is represented by the relation between the mechanical properties and the degradative properties of the scaffold. Scaffolds in fact are projected to degrade in the same regeneration time of the tissue. This time can change significantly depending on the tissue (from few weeks for skin implant to several month for bones). A faster degradation could cause weak adhesion of the new cells while, in case of slower degradation, the permanence of the scaffold can compromise the correct regeneration. In addition, it results quite obvious that the mechanical properties of the scaffold must imitate as much as possible those of the tissue, in terms of stiffness, tensile strength and resistance to compression. Is also desirable for the scaffold to maintain its properties unchanged as much as possible during the beginning of the degradation process.
A third requirement is that, due to their utilisation in the human body, the materials used for the realisation of scaffolds must be biodegradable (must be degraded by physiologic processes and at physiologic conditions) and biocompatible (its degradation products must not trigger adverse reaction by the human organism).
Finally is preferable to use materials that allow the modelling of scaffolds in complex three-dimensional shapes and that are easily sterilizable.
It results really hard to find a material that fulfils all the requirements listed above. Biologically derived polymers, like proteins or polysaccharides, may seem the more obvious choice. Examples of usable polymers are collagen [6], hyaluronic acid [15], oligopeptides [16], chitosan [17] or alginate [18]. It’s also possible to reticulate them to enhance their resistance to degradation. However some of these polymers, like collagen or hyaluronic acid, despite their biocompatibility may present in some cases a potential immunogenicity [19] [20]. In addition their mechanical properties are often low and their processing
7 can be quite difficult. Finally the modification of their structure to obtain specific properties, like better degradability or workability, presents some problems of difficult solution. As an example the only wide accepted method to tailor the degradative properties of collagen is its chemical reticulation with glutaraldehyde. Nevertheless, the persistence of glutaraldehyde in the cross-linked collagen can have a citotoxic effect when the collagen is used in the human body [19]. Another widely used natural polymer, chitosan, has nearly no solubility in water or organic solvents. To enhance its workability it is necessary to make it soluble by modifying it with methyl or ethyl acetate. This reactions, however, require the use of organic solvents like methanol together with acetic acid, both of which can lead to citotoxicity if they are not removed from the chitosan. In addition, also for chitosan a cross-linking with glutaraldehyde or formaldehyde is necessary to enhance its mechanical properties, with negative consequences on its biocompatibility [21].
1.2.1 Scaffold manufacture
The macro- and micro-structure of a scaffold strongly affect the adhesion and proliferation of cells together with the mechanical and degradative properties. So the structure control is extremely important. Depending by the properties which are desired, it is possible to choose between different methodologies that can be applied to obtain the best result. During the last decade, several methods for the manufacturing of scaffolds have been developed
Solvent casting and particle leaching: for this methodology the polymer is dissolved in a solvent and casted in a three dimensional mold, filled with porogen particles. The solvent is afterwards removed by evaporation. The particles dimension are related to the dimension of the pores that we want to obtain and can be inorganic (like NaCl), saccharose, gelatine or paraffine. After solvent evaporation the particles are removed using a suitable solvent. The advantage of this method is the ease of fabrication and the good control of the pores size. The disadvantage is the impossibility to obtain scaffold with a thickness under 2 mm, the use of tossic organic solvents and the possibility of agglomeration of the particles. This is the older and
8 most simple technique, and it has been successfully used for the realization of porous scaffolds with complex 3D structures, like ear or bone-shaped scaffolds (fig. 1.4), using poly (L-Lactic Acid) (PLLA) [22], poly-(D,L Lactide-co- Glycolide) (PLGA) [23], and with poly(dimethylsiloxane) (PDMS) [24].
Figure 1.4: 3D scaffolds of PLAG obtained by solvent casting and particle leaching [23]
Freeze drying: in this case the polymer solution is cooled under the melting point of the solvent (usually water), with the formation of ice crystals and the subsequent aggregation of the polymer in the interstitial space. The solvent is then removed by sublimation followed by a progressive slightly warming, with the formation of the pores. This method allows to obtain highly porous structures, like the one that can be observed in figure 1.5. The pores structure depends by the conditions of the process (freezing rate, concentration, pH and partial pressure). In literature are reported examples of scaffolds made by freeze-drying techniques using collagen[25], gelatine [26] and fibroin [27].
Figure 1.5: SEM image of the cross-section of a fibroin scaffold made by freeze drying process [27]
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Supercritical-fluid: in this method the polymer is saturated with a gas at supercritical conditions, forming a polymer-supercritical fluid solution. The pressure is than decreased, allowing the formation of gas bubbles in the polymer. Usually is used the CO2
for the process, due to the relatively mild supercritical conditions (31 °C and 73.8 bar), the low toxicity and no flammability. The main advantage of this technique is the absence of solvents, but it does not allow the formation of an interconnected structure and leads to the formation of large pores. The supercritical fluid-based technology has been used for the realization of scaffold made of amorphous polymers like poly-(D,L Lactic Acid) (PDLLA) and PLGA [28] or hyaluronic acid [29].
Figure 1.6 Discs of PGLA obtained by supercritical fluid technology [28]
Phase separation techniques: these are a group of methodologies that involves the formation of a thermodynamical instability in a polymer solutions. The most used methods is the Thermally Induced Phase Separation (TIPS). It consists in the rapid cooling (or heating) of a polymer solution under (or over) the upper (or lower) critical solution temperature, with the consequent separation of the polymer from the solution. Adjusting the process parameters is possible to control the structure of the scaffold, even if this control may result quite hard. Moreover this methodology presents the disadvantage of use organic solvents. Another example is represented by the wet spinning: the polymer solution is poured through an orifice in a nonsolvent, that leads to the precipitation of the polymer and the formation of a continuous fibre, like the one reported in figure 1.7. It is possible to deposit the fiber on a support to form a three dimensional non-woven mesh. This method is usable for a wide range of polymers and allow the co-precipitation of the polymer with additives, but still
10 presents the problem of use organic solvents. This method allowed the production of irregular 3D scaffolds with several types of polymer like chitosan [30], poly-caprolactone (PCL) [31], PLLA and PLGA [32].
Figure 1.7: SEM image of wet spun PLGA fibre [32]
Fiber bonding: there are several methods to combine or bond different types of fibers. An example is the immersion of a polymer fyber in a solution of a different polymer, evaporate the solvent and heat over the melting point of the two polymers. The polymer with the lower melting point melts and fill the voids of the second polymer, creating a porous foam. Another example is the electrospinning of two polymer solutions.
Molding techniques: they consist in pouring the polymer and a porogen material in a mold with a suitable shape and then heat it. After the solidification the particles are removed by leaching and the scaffold is dried. It is also possible to use injection molding to obtain 3D-scaffolds. This techniques allow an high control of the macro-structure and the porosity, but imply high temperature and the possible permanence of particles. The injection molding has been used to produce scaffold based on starch fibres mixed with ethylene vinyl-alcohol (EVA) and with cellulose acetate (CA), obtaining a structure with interconnected micropores like the one reported in figure 1.8 [33].
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Figure 1.8 : SEM image of a EVA scaffold obtained by injection molding (a) and image of the interconnected porous structure (b) [33]
Solid free form (SFF): in this technique a three-dimensional model is created using a design software, then is built layer-by-layer with a SFF machine. So during the process each layer-by-layer is deposited on the lower one, allowing a really good structure control. Usually the methods used are 3D-printing, stereolitography, selective laser sintering or extrusion based systems. The advantage are the structure and porosity control and the repeatability. These techniques however involve use of organic solvents, low dimension of pores, high temperature and a limited range of materials. This methods can be used for the realization of scaffolds with several polymers, as poly(ethylene-oxide)/poly(propylene-oxide) (PEO–PPO–PEO), PLLA, PCL or poly(hydroxybutyrate-cohydroxyvalerate) (PHBV), allowing to obtain scaffolds with an highly complex structure and
12 reproductions of human bones or organs [34] [35], like those reported in figure 1.9.
Figure 1.9: three-dimensional model of a human femoral condyle and sintered PHBV nanocomposite proximal femoral condyle scaffold [35]
1.3 Biodegradable polymers
The necessity to find versatile materials for the realisation of tissue engineering scaffolds is not always satisfied by biologically-derived materials. For this reason, in the last decades, the attention of researchers has been focused on synthetic polymeric materials. There are several reasons to prefer this kind of polymer to biologically derived polymers. The main reason is represented by the possibility to have an high control on their molecular structure, so obtaining a huge range of mechanical ad degradative properties. This can be done acting on four factors:
The choice of monomer(s): it’s obvious that depending on the monomer the resulting polymer will have different features, going from plastic materials, to elastomers, to resins. Combining two different monomers and acting on their relative concentrations it is also possible to obtain copolymers and widen extensively the range of obtainable mechanical and degradative properties.
The molecular weight: dosing the initiator amount it is possible to adjust the molecular weight of the polymer (or the copolymer), which is strictly related with the degradative properties.
The cristallinity: at the same way, acting on the co-monomers is possible to enhance or diminish the cristallinity grade of the
13 copolymer, with a remarkable influence on the mechanical and degradative properties (crystalline fraction are harder to degrade).
Presence of additives: micro- or nano-fillers, anti-oxidants, growth factors or drugs can significantly improve the scaffold features in many different ways.
In addition to this, synthetic polymeric materials presents other attractive features for the realisation of tissue engineering scaffolds: they can be easily functionalized, they are easier to process in complex shapes, they are easier to sterilize and they are often cheaper than biologically derived polymers.
For the reasons listed above, aliphatic polyester are the most used material for the realization of tissue engineering scaffolds. Contrarily to aromatic polyesters (like for example polyethylene terephtalate or polyethylene naphtalate) they are in fact easily degradable at physiological conditions by the human enzymes [36] [37]. Nevertheless, their mechanical and thermal properties are often comparable to those of commonly used polymeric materials like polyethylene, polystyrene and polyethylene terephtalate [38], as reported in the table in figure 1.10.
Figura 1.10: Comparison of thermal and mechanical properties of common biodegradable polymers with PE, PS and PET [38]
The enzymatic degradation of aliphatic polyesters is possible because of the higher chain-flexibility and hydrophilicity of aliphatic polyesters, that make easier the contact between the chain and the active group of the enzyme [39]. In the biomedical field there are mainly three monomers that are used:
Glycolic acid (GA): glycolic acid can be polymerized to obtain
14 cristallinity (45-55%) and a glass transition temperature of 35-4 °C and an high melting point (225-230 °C). Because of its high cristallinity PGA has high strength and modulus (7 GPa) [40]. It can be processed with the common techniques and, due to its hydrophilicity, it is degraded in relatively low time (2-4 weeks) [37]. It is commonly used for degradable sutures and for bone fixing, and it can be copolymerized with other monomers to improve its properties. PGA is obtainable starting from the glycolic acid, but is more often synthesized from its dimeric ester, the glycolide (Figure 1.11).
Figure 1.11: Structure of Glycolic Acid
Lactic acid (LA): lactic acid exists in two different stereoisomer forms:
D-Lactic Acid and L-Lactic Acid (the natural form). Consequently Poly-Lactic Acid (PLA) can exist in three different forms: poly-D-Poly-Lactic Acid (PDLA), poly-L-Lactic Acid (PLLA) and poly-DL-Lactic Acid (PDLLA).
PDLA has an high cristallinity, which makes it poorly degradable and workable. For this reason it is not employed for the realization of tissue engineering scaffolds. PLLA and PDLLA instead find a wide range of uses in medicine for the realization of dental prostheses, bone fixation devices and tissue engineering scaffolds.
PLLA is less crystalline (37%) and more hydrophobic respect to PGA. Its degradation times are longer than those of PGA, and can reach up to two years because of the presence of an optical active carbon [39]. This makes PLLA a suitable material for semi-permanent implants, which must remain in the human body for long periods of time, like orthopaedic fixation devices or ligament replacements [41].
To better the heat resistance and the mechanical properties of PLLA, it is possible to mix it with PDLA in a concentration between 3 and 50%. To
15 decrease its glass transition temperature, better its workability and decrease its degradation times, it is possible instead to synthesize a copolymer of Poly-L-Lactic acid-co-D-Lactic acid. The presence of the two stereoisomers prevents the copolymer to organize in regular domains, lowering the cristallinity and so the processing temperature and the degradation times. PDLLA is then used for the realization of sutures and short terms implants. PLLA can be also copolymerized with other hydroxy-esters like glycolic acid or ε-caprolactone to obtain copolymers with a wide range of mechanical and degradative properties.
PLLA and its copolymers are workable with the common industrial processes, to obtain materials with degradative and mechanical properties (compressive strength of 40 MPa, modulus of 2.7 GPa [39]) suitable for bone tissue and organs repairing. Like PGA, PLLA and PDLLA are obtainable from the lactic acid (D or L) or from the dimeric esters, the L-Lactide and the D-L-Lactide (Figure 1.12).
Figure 1.12: Structure of L-Lactide
ε-caprolactone (CL): it is an hydroxyl-ester derived from the
ω-hexanoic-caproic acid (Figure 1.13). Its polymerization gives an highly hydrophobic polymer, with low cristallinity, a low melting point (about 60 °C) and low glass transition temperature (-60 °C). It is an elastic polymer, with an high breaking elongation (up to 120% [39]) but with poor modulus and tensile strength. Because of its low hydrophilicity it results extremely difficult to degrade for the human enzymes, and its permanence in the human body is often more than two years (it depends from the formulation). It is used in medicine for the realization of sutures and long term implants.
16 Like the other two monomers, also ε-caprolactone can be copolymerized with other hydroxy-esters to reduce its degradation times or to improve its mechanical properties.
Figure 1.13: Structure of ε-Caprolactone
These three monomers are not the only available for the manufacture of tissue engineering scaffolds, but respect to the others they present several advantage.
First of all they are easy copolymerizable to obtain block and random copolymers. Block copolymers are not usable for the production of devices, because of the existence of a phase separation at a microscopic level that worsen the properties of the copolymer respect to the homopolymer. Block copolymers are anyway of great interest because they can act as compatibilizers for blends of the two homopolymers [42]. Random copolymers and ter-polymers can also be obtained in a wide range of composition, with a remarkable variety of properties. Examples are poly(lactide-co-glycolide) (PLGA), an amorphous copolymer used for sutures, drug delivery and scaffolds, or poly(D,L -lactide-co-caprolactone), that can be used to obtain materials for bone scaffold with high degradation times [40].
Secondly they have been approved by FDA for utilization in the human organism for several years now. They are also produced at an industrial level, they are processable with common industrial process and they are cheaper respect to biologically derived polymers.
Their main disadvantage remains the production of acid species during their degradation, with the result of a possible toxicity for the cells. Their degradation in the physiologic environment consist in the action of enzymes called esterases, especially the lipases [39] [43]. These enzymes are responsible for the scission of the triglycerides to glycerol and fatty acids, and have the peculiarity of acting at the substrate-water interface
17 (even if the mechanism is yet not clear) [43]. It is supposed that the same mechanism is performed in the scission of aliphatic polyesters.
In the first degradation step the esterases are adsorbed on the polymer, then the enzyme proceed to the random scission or to a zipper-type scission of the polymer chain, leading to soluble low molecular weight oligomers and monomers [39].
1.3.1 Synthesis of PGA, PLA and PCL: Ring Opening
Polymerization
For the polymerization of PGA and PLA there are two different ways: direct condensation and pseudo-anionic Ring Opening Polymerization (ROP) or coordination-insertion ROP.
The direct condensation starts from the monomers, and can be performed at melt state or in solution. In the first reaction step the monomer (GA or LA) is heated at environmental pressure up to 175-180 °C for GA and about 200 °C for LA. In this step there is the formation of low oligomers and water that must be removed by distillation. In the second step the pressure is lowered at about 100 mmHg to promote the water removal and the condensation of the oligomers to high weight polymers. For the PLA this method allow to obtain average molecular weight of about 130 kDa, while for PGA only low molecular weight polymer is obtainable. For the ε-CL there are no examples in literature of direct condensation. The pseudo-anionic ROP is a widely used reaction for the industrial synthesis of several polymers. It is defined as “anionic” because it involves the use of nucleophilic reagents (mostly organometals) as initating systems. It proved to be extremely efficient for the polymerization of monomers containing polarized bonds, like the ester bond [44]. Nowadays it is the most used method for the synthesis of PGA, PLA and PCL and of the respective copolymers. It is a “pseudo-anionic” polymerization which proceeds with the coordination of the monomer with the active species (usually an Al, Zn or Sn compound), followed by the insertion of the monomer between the initiating system and the growing polymer chain [45]. During the years several catalytic systems have been proposed: Al compounds like the aluminium triflate ((CF3SO3)3Al) or isopropoxide (C9H21O3Al) allow the best control on
18 polydispersity and best yields, but at the same time have low reactivity and leads to molecular weight lower than 105 Da. In addition Al ions are not metabolizable by the human organism.
Nowadays the most used catalyst are based on transition metal like Zn, Ti, Zr and Sn, due to their low tossicity, high reactivity and low polydispersity. Among them Tin (II) 2-ethyl-hexanoate (or tin Octoate, SnOct2) is the most used one. In combination with a primary alcohol as
initiator, this system guarantees an high activity, good yields, low polydispersity and a good linear correlation between its concentration and the molecular weight of the polymer.
It presents the only disadvantage of remaining incorporated into the polymer, and since its concentration in a device must be under 20 ppm, it must be removed from the polymer through an acid washing.
Although this initiating system has been used for several years, the catalytic mechanism for the ROP is still not clear. The first mechanism of catalysis was proposed by Kricheldorf et al [46], and implies the simultaneous coordination of the alcohol and the monomer with the metal centre, followed by the ring opening and the coordination of the chain with the SnOct2 as illustrated in figure 1.14.
Figure 1.14: Reaction mechanism proposed by Kricheldorf [46]
The second mechanism was proposed more recently by Kowalsky et al [47], and implies the reaction of tin octoate with the primary alcohol to form a metal alkoxide, followed by the insertion of the monomer between the metal centre and the alcohol, as illustrated in figure 1.15. Recent studies however seem to confirm the hypothesis of Kowalsky [48] [49]. It was seen in fact that the polymerization rate of two
19 reactions, one catalysed with Sn(Oct)2 and n-buthanol, and another with
Sn(OBu)2 and n-octane, was quite the same, while the only Sn(Oct)2 had
a much lower activity.
Figure 1.15: Reaction mechanism proposed by Kowalsky [47]
This suggests that the first step of catalysis is the formation of tin buthoxide. Moreover the best results in terms of catalytic activity are when the primary alcohol is added in a stoichiometric quantity (1:2).
The ROP catalyzed by the tin octoate is a living polymerization, which means that it can advance theoretically until there is available monomer in the reaction environment. The only possible termination reaction so is the hydrolysis of the catalyst-polymer bond by the addition to the reaction mixture of a termination agent. So pseudo-anionic ROP allows to obtain polymers with an high molecular weight (up to 106 Da) with low polydispersity.
Despite this, there are several factors that can limit the final molecular weight: since Sn(Oct)2 is a trans-esterification catalyst, it can promote the
inter- or intra-molecular trans-esterification reactions reported in figure 1.16 a and b (back-biting) [44].
20
Figure 1.16 b: Reaction scheme of inter-molecular transesterification [44]
Trans-esterifications reactions have the consequence of a reduction of the molecular weight and of a broadening of polydispersity.
The termination reaction can occur also in presence of traces of atmospherical oxygen or moisture, which can hydrolyze the catalyst-chain bond. Also in this case the consequence is a lowering of the molecular weight.
A third factor that must be considered is the presence of an excess of primary alcohol respect to the stoichiometric quantity. The eventual excess can in fact act as a chain-transferring agent, also in this case limiting the molecular weight.
1.4 Bionanocomposite
Even if acting on the parameters that we have seen above, there are several limitations on the physical and chemical properties obtainable by polymeric materials. Because of this reason in most cases polymers are added with organic and inorganic additives to improve their mechanical, thermal or degradative behaviour or to confer to the material particular properties. The resulting materials is composed by a dispersion of particles in a polymer matrix, and takes the name of composite materials, Example are the inorganic fillers, which enhance the elastic modulus making the material less deformable and give it anti-flame properties [50].
21 However the improvement of the properties of the composite materials is really low or requires high quantity of filler. This problem is due the low interfacial interaction between the polymer matrix and the filler, which leads to a poor and not homogenous dispersion of the filler. So it is important to maximize the interaction between the filler and the polymer, increasing the aspect ratio of the filler particles. About this, it was of extreme importance the in-depth study, in the last twenty years, of the nano-particles.
Due to their low dimension and their high aspect ratio, nano-particles allow to obtain highly homogenous dispersion in polymeric matrices. The result is an high interaction grade and consequently an high improvement of the properties of the resulting nanocomposite, even at low concentration of nano-filler [51].
Figure 1.17: Scheme of the effect of a nano-filler in a polymer matrix [51]
Starting from early 1990s there have been lots of studies and applications of polymeric nanocomposites. Materials with enhanced or “smart” properties have been obtained by dispersion of metal nanoparticles, carbon nanotubes, graphene and layered silicates [51].
The enhanced properties of nanocomposites makes them a suitable material for tissue engineering, provided that they possess the requisites of biodegradability and biocompatibility. Combining a biodegradable polymeric matrix with a nano-filler allow to obtain a particular class of nanocomposites called bionanocomposites [52]. Bionanocomposites can be obtained by the dispersion of inorganic particles in a natural polymer. Nature employs this kind of materials for the realization of hard tissue in both invertebrates (seashells are made of dispersion of nano-particles of CaCO3 in a protein matrix) and vertebrates (bones are made of a protein
22 matrix combined with hydroxylapatite, a mineral form of calcium apatite). Bionananocomposite are also obtainable synthetically: examples are the combination hydroxyapatite and collagen [53] or chitosan [54] for the repairing of bone tissue, or the incorporation of a drug and magnetic nanoparticles in poly(hydroxyethyl-methacrylate) or in PLA to make drug delivery systems [54]. For the manufacture of tissue engineering scaffolds it is obviously necessary that the nano-filler be biocompatible, especially considering that several types of nano-particles have proven to be particularly dangerous for human health. It results so of great importance to find a nanofiller which is not toxic for human body or whose toxicity is minimal.
1.4.1 Cellulosic nanofillers
Among the several types of nano-fillers, cellulose based nano-fillers present different advantages. They are obtainable by renewable resources, they are biodegradable and they can be obtained by an almost infinite number of sources. Moreover, first studies on the possible toxic effect of cellulose nano-crystals have given good results [55], showing good biocompatibility and hemocompatibility and low foreign body responses [56]. Nevertheless, the best feature of cellulose based nano-fillers is their ease of functionalization. The huge abundance of hydroxilic groups on their surface in fact make possible to chemically link on the surface on the filler a wide range of functional groups. This feature results of great usefulness since, because of the high hydrophilicity of cellulose, its dispersion in apolar matrices can result quite difficult to obtain. For this reason the number of studies on this particular type of filler is growing constantly in the last 10 years.
Among cellulose based nano-fillers, the most employed are Cellulose Nano-Fibres (CNF). They can be obtained either by a bottom-up and a top-down approach. The first one consists in the biosynthesis of ribbon-shaped fibrils of cellulose by bacteria of Acetobacter species [57]. The obtained fibres take the name of Bacterial Cellulose (BC), with a width from 20 to 80 nm and a length up to several micrometers. BC has the main advantage of high purity, but have also an high cost and are obtainable in low quantities.
23 CNF are also obtainable by sonication of natural cellulosic fibres (top down method). CNF obtained by sonication have the same structure of BA, but are lees pure and contains a relatively high fraction of hemicellulose [57].
CNF have received a great deal of attention in the field of bionanocomposites, being successfully incorporated in polymer matrices like PVA [58], polyurethane [59], PLA [57] etc., showing a good improvement of the mechanical properties of the resulting nanocomposites.
Figure 1.18: CNF dispersed in PVA [58]
CNF nevertheless presents the main disadvantage of having an high fraction of amorphous regions. This regions organize themselves in disordered ribbons that lower the density respect to the crystalline regions, worsening the mechanical properties of the final nanocomposites [52] [60-61]. However it is possible to chemically decompose these amorphous regions by an acid attack leaving unchanged the crystalline regions, obtaining highly crystalline nano-cellulose with higher mechanical and chemical properties.
1.4.2 Cellulose Nanowhiskers
Another kind of cellulose based fillers is represented by nano-crystals of cellulose and Cellulose Nano-Whiskers (CNW). CNW are nano-fibrils of cellulose, with an average thickness from 5 to 15 nm and a length from 100 nm to 1 μm [52]. They can be easily obtained by the hydrolysis of different types of cellulosic biomasses in acid conditions. H+ ions can in fact penetrate the amorphous regions of cellulose fibres, leading to their degradation and allowing to obtain highly crystalline cellulose fibrils [60]. It has been seen that the reaction time strongly affects the final structure of CNWs, since increasing the reaction time is
24 possible to obtain shorter crystals with a lower dimension dispersity [62]. Too high times although lead to complete degradation of the CNWs to glucose. On the other hand too low reaction times lead to only partly degraded aggregates of nanofibers.
Usually the process is performed in hydrochloric or sulphuric acid, with a preference for sulphuric acid which leads to more stable aqueous suspensions of CNWs thanks to the higher surface charge [63-64]. Acid hydrolysis is a really low cost process that allows so to obtain CNWs with an high purity grade and high cristallinity. The structure of obtained CNWs will be obviously different depending by the nature of the initial wood pulp, as can be seen in figure 1.19
Figure 1.19: CNWs obtained by tunicin (a), ramie (b), cotton (c), sugar beet (d), micro crystalline cellulose (e), and bacterial cellulose (f) [52]
CNWs present quite interesting properties for their application in the production of bionanocomposites: together with their biodegradability and biocompatibility, their low cost, large availability and low density, they also show an high geometrical aspect ratio, which traduces in a large interfacial surface with the matrices, [63]. They also posses extremely high mechanical properties, like an estimated tensile modulus between 130 and 250 GPa [52] and a strength of 10 GPa [63]. In addition to the improvement of the mechanical properties, CNWs offers another interesting possibility. A recent study has in fact highlighted that the presence of CNWs in low concentration delay the degradation process of a PLLDA nanocomposites and enhance its thermal stability [65].
On the other hand, CNWs have two main drawback: the first one is the impossibility to predict the final properties of the nanocomposites. The
25 second, and more important one, is their high hydrophilicity that make them hardly dispersible in apolar polymeric matrices.
To overcome this limitation the most simple way is to obtain CNWs dispersion in water-soluble polymers. The dispersion of CNWs in PVA or hydroxypropyl cellulose (HPC), for example, allowed to obtain transparent films with an enhanced storage modulus, tensile strength and yield stress respect to the native polymers, and a lower breaking
elongation [66-68].
However water has a plasticizing effect on the polymer that can worsen its properties and it is extremely difficult to extract from the nanocomposites. In addition, the prerogative of water solubility limits the choise of polymeric matrices to only a few.
The second option is to make the CNWs more compatible with the apolar matrix through a functionalization, that can be non-covalent or covalent. One method of non covalent fuctionalization implies the preparation of a masterbatch formed by the CNWs and a water soluble polymer. The interaction between the polar groups of the polymer and the CNWs stabilize them preventing their aggregation. The obtained masterbatch can be then mixed with a more apolar matrix to obtain a nanocomposite. To this purpose both PEG and Polyethylene-Vinylalcohol have been used to stabilize dispersion of CNWs in PLA matrices [69-70]. The effect was in both cases an improvement of tensile strength and modulus, better barrier properties and a good nanometric dispersion of the CNWs.
Non covalent functionalization can be achieved also by the use of dispersing agents, like for example surfactants. There are reported in literature different examples of a treatment of CNWs with the acid phosphate ester of ethoxylated nonylphenol followed by freeze-drying. The CNWs which have been submitted to this procedure were then
26 dispersed in apolar matrices like isotactic polypropylene or PLA. In both cases the TEM analysis showed a better dispersion of the CNWs in the matrix and enhanced mechanical properties (improved tensile strength and elongation at break) [71-72].
The second way to obtain the compatibilization of CNWs with apolar matrices is their covalent functionalization. This possibility is due to the extreme abundance of hydroxylic groups on the surface of cellulose, that can chemically react with a wide range of compounds to obtain functionalized particles.
The most common example of this kind of functionalization is the acetilation of cellulose to obtain cellulose acetate. The reaction has been performed in mild conditions with vinyl acetate to obtain organophilyc CNWs. It was seen how after at least two hours of reaction, the obtained CNWs gave stable dispersion in organic solvents like THF [73]. So this kind of modified CNWs are suitable for the production of bionanocomposites by solution casting with apolar polymers.
Figure 1.21: Reaction scheme of the acetylation of cellulose
Figure 1.22: Dispersion of acetylated cellulose in different organic solvents [73]
There is however another possibility of functionalization, which leads to the best results at all. Is possible to chemically modify the CNWs with low molecular chains of the same polymer where we want to disperse them.
27
1.4.3 Polymer Grafting on CNWs
The presence of a suitable polymer chain on the surface of CNWs can in fact improve the interfacial adhesion, allows a better dispersion and decreases the water absorption.
The hydroxyl groups on the surface of CNWs can act, in the presence of a catalyst, as initiating agents for a polymerization reaction. The possibility of growing polymeric chains directly on the surface of cellulose nanowhiskers takes the name of grafting from, and nowadays is maybe the most promising method for the production of polymer-CNWs bionanocomposites. There are several examples of this technique in recent literature. Grafting from has been performed with styrene by an atom transfer radical polymerization (ATRP), obtaining well dispersed modified CNWs in polystyrene with an high stability to hydrolysis [74]. Most importantly, the hydroxyl groups on the surface of CNWs can act as initiating agent for a ROP reaction, giving the possibility of growing biodegradable polymers like PLA, PGA or PCL on the surface of nanowhiskers. The first attempt to perform a grafting from reaction on a solid cellulose substrate (filter paper) was performed in 2005 by Hanfrén et al. using tartaric acid as initiator [75]. In the same year another research group succeeded in a grafting from reaction of ε-CL on microfibrillated cellulose [76]. Since then several studies have been performed on the possibility of growing biodegradable polyesters on the surface of CNWs and on their possible use in biodegradable nanocomposites. CNWs modified with PCL have successfully been used has compatibilizers for cellulose bi-layer laminates [77]. PCL-modified CNWs obtained by ROP increased the ductility and the strength of the composite respect to the native polymer [78], and the same improvement of the mechanical properties was seen also when they were mixed with PLLA [79]. PLLA-modified CNWs were also used for the production of a nano-composite, which resulted in a material with both higher elongation and strength respect to pure PLLA [80].
The possibility to chemically modify CNWs with excellent results, together with the possibility to easily copolymerize LA and ε-CL, offers therefore the possibility to obtain composite materials with a virtually infinite range of mechanical and degradative properties. Nevertheless,
28 there are only few examples in literature of modification of CNWs with random or block copolymers of LA and ε-CL [81].
Figure 1.23: Scheme of a grafting from reaction on cellulose
Due to the impossibility of determining the concentration of the initiator (the hydroxyl groups on the surface of CNWs) it results extremely difficult to have a good regulation of the molecular weight of the polymer [81]. Secondly it could be really hard to regulate the structure and the concentration of the comonomers in a copolymer obtained by a grafting from approach. To overcome this problem it is possible to modify CNWs presynthesising a polymer and then linking it to the hydroxyl groups, with an approach that takes the name of grafting to. Differently from grafting from, there are few examples of this method reported in literature. One method taken in consideration was to obtain a polymer by a living polymerization and then add the living polymer to the CNWs; an example is the surface modification of cellulose powder with poly (2-Methyl-2-oxazoline) obtained by living polymerization [82]. However it results more simple to use click chemistry reactions [83] or coupling agents like dianhydrides or diisocyanates [84]. This method although implies the necessity to have on the termination of the polymer chain a functional group capable to react with the coupling agent, like an hydroxyl group.
The latter method listed above was used to successfully modify the surface of CNWs with block copolymer of LA and CL [81]. Different low molecular weight copolymers were previously synthesized by a ROP reaction, then linked to the CNWs surface using toluene- diisocyanate.
29
Figura 1.24: Scheme of a grafting to reaction on cellulose
The resulting nanocomposites showed an increase in the glass transition temperature and high dispersion of the CNWs in the polymer matrix, reported in figure 1.25.
Figure 1.25: TEM image of block P(LLA-CL)-modified CNWs [81]
On the other hand, the attempt to modify the CNWs surface with a random copolymer of LA and CL has not been performed yet.
The grafting to approach, despite the advantages listed above, presents two main disadvantages. The first is the necessity to have a copolymer with a functionalizable group on the termination. The second disadvantage is that the terminal groups must be available for the reaction with the linking agent, that is not always possible in polymeric system which have an high viscosity and so a reduced molecular mobility.
Despite this two limitations, which can be overcome using low molecular weight polymers, the grafting to approach is one of the most promising techniques for the modification of CNWs with copolymers with
30 controlled structure and comonomers concentration, and should be explored in other research works.
31
1.5 Aim of the work
The aim of this PhD work is the realization of a bionanocomposites based on functionalized Cellulose Nanowhiskers and biodegradable polymers, usable for the realization of a tissue engineering scaffold. The first part of the work will be focused on the synthesis of L-Lactic Acid (LLA) and ε-Caprolactone (CL) random copolymers by Ring Opening Polymerization (ROP). Two different concentration of the two comonomers, 60% LLA/ 40% CL and 70%LLA/ 30% CL will be taken in consideration for the synthesis of the copolymer. A particular attention will be focused on the increase of the copolymer randomness. For this purpose, different reaction conditions will be tested, using three different catalysts: the commonly used ROP catalyst Tin (II) ethyl-hexanoate (SnOct2), the Bismuth Subsalicylate (BiSS) and the organic catalyst
Dimethylamminopyridine (DMAP). The obtained copolymers will be characterized by FT-IR spectroscopy, 1H-NMR spectrometry, thermal analysis and Gel Permeation Chromatography to determine the copolymer which best fulfil the necessary features for the functionalization of CNWs.
The second part of the work will be the CNWs functionalization with the chosen copolymer. The functionalization reaction will be performed with a method developed in a previous research work of our group. The effective success of the functionalization reaction will be verified by solvent extraction and thermal analysis. The obtained functionalized CNWs will be employed for the realization of a bionanocomposites through solution casting. The thermal and degradative properties of the obtained bionanocomposites will be tested.
The third part of the work will be focused on the realization of a tissue engineering scaffold by an additive manufacturing technique Tests to obtain a tissue engineering scaffold containing polymer functionalized CNWs will be performed, using an innovative method named Computer Assisted Wet Spinning (CAWS). The influence of the modified nanofiller on the manufacturing process and its dispersion in the polymeric system will be verified.
32
2. Results and Discussion
2.1 Statistical P(LLA-CL) Copolymers
The first part of the research work has been focused on the synthesis of the polymers to be used for the modification of cellulose nanowhiskers. The structure and molecular mass of the polymer is in fact of primary importance for the success of the polymer grafting on the CNWs and for the final properties of the resulting bionanocomposites.
The synthesis of the polymers was performed by Ring Opening Polymerization reaction, starting from two monomers: the L-Lactide (the cyclic dimeric form of LLA) and the ε-caprolactone. In order to fine-tune LLA-CL copolymers with properties which are suitable for our purpose, it was necessary to test different types of initiating systems and reaction conditions. The parameters, that have a marked influence on the structural properties of the copolymers and have been taken in consideration, were the relative concentration of LLA and CL, the average molecular weight of the copolymers and the distribution of the two co-monomers in the polymer chain.
2.1.1 Comonomers relative concentration
In fig 2.1 it is reported the scheme of the four propagation reactions that occur in the copolymerization of two generic monomers m1 and m2
Figure 2.1: Reaction scheme of copolymerization of two monomers
It is also possible to define the reactivity ratios as and
33 Depending on the reactivity ratio there are four limiting cases:
If r1 and r2 >> 1 the two monomers will react only with
themselves and there will be the formation of the two homopolymers in the reaction environment
If r1 and r2 > 1 every monomer reacts preferably with itself, so
there will be the formation of random copolymer with longer sequences (blocks) of the comonomer in excess in the feed.
If r1 and r2 ~ 0 every monomer will react preferably with the
other, leading to a perfectly alternate copolymer
If r1 and r2 ~ 1 both monomers will react indifferently with
themselves or with the other monomer, leading to a random copolymer.
The last case is r1 >> 1 and r1>> r2, in this case one monomer is more
reactive than the other. In a batch polymerization process, at the beginning of the reaction the copolymer chains will contain long sequences of the more reactive comonomer interspersed by single units of the second comonomer. When the first monomer is depleted, the less reactive monomer will begin to react leading to more random copolymer sequences. If the more reactive comonomer is in a molar excess respect to the other, there will be the probable formation of blocks.
In our case LLA is much more reactive than CL, so the active group of the growing chains will react preferably with the first one, leading to copolymers with higher content of LLA respect to the reaction mixture. Reaction temperature plays so an important role: it was seen in a recent study [85] that when reaction temperature is below 120 °C the reactivity of CL is nearly negligible and there is the formation of only PLLA [86]. Rising the temperature up to about 140° it is however possible to enhance the reactivity of the CL, obtaining a copolymer with a composition rather similar to that of the reaction mixture. At higher temperatures the composition of the copolymer becomes practically the same as that of the mixture, but at the same time there is a decrease of the activity of the catalyst [85].
The relative concentration of the two co-monomers is of great importance because it results in a different thermal and mechanical
34 behaviour of the obtained copolymers. The glass transition temperature (Tg) is one of the affected properties.
The theoretical Tg of a copolymer is determinable by the Flory-Fox equation (eq. 2.1)
eq 2.1 where
w1 and w2 are the weight fraction of the two comonomers inserted
in the copolymer structure
Tg1 and Tg1 are the glass transition temperature of the
homopolymers obtained by comonomers 1 and 2
The Flory-Fox equation is valid only for totally amorphous random copolymers, nevertheless it allows to obtain a relatively good estimation of the final glass transition temperature of the copolymer.
Since PLLA at room temperature is a glassy polymer, with a Tg of about 65 °C, while PCL is a viscoelastic polymer, with a Tg under room temperature (-60 °C), the Tg of the resulting copolymer could vary in a quite wide range. For example the theoretical Tg for a 50/50 mol/mol copolymer of LA and CL, calculated with Flory-Fox equation, is -9.8C, while for a 70/30 copolymer it raises up to 19.3 °C
The mechanical properties of the copolymer will be obviously influenced too by the two comonomers composition. A copolymer with an high concentration of LLA will result in an hard and stiff material, while high concentration of CL will lead to a more elastic and flexible polymer. This characteristics will obviously influence the Young modulus of the final materials, its breaking elongation and mechanical resistance.
Degradative properties will be influenced by the copolymer composition too. PLLA has an higher hydrophilicity respect to PCL [39], so a copolymer rich in LLA will be easier to degrade respect to a copolymer containing high concentration of CL (that is more hydrophobic).
2.1.2 Molecular weight
A second important factor that must be considered is the average molecular weight of the copolymers. Normally, for the manufacture of
35 biomedical devices, polymers with an average molecular weight between 10,000 and 200,000 kDa [87] are employed, however the optimal weight to obtain a well workable polymer is around 60,000. It is therefore important to consider also that a too high molecular weight implies an higher viscosity of the polymer solutions, and so a worse reactivity of the hydroxilic terminations which are used for functionalization and graft reactions.
It is possible to adjust the average molecular weight acting on the concentration of the catalyst and of the initiator (the primary alcohol). Maintaining a catalyst to initiator ratio of 1:2 in fact, the average molecular weight of the polymer decreases increasing the concentration of initiating system. According to Kowalski et al [47] to form an active specie two molecules of alcohol must coordinate with the Sn(Oct)2. The
growth of the polymer chains starts on the active centre formed on the metal catalyst, so increasing the number of active centre an higher number of growing chains is formed and the molecular weight decreases. Increasing further the amount of alcohol, it acts as a terminating agent and the molecular weight falls.
In a previous work of our research group [81] a good linear correlation between the average molecular weight and the initiator concentration was found for a quite wide ratio range
2.1.3 Randomness factor
In addition to the relative co-monomers composition in the copolymer and the average molecular weight, for the purposes of this study it is important to consider also the co-monomer distribution in the polymer chain. As said above in normal ROP reaction conditions LLA is more reactive than CL, so the copolymers will basically contain relatively long sequences of LLA interrupted by isolated CL units or CL short sequences. Longer LLA sequences may organize themselves in crystalline domains, raising also the Tg of the copolymer and making it stiffer and less degradable. Crystalline materials are less hydrophilic than amorphous, so they can hardly be swollen by water and attacked by hydrolytic enzymes and are less susceptible to hydrolysis [88]. To have a material that degrades in a uniform way it is so desirable to have a low cristallinity. This is obtainable by a structure as “disordered” as possible, with a random distribution of the two co-monomers.
36 It is possible to describe the statistical distribution of the co-monomers in the polymer chain with a factor that takes the name of randomness
character (R) [89]. This value indicates the ratio between the alternating
copolymer sequences and those of the two homopolymers in the polymer chains and it is determinable by eq 2.4
lLA=
;
lCL=eq 2.2 lLA(random)=
;
lCL(random)= eq 2.3 R= ==
eq 2.4
where lLA and lCL are the average lengths of LA and CL homopolymer
sequences and lLA(random) and lCL(random) are the average length of random
fractions in the copolymer.
So for R=0 the system it is composed by a mixture of the two homopolymers, while for R=1 the copolymer will be perfectly alternate [89].
The relative concentration of the two co-monomers are determinable by
1
H-NMR analysis. As example the typical 1H-NMR spectrum of a LLA-CL copolymer is reported in fig. 2.2
37
Figure 2.2: Typical 1H-NMR spectrum of a P(LLA-CL) copolymer
In the enlarged portions of the spectrum it is possible to see three different signals attributable to the LLA monomeric units (a) and to the CL monomeric units (b nd c).
In table 2.1 are reported the chemical shift values with their relative attribution.
