DESIGN OF POLYESTER ARCHITECTURES AND POROUS …kth.diva-portal.org/smash/get/diva2:14207/FULLTEXT01.pdf · architectures. In many applications the materials’ three-dimensional

DESIGN OF POLYESTER

ARCHITECTURES AND POROUS

SCAFFOLDS

Karin Odelius

AKADEMISK AVHANDLING som med tillstånd av Kungliga Tekniska Högskolan i Stockholm framlägges till offentlig granskning för avläggande av teknisk licenciatexamen fredagen den 25 november 2005, kl. 11.00 i Salongen, KTHB, Osquars backe 31, KTH, Stockholm. Avhandlingen försvaras på svenska.

ABSTRACT The use of synthetic materials for tissue and organ reconstruction, i. e. tissue engineering, has become a promising alternative to current surgical therapies and may overcome the shortcomings of the methods in use today. The challenge is in the design and reproducible fabrication of biocompatible and bioresorbable polymers, with suitable surface chemistry, desirable mechanical properties, and the wanted degradation profile. These material properties can be achieved in various manners, including the synthesis of homo- and copolymers along with linear and star-shaped architectures. In many applications the materials’ three-dimensional structure is almost as important as its composition and porous scaffolds with high porosity and interconnected pores that facilitate the in-growth of cells and transportation of nutrients and metabolic waste is desired. In this work linear and star-shaped polymers have been synthesized by ring-opening polymerization using a stannous-based catalyst and a spirocyclic tin initiator. A series of linear copolymers with various combinations of 1,5-dioxepane-2-one (DXO), L-lactide (LLA) and ε-caprolactone (CL) have been polymerized using stannous octoate as catalyst. It is shown that the composition of the polymers can be chosen in such a manner that the materials’ mechanical and thermal properties can be predetermined. A solvent-casting and particulate leaching scaffold preparation technique has been developed and used to create three-dimensional structures with interconnected pores. The achieved physical properties of these materials’ should facilitate their use in both soft and hard tissue regeneration. Well defined star-shaped polyesters have been synthesized using a spirocyclic tin initiator where L-lactide was chosen as a model system for the investigation of the polymerization kinetics. Neither the temperature nor the solvent affects the molecular weight or the molecular weight distribution of the star-shaped polymers, which all show a molecular weight distribution below 1.19 and a molecular weight determined by the initial monomer-to-initiator concentration. Keywords: ring-opening polymerization, porous scaffold, L-lactide, 1,5-dioxepane-2-one, ε-caprolactone, spirocyclic initiator, star-shaped polyester

SAMMANFATTNING Nedbrytbara material för vävnadsersättning har blivit ett lovande alternativ till de implantat som används i dag och kan komma att lösa många av dagens brister. Utmaningen för de syntetiska materialen ligger i design och storskalig tillverkning av biokompatibla, bioresorberbara polymerer med önskvärd ytkemi, nedbrytningsprofil och mekaniska egenskaper. Olika materialegenskaper kan åstadkommas genom syntes av homo- och sampolymerer eller genom syntes av linjära eller stjärnformade arkitekturer. I många situationer är materialets tredimensionella struktur nästan lika viktig som polymerens sammansättning. Det som eftersträvas är strukturer med hög porositet och sammankopplade porer, som bland annat möjliggör inväxt av celler och transport av näringsämnen och avfallsprodukter. I arbetet har linjära och stjärnformade polymerer syntetiserats genom ringöppningspolymerisation, med hjälp av en tennbaserad katalysator och en spirocyklisk tenninitiator. En serie linjära sampolymerer med olika kombinationer av 1,5-dioxepan-2-one, L-laktid och ε-kaprolakton har syntetiserats med tennoctoate som katalysator. Sammansättningen hos sampolymeren kan väljas på ett sådant sätt att de mekaniska och termiska egenskaperna kan förutses. En metod för framställning av tredimensionella porösa strukturer med sammankopplade porer har utvecklats och använts, där strukturernas fysiska egenskaper ska möjliggöra användning för både hård- och mjukvävnadsregenerering. Väldefinierade stjärnformade polyestrar har syntetiserats med en spirocyklisk tenninitiator. L-laktid valdes som modellsystem för en undersökning av polymerisationskinetiken. Vi visade att varken temperaturen eller lösningsmedlet påverkar molekylvikten eller molekylviktsfördelningen hos de stjärnformade polymererna. Alla polymerer har en molekylviktsfördelning under 1,19 och en molekylvikt som bestäms av den initiala monomer-till-initator-koncentrationen.

LIST OF PAPERS

This thesis is a summary of the following papers: I “Elastomeric Hydrolysable Porous Scaffolds: Copolymers of aliphatic

polyesters and a polyether-ester”, Odelius K., Plikk P., and Albertsson A.-C., Biomacromolecules, (2005), 6(5) 2718-2725

II “Versatile and controlled synthesis of resorbable star-shaped polymers using

a spirocyclic tin initiator – reaction optimization and kinetics”, Odelius K., Finne, A. and Albertsson A.-C., J. Polym. Sci., Part A: Polym. Chem. Accepted for publication (2005)

TABLE OF CONTENTS

1 PURPOSE OF THE STUDY..............................................................................1

2 INTRODUCTION ...............................................................................................2

2.1 BACKGROUND ................................................................................................2 2.1.1 Ring-opening polymerization ..................................................................3 2.1.2 Polymers..................................................................................................4

2.2 TISSUE ENGINEERING.....................................................................................6 2.2.1 Porous Scaffolds .....................................................................................6 2.2.2 Preparation Techniques..........................................................................7

2.3 INITIATORS .....................................................................................................8 2.3.1 Tin (II) 2-ethylhexanoate.........................................................................8 2.3.2 Tin (IV) alkoxides..................................................................................11

2.4 STAR-SHAPED POLYMERS.............................................................................12

3 EXPERIMENTAL ............................................................................................14

3.1 MATERIALS...................................................................................................14 3.2 INITIATORS....................................................................................................14

3.2.1 Stannous 2-ethylhexanoate....................................................................14 3.2.2 Spirocyclic tin initiator .........................................................................14

3.3 MONOMERS ..................................................................................................15 3.4 POLYMERIZATION .........................................................................................15

3.4.1 Copolymers ...........................................................................................15 3.4.2 Star-shaped polymers with a spirocyclic tin initiator ...........................15

3.5 SCAFFOLD PREPARATION TECHNIQUE ..........................................................16

4 CHARACTERIZATION ..................................................................................17

4.1 NMR ..............................................................................................................17

4.2 SEC................................................................................................................17 4.3 DSC ...............................................................................................................18 4.4 TENSILE TESTING ..........................................................................................18 4.5 SEM ...............................................................................................................18 4.6 POROSITY DETERMINATION..........................................................................19 4.7 UV-VIS...........................................................................................................19

5 RESULTS AND DISCUSSION........................................................................20

5.1 COPOLYMERIZATION BY STANNOUS OCTOATE .....................................20 5.1.1 Polymerization conditions.....................................................................20 5.1.2 Polymer Characteristics .......................................................................21 5.1.3 Scaffold Preparation Technique ...........................................................26

5.2 STAR-SHAPED POLYMERS.............................................................................31 5.2.1 Synthesis of the spirocyclic tin initiator ................................................31 5.2.2 Polymerizations.....................................................................................32 5.2.3 Temperature effect ................................................................................36 5.2.4 Solvent effect .........................................................................................37 5.2.5 Kinetics..................................................................................................41 5.2.6 Thermal properties................................................................................44

6 CONCLUSIONS................................................................................................45

7 FUTURE WORK...............................................................................................46

8 ACKNOWLEDGEMENTS ..............................................................................47

9 REFERENCES ..................................................................................................48

ABBREVIATIONS

LLA L-lactide DLA D,L-lactide CL ε-caprolactone DXO 1,5-dioxepan-2-one PLLA Poly(L-lactide) PDLLA Poly(D,L-lactide) PCL Poly(ε-caprolactone) PDXO Poly(1,5-dioxepan-2-one) PLA Poly(L-lactide) and/or poly(D,L-lactide) SnOct2 Stannous octoate DSC Differential Scanning Caliometry SEC Size Exclusion Chromatography NMR Nuclear Magnetic Resonance SEM Scanning Electron Microscope ROP Ring-Opening Polymerization Mn Number average molecular weight Mw Weight-average molecular weight MWD Molecular Weight Distribution [M]/[I] Monomer-to-Initiator ratio Tg Glass transition temperature Tm Melting temperature

————— Purpose of the study —————

1

1 PURPOSE OF THE STUDY

ROP is a versatile polymerization method for e. g. synthesizing aliphatic polyesters from lactones and lactides. Good control over the polymerization is essential, and the polymerization kinetics and mechanism needs to be fully understood. Today, these bioresorbable materials are being investigated extensively for biomedical applications such as tissue engineering. It is established that there are numerous parameters that influence these materials’ interactions with the surrounding tissue into which they are implanted. These parameters include the composition of the polymer, the physico-chemical characteristics of the material, the shape of the implant, leakage of low molecular weight molecules and degradation products, and the effect of the sterilization method on the material and so on. In this study the synthesized linear and star-shaped materials are intended for use in tissue engineering applications. The specific purposes of this study:

I. To create an array of copolyesters of aliphatic esters and an ether-ester and to tailor make porous scaffolds by considering the materials unique physical properties and specific degradation profiles. The scaffolds should have such diverse mechanical properties that they can be used both for soft and hard tissue regeneration.

II. To investigate the polymerization kinetics for the synthesis of star-shaped aliphatic polyesters using a spirocyclic tin alkoxide initiator in order to gain a deeper understanding of the material synthesis.

—————Introduction—————

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2 INTRODUCTION

2.1 BACKGROUND

If a bodily organ or a tissue for some reason looses its function, up to 30 or 40 years ago a surgeon had three choices to replace its function. An organ or tissue could be transplanted from the same body (autograft) from one human to another (allograft) or from one species to another species (xenograft). However all three methods have their limitations. In autograft transplant donor site morbidity can become an issue and for the other methods donor availability, immunocompatibility, tissue preservation and function reconstitution are potential problems that need solutions. The use of synthetic materials for tissue and organ reconstruction has therefore become a promising alternative. The material of choice can be metallic, ceramic, polymeric, or a composite, and it is either biostable or bioresorbable. Obviously all materials placed in the human body are influenced by their sometimes harsh environment, so biostable is here a physiologically inert material that causes only minimal immunological response to the surrounding tissue and can retain its properties for years or decades e. g. poly(ethylene) and poly(methyl metacrylate). Bioresorbable materials are by definition solid materials and devices which show bulk degradation and further resorb in vivo. Successful bioresorption means the total elimination of a material and its degradation products. In many cases bioresorbable or temporary implants are better alternatives to biostable ones, mainly because removal by surgery is never needed. General demands on bioresorbable materials include that the material must be non-mutagenic, non-antigenic, non-carcinogenic, non-toxic, non-teratgenic, antiseptic, and tissue compatible. They should cause minimum morbidity and degradation products should be water soluble small molecules. Polylactides and polylactones are bioresorbable aliphatic polyesters used in many applications in the medical field1. In the 1960s PLA was investigated as a potential material for tissue replacement2.


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Today applications of aliphatic polyesters include resorbable sutures3, bone fixation4-

6, drug delivery7, nerve guides8, cartilage6,9 etc.

2.1.1 Ring-opening polymerization

The synthesis of polylactides and polylactones can be achieved either by a polycondensation reaction of an alcohol and an acid or through ROP of cyclic ester monomers. The development of ROP of aliphatic esters started already in the 1930s10,11 and today ROP is used almost exclusively. Partly because of the difficulties in achieving high molecular weights and desired end-groups by polycondensation reactions and partly because of the difficult in preparing well-defined copolyesters. ROP can be performed in bulk or solution (chloroform, toluene, dichloromethane, etc.), emulsion12 or dispersion13,14. In bulk, the temperature is generally in the range of 100-180 °C, whereas the polymerization in solution is normally performed at much lower temperatures. Many catalysts and initiators have been used and the reaction can precede through a large number of different polymerization reactions, i. e. radical, anionic or cationic polymerization or by a coordination-insertion polymerization. In this study, LLA, CL and DXO have been polymerized using a stannous based catalyst and an initiator by coordination-insertion mechanisms both in bulk and solution. ROP of lactones and lactides with organometallic initiators lead to inter- and intra-transesterification reactions especially with stannous containing initiators; thus yielding higher MWDs. More specifically, intermolecular transesterification reactions prevent the formation of block copolymers and intramolecular transesterification reactions, back-biting, lead to polymer degradation. The reaction parameters that influence the transesterification reactions are temperature15, reaction time16, type and concentration of the catalyst or initiator17 and the nature of the lactone or lactide18. In 1993 the first articles on the enzymatic polymerization of polyesters were published19,20 and since then lipase catalyzed polymerization of lactones and lactides has gained increasing attention. The enzymes work by a quite generous lock-key principle that recognizes even unnatural substrates in vitro. Advantages include non-toxic catalysts, the possibility to reuse the catalyst, enantioselectivity and stereoselectivity along with mild reaction conditions (pH, temperature and pressure). Problems in achieving low MWD, high yields, and molecular weight above 100.000 g/mol are the three major downsides21,22. Numerous lactones and lactides have been polymerized enzymatically, more recently our group presented the lipase CA catalyzed polymerization of an ether-ester, DXO23. A new and interesting application


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of enzymatic catalyzed polymerization is shown by the direct miniemulsion synthesis of nanoparticles by lipase PS catalysis of lactone intended for drug delivery24.

2.1.2 Polymers

Scheme 2.1. Ring-opening polymerization of a) DXO b) LLA and c) CL.

O

O

O

O

OHO

O

O

O

Hn

O

O

OHO

O

Hn

O

O

O

OH OO

O

Hn

PDXO PLLA

PCL

Table 2.1. Material characteristics.

PDXO PLLA PCL

Tg [ºC] -36 – (-39) 55 – 60 -55 – (-60)

Tm [ºC] – 175 – 190 55 – 60

Tensile strength [MPa] – 28 – 50 2.0 – 6.0

Elongation-at-break [%] – 16 80

Wetting properties Hydrophilic Hydrophobic Hydrophobic Total mass loss upon degradation [months]

– 36 – 48 24 – 36

Poly(1,5-dioxepan-2-one) PDXO is an amorphous polymer that has poor mechanical properties. It is however highly hydrophilic and upon copolymerization or cross-linking becomes an interesting material for biomedical applications. Several methods for cross-linking PDXO have been performed, including copolymerization with a multifunctional cross-linking agent25,26, functionalization with unsaturation and subsequent UV cross-linking27 or cross-linking using carboxylic acid chloride in a one-step reaction28. All of the networks types are elastic, swell in chloroform and have high Tgs. Differences between them include a varying ability to control the length between cross-links and the number of steps needed to create the networks, where the latter system is the most eminent. Networks formed by copolymerization with a multifunctional cross-linking agent have been used as substrates for grafting reactions. Our lab has shown that

a) b)

c)


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acrylamide can be grafted on cross-linked PDXO and that the degradation rate of the grafted materials is higher than the untreated material29,30. Polylactide and poly(LLA-co-DXO) Polylactide is a poly(α-hydroxy acid) commonly synthesized by ROP of lactide, the cyclic dimmer of lactic acid. Three different forms of lactide exist, namely the two stereoisomers L-lactide and D-lactide, and a racemic form D,L-lactide or meso-lactide. Upon polymerization the different stereoisomers give rise to polymers with very different physical and mechanical properties, e. g. PDLLA is amorphous while PLLA is a semicrystalline polymer. The lactide monomers are often copolymerized with various other monomers in order to tailor the polymer properties. The sequence lengths of the monomers in the copolymer in turn also influence the material properties strongly. Copolymerization of LLA with DXO by SnOct2 gives copolymers with a blocky microstructure. These copolymers have lower stiffness and higher elasticity as compared to homopolymers of LLA31. Two microblock copolymers of 20% DXO and 80% of either LLA or DLA were investigated looking at their in vitro degradation behavior and their in vivo characteristics. It was shown that the degradation was fastest for the amorphous poly(DXO-co-DLA) copolymer32,33 and that this copolymer showed a smaller foreign body response of the two34. Both in vitro and in vivo degradation was shown to proceed via a hydrolytic cleavage of the ester bond32-34. Completely different material properties can be achieved when triblock copolymers of DXO and LLA are synthesized. Triblock copolymers possess unique properties due to microphase separation of the hard crystalline domains dispersed in a continous amorphous matrix. They are often thermoplastic elastomers with many physical properties similar to the rubbers i. e. softness, resilience and flexibility. Poly(LLA-co-DXO) portray these thermoplastic elastic properties35, containing both hydrophilic and hydrophobic parts, which upon heat treatment phase separate into fiber-like structures. These morphologies can be varied by altering the molecular weight, monomer composition and casting method giving rise to a variety of structures that possibly promote cell-growth36. Poly(ε-caprolactone) and poly(CL-co-DXO) Poly(ε-caprolactone) is a poly(ω-hydroxyalkanoate) that is biodegradable by hydrolytic ester bond cleavage. Its main degradation product is hydroxyhexanionic acid that subsequently is degraded in the human body. As a homopolymer PCL is a semi-crystalline polymer with a degree of crystallinity around 50%. We have reported on several studies of the copolymerization of CL and DXO. When SnOct2 is used as a catalyst a random copolymer is created37. Using aluminum


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isopropoxide as initiator and by addition of subsequent monomer yields a triblock copolymer with a MWD between 1.1 and 1.331. By using a five membered tin initiator and allowing the first monomer, DXO, to react to full conversion followed by the addition of CL, multiblock copolymers were created38.

2.2 TISSUE ENGINEERING

Public health care has reached a level where body repair by tissue or organ transplantation is a standard method to treat patients. Thus, the restricting factor in restoring lost function is not surgery but rather the limited access to suitable human donors along with practical and ethical issues. There is therefore an urgent need to attain implantable materials from other sources, and tissue engineering has become a promising alternative that does not have the shortcomings of current therapies39-41.

2.2.1 Porous Scaffolds

The goal of tissue engineering is to augment, replace, or restore complex human tissue functions by combining synthetic and living components in the correct environmental conditions. This can be achieved either by culturing cells in vitro for transplantation as an auto transplant or by guiding cells in vivo to regenerate tissue (guided tissue regeneration). Porous three-dimensional scaffolds play an important role in directing cells to form new organs. Since the human body is such a complex system, an assortment of materials with varying chemical compositions42 and three-dimensional structures must be created to meet all these needs. The challenge is now to design and fabricate the scaffolds according to their intended application. Design criteria include43:

• suitable surface chemistry for cell attachment, proliferation and differentiation,

• suitable mechanical properties and degradation profile, along with a reproducible manufacturing technique,

• biocompatibility and bioresorabability, where neither the polymer nor the degradation products should provoke an unwanted inflammatory response, and

• high porosity with interconnected pores for cell growth and transportation of nutrients and metabolic waste.

The function of 3D scaffolds is to provide a framework for cells to attach, proliferate, and form an extracellular matrix, and they may also serve as carriers for growth factors and/or bimolecular signals. Ideally the scaffolds should degrade in vivo so that


7

the space at the appropriate time is replaced with host tissue44. Three-dimensional scaffolds have been used for tissue engineering of nerve8, skin45, bone5,6 and cartilage6,9,46 etc.

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Figure 2.1. The 3D scaffold is created and cells seeded in vitro in a static culture. The cell growth then proceeds in a dynamic environment, followed by a bioreactor after which the implant is surgically transplanted and host tissue can remodel the function in vivo. There are generally two different strategies of the in vitro design of porous scaffolds. They differ in their approach to the choice of material properties. In the first strategy the material properties and the degradation rate of the material has to be chosen in such a way that they are retained until the tissue engineered transplant is fully remodeled by the host tissue and it can take over the structural role. In the second approach the materials’ mechanical properties only has to be retained until the material is placed in a bioreactor47. See Figure 2.1.

When we summarize results from different research groups it becomes obvious that the cell source, material choice and culture form all are very important in tissue engineering and that the results are dependent on each one of them separately.

2.2.2 Preparation Techniques

Several methods have been developed and employed in order to create 3D porous scaffolds. The techniques include fabrication of fiber meshes6,48, freeze-drying49,50, gas foaming51, phase separation49,50,52, fused deposition modelling53, three-dimensional printing techniques54 and particulate leaching55,56, as well as combinations of these techniques46,57. Each method has its advantages and disadvantages, however they all strive for good control over the size distribution of


8

the created pores and the interconnectivity of them, thereby giving predictable structural properties that are very important in tissue engineering47,49,57. Fiber meshes. This structure consists of fibers that are either non-woven or woven/knitted and they have the advantage of a large surface area for cell attachment and diffusion of nutrients and waste products. The drawback of the fiber meshes is their lack of mechanical strength, and they are today only used as non-woven fiber meshes for the second strategy applications6,48. Freeze-drying. This method involves freeze-drying a polymer solution in a suitable solvent. Phase separation takes place upon cooling, and the removal of the solvent by sublimation results in a porous morphology49,50. This method is however both user and technique sensitive, and interconnectivity can be questioned along with the usefulness of the created pore size range (5-80 µm most common). Gas foaming. This technique involves equilibrating polymer discs with high pressure CO2 gas by which a polymer/gas is formed, creating pores. When the gas pressure is decreased, the gas molecules cluster, creating macro-pores. The porosity and pore structure can be changed by varying the amount of gas dissolved in the polymer, the rate and type of the gas clustering, and diffusion rate. The technique, however, often results in a closed cellular structure within the scaffold51. Three-dimensional printing. This technique is a solid-fabrication technique which produces components by inkjet printing a binder into sequential powder layers. This is a very promising technique that as of now has not been thoroughly investigated; the one major drawback is the costly equipment54. Particulate leaching. The solvent casting and porogen leaching technique involves the casting of a mixture of polymer solution and porogen in a mold, drying the mixture, and following this by a leaching of the porogen with water to generate pores 55,56. The technique allows for easy structure manipulation by controlling the amount and fraction of the porogen. Porogens include sodium chloride, ammonium hydrogen carbonate, or carbohydrates. It is well established that the polymer salt composite has a tendency to adhere to a glass mould, so that the samples must be formed after leaching. In this study we therefore aim at developing a technique to solve this adherence problem.

2.3 INITIATORS

2.3.1 Tin (II) 2-ethylhexanoate

Tin (II) 2-ethylhexanoate or SnOct2, is the most widely used catalyst for the synthesis of polylactides and polylactones both in technical production and for research purposes. Reasons include a high efficiency and versatility, easy handling and its solubility in most common solvents and cyclic ester monomers, and its approval by


9

the American Food and Drug Administration (FDA) as a food additive. Unfortunately SnOct2 chemistry also has its downsides, including difficulty in controlling the molecular weight of the polymer since any presence of water or other hydroxyl functionality is likely to initiate the system along with large amounts of transesterification reactions58,59. The mechanism through which the polymerization takes place has over the past decades been under debate and was for a long time considered to proceed through a cationic polymerization mechanism. It has however become evident that such a mechanism cannot describe the reaction; the reasons include the high optical purity (above 99% at 150 ºC) of the final PLLA. Today the reaction is instead considered to take place through a non-ionic coordination-insertion mechanism and there are primarily two different mechanisms proposed60-63. The role of SnOct2 in the polymerization is therefore still discussed, and depending on the proposed mechanism SnOct2 is called either catalyst or initiator. We considered SnOct2 as a catalyst. The mechanism that was proposed first is, the activated monomer mechanism, in which the monomer coordinates with SnOct2 and thereby becomes activated, Scheme 2.260-62. The reaction thereafter proceeds by a nucleophilic attack by an alcohol. The reaction is considered to be very complex, and it is likely that there are many initiating species present; the nature of the species present depending on the reaction temperature64. In the second proposal SnOct2 is believed to react with compounds containing hydroxide groups thus giving the actual initiator i. e. tin (II) alkoxide or hydroxide. The chain growth reaction is then assumed to proceed via monomer insertion, similar to that of other metal alkoxide active centres63 Scheme 2.3. Penczek et. al. recently showed that the rate of polymerization of lactides and lactones in the presence of hindered amines (proton traps) is either exactly the same or exceeds the rate in the absence of these amines. This indicates that the reaction mechanism involving active species is incorrect along with the proposed cationic reaction mechanism65. Proof for the mechanism involving the formation of tin alkoxide as an intermediate was recently given when octanoic acid was isolated and the presence of Sn-alkoxides was shown by MALDI-TOF mass spectrometry66,67.


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I. Activated monomer mechanism Scheme 2.2. Activated monomer mechanism.

SnOct2 + R-OH + LLA (1)

Sn

O

OO

OO R

H

O

O

O

O II. Tin alkoxide mechanism Scheme 2.3. Tin alkoxide mechanism.

SnOct2 + R-OH OctSnOR + OctH (2)

RO

Sn

O

O

O

O

O

O It is well known that polymerizations with SnOct2 are initiated by hydroxyl-containing compounds. These hydroxyl-groups can either be purposely added to the reaction mixture or appear as impurities in the monomer or catalyst itself as a result of the production method and the fact that SnOct2 is extremely hygroscopic68. It has been shown that when SnOct2 is distilled to remove 2-ethylhexanoic acid, new acid


11

will appear shortly after the distillation60. This addition of hydroxyl-groups reduces the final molecular weight of the polymer69. The molecular weight of the produced polymer is also influenced by the catalyst concentration where the molecular weight increases with decreasing catalyst concentration60,68 and by the reaction temperature were a low temperature yields a polymer with high molecular weight60. The addition of an alcohol to the reaction mixture makes the activation of initiation easier compared to neat SnOct2

61, with varying reaction rates depending on the nature of the added alcohol64. Anhydrous octanoic acid for example decreases the rate of propagation without affecting the final molecular weight63.

2.3.2 Tin (IV) alkoxides

Tin (IV) alkoxides are generally synthesized by the reaction of an alcohol and dibutyltin oxide, dibutyltin dichloride70 or dibutyltin diethoxide71. They are known to form cycles during synthesis and exist as monomers and dimers71. The reaction mechanism through which the polymerization proceeds is thought to be a coordination-insertion mechanism in which the acyl-oxygen in the lactone or lactide is cleaved while the configuration is retained. Depending on the structure of the alcohol, initiators of varying form and functionality can be synthesized. An ethylene glycol segment72 (Scheme 2.4 I), unsaturation incorporated into the initiator73 (Scheme 2.4 II & III) and star-shaped initiators are some examples of this74-76 (Scheme 2.4 IV). Scheme 2.4. I. 1,1,6,6-tetra-n-butyl-1,6-distanna-2,5,7,10-tetraoxacyclodecane II. 1,1-di-n-butyl-stanna-2,7-dioxacyclo-4-heptene III. 9,9,20,20-tetrabutyl-8,10,19,21-tetraoxa-9,20-distannadispiro[5.5.5.5.] docosa-2,14-diene IV. Spirocyclic tin initiator.

O

OSn

O

Sn

O

O

SnO

Sn

OO

OO

O

O

O

O

Snm

n

o

p

(m + n + o + p = 15)

I. II. III.

IV.

(m + n + o + p = 0)

Kricheldorf et. al. were the first to use these initiators for ROP of lactones and lactides. The reactions were mainly performed in bulk at high temperatures, yielding stereoregular polymers with low molecular weight, broad MWD and


12

monomer/initiator ratios that did not parallel the molecular weight of the resulting polymers. The problem of low control over the polymerization was solved by using solution polymerization performed at temperatures in the range of 40 – 60 ºC72. The synthesis of star-shaped polymers using tin (IV) alkoxides was introduced by the same group. A spirocyclic tin initiator was synthesized from pentaerythritol and dibutyltin oxide75 enabling the creation of defined star-shaped polymers. It had however one major drawback; the initiator has a very low solubility in all common inert organic solvents. This problem was overcome by the introduction of ethoxylated pentaerythritols that make it possible to create well-defined star-shaped homo- and copolyesters74,76 and, when required, subsequent cross-linking of them28,77.

2.4 STAR-SHAPED POLYMERS

Today it is possible to achieve numerous sophisticated architectures of polymers, each class being associated with a unique set of properties. By taking advantage of these advanced structures, such as star-shaped polymers and dendrimers, it is possible to tailor the material properties to an even greater degree than is possible with conventional linear polymers. The challenge of finding a material with the proper characteristics for a given biomedical application is still present. One possibility may be to use star-shaped polymers. Their physical properties are different from their linear analogues of the same molecular weight and composition, i. e. their hydrodynamic radius is smaller, their melt viscosity is reduced, and the large number of end groups reduces their crystallinity. It has also been shown that their degradation rate is altered78,79. The synthesis of star-shaped polymers is generally achieved by one of two approaches, namely either the “arm-first route” in which the polymer arms are coupled to a multifunctional coupling agent, or the “core-first route” based on a multifunctional core as initiator. This is similar to dendrimer chemistry where one talks about two fundamentally different synthetic routes; namely (i) the convergent route where the macromolecule is built from the outside and (ii) the divergent route in which the macromolecule is built from the inside and out. The two strategies for creating star-shaped polymers have pros and cons; the main concern is the degree of substitution of the multifunctional core. If the arm first route is chosen, the large functionality of the core can lead to coupling problems of the arms, where the last arm/arms can be difficult to attach because of steric hindrance. On the other hand, in the core first route the reactivity of the last unreacted arm/arms is lower than that of the arm that reacts first. In other words, both approaches may lead to unreacted sites on the core and hence incomplete substitution. Luckily, using appropriate systems this problem can be suppressed and stars with the desired amount of substitution can


13

be achieved. For polyesters, the core-first route is generally adopted using a tri-80, tetra80-83 or multifunctional hydroxyl-terminated core84,85 together with SnOct2 a as catalyst. Hydroxyl-terminated cores have also been used in similar approaches with other catalysts, such as the non-toxic bismuth(III) acetate86 and stannous acetyl acetonate87. As described above, a more “sophisticated” system using tin (IV) alkoxide chemistry with a spirocyclic tin initiator gives even better control of the system. The problems associated with SnOct2 chemistry are overcome, making it possible to create well-defined star-shaped homo- and copolyesters74,76.

—————Experimental—————

14

3 EXPERIMENTAL

3.1 MATERIALS

Dibutyltin oxide (Sigma-Aldrich, Germany) and pentaerythritol ethoxylate, 15/4 EO/OH (Sigma-Aldrich, Sweden), were used as received. Diethyl ether (LabScan, Sweden) was dried over molecular sieves, and toluene (Merck, Germany) was dried over an Na-wire. Dichloromethane (Labora, Sweden), chloroform (Labora Chemicon, Sweden) and toluene were dried over calcium hydride (Sigma-Aldrich, Sweden), and chlorobenzene (BDH, Germany) was dried over phosphorus pentoxide (VWR, Sweden) for at least 24 hours and then distilled at reduced pressure under an inert atmosphere prior to use. Sodium chloride (NaCl) (Fischer chemicals, Germany) was used after separating the agglomerates by grinding in a mortar. All other chemicals were used as received.

3.2 INITIATORS

3.2.1 Stannous 2-ethylhexanoate

Stannous 2-ethylhexanoate, SnOct2 (Sigma-Aldrich, Sweden), Scheme 3.1 was distilled under reduced pressure at 175 ºC before use. Scheme 3.1. SnOct2.

SnO

O O

O

3.2.2 Spirocyclic tin initiator

Dibutyltin oxide and pentaerythritol ethoxylate were weighed into a round-bottomed flask and suspended in dry toluene. The reaction vessel was heated to 135 ˚C and


15

refluxed until the theoretical amount of water was eliminated in a Dean & Stark trap. The solution was cooled in the refrigerator and the product precipitated. The initiator, Scheme 3.2, was dried under reduced pressure for 24 h. Scheme 3.2. Spirocyclic tin alkoxide.

Sn

OO

OO

O

O

O

O

Snm

n

o

p

(m + n + o + p = 15)

3.3 MONOMERS

L-lactide, LLA, (Serva Feinbiochemica, Germany) was recrystallized from toluene three times, dried at room temperature under vacuum for 48 hours and stored in an inter atmosphere before use. ε-caprolactone, CL, (Sigma-Aldrich, Sweden) was dried over calcium hydride for 24 hours at room temperature, was distilled at reduced pressure and stored in an inert atmosphere before use. 1,5-dioxepan-2-one (DXO) was synthesized according to literature88. The DXO was then purified by recrystallizing it from diethyl ether three times, distilling it twice, followed by drying over CaH2 for 24 hours and a final dry distillation under reduced pressure.

3.4 POLYMERIZATION

3.4.1 Copolymers

The desired amounts of monomer, co-initiator and catalyst were weighed into a previously silanized 25 ml round-bottom flask under nitrogen atmosphere inside a drybox (Mbraun MB 150B-G-I). The flask was fitted with a magnetic stirrer bar and was sealed with a three-way valve. The polymerizations were started by immersing the flask into a thermostated oil bath (110 ºC) and preceded for 10 h. The polymer was precipitated in a mixture of cold hexane and methanol (95:5).

3.4.2 Star-shaped polymers with a spirocyclic tin initiator

Polymerization was carried out in a silanized round-bottom flask with a magnetic stirring bar closed by a three-way valve. LLA and the initiator were weighed and added to the reaction vessel in a glovebox (Mbraun MB 150B-G-I) purged with


16

nitrogen. The desired amount of distilled solvent was transferred to the reaction vessel by a flamed syringe under anhydrous conditions. The reaction vessel was stirred for a few minutes to achieve a homogeneous reaction mixture after which it was immersed in a thermostated oil bath preheated to the reaction temperature. The temperature was held constant (± 1 ºC) using an Ikatron ETS D3 temperature regulator. Samples for 1H NMR and SEC assessment were withdrawn from the reaction vessel using a flamed syringe while flushing with nitrogen gas. When the reaction had reached full conversion the product was precipitated in cold ether, evaporated and dissolved in chloroform after which it was precipitated in cold hexane/methanol (90:10).

3.5 SCAFFOLD PREPARATION TECHNIQUE

The copolymers were dissolved in chloroform (CHCl3) to form a 5 wt% homogeneous solutions and were subsequently poured over NaCl in moulds with a diameter of 9 cm. Each mixture was thoroughly blended using a glass rod and evenly distributed over the bottom of the mould. The mixture was slowly air-dried under a lid for 7 days, after which the scaffold was allowed to dry freely for 7 days, to ensure complete chloroform evaporation. A two-step leaching process was thereafter performed on the now solidified composite. The scaffold and mould were first immersed in methanol for approximately 20 minutes to wet and swell the polymer and, in most cases, loosen it from the mould. The free samples were thereafter die-cut into their desired shape and immersed in de-ionized water to dissolve the salt particles. The water was changed after each 20 minutes period during one hour and then once every hour for five hours, followed by a water exchange 2-3 times every 24 hours for four days. The porous scaffold was thereafter dried to constant weight in vacuum and thereafter for another 3 weeks to minimize the amount of residual solvent. In cases where the scaffolds were fixed to the moulds, the scaffolds loosened after three days of water immersion and the same leaching procedure as for the other scaffolds was adopted. A silver nitrate test, SEM and gravimetric measurements were also used to confirm total salt leaching.

————— Characterization —————

17

4 CHARACTERIZATION

4.1 NMR

Copolymers. The chemical compositions of the copolymers and the degree of monomer conversion were determined by 1H NMR spectroscopy, comparing the relative intensities of the peaks originating from the comonomers and the resonance peaks from the monomer and polymer. The monomer sequence was determined by 13C NMR spectra. 1H NMR and 13C NMR were obtained using a Bruker AC-400 Fourier-Transform Nuclear Magnetic (FT-NMR) operating at 400 MHz and 110.61 MHz respectively. Star-shaped polymers. 1H NMR was used to determine the monomer conversion and molecular weight, using a Bruker Avance 400 MHz NMR instrument, operating at 400.13 MHz. In both cases deuteron-chloroform (CDCl3) was used as solvent. Non-deuterated chloroform was used as an internal standard (δ = 7.26 ppm).

4.2 SEC

SEC was used to monitor the molecular weights and the MWDs of the polymers during and after polymerization. The polymers were analyzed with a Waters 717 plus autosampler and a Waters model 510 apparatus equipped with two PLgel 10 µm mixed B columns, 300 x 7.5 mm (Polymer Labs., UK). Spectra were recorded with an PL-ELS 1000 evaporative light scattering detector (Polymer Labs., UK). Millenium32 version 3.05.01 software was used to process the data. Chloroform was used as eluent, at a flow rate of 1.0 mL/min. Polystyrene standards with a narrow MWD in the range of 580-400.000 g/mol were used for calibration.


18

4.3 DSC

The thermal properties of the synthesized polymers were investigated using a DSC (Mettler Toledo DSC 820 module) under nitrogen atmosphere. To erase the thermal history, the specimens were heated from 25 ºC to 180 ºC, held there for 2 minutes, and then cooled to 25 ºC at a rate of 10 ºC/min. The second scan was used to record the heat of fusion at a heating rate of 10 ºC/min. The melting temperatures, Tm, were noted as the maximum values of the melting peaks and the midpoint temperature of the glass transition was determined as the glass transition temperature, Tg. When evaluating the crystallinity of the copolymers, it was assumed that the only contribution to the heat of fusion in the poly(DXO-co-LLA) and poly(DXO-co-CL) was from the PLLA and PCL. PDXO has earlier been shown to be a fully amorphous polymer having a Tg between -35 and -40 ºC89.

4.4 TENSILE TESTING

The tensile testing of the copolymers was performed with an Instron 5566 equipped with pneumatic grips. The tensile measurements were performed with a load cell with a maximum of 0.1 kN at a crosshead speed of 50 mm/min according to ASTM D 638M-89. The films had a thickness of approximately 1.5 mm, a width of 4 mm and a gauge length of 35 mm. They were preconditioned before testing according to ASTM D618-96 (40 h at 50 ± 5 % relative humidity and 23 ± 1 ºC). Five samples from the same porous scaffold were tested for each polymer and the average thickness of each sample was calculated from five different measurements with a Mitutoyo micrometer. The load at break was chosen as the reference point, since the materials did not yield and in many cases were too weak to induce total stop of the driving motor when breaking.

4.5 SEM

Three sample pieces were randomly chosen from each porous scaffold. To ensure minimum loss of structure, the scaffold was immersed in liquid nitrogen and fractured for view of the sample cross-section. Pore size, porosity, efficiency of the salt leaching method and surface characteristics were evaluated by means of a JEOL JSM-5400 SEM using an acceleration voltage of 15 kV. The samples were mounted on metal studs and sputter-coated with gold-palladium (60 %/40 %) using a Denton Vacuum Desk II cold sputter etch unit operated at 45 mA for 3x15 s.


19

4.6 POROSITY DETERMINATION

The porosity, P, of the scaffolds was determined by measuring the dimensions and mass of the scaffold and was calculated as:

1001 ×⎥⎦

⎤⎢⎣

⎡−=

d

dP p (4.1)

where dp = (mp/Vp) is the scaffold density and d is the density of a non-porous film fabricated using the same technique as that used for the scaffold fabrication.

4.7 UV-VIS

To ensure total NaCl leaching, a silver nitrate test was performed, in which the presence of chloride ions in the leaching water is detected by precipitation of AgCl, using a WPA UV-Vis spectrophotometer version 1.6, after the instrument had been referenced with de-ionized water and AgNO3.

————— Results and discussion —————

20

5 RESULTS AND DISCUSSION

5.1 COPOLYMERIZATION BY STANNOUS OCTOATE

Since the human body is such a complex system, a large variety of materials with different characteristics are needed in order to fulfill all conceivable tissue engineering applications. By copolymerization, properties such as degradation rate, hydrophilicity and mechanical properties can be tailored and predicted. In order to achieve an appropriate foreign body reaction in vivo in addition to being a non-toxic material the mechanical properties should mimic the tissue environment in which the material is placed. Studies have also indicated that crystalline materials can have adverse effects.

5.1.1 Polymerization conditions

Three different types of copolymers, poly(DXO-co-LLA), poly(DXO-co-CL) and poly(CL-co-LLA), were synthesized as shown schematically in Scheme 5.1. The reactivity’s of the three monomer pairs varies considerably when stannous octaoate is used as catalyst, giving rise to different copolymer types Table 5.1. Table 5.1. Reactivity factors for copolymerization at 110 ºC in bulk.

Monomer 1 Monomer 2 r1 r2 Sequence structure

DXO LLA 0.1 10 Multiblock31

DXO CL 0.6 1.6 Random90

CL LLA 0.36 42 Multiblock91


21

Scheme 5.1. Schematic polymerizations of poly(DXO-co-LLA), poly(DXO-co-CL) and poly(CL-co-LLA).

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

OO

O OO

O OO

O

O

H

O

O

H a

b

c

d

OO

OO

OO

O

O

H

O

O

H a

b

c

d

O OO

O OO

O

O

O H

O

O

O b

c

d

H a

SnOct2 normally acts as a catalyst starting the polymerizations from hydroxyl functional impurities in the system68. In this case a dry and clean system was used with ethylene glycol as co-initiator, to ensure better control over the number of chains created and the MWD. In all cases, the polymerization temperature was 110 ºC, [Monomer]/[Sn(Oct)2] ≈ 600, MnTheor = 80,000 g/mol and the polymerization time was set to 10 h. The reaction time was chosen to obtain high conversions and at the same time to hinder transesterification reactions that lead to higher MWDs. Full conversion was however never reached, probably due to the limited diffusion in bulk polymerizations and to the short reaction times.

5.1.2 Polymer Characteristics

The composition, molecular weight, MWD and sequence lengths of the synthesized polymers are all given in Table 5.2 and are discussed below. The abbreviated names of the copolymers are given by the polymer composition e. g. 14D86L contains 14 mol% DXO and 86 mol% LLA. Chemical composition. To evaluate the chemical composition of the copolymers the integrated ratios of the intensities of the 1H NMR peaks were used. Thereafter the weight percent of the different components was determined according to:

iiji

iii MxMx

Mxwtw

×+×−××

=)100(

100%)( (5.1)

where xi is the molar fraction of component i and Mi and Mj are the molecular weights of components i and j respectively. In the case of LLA, MLLA was set to 72.1 g/mol i. e. the molecular weight of the half lactide unit. This value was chosen since


22

SnOct2 gives rise to transesterification reactions possibly giving half of the monomer length as the repeating unit. The results show that the incorporation of the less reactive monomer in the copolymer is lower than expected, thus an excess of the less reactive monomer must be added in order to reach the desired copolymer compositions. Table 5.2. Molecular weight and composition characterization of poly(DXO-co-LLA), poly(LLA-co-CL), and poly(LLA-co-CL).

Mole ratio in Feed (%)

Polymer Composition (%)a Sequence length Polymer

name DXO LLA CL DXO LLA CL

Mnb MWDb

LDXO LLLA LCL

14D86L 25 75 - 14 86 - 133600 1.29 2.0 12.9 -

23D77L 40 60 - 23 77 - 144400 1.29 3.1 9.9 -

33D67L 50 50 - 33 67 - 48400 1.34 3.7 5.5 -

48D52L 65 35 - 48 52 - 109400 1.23 4.1 3.4 -

61D39L 75 25 - 61 39 - 34700 1.19 8.0 3.3 -

87D13L 90 10 - 87 13 - 131400 1.33 10.6 1.0 -

26D74C 25 - 75 25 - 75 186800 1.35 1.4 - 5.0

50D50C 50 - 50 50 - 50 114800 1.25 2.7 - 1.6

75D25C 75 - 25 75 - 25 41800 1.49 4.1 - 1.4

82C18L - 10 90 - 18 82 155900 1.27 - 1.4 7.1

54C46L - 25 75 - 46 54 163800 1.27 - 2.7 3.6

34C66L - 40 60 - 66 34 125200 1.24 - 5.9 3.6

23C77L - 50 50 - 77 23 138800 1.34 - 6.5 2.9

6C94L - 75 25 - 94 6 140500 1.28 - 16.7 2.0

100L - 100 - - 100 - 103500 1.11 - - -

100C - - 100 - - 100 172200 1.29 - - - aMolar composition of the copolymers determined by 1H NMR at δD = 3.65 ppm, δL = 5.13 ppm and δC = 1.37 or 2,30 ppm bdetermined by SEC (using polystyrene standards).

Molecular weight and molecular weight distribution. The molecular weights of the polymers were in most cases higher than expected. This is seen when calibration with polystyrene standards has been used for this type of polymer and, as described by Kricheldorf et. al., a two-fold increase in molecular weight is common92. Therefore the SEC results are mainly used qualitatively. However, the results indicate that high molecular weight copolymers with MWDs, ranging from 1.1 – 1.49 have been produced.


23

Sequence lengths. 13C NMR is more sensitivity to sequence effects than 1H NMR58 and was therefore used for the sequence analysis, looking in the carbonyl region. Typical spectra are shown in Figure 5.1, where the homopolymer carbonyl peaks for all copolymers are at the highest and lowest fields in the spectra. Figure 5.1. Expanded 13C NMR of the carbonyl region a) poly(DXO-co-LLA) b) poly(DXO-co-CL) c) poly(LLA-co-CL). For the poly(DXO-co-LLA) eight different triads can be distinguished were e. g. IDDD is short for the carbonyl peak corresponding to a monomer sequence of DXO-DXO-DXO and the sequence length was determined using the following equations:

1+++=

LDLDDL

LDDDDDD II

IIL (5.2) 1+

++

=DLDLLD

DLLLLLL II

IIL (5.3)

where e. g. IDLD is the intensity of the peak representing the DLD triad90. The sequence lengths of poly(DXO-co-CL)38 and poly(LLA-co-CL)58,93 were determined in the same manner. In the case of poly(DXO-co-CL), the 13C NMR spectra showed four different peaks, corresponding to the homopolymer peaks and two dyad peaks, i. e. the transitions between DXO and CL and vice versa. This necessitated a small alteration in the standard equation used for poly(DXO-co-LLA) as follows:

11 +=+=DC

CC

CD

CCC I

I

I

IL (5.4) 11 +=+=

CD

DD

DC

DDD I

I

I

IL (5.5)

In the case of poly(LLA-co-CL) the carbonyl signals LLA-LLA-CL and CL-LLA-LLA overlap, yielding equations:

1+=CCL

CCCC I

IL (5.6) ( )

( ) 12/

2/ +++++=

CLLLLCCLC

CLLLLCLLLL III

IIIL (5.7)

171.0171.4171.8172.2172.6173.0173.4173.8 171.0171.4171.8172.2172.6173.0173.4173.8

CC

CD

DC

DDb)

171.0171.4171.8172.2172.6173.0173.4173.8 171.0171.4171.8172.2172.6173.0173.4173.8 171.0171.4171.8172.2172.6173.0173.4173.8

CC

CD

DC

DDb)

168.5169.0169.5170.0170.5171.0171.5172.0172.5173.0173.5174.0

168.5169.5170.5171.5172.5173.5

CCC

CCL

CLC

LLC+CLL

LLLc)

168.5169.0169.5170.0170.5171.0171.5172.0172.5173.0173.5174.0

168.5169.5170.5171.5172.5173.5 168.5169.5170.5171.5172.5173.5

CCC

CCL

CLC

LLC+CLL

LLLc)

169.2169.4169.6169.8170.0170.2170.4170.6170.8171.0171.2171.4169.2169.6170.0170.4170.8171.2

DDD

LDD

DDL LDLLLD

DLL

LLL

DLD

a)

169.2169.4169.6169.8170.0170.2170.4170.6170.8171.0171.2171.4169.2169.6170.0170.4170.8171.2 169.2169.6170.0170.4170.8171.2

DDD

LDD

DDL LDLLLD

DLL

LLL

DLD

a)


24

It is here shown, Table 5.2, that the monomer composition in the feed greatly affects the sequence lengths of the copolymer blocks. Similar results have been found by other groups, e. g. Choi et. al.94 who found LL values of 7.5, 3.7 and 3.4 when the feed ratios of LLA/CL were 80/20, 65/35 and 50/50 respectively at 140 ºC. It has previously also been shown that an increase in temperature increases the likelihood for transesterification reactions and affects the reactivity’s of the monomers, thereby decreasing the block lengths91. The block lengths presented here for a monomer feed ratio of LLA/CL 50/50 agree well with values previously reported95,96. Thermal Properties. The Tg, melting temperature and crystallinity of the copolymers were determined and compared using DSC, in order to establish how changes in composition alter their thermal properties, Table 5.3. Table 5.3. Thermal properties of poly(DXO-co-LLA), poly(LLA-co-CL) and poly(LLA-co-CL)

∆Hm [J/g] Polymer name Tg

a [˚C] Tm [˚C] LLA CL

Xc [%]

14D86L 23.2 142.6 22.6 - 24.3

23D77L 3 132 18.6 - 20.0

33D67L -5.2 - - - -

48D52L -20.3 - - - -

61D39L -26.1 - - - -

87D13L -34.7 - - - -

26D74C -60.5 (-61.6) 36.8 - 77.2 55.3

50D50C -57.1 (-54.7) 15.3 - 0.8 0.56

75D25C -48.5 (-47.1) - - - -

82C18L -51.7 43.9 - - -

54C46L -44.6 41.1 - - -

34C66L -28.3 - - - -

23C77L -9.1 - - - -

6C94L 28.1 154.2 - - -

100L 53.8 173.3 64.1 - 68.9

100C -68.7 57.5 - 81.0 58.1 a The number in parentheses is calculated by the Fox equation

A single glass transition could be observed in all thermograms located between the Tgs of the corresponding homopolymers indicating one continuous amorphous phase. In other words it was seen that the short sequence lengths in all copolymers are not long enough to induce two separate amorphous phases, Table 5.3 and Figure 5.2, as can be seen for lower conversions97.


25

For the random copolymers of poly(DXO-co-CL), the Tg values increase linearly with an increasing amount of DXO consistent with the Fox equation:

2

2

1

11

ggg T

w

T

w

T+= (5.8)

where w1 and w2 represent the weight fractions of the respective monomers and Tg1 and Tg2 the Tgs of homopolymers PDXO and PCL. As can be seen in Table 5.2 the experimental values are very close to the theoretical values. For the copolymers poly(DXO-co-LLA) and poly(LLA-co-CL), the Tg increases with increasing LLA content, although not linearly and the Fox equation does not apply. This non-linearity is because, at very high and very low ratios of one of the monomers, the Tg is not intermediate between those of the two homopolymers, but is instead increasingly affected by the monomer in abundance.

-80

-55

-30

-5

20

45

70

0 25 50 75 100% LLA

Tg[˚C]

-80

-70

-60

-50

-40

-30

0 25 50 75 100% DXO

Tg[˚C]

Figure 5.2. Tg measured by DSC a) as a function of the ratio of LLA for (○) poly(DXO-co-LLA) and (∆) poly(LLA-co-CL) b) as a function of DXO content for (□) poly(DXO-co-CL) and theoretical values calculated by the Fox equation is shown by the straight line. The crystallinity, Xc, was calculated according to96:

0f

fc H

HX

∆∆

= (5.9)

where ∆Hf (J/g of the crystalline polymer) is the enthalpy of fusion of the specimen and ∆H0

f is the enthalpy of fusion of a 100 % crystalline polymer. For PCL and PLLA ∆H0

f is 139.5 J/g98 and 93 J/g99 respectively.

a) b)


26

For copolymers of poly(DXO-co-LLA), the crystallinity depends on the block length of LLA, the crystallinity decreases with increasing DXO content which in turn yields shorter LLA blocks. When the proportion of the DXO exceeds 24 %, the copolymer becomes amorphous. For poly(DXO-co-CL), the degree of crystallinity depends on the length of CL sequences, which decreases with increasing DXO content. The CL sequences, however need not be as long as the LLA blocks to crystallize, Table DSC. Nevertheless, the crystallinity decreases to almost zero when the mole ratio of DXO is raised from 25 to 50 mole%. At DXO molar ratios above 50 % the copolymer is amorphous. In poly(LLA-co-CL), crystals are built either by LLA or CL depending on the composition. It was seen that when the molar ratio of CL was about 50 % or higher, the melting temperature of the crystals was closer to that of the crystals in the homopolymer, implying that the crystals formed are from the CL blocks. In contrast, when the LLA proportion was high, the melting temperature was close to that of pure PLLA and the LLA blocks are thus most likely responsible for the crystallinity. For both poly(DXO-co-CL) and poly(DXO-co-LLA), the Tm decreases with increasing DXO content, due to a shortening of the crystalline blocks and thus the formation of smaller and more imperfect crystals with a lower melting point.

5.1.3 Scaffold Preparation Technique

Tissue engineering is a research field often relying on three-dimensional porous structures fabricated and used as scaffolds in cell generation. The advantages of the porous structure include flow of nutrition’s and waste products through the structure and it enables the in growth of cells. The scaffolds are designed to mimic the extracellular matrix produced by human cells and to facilitate the initial cell attachment and subsequent tissue formation in vitro or in vivo. In this study, we use a solvent casting and porogen-leaching technique to construct porous scaffolds, when possible, of the three series of copolymers created. The choice of method was based on the fact that solution casting and particulate-leaching method is a versatile method that can be applied to materials with varying mechanical properties. It is well established that the polymer salt composite has a tendency to adhere to a glass mould, so that the samples must be formed after leaching. Methanol has here been used to solve this problem. Immersing the scaffold in methanol until it is totally soaked facilitates the separation of the scaffold from the mould, and enables the design of scaffolds with different shapes and forms to be studied without destroying or damaging the porous structure. This is because methanol has the ability to wet and slightly swell the polymers without leaching the salt. Unfortunately, all copolymers


27

cannot be used as scaffold materials. More specifically, for the copolymers of poly(DXO-co-LLA), a large loss of structure was seen when the amount of DXO reached 61 mol%. These copolymers first adhered to the mould, and after complete salt leaching, the scaffold structure collapsed completely. The same phenomenon was seen for the poly(DXO-co-CL), for which the problem occurred at even lower contents of DXO. More interestingly, 54C46L, a copolymer of poly(LLA-co-CL), lost it structure in a different manner. This copolymer was also first fixed to its mould, but it could easily be removed and leached where after it folded and fixed to itself. No explanation of this phenomenon can be found in the chemical structure of this particular copolymer, but the polymer obviously became too sticky. Table 5.4. Porous scaffold characteristics.

Polymer name Scaffold Stability

Salt leaching efficiency

[days]b

Pore size of largest occurrence pores [µm]c

Porosity [%]

14D86L + + 2 20-100 92.6 23D77L + + 3 20-140 93.4 33D67L + + 1 20-200 92.2 48D52L + 4 20-200 90.5 61D39L - 2 - -

87D13L - 2 - -

24D76C + 3 40-160 91.1 50D50C - 2 - - 75D25C - 2 - -

88C18L + + 4 20-200 91.5 54C46L - 2 - - 34C66L + 4 20-200 94.5 23C77L + + 2 20-120 94.6

6C94L + + 4 20-100 90.7

100L ± 2 20-160 89.2

100C + + 2 0-120 91.5 a The stability of the scaffold after salt leaching is denoted as: ++ no fragmentation and total form stability, + no fragmentation but adhere to mould, ± a few fragments comes off and form stability, - the scaffold form is not preserved b Determined by UV-VIS, and measured as the number of days after which the salt concentration was lower than 0.001 mg salt per ml water at all wavelengths c determined by SEM picture evaluation.

An ongoing discussion into the possible disadvantages of creating porous scaffolds using salt particulate leaching techniques has been concerned with whether or not the pores are interconnected and whether the regular shape of the created pores leads to problems in cell seeding and cell culture. It is here shown that the porous scaffolds manufactured by our technique, Figure 5.3, have interconnected pores, so that growth


28

throughout the scaffolds should be possible. It can also be seen that the scaffolds feature regular structures with homogeneously distributed pores and that the shape and structure of the pores are influenced by, but not completely determined by, the salt particles used. This can be seen as small variations in the shape of pores. The size distribution of the pores, Table 5.4, has been evaluated using image analysis, in which their diameter was plotted against their occurrence. It was seen that the ground salt gave rise to small variations in the pore diameter. Generally, the pore size is between 20 and 300 µm, the largest amount of pores being in the 20-160 µm range, and the porosity of the scaffolds was in the range of 89.2 to 94.6 % (equation 4.1). As can be seen in Figure 5.3 (examples of surface micrographs) the surface topography of the porous scaffolds show equivalent porosities and interconnectivities as the cross-sections. Figure 5.3. SEM micrographs of 33D67L, 24D77C and 23C77L

Cross section

Topography

TopographyCross section

Cross section

Topography

TopographyCross section

33D67L

24D76C

23C77L


29

Total salt leaching was determined by measuring the concentration of AgCl precipitate in the leaching media. Concentrations as low as 0.001 mg/ml can be detected and, as can be seen in Table 5.4, for most polymer scaffolds the concentration of precipitate in the water was lower than 0.001 mg/ml after two days of salt leaching. In all cases, no salt could be detected after four days of leaching. SEM micrographs showed no visible salt residues and gravimetric measurements confirmed total salt leaching. Table 5.5. Tensile properties of the porous scaffold.

Polymer name

Stress at max load

[MPa]

Strain at max load

[%]

Modulus [MPa]

14D86L 0.53 ± 0.2 11 ± 4.1 13 ± 0.55

23D77L 0.33 ± 0.03 58 ± 5.4 1.5 ± 0.21

33D67L 0.19 ± 0.02 109 ± 4.2 0.31 ± 0.05

48D52L 0.06 ± 0.01 113 ± 20 0.12 ± 0.01

24D76C 0.64 ± 0.20 15 ± 6.3 13 ± 2.2

82C18L 0.46 ± 0.04 53 ± 9.2 4.3 ± 0.65

34C66L 0.09 ± 0.01 176 ± 21 0.58 ± 0.49

23C77L 0.14 ± 0.04 87± 5.5 0.24 ± 0.05

6C94L 0.23 ± 0.04 4.5 ± 0.41 11 ± 2.4

100L 0.12 ± 0.04 1.4 ± 0.48 9.6 ± 0.48

100C 0.59 ± 0.08 9.6 ± 1.7 16 ± 2.4

Tensile testing. The porous scaffolds were investigated by tensile testing in order to determine how variations in the copolymer composition alter the mechanical properties of the materials. As mentioned above, the copolymers created here showed a single Tg indicating one amorphous phase, and in most cases, a crystalline region of one of the monomers. These soft and hard parts are intertwined through chain segments incorporated in both regions and thus create thermoplastic elastomeric like properties95.


30

0

0,2

0,4

0,6

0,8

0 25 50 75 100

% LLA

Str

ess

at m

ax lo

ad [

MP

a]

0

50

100

150

200

250

0 25 50 75 100

% LLA

Str

ess

at m

ax lo

ad [

%]

Figure 5.4. a) Maximum stress as a function of LLA content in (○) poly(DXO-co-LLA) and (∆) poly(LLA-co-CL) b) Maximum strain dependent a function of LLA content in (○) poly(DXO-co-LLA) and (∆) poly(LLA-co-CL). In poly(DXO-co-LLA), the hard domain would be made up of LLA moieties and it was seen that, when the proportion of LLA in the scaffolds was decreased, thereby increasing the amount of supple segments in the chains and decreasing the crystallinity, the material became more flexible with a resulting reduction in strength but an improvement in the maximum elongation, Figure 5.4. In the poly(DXO-co-CL), only one type of scaffold could be examined by tensile testing due to the collapse of the porous structure after leaching. This scaffold was hard and brittle with a low elongation and a high tensile strength compared to the poly(DXO-co-LLA) scaffolds with similar molar ratios of DXO in the copolymer. In comparison with pure PCL scaffolds, the strength is similar but the elongation at break and flexibility are higher, while the modulus is somewhat lower, Table 5.5. In general, the mechanical properties of porous scaffolds never reach those of a non-porous material since pores create voids which are similar to fractures. Scaffolds constructed of poly(LLA-co-CL) exhibited a more complex behavior with variation in the LLA content. When the proportion of LLA was low, the scaffolds were strong and fairly flexible. When the LLA content was increased up to 66 %, the strength decreased, whereas the flexibility increased considerably. However, when the LLA content was increased even more the flexibility again decreased. At 94 % LLA, the material was brittle and had tensile properties that are slightly enhanced, but which were almost identical to those of pure PLLA. In general, a PCL homopolymer has a higher tensile strength than a copolymer of poly(LLA-co-CL), Table 5.5 and Figure 5.4, because the crystals in the copolymer are disturbed by the incorporation of another monomer. This would contribute to the reduced flexibility of

a) b)


31

the homopolymer compared to most of the copolymers. The higher modulus of the copolymers compared to scaffolds of PLLA homopolymer is explained by cracks induced in the very brittle PLLA scaffolds by die cutting or it might merely be an artifact of the different characteristics of the polymers.

5.2 STAR-SHAPED POLYMERS

Star-shaped aliphatic polyesters portray physical properties quite different from their linear analogues and are therefore interesting to evaluate for usage in tissue engineering applications. Together with linear polyesters they offer us an large array of materials with properties that can be tailored for a desired application. To enable this customization, the polymerization kinetics must be well understood and control over the polymerization must be extensive.

5.2.1 Synthesis of the spirocyclic tin initiator

A spirocyclic tin initiator was successfully synthesized by allowing dibutyltin oxide and pentaerythritol ethoxylate (15/4 EO/OH) to react as described earlier76. The fifteen ethylene oxide units of pentaerythritol ethoxylate are divided between four arms, so the number of units on each arm is not equal, Figure 5.5. 1H NMR analysis of the initiator shows signals for the methylene protons in the ether chain at chemical shifts of 3.52 – 3.68 and the methylene groups from the four butyl tails at 0.91.


32

ppm (t1)1.02.03.04.05.0

t = 4 min

ppm (t1)1.02.03.04.05.0

t = 55 min

ppm (t1)1.02.03.04.05.0

t = 171 min

ppm (t1)1.02.03.04.05.0

t = 291 min

ppm (t1)1.02.03.04.05.0

t = 483 min

ppm (t1)1.02.03.04.05.0

t = 687 min

ppm (t1)1.02.03.04.05.0

t = 1286 min

ppm (t1)1.02.03.04.05.0

t = 3276 min

Figure 5.5. 1H NMR spectra of reaction mixture at different time intervals and peak assignment of PLLA initiated by a spirocyclic tin initiator at [M]0/[I] = 100.

5.2.2 Polymerizations

ROP of LLA initiated by a spirocyclic tin initiator was investigated in dichloromethane, chloroform, toluene and chlorobenzene. The reactions were performed at temperatures just below the boiling point of the solvent and, when possible, also at 40, 60, 80, 110 and 130 ºC, Table 5.6. Subsequently, a thorough investigation of the kinetic behavior of the initiator was performed to gain information on the function of the initiator in the ROP. Aliquots were withdrawn from the polymerizations at regular time intervals and analyzed by SEC and 1H NMR to monitor the conversion and the MWD.

Sn

OO

OO

O

O

O

O

Snm

n

o

p

O

O

O

O

*

OO

OO

* OO

OO

OH

OH

O

O

OOm

n

x

x

g’, h’, i’, j’

a

b

a’

b’

k’ l’

c de f

(m + n + o + p) = 15

Sn

OO

OO

O

O

O

O

Snm

n

o

p

O

O

O

O

*

OO

OO

* OO

OO

OH

OH

O

O

OOm

n

x

x

g’, h’, i’, j’

a

b

a’

b’

k’ l’

c de f

(m + n + o + p) = 15 5.17a’

0.91f

1.37e

1.51d

1.54c

1.58b’

1.68b

3.41k’

3.52j’

3.57i’

3.62h’

3.67g’

4.28l’

5.02a

δ (ppm)Proton

5.17a’

0.91f

1.37e

1.51d

1.54c

1.58b’

1.68b

3.41k’

3.52j’

3.57i’

3.62h’

3.67g’

4.28l’

5.02a

δ (ppm)Proton


33

0

20

40

60

80

0 750 1500 2250Reaction time [min]

Co

nve

rsio

n [

%]

0

20

40

60

80

100

0 1500 3000Reaction time [min]

Co

nve

rsio

n [

%]

0

20

40

60

80

100


Co

nve

rsio

n [

%]

0

20

40

60

80

100


Co

nve

rsio

n [

%]

Figure 5.6. Conversion as a function of reaction time for the polymerization of LLA with [M]0/[I] = 100 at (○) T = 40 ºC (□) T = 60 ºC (∆) T = 80 ºC (◊) T = 110 ºC (×) T 130 ºC in a) dichloromethane b) chloroform c) toluene d) chlorobenzene. The conversion curves show that the polymerizations were initiated instantaneously. They were thereafter allowed to proceed until the conversion curves reached their plateau values, yielding different reaction times, Figure 5.6 and Table 5.6. It was concluded by 1H NMR that all arms of the initiator participate in the reaction and it is assumed that the reaction proceeds equally on all arms. Figure 5.7 illustrates how the Mn increases linearly during the conversion and how the MWD is narrow through the entire interval i. e. under 1.1. These results are unique in the synthesis of star-shaped polymers and are comparable to results of dendrimer synthesis. Other groups, using systems with a core of pentaerythritol and SnOct2 as catalyst have found MWD in the vicinity of 1.2-1.3 which is here considered to be much higher.

a) b)

c) d)


34

0

4000

8000

12000

16000

0 25 50 75

Conversion [%]

Mn

[g

/mo

l]

1

1,2

1,4

MW

D

0

5000

10000

15000

20000

25000

0 25 50 75 100

Conversion [%]

Mn

[g

/mo

l]

1

1,2

1,4

MW

D

0

5000

10000

15000

20000

0 25 50 75 100

Conversion [%]

Mn

[g

/mo

l]

1

1,2

1,4

MW

D

0

5000

10000

15000

0 25 50 75 100Conversion [%]

Mn

[g

/mo

l]

1,00

1,20

1,40

MW

D

Figure 5.7. Relationship between the number-average molecular weight measured by SEC (●), molecular weight distribution (○) and the monomer conversion for the polymerization of LLA at 40 ºC and [M]0 = 0.5 in a) dichloromethane b) chloroform c) toluene and d) chlorobenzene. The molecular weights of the polymers during polymerization and after the conversion plateau was reached were determined both by SEC and by 1H NMR. In the SEC analysis, linear polystyrene standards were used to calibrate the system. It has previously been reported that these standards overestimate the molecular weights of linear polyesters by a factor of almost two92. It is also a well established fact that, since SEC discriminates the polymers on the basis of their hydrodynamic volume, the measured molecular weights of star-shaped polymers will therefore be lower than their linear analogues. The molecular weights based on SEC results presented in Table 5.6 are therefore a slight overestimation of the “true” molecular weights, but they are considered to be a good approximation of the molecular weights achieved and to be valid for comparing the different systems. It has also been shown that the MWDs of the individual arms of the star-shaped polymers can be broader than the values found for the complete star-shaped polymers. This effect was not however

a) b)

c) d)


35

investigated here, but it is probable that the MWDs are not the exact values. In three of the polymerizations, the reaction was not allowed to proceed to full conversion because of their extensive reaction times. The 1H NMR peaks of the initiator and the monomer/polymer all shift when polymerization occurs, as is seen in Figure 5.5. This is especially true of the methylene protons in the ethylene oxide unit connected to PLLA, where the two proton groups are shifted from the cluster of initiator protons at 3.59-3.71 (not shown) in the pure initiator, to 4.28 and 3.41 denoted l’ and k’ respectively, Figure 5.5. This downfield shift of the protons is commonly seen in these systems75,76, since an α-ester group is more electron-withdrawing than an α-ether group. The shift of the rest of the methylene protons in the ethylene oxide units remains almost unchanged after polymerization. 1H NMR was used to determine the absolute values of the molecular weights of the polymers by comparing the signals from the methine protons in PLLA and the methylene protons from the last repeating unit in the ethylene oxide, denoted a’ and k’ respectively. The molecular weights determined by 1H NMR are considered to be accurate in the low region of molecular weights, but their intensities decrease with increasing [M]/[I], due to the weakening of the initiator signal compared to the methine signal. Table 5.6. Ring-opening polymerization of LLA with [M]0/[I] = 100 and [M]0 = 0.5 M.

Solvent Temperature [ºC]

Reaction time [min]

Conversion [%]a

Mn b

[g/mol] MWD

b

Mn NMR c [g/mol]

Mn,theory d

[g/mol]

Dichloromethane 40 1955 64.7 11800 1.06 10700 9300 Chloroform 40 3394 84.1 21100 1.06 13600 12100 Chloroform 60 4218 97.7 25400 1.07 17900 14100

Toluene 40 1667 99.4 21100 1.09 15800 14300 Toluene 60 1595 97.3 23900 1.07 17300 14000 Toluene 80 548 99.2 16600 1.08 16200 14300 Toluene 110 188 96.0 19400 1.11 16100 13800

Chlorobenzene 40 542 70.1 14100 1.05 10100 10100 Chlorobenzene 60 321 99.3 19300 1.06 13700 14300 Chlorobenzene 80 191 96.7 21200 1.08 16000 14000 Chlorobenzene 110 104 94.2 20700 1.09 14700 13600 Chlorobenzene 130 57 90.1 22400 1.09 13800 13000 a Calculated from 1H NMR on crude reaction mixture. b Determined by SEC analysis. c Determined by 1H NMR d Theoretical average-number molecular weight, Mn = [M]/[I]·MLLA·conversion.


36

5.2.3 Temperature effect

The effect of temperature on the polymerization was investigated by comparing the reaction at different temperatures, while keeping all other reaction conditions unchanged. An increase in temperature usually reduces the induction period before the polymerization starts and yields polymers with higher MWDs. These systems do not however possess an induction period and a temperature effect is thus not seen in this sense. The MWD of the polymers is not affected by the increase in temperature, as is seen in Figure 5.8, indicating that the number of inter- and intramolecular transesterification reactions is not increased by raising the reaction temperature. If intermolecular transesterification reactions occurred frequently in the reaction, chains of different lengths would be created and the MWD would be increased. Similarly, backbiting reactions in the polymer chains would lead to polymers with varying arm lengths, giving polymers with different hydrodynamic volumes measured by SEC and thus a broader MWD.

1

1,05

1,1

1,15

1,2

0 25 50 75

Conversion [%]

MW

D

1

1,05

1,1

1,15

1,2

0 25 50 75 100

Conversion [%]

MW

D

1

1,05

1,1

1,15

1,2

0 25 50 75 100

Conversion [%]

MW

D

1,00

1,05

1,10

1,15

1,20

0 25 50 75 100Conversion [%]

MW

D

Figure 5.8. Relationship between the molecular weight distribution and the conversion for the

a) b)

c) d)


37

polymerization of LLA with [M]0 = 0.5 at (○) T = 40 ºC (□) T = 60 ºC (∆) T = 80 ºC (◊) T = 110 ºC (×) T 130 ºC measured by SEC in a) dichloromethane b) chloroform c) toluene d) chlorobenzene. We have previously reported for similar but linear systems that the temperature affects the Mn determined by SEC72. This effect was not seen in these systems, which all showed equivalent results for the polymerization at different temperatures both in SEC and by 1H NMR analysis, Figure 5.9. The difference in Mn is generally attributed to transesterification reactions and it is here concluded that the temperature does not influence the number of transesterification reactions in this system.

0

2000

4000

6000

8000

10000

12000

0 25 50 75 100

Conversion [%]

Mn

[g

/mo

l]

0

4000

8000

12000

16000

20000

0 25 50 75 100

Conversion [%]

Mn

[g

/mo

l]

0

4000

8000

12000

16000

20000

0 25 50 75 100

Conversion [%]

Mn

[g

/mo

l]

0

4000

8000

12000

16000

20000

0 25 50 75 100

Conversion [%]

Mn

[g

/mo

l]

Figure 5.9. Number average molecular weight as a function of LLA monomer conversion measured by 1H NMR for reactions with [M]0/[I] = 100 at (○) T = 40 ºC (□) T = 60 ºC (∆) T = 80 ºC (◊) T = 110 ºC (×) T 130 ºC in a) dichloromethane b) chloroform c) toluene and d) chlorobenzene.

5.2.4 Solvent effect

The four solvents, dichloromethane, chloroform, toluene and chlorobenzene, have different dielectric constants (see Table 5.7) and were used in order to investigate the

a) b)

c) d)


38

solvent effect on the polymerization rate, molecular weight and MWD for the different systems. The solvents in question are frequently used in the ROP of lactides. These solvents enable both the effect of solvent coordination to the active site of the polymerization, and the effect of the aromaticity and aromatic substituents of the solvent on the reaction rate to be investigated. Table 5.7. Boiling points, dielectric constants of the solvents and the half polymerization time100.

Solvent Boiling point [ºC]

D [40 ºC]

t½ at 40 ºC [min]

t½ at 60 ºC [min]

t½ at 80 ºC [min]

t½ at 110 ºC [min]

t½ at 130 ºC [min]

Dichloromethane 40 8.999 1644 - - - - Chloroform 61 4.803 1420 295 - - - Toluene 110 2.387 320 115 40 17 - Chlorobenzene 131 1.166 341 75 30 10 8

In Table 5.7, it is evident that the dielectric constant (D) of the solvent greatly influences the reaction rate, where the reaction time to reach half the desired molecular weight, the half polymerization time (t½), decreases with decreasing D. This trend applies in all cases except for the reactions at 40 ºC, where the polymerization proceeds faster in toluene than in chlorobenzene. The difference is however minor and is considered to be within the margin of error. The order of increasing reaction rate of the investigated solvents is thus dichloromethane < chloroform < toluene < chlorobenzene.


39

0

0,5

1

1,5

2

0 1000 2000 3000 4000Reaction time [min]

-ln

([M

]/[M

] 0)

0

1

2

3

4

0 500 1000 1500 2000Reaction time [min]

-ln

([M

]/[M

] 0)

0

0,5

1

1,5

2

2,5

3


-ln

([M

]/[M

] 0)

0

0,5

1

1,5

2

2,5

3


-ln

([M

]/[M

]0)

Figure 5.10. Semi-logarithmic plot of LLA monomer conversion versus reaction time with [M]0/[I] = 100 in (●) dichloromethane (■) chloroform (▲) toluene (♦) chlorobenzene at a) 40 ºC b) 60 ºC c) 110 ºC d) 130 ºC. In Figure 5.10 the semilogarithm of the instantaneous monomer concentration is (-ln([M]/[M]0)) is plotted versus the reaction time, where [M]0 is the initial LLA monomer concentration and [M] the unreacted LLA concentration at a given time t. The linearity of the plot shows that the propagation was first order with respect to lactide for all the systems and that no induction period is seen. At 40 ºC two groups of reactions are seen. Polymerization in the aromatic solvents proceeds much faster than in the other two, which is attributed to a lower degree of aggregation in these systems because of the larger solvent molecules. The difference in reaction time with the chlorinated solvents is considered to be an effect of their difference in dielectric constant (D). Similar results have been noted in bimetallic alkoxide systems101 and in aluminum alkoxide systems17.

a) b)

c) d)


40

The molecular weight of the synthesized polymers during the polymerizations was monitored by 1H NMR and SEC and it was observed that all polymers showed similar molecular weights at a given degree of conversion (Figure 5.11). It is therefore concluded that the polarity of the solvents in this study does not affect the molecular weights of the polymers. All plot of molecular weight versus conversions were linear, indicating that all reactions proceed through the same reaction mechanism. The MWDs of the systems were equivalent, showing that solvent does not influence the number of chains initiated.

0

5000

10000

15000

20000

25000

0 25 50 75 100Conversion [%]

Mn

[g

/mo

l]

0

5000

10000

15000

20000

25000

30000

0 25 50 75 100Converision [%]

Mn

[g

/mo

l]

0

5000

10000

15000

20000

25000

0 25 50 75 100Conversion [%]

Mn

[g

/mo

l]

0

5000

10000

15000

20000

25000

0 25 50 75 100

Conversion [%]

Mn

[g

/mo

l]

Figure 5.11. Number average molecular weight (Mn) as a function of LLA monomer conversion measured by SEC with [M]0/[I] = 100 in (●) dichloromethane (■) chloroform (▲) toluene (♦) chlorobenzene at a) 40 ºC b) 60 ºC c) 80 ºC d) 110 ºC. Since the solvent and temperature used affected only the reaction rate and not the molecular weight or MWD, the choice of solvent for the rest of the study was governed by the desire to use the most environmental-friendly system. All the subsequent studies were performed in toluene at 110 ºC.

a) b)

c) d)


41

5.2.5 Kinetics

Figure 5.12 shows the plot of –ln([M]/[M]0) versus the reaction time, t, for the polymerization of LLA in toluene at 110 ºC using the spirocyclic tin initiator. The polymerization was first order with respect to monomer. No induction period was observed and the linearity of this plot for all systems illustrates that no termination reactions occurred during the polymerization and thus that the number of propagating chains remained constant throughout the reaction. It has previously been shown by Teyssie et. al. that neither the solvent and temperature nor the metal and alkoxide groups used affected the linearity of these plots and they are thus first order in monomer101.

0

1

2

3

4

0 200 400 600 800

Reaction time [min]

-ln

([M

]/[M

] 0)

Figure 5.12. Semi logarithmic plot of monomer conversion versus reaction time for reactions in toluene with [M]0/[I] = 100 at (○) T = 40 ºC (□) T = 60 ºC (∆) T = 80 ºC (◊) T = 110 ºC. Table 5.8. Ring-opening polymerization of LLA in toluene with at 110 ºC with [M]0 = 0,5 M.

[M]0/[I] Reaction time [min]

Conversion [%]a

Mn b

[g/mol] MWDb Mn NMRc [g/mol]

Mn theoryd [g/mol]

50 49 95.2 10800 1.14 6900 6900

200 185 94.2 56800 1.19 34700 27100

400 257 95.2 97300 1.14 65500 54500 a Calculated from 1H NMR on crude reaction mixture. b Determined by SEC analysis. c Determined by 1H NMR. d Theoretical average-number molecular weight, Mn = [M]/[I]·MLLA·conversion.

Aggregation of the active center in the metal-alkoxide-initiated ROP of lactides and lactones is a commonly reported phenomenon that influences the reaction e. g. the control of the molecular weight101. The degree of aggregation has been shown to depend on the substituents on the metal atom, the solvent used and on added


42

complexing agents such as amines or alcohols. Commonly the reactivities of aggregated and non-aggregated species usually differ extensively; i. e. the kinetics of the system is affected. The propagation usually proceeds much faster via the non-aggregated species, and the aggregated species can be considered to be temporarily terminated101,102. The proposed kinetic schemes describing these systems are:

(5.10)

∗+

∗ ⎯→⎯+ 1nk

n PMP p (5.11)

where M, Pn* and (Pn*) denote the monomer, the non-aggregated center and the aggregated center respectively. m is the degree of aggregation, Kda is the aggregation equilibrium constant and kp is the propagation rate constant. A method of determining the degree of aggregation from the plot of ln(kapp) versus ln[I]0 was proposed by Penczek et. al, (where the apparent rate constant kapp = (ln[M]/[M]0)/t and [I]0 is the initial concentration)103. It is stated that if the polymerization is first order with respect to initiator the number of active sites is independent of the initiator concentration and the plot is linear, Table 5.8 and Figure 5.13.

0

1

2

3

0 2000 4000 6000Reaction time [s]

-ln

([M

]/[M

]0)

Figure 5.13. Semi logarithmic plot of monomer conversion versus reaction time for reactions in toluene at 110 ºC with [M]0 = 0,5 with (♦) [M]0/[I] = 49 (∆) [M]0/[I] = 201 (□) [M]0/[I] = 410. When the polymerization proceeds via reversible aggregation, however, the plot is often curved, due to a change in the proportions of aggregated and non-aggregated species. This effect was not seen here and the relationship between aggregation and non-aggregation is considered to be uniform throughout the reaction, showing that the formation of polymer does not influence the reaction rate.

∗nmP( ) daK

mnP ∗ ∗nmP( ) daK

mnP ∗


43

For these systems and for polymerizations in which a fast and reversible aggregation of the active centers takes place, the following equation is proposed:

( ) ( ) [ ] ( ) mappp

mpda

mapp kIkkKmk −−− +−= 0

11 / (5.12)

The apparent rate constant was determined from the slope of the linear relationship in Figure 5.13, i. e. kapp = -(ln([M]/[M]0)t, for different initiator concentrations [I]o. To determine the reciprocal of the external order in initiator, i. e. the degree of aggregation of the active species, ln(kapp) was plotted versus ln([I]0) as in Figure 5.13. The slope, m, gives a degree of aggregation of about 4/5 throughout the entire investigated interval ([I]0 ≈ 1-10 mmol L-1). This value is comparable to the values found by our group previously for a similar but linear system, were m was found to decrease from 4/3 at [I]0 ≈ 5 mmol L-1 and higher to 1/2 at [I]0 ≈ 2 mmol L-1 and lower104. It is commonly seen that this type of plot shows two different steps attributed to a variation in the ratio of aggregated and non-aggregated species with varying initiator concentration. No such change is seen here, showing that there is no difference between aggregation and non-aggregation with different initiator concentrations in the investigated interval.

-8

-7,5

-7

-6,5

-6

-5,5

-7 -6 -5 -4ln([I]0) [mol x L-1]

ln(k

app)

[s

-1]

Figure 5.14. External orders in initiator. The polymerizations of LLA were conducted at 110 ºC with [M]0 = 0,5 M in toluene.


44

Table 5.9. Molecular weight for the polymerization of LLA in toluene at 110 ºC and [M]0 = 0,5 M.

[M]0/[I]a Reaction time [min]

Conversion [%]a

Mn b

[g/mol] MWDb Mn NMRc Mn theoryd

[g/mol] 50 50 94.8 16200 1.13 7200 6800 100 100 93.8 27500 1.14 14400 13500 200 185 94.7 57000 1.12 34900 27300 340 260 94.8 96300 1.19 81100 54600

a Molar feed ratio b Determined by SEC analysis. , c determined by 1H NMR d Theoretical average-number molecular weight, Mn = [M]/[I]·MLLA·conversion.

5.2.6 Thermal properties

To investigate the effect which four arms have on the thermal properties of the polymers, DSC characterization was performed. The results are given in Table 5.9 and Table 5.10. It is here seen that the Tg and the melting temperature of the star-shaped polymers increase with increasing arm length, as expected. The melting temperature did not reach the value for linear PLLA of the same total degree of polymerization, suggesting that smaller and less perfect crystals76,80,105 were created. This was confirmed by the fact that the maximum melting temperature of the second scan was also lower than that of the first heating scan. The formation of smaller and less perfect crystals is attributed to the increase in the number of end groups that can disrupt the crystal formation and also to the more complex three-dimensional structure. The degree of crystallinity increases with increasing arm length since it is possible for the polymers to create larger crystals because of their longer arms. Table 5.10. Thermal properties of the star-shaped polymers.

[M]0/[I]a Tg [ºC] Tm [ºC] ∆Hm [J/g] Xc [%]b

50 16.9 124.6 6.7 7.26 100 23.4 136.1 38.6 41.49 200 25.4 145.7 46.4 49.87 340 37.8 155.4 59.6 64.10

a Molar feed ratio b The crystallinity was calculated according to Xc = ∆Hf/∆Hf0, where ∆Hf

0 = 93 J/g 98

————— Conclusions —————

45

6 CONCLUSIONS

Using SnOct2, a series of copolymers with various combinations of DXO, LLA and CL was synthesized by ROP. The composition of the copolymer can be chosen in such a manner that the mechanical and thermal properties of the materials can be predetermined. A new solvent-casting and particulate leaching scaffold preparation technique was developed and used to create three-dimensional structures with interconnected pores. It is believed that the large range of properties made available by these copolymers show that it is possible to fabricate scaffolds to meet a range of different applications within the body. Star-shaped polyesters were successfully synthesized with monomer/initiator ratios that parallel the molecular weight and narrow MWD using a spirocyclic tin initiator. The temperature and the solvent used do not influence the achieved molecular weight nor the MWD. An increase in temperature or a decrease in dielectric constant of the solvent increases the reaction rate considerably, and the reaction rate increased in the order dichloromethane < chloroform < toluene < chlorobenzene.

————— Future work —————

46

7 FUTURE WORK

We have investigated the polymerization kinetics of a spirocyclic tin initiator with LLA and shown that four-armed star-shaped homopolymers can be synthesized in toluene at 110 ºC. An investigation of the effect of the number of arms on the morphology, mechanical properties and degradation rate is the next logical step. Since the materials are very monodisperse and thereby quite unique, the influence of the end-groups can be investigated. The star-shape of the polymers should also yield interesting properties in drug delivery applications compared with their linear analogues. Finding a material with the appropriate degradation characteristics and mechanical properties for skin regeneration should also be possible by copolymerizing LLA, CL, DXO and TMC. Hydrogels are three-dimensional water-swellable networks which absorb and retain water without dissolving. Generally, hydrogels are preferred in medical applications because of their similarity to natural living tissue. It would therefore be interesting to create hydrogels using various cross-linking chemistries with linear and star-shaped prepolymers and when necessary incorporating macro pores into the system to further enhance the usefulness of the hydrogels. We have an ongoing collaboration with Prof. Gunnar Kratz at Linköpings University hospital and Prof. Urban Lindgren at Huddinge University hospital to investigate the materials’ interactions with cells in vitro. As described above, aliphatic polyesters are often used in tissue engineering and it is well known that their degradation rate and interactions with tissue is beneficiary for tissue regeneration. It has, however, been shown that the materials’ in vitro characteristics do not always portray the interactions the materials have in vivo. We therefore wish to investigate the materials in vivo interactions in vivo directly, focusing on the samples’ mechanical characteristics, cross-linking density and surface properties.

————— Acknowledgements —————

47

8 ACKNOWLEDGEMENTS

First of all I would like to tank my supervisor Ann-Christine Albertsson for accepting me as her PhD student and for encouraging me and supporting me throughout my work. Thank you for your scientific guidance and for giving me the opportunity to attend conferences and thereby meeting interesting scientists from all over the world. The Foundation for Strategic Research is thanked for financial support of this work. I would also like to thank all senior scientists at our department for making it such a nice place to work at. All members of the administrative staff are also thanked for their endless patience and help. I thank all PhD students who are or have been at our department for contributing to the good atmosphere here. Finally I would like to thank my friends, at work and outside, and my family for everything. - Thank you!

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