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Degradable Porous Scaffolds from Various L-Lactide and Trimethylene Carbonate Copolymers Obtained by a Simple and Effective Method Therese Tyson, Anna Finne-Wistrand, and Ann-Christine Albertsson* Fibre and Polymer Technology, School of Chemical Science and Engineering, Royal Institute of Technology, SE-100 44, Stockholm, Sweden Received September 18, 2008; Revised Manuscript Received October 30, 2008
A simple and effective method of fabricating scaffolds with open pore structures was successfully used on several copolymers. The method, which is straightforward and fast, was developed to overcome problems such as low pore interconnectivity and to achieve thick three-dimensional scaffolds. Copolymers are of particular interest because it is possible to tune their mechanical and degradable properties, and in this work, copolymers of L-lactide (LLA) and trimethylene carbonate (TMC) were synthesized through ring-opening polymerization. The copolymers formed had molecular weights ranging from close to 60000 g/mol to over 300000 g/mol and they were composed of 12-55 molar percentages of TMC and 88-45 molar percentages of LLA. The synthesized copolymers were evaluated as scaffold materials using a combined phase separation and particulate leaching technique, in which sugar templates were used as the leachable porosifiers. Differences in molecular weights, molar compositions, and degrees of crystallinity were all factors that influenced the properties of the prepared scaffolds. The copolymers with high LLA contents and high degrees of crystallinity were best suited for the scaffold fabrication technique used and gave degradable scaffolds with interconnected pores.
Introduction Biodegradable porous scaffolds have to fulfill several functions to be able to work effectively in a tissue engineering application, and this makes demands on both the polymeric material used and on the design of the porous structure. The material has to have the desirable mechanical properties and the desired degradation profile, while being biocompatible and nontoxic and having appropriate surface properties for cell attachment, proliferation, and differentiation. The threedimensional scaffold should also be able to permit cell growth inside its porous structure and support the growing tissue. To achieve this, the scaffold should have a high degree of interconnected pores of the right size. The scaffold must allow vascularization for a sufficient transport of nutrients and waste products to and from the cells which are in charge of rebuilding the new tissue. Poly(L-lactide) (PLLA) is one of the most commonly used polymers in biomedical applications such as porous scaffolds, because it is biocompatible and degrades in vivo to lactic acid, which is in turn metabolized in the body. PLLA is however highly crystalline and rigid at body temperature, and L-lactide (LLA) has, therefore, been copolymerized with several other monomers to obtain more flexible materials. Another advantage of incorporating different monomers into the PLLA chain is the tuneable degradation profile.1,2 Our group has previously functionalized PLLA3 and copolymerized LLA with -caprolactone (CL), 1,5-dioxepan-2-one (DXO), and trimethylene carbonate (TMC).1,4–7 Recently, the synthesis of elastic A-B-A type block copolymers with a soft amorphous middle block consisting of TMC and DXO and two hard semicrystalline PLLA terminal blocks has also been reported.4 These materials were synthesized using the cyclic five-membered tin alkoxide * To whom correspondence should be addressed. Tel.: +46-8-790 82 74. Fax: +46-8-208477. E-mail:
[email protected].
initiator 1-di-n-butyl-1-stanna-2,5-dioxacyclopentane. They all showed highly elastic behavior and have potential applications in soft tissue engineering.4 In the present work, we have used different copolymers of LLA and TMC in the fabrication of porous scaffolds. Porous scaffolds can be prepared using a range of different methods, for example, solvent casting and particulate leaching,8 gas foaming,9 and phase separation.10 Solvent casting and particulate leaching is a commonly used method which is simple and permits a sufficient control of the pore size and porosity through the particle size and the particle/polymer ratio. There are, however, some drawbacks with this method, including limitations in pore interconnectivity, difficulties in preparing thick three-dimensional scaffolds, the formation of a solid surface side and a slow salt leaching. In an attempt to overcome these problems, three-dimensional porous scaffolds have in this work been fabricated using a combined phase separation and particulate leaching technique, in which sugar templates were used as the leachable porosifiers. Preparations of porous scaffolds using sugar or sugar templates in different modified particulate leaching techniques have previously been reported in the literature and have resulted in scaffolds with high porosities and well interconnected pore networks.11–14 In this work, copolymers with different LLA and TMC contents and molecular weights were synthesized using either stannous octoate (Sn(Oct)2) and ethylene glycol or the five-membered cyclic tin alkoxide initiator 1-di-n-butyl-1-stanna-2,5-dioxacyclopentane, and the copolymers were evaluated as potential scaffold materials. Both of these initiator systems are of interest because we have shown that it is possible to synthesize materials with low residual amounts of tin using both Sn(Oct)2 and the cyclic tin alkoxide initiator.15,16 The aim of this work was to develop a simple and fast method to prepare interconnected porous scaffolds for tuneable degradable polymers. Our hypothesis was that factors such as the molecular weight, the thermal properties,
10.1021/bm801052m CCC: $40.75 2009 American Chemical Society Published on Web 12/08/2008
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Scheme 1. Two Monomers Used in the Copolymerization Reactions: L-Lactide (LLA) and Trimethylene Carbonate (TMC)
Scheme 2. Example of a Poly(LLA-co-TMC) Copolymera Figure 1. Procedure for making porous scaffolds.
a Synthesized using either (i) Sn(Oct)2 together with ethylene glycol or (ii) 1-di-n-butyl-1-stanna-2,5-dioxacyclopentane.
and the degree of crystallinity affect the quality of the fabricated scaffolds and need to be optimized to achieve a useful scaffold for tissue engineering.
Experimental Section Materials. L-Lactide (LLA) was purchased from Sigma-Aldrich, Sweden, and was purified by recrystallization in dry toluene (dried over Na-wire) three times and dried under reduced pressure at room temperature for 24 h. Trimethylene carbonate (TMC) was kindly supplied by Radi Medical Systems AB, Sweden, and was used as received. The chemical structures of the two monomers LLA and TMC are shown in Scheme 1. Stannous octoate (Sn(Oct)2) was purchased from Sigma-Aldrich, Sweden, and dried over molecular sieves before use. 1-di-n-butyl-1-stanna-2,5-dioxacyclopentane was synthesized previously by our research group, as described in the literature.17,18 Chlorobenzene (VWR) was dried over P4O10 (VWR) and distilled at reduced pressure in an argon atmosphere before use. N-Methyl-2pyrrolidone (NMP; Merck), hexane (Fisher Chemicals), methanol (Scharlau), diethyl ether (Prolabo), and chloroform (Fischer Chemicals) were used as received. Polymerizations. In this work, six different poly(L-lactide-cotrimethylene carbonate) (poly(LLA-co-TMC)) copolymers with different LLA and TMC contents and molecular weights were synthesized. The chemical structure of these materials is shown in Scheme 2. Two types of copolymerization were carried out: (i) five bulk polymerizations in which Sn(Oct)2 and ethylene glycol were used and (ii) one polymerization in a concentrated chlorobenzene solution (5 M with respect to each monomer concentration) in which the cyclic tin alkoxide 1-di-nbutyl-1-stanna-2,5-dioxacyclopentane was used as initiator. In the five Sn(Oct)2 polymerizations, the desired amounts of TMC, Sn(Oct)2, and ethylene glycol were weighed into previously silanized two- or three-necked flasks equipped with magnetic stirrers, and LLA was weighed into regular round-bottomed flasks, all under a nitrogen atmosphere inside a glovebox (MBraun MB 150B-G-I, Germany). The polymerizations were started by immersing the two- or three-necked reaction vessel in 140 °C thermostatted oil baths. TMC was allowed to polymerize until a monomer conversion of at least 80% was reached, which was determined by proton nuclear magnetic resonance (1H NMR) spectroscopy. LLA was then added to the reaction vessels under an argon atmosphere. In four out of five cases, the temperature was kept at 140 °C also during the second reaction step, but in one case, the temperature was raised to 180 °C after LLA had been added. Each polymerization was terminated after a total reaction time of 72 h. The polymers formed were dissolved in chloroform and precipitated in a mixture of cold hexane and methanol (95:5).
The copolymerization performed using the cyclic tin alkoxide initiator has previously been described in more detail.4 Briefly, the desired amount of TMC and the cyclic tin alkoxide initiator were weighed into a previously silanized three-necked flask and LLA was weighed into a regular round-bottomed flask, both under a nitrogen atmosphere inside a glovebox. Previously distilled chlorobenzene was then transferred to the three-necked reaction vessel with a flame-dried syringe through a rubber septum under an inert atmosphere. The reaction vessel was connected to a mechanical stirrer and the polymerization was started by the immersion of the three-necked reaction vessel in an 80 °C thermostatted oil bath. TMC was allowed to polymerize until almost complete conversion, which was determined by 1H NMR analysis. LLA was then added to the reaction vessel under an argon atmosphere together with distilled chlorobenzene, which was transferred as described earlier. The temperature in the thermostatted oil bath was kept at 80 °C also during the second reaction step and the polymerization was allowed to proceed until almost complete monomer conversion had taken place. The formed polymer was dissolved in chloroform and precipitated in cold diethyl ether. Preparation of Sugar Templates. Sugar templates were prepared from commercially available granulated sugar (Dan Sukker), which was moistened with 2% (w/w) deionized water and closely packed in circular plastic molds. The sugar was left in the molds until dry, after which the sugar templates formed were carefully removed from the molds and used to model interconnected porous scaffolds. The circular sugar templates had a thickness of 3 mm and a diameter of 12 mm. Preparation of Porous Scaffolds. Porous scaffolds were prepared using a simple and effective phase separation and particulate leaching technique in which the previously prepared sugar templates were used as the leachable porosifiers. The polymers that were used to make the porous scaffolds were the six synthesized poly(LLA-co-TMC) copolymers with different LLA and TMC contents and molecular weights (chemical structure shown in Scheme 2). Figure 1 shows the scaffold fabrication method used in this work. The copolymers were dissolved in NMP at 100 °C with a polymer concentration of 5-15% (w/w). The polymer solutions were then dropped onto the sugar templates (1) so that all the spaces within the sugar were filled (2). Excess polymer solution was removed from the sugar templates using tissue paper and the sugar structures filled with polymer solution were then placed at the bottom of large beakers filled with deionized water of different temperatures (3). The sugar structures were left in the beakers for a couple of hours while the polymer was allowed to phase separate and the sugar was slowly dissolved. During this time, porous scaffolds were created and rose to the water surface (4). The deionized water in each beaker was then changed and the scaffolds were left in the new water overnight to ensure adequate sugar leaching. The porous scaffolds were thereafter left to dry in a fume cupboard. Nuclear Magnetic Resonance (NMR). The degree of monomer conversion during and after the polymerizations and the chemical compositions of the final copolymers were determined by 1H NMR by comparing the relative intensities of the different monomer and polymer peaks. Carbon nuclear magnetic resonance (13C NMR) spectroscopy was used to investigate the microstructure of the copolymers. All NMR spectra were recorded with a Bruker Avance 400 NMR instrument equipped with a BBFO probe, z-gradient and ATM unit (autotuning
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Table 1. Six Synthesized Poly(LLA-co-TMC) Copolymers polymer composition (%)a
thermal propertiesc
polymer
LLA
TMC
Mn (g/mol)b
1 2 3 4 5 6
65 88 43 88 85 45
35 12 57 12 15 55
80000 314100 56500 84200 68400 74000
PDIb
Tg1 °C
Tg2 °C
Tm1 °C
1.5 1.3 1.4 1.3 1.3 1.4
-21.5
26.0 50.2 18.6 39.6 41.8 49.0
153.2 174.4 149.4 163.8 156.3 164.0
-22.7 -11.6
Tm2 °C
150.8 156.7
degree of crystallinity (%) 16.6 28.7 9.5 34.6 16.5 10.3
The molar composition of the copolymers determined by 1H NMR at δPLLA ) 5.17 ppm and δPTMC ) 2.03 ppm. b The number average molecular weight (Mn) and the polydispersity index (PDI) determined by SEC, using narrow polystyrene standards. c The glass transition temperature (Tg), melting temperature (Tm), and degree of crystallinity, determined by DSC during the second heating. For three of the synthesized copolymers, two glass transitions were observed, and for two copolymers, a bimodal melting peak was found. a
and autoshimming). The instrument was operating at 400.13 and 100.62 MHz for 1H NMR and 13C NMR, respectively. Standard Bruker pulse programs were used and the experiments were carried out using ICONNMR software. Deuterated chloroform (CDCl3) was used as solvent and nondeuterated chloroform was used as an internal standard (δ ) 7.26 ppm for 1H NMR and δ ) 77.0 ppm for 13C NMR). Size Exclusion Chromatography (SEC). The molecular weights and the polydispersity index (PDI) of the synthesized copolymers were determined using SEC. A Waters 717 plus autosampler and a Waters model 515 apparatus equipped with three PL gel 10 µm mixed B columns, 300 × 7.5 mm (Polymer Laboratories, U.K.) connected to an IBM-compatible PC were used. Spectra were recorded with a PLELS 1000 evaporative light scattering detector (Polymer Laboratories, U.K.). Millenium 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 and polystyrene standards with narrow molecular weight distributions were used for calibration. Differential Scanning Calorimetry (DSC). The thermal properties of the synthesized copolymers were investigated using DSC. A Mettler Toledo DSC 820 module was used under a nitrogen atmosphere with a nitrogen flow of 80 mL/min and a heating/cooling rate of 10 °C/min. The samples were first heated from 25 to 200 °C and then cooled to -40 °C to erase the thermal history of the polymers. The samples were then heated again from -40 to 200 °C and it was this second heating scan that was used to evaluate the thermal properties. Scanning Electron Microscopy (SEM). The porosity and the surface characteristics of the porous scaffolds were evaluated using a Hitachi S-4300 FE-SEM scanning microscope. The samples were cut and mounted on metal studs and sputter-coated with gold-palladium (Agar HR Sputter Coater) for 45 s. SEM micrographs of one scaffold from each polymer are presented in this paper. Each synthesized copolymer was used to make a large number of scaffolds, but little or no visual differences were found in the scaffolds prepared from the same material. Determination of Porosity and Pore Size Distribution. The porosity, the pore volume and the pore size distribution of the porous scaffolds made from one of the synthesized poly(LLA-co-TMC) copolymers were studied at the Swerea IVF Keraminstitutet in Mo¨lndal, Sweden. The porosity was determined using a water penetration technique in accordance with SS-EN-623-2 and the pore volume and the pore size distribution were determined using a mercury porosimeter (Micromeritics AutoPore III 9410).
Results and Discussion Synthesized Copolymers. Six different poly(L-lactide-cotrimethylene carbonate) (poly(LLA-co-TMC)) copolymers were synthesized and their chemical structure is shown in Scheme 2. Five of these polymerizations were performed in bulk with stannous octoate (Sn(Oct)2) and ethylene glycol, and the sixth polymerization was performed in a concentrated chlorobenzene solution with the cyclic tin alkoxide initiator 1-di-n-butyl-1stanna-2,5-dioxacyclopentane. The Sn(Oct)2 polymerizations are
for the sake of simplicity hereafter referred to as polymerizations 1-5 and the cyclic tin alkoxide initiated polymerization is referred to as polymerization 6. In polymerizations 1-5, the theoretical Sn(Oct)2/monomer molar ratios were 1:20000 and ethylene glycol was added to give a theoretical molecular weight of 80 000 g/mol. In polymerization 6, the theoretical degree of polymerization (DP) of the poly(trimethylene carbonate) (PTMC) middle block was 400 and the two poly(L-lactide) (PLLA) side blocks each had a theoretical DP of 100, giving a total theoretical molecular weight of almost 70000 g/mol. In all six polymerizations, LLA was added to the reaction vessel in a second reaction step after the first monomer TMC had reached a monomer conversion of at least 80%. LLA was added before complete TMC conversion so that the effect of TMC units in the PLLA blocks could be studied. If TMC units are allowed to react into the PLLA blocks, it is expected that the thermal properties and the degrees of crystallinity will be affected, and the influences of these properties on the fabricated scaffolds were studied. When polymers 1 and 4 were synthesized, the TMC conversion had reached between 84 and 86% when LLA was added, whereas in the other polymerizations, TMC conversion had reached over 96% at this stage. The total reaction time of polymerizations 1-5 was 72 h. Polymerization 6 was allowed to react for just over 10 h, when both monomers had reached almost full conversion. The data for the six polymerizations are summarized in Table 1. Table 1 shows that six polymers of different compositions and different molecular weights were successfully synthesized. The deviations from the theoretical molecular weights are explained by the fact the polymerizations were terminated before full conversion and polystyrene standards were used for calibration in the size exclusion chromatography (SEC) analysis. The chemical compositions of the synthesized copolymers were determined from proton nuclear magnetic resonance spectroscopy (1H NMR) and the relative intensities of two polymer peaks at δPLLA ) 5.17 ppm and δPTMC ) 2.03 ppm. The molar percentage of TMC in the synthesized polymers varied between 12 and 57% and LLA made up the rest. Carbon nuclear magnetic resonance spectroscopy (13C NMR) was used to study the microstructure of the copolymers. The carbonyl region of the spectra showed sharp single peaks around 169.7 ppm corresponding to the LLA blocks and sharp single peaks around 154.8 ppm corresponding to the TMC blocks. The relative intensities of these two peaks corresponded well with the molar percentages of each monomer determined by 1H NMR. Small peaks were observed around 170.1 and 154.5 ppm, corresponding to the linking groups between the two blocks.19–21 The intensity of these peaks was about 10% of that of the LLA block peak. The thermal properties, that is, the melting temperature (Tm), the glass transition temperature (Tg), and the degree of crystal-
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linity, of the synthesized copolymers were investigated using differential scanning calorimetry (DSC). The heating-coolingheating cycle was set to range from -40 to 200 °C. The thermal properties of the synthesized copolymers were assumed to be detectable within this temperature interval, since the homopolymer poly(L-lactide) (PLLA) has a Tm and a Tg of about 170-180 °C and 55-60 °C, respectively, and the homopolymer poly(trimethylene carbonate) (PTMC) has a Tg of about -17 °C. Tm was determined from the DSC curves as the temperature at the maximum of the melting peak, and Tg was determined as the midpoint temperature of the glass transition. The approximate crystallinities were calculated according to:
wc )
∆Hf ∆H0f
× 100
(1)
where ∆Hf is the heat of fusion of the sample and ∆Hf0 is the heat of fusion of 100% crystalline PLLA. When evaluating the degrees of crystallinity of the synthesized copolymers, it was assumed that the only contribution to the heat of fusion was from semicrystalline PLLA segments. The value of ∆Hf0 used in the calculations was 93 J/g.22 As seen in Table 1, the two polymers with the highest LLA contents, that is, polymers 2 and 4 containing 88 molar % LLA, had the highest degrees of crystallinity, 29 and 35%, respectively. Polymers 1 and 5 had similar crystallinities of about 16% and their LLA contents were 65 and 85 molar %, respectively. The two polymers with LLA contents below 50 molar %, that is, polymers 3 and 6, had degrees of crystallinity of about 10%. As previously mentioned, TMC conversion of 84-86% was reached in polymerizations 1 and 4 before LLA was added to the reaction vessel. Nonreacted TMC monomers were expected to react into the PLLA blocks, decreasing the degree of crystallinity, but this effect on the degree of crystallinity cannot be established, as seen in Table 1. Polymer 6 was synthesized using the cyclic tin alkoxide initiator 1-di-n-butyl-1-stanna-2,5-dioxacyclopentane as described in an earlier publication from our group, and the properties of polymer 6 were comparable to the previously reported results.4 The melting peaks of both polymers 4 and 6 were bimodal, with a second peak at 150.8 and 156.7 °C, respectively. Polymers 1, 3, and 6, which had the three lowest LLA contents, had two observable glass transition temperatures, unlike the other polymerizations. The first Tg corresponding to the PTMC block was between -22.7 and -11.6 °C and the second Tg corresponding to the PLLA blocks was between 18.6 and 49.0 °C. Porous Scaffolds. As described in the Experimental Section, porous scaffolds were prepared using a simple and effective phase separation and particulate leaching procedure in which circular sugar templates were used as the leachable porosifiers. The six synthesized poly(LLA-co-TMC) copolymers with different LLA and TMC contents and molecular weights, described in Table 1, were evaluated as potential scaffold materials with regard to their different molecular weights, TMC contents, and methods of synthesis. The polymers were dissolved under heating in N-methyl-2pyrrolidone (NMP). In all cases but one, the polymer concentration in NMP was 15% (w/w). When polymer 2 was used, this concentration had to be adjusted to 5% (w/w) due to a much higher polymer solution viscosity. As can be seen in Table 1, polymer 2 had a much higher molecular weight than the other polymers. If the polymer-NMP solution was too viscous, it
Figure 2. SEM micrographs of the inside of porous scaffolds fabricated from (a) polymer 2, (b) polymer 4, and (c) polymer 5.
simply stayed on top of the sugar template and was not able to fill the inside of the sugar template as required for the method to be used. After the sugar templates had been filled with the heated polymer solutions, they were placed in beakers with deionized water of three different temperatures: room temperature (RT), 40, and 60 °C. When comparing the different fabricated scaffolds, it was clear that RT in almost all cases resulted in scaffolds that were ragged or had sunken, especially toward the middle. An explanation of this behavior is the lower degree of crystallization that should arise when the polymer solution, heated to 100 °C, is quickly brought down to the lower temperature of the water. No difference between 40 and 60 °C was evident. The Tg values of the synthesized copolymers were in some cases above 40 °C, but no significant differences in the fabricated scaffolds due to the difference in water temperatures could be observed. Comparing Different Molecular Weights. Polymers 2, 4, and 5 are copolymers with a very similar composition, as shown in Table 1. The TMC content in these three polymers varied between 12 and 15%, giving LLA contents of 88-85%. The molecular weights of polymers 4 and 5, however, differed greatly from the much higher molecular weight of polymer 2. This was shown by SEC, but was also noticeable during the scaffold fabrication because the polymer 2 solution was much more viscous than the other polymer solutions and the concentration of polymer 2 in NMP had to be decreased from 15 to 5% (w/w). Polymers 4 and 5 both gave porous scaffolds that felt hard and durable upon handling due to the high LLA content in these polymers. Poly(L-lactide) (PLLA) is known to have both a high crystallinity and a high rigidity. The fabricated porous scaffolds were analyzed using scanning electron microscopy (SEM), as described in the Experimental Section, and some of these results are shown in Figure 2. The SEM micrographs in Figure 2 show that highly porous scaffolds with interconnected pores were successfully fabricated using both polymer 4 and polymer 5.
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Polymer 2 has a molar composition similar to that of polymers 4 and 5 as well as a high molecular weight, which is often expected to result in good mechanical properties. However, this polymer was not as well suited to the scaffold fabrication method used as the other two. Polymer 2 gave porous scaffolds which felt much softer upon handling than those made from polymers 4 and 5 and, as shown in the SEM micrographs in Figure 2, the scaffold walls in this scaffold were thin and thready. This is due to the lower polymer concentration in NMP. A polymer with too high a molecular weight results in a polymer-NMP solution that is too viscous for the method to be used. At the same time, too low a polymer concentration seems to result in thin scaffold walls that are not able to support the scaffold during handling and cutting, and this would most certainly also cause problems during possible insertion of the scaffold in vivo. The results presented in Figure 2 show that it is possible to successfully use poly(LLA-co-TMC) copolymers composed of about 12-15% TMC units and with molecular weights of around 70000-80000 g/mol in the fabrication of interconnected porous scaffolds. A high molecular weight is often desirable in many polymer applications due to the mechanical properties of the polymer, but this is not necessarily an adequate requirement when using this method to fabricate porous scaffolds. Attention must also be paid to on the concentration of the polymer solution during fabrication since this also affects the mechanical properties of the scaffold. Comparing Different TMC Contents. As mentioned above, polymers 4 and 5 are copolymers with very similar compositions, and the same can be said of polymers 3 and 6. Polymers 4 and 5 have low TMC contents of 12-15%, while polymers 3 and 6 have higher TMC contents of 55-57%. In between these polymer sets is polymer 1, which was synthesized with a TMC content of 35%. Polymers 4 and 5 both gave porous scaffolds that felt hard and durable upon handling due to their high LLA contents. The SEM micrographs in Figure 2 show that porous scaffolds with interconnected pores were successfully fabricated using both polymer 4 and polymer 5. The SEM micrographs of the porous scaffolds prepared from polymers 3 and 6 are shown in Figure 3. Both of these polymers contained more than 50% TMC, and this was expected to bring more softness and flexibility to the materials.4 These materials gave scaffolds that felt both soft and flexible upon handling, but the scaffold structures of polymer 3 were completely collapsed. By visual observation, it was clear that the use of polymer 3 in the fabrication of porous scaffolds had not been successful because there had been a large loss in both scaffold shape and thickness. This was later confirmed by SEM analysis. As can be seen in Figure 3, the scaffold made from polymer 3 has collapsed and no open pore structure is observed. Polymer 6, on the other hand, resulted in better quality scaffolds with interconnected pore structures. There was a slight visual indication that a few of these scaffolds had a slight tendency to collapse in the middle of the structure, but not to the same extent as the polymer 3 scaffold. These results show that two polymers with very similar molar compositions, polymer 3 and polymer 6, produce very different scaffolds. The polymer-NMP solution concentration during the scaffold fabrication was 15% (w/w) for both polymers. However, the most significant difference between these two materials is in the methods used to synthesize them. While polymer 3 was synthesized using Sn(Oct)2 and ethylene glycol, polymer 6 was formed using a cyclic tin alkoxide as initiator. The 13C NMR spectra of these two copolymers differed in that the spectrum of polymer 3 contained several smaller peaks that were not observable in the spectrum of polymer 6.
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Figure 3. SEM micrographs of porous scaffolds fabricated from (d) polymer 1 (the inside of the scaffold), (e) polymer 3 (the top surface of the scaffold due to structural collapse), and (f) polymer 6 (the inside of the scaffold).
The degrees of crystallinity in these two materials were both around 10%, but the thermal properties Tg and Tm differed more widely. The Tg values of polymer 3 are both below RT, bringing the polymer to its rubbery state when placed in the water beakers, whereas the Tg values of polymer 6 are higher. The small but still noticeable, difference in molecular weight between polymers 3 and 6 is also a possible explanation of why polymer 6 was better suited for this fabrication method than polymer 3. Polymer 1 resulted in porous scaffolds which were slightly softer than those of polymers 4 and 5, but they felt hard and durable upon handling compared to that of polymer 3. As can be seen in Figure 3, there seem to exist, at least locally, thin and thready scaffold walls in this scaffold. The pore structure also has a slightly rougher and more irregular appearance than those observed for polymers 4, 5 and 6. Nevertheless, the SEM micrographs show that the scaffold fabricated from polymer 1 has a porous structure. The results obtained from both visual observations and SEM indicate that copolymer 1 and 6 containing between 35 and 55% TMC can be successfully used in this method to form porous scaffolds for tissue engineering applications, with no visual tendency toward collapse of these scaffolds. A comparison of the SEM results with those obtained from the DSC-measurements suggests that poly(LLA-co-TMC) copolymers with higher LLA contents, and hence, in this case, higher degrees of crystallinity, are better suited for the fabrication of porous scaffolds using this combined phase separation and sugar template leaching technique. Other factors such as molecular weight and thermal properties also play important roles. As can be seen in several of the SEM micrographs in Figures 2 and 3, not only do the scaffolds have large pores created from the extracted sugar but the scaffold walls are also porous. These smaller pores are created during the phase separation that occurs
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during fabrication. A porous scaffold made from polymer 5 was sent to the Ceramics Institute in Mo¨lndal, Sweden, for determination of its porosity, pore volume, and pore size distribution. When a water penetration technique was used, the porosity was measured as 79 vol % and using a mercury porosimeter calculated to 91 vol %. The mercury porosimeter analysis also resulted in a pore volume of 5.3 cm3/g and pore diameters of about 250 and 0.6 µm. The fact that the scaffold contained one set of larger pores and one set of smaller pores is in agreement with the pore structure observed in the SEM analysis. The Hgtechnique used on this scaffold is, however, only able to detect pores smaller than 350 µm and the porous scaffolds prepared in this work certainly also have pores larger than that. This is evident from visual observations, from SEM analysis, and also from the fact that granulated sugar used to make the sugar templates has an average crystal size of 400-600 µm.
Conclusions Porous scaffolds were successfully prepared from poly(Llactide-co-trimethylene carbonate) copolymers with both high and low TMC contents, using a simple and effective phase separation and particulate leaching technique, in which sugar templates of chosen size and shape were used as the leachable porosifiers. When comparing five copolymers synthesized using Sn(Oct)2 and ethylene glycol, it was found that a higher LLA content and a higher degree of crystallinity resulted in the most promising porous scaffolds. Other factors such as the molecular weight and thermal properties also played important roles when using this fabrication process. A polymer with too high a molecular weight is not suited for the method, due to the high viscosity of the polymer-solvent solution. One polymer was synthesized using a five-membered cyclic tin alkoxide initiator. This polymer was better suited for this scaffold fabrication technique than the copolymer synthesized using Sn(Oct)2, which had both a similar molar composition and similar degree of crystallinity. The differences between these two materials were that the molecular weights and the thermal properties Tg and Tm were all higher for the better-suited material. The temperature of the water in which the polymers were solidified and the sugar leached out also affected the final structure of the scaffolds. A water temperature of about 25 °C resulted in rugged and sunken scaffolds, whereas water temperatures of 40 or 60 °C gave scaffolds with interconnected pore structures. Scanning electron microscopy (SEM) was used to study the porosity and the surface characteristics of the scaffolds, and the scaffolds were shown both to be highly porous and to possess interconnected
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pores. In addition to the large pores created by the leached sugar templates, SEM and a mercury porosimeter analysis also revealed that smaller pores, less than 1 µm in size, were present in an analyzed scaffold. Acknowledgment. The authors thank the Swedish Research Council, Grant No. 2005-6082, for financial support during this work. Dr. Torbjo¨rn Mathisen is gratefully thanked for his scientific guidance throughout this work.
References and Notes (1) Ryner, M.; Albertsson, A.-C. Biomacromolecules 2002, 3, 601–608. (2) Stridsberg, K.; Albertsson, A. C. Polymer 2000, 41, 7321–7330. (3) Finne, A.; Albertsson, A.-C. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 444–452. (4) Andronova, N.; Albertsson, A.-C. Biomacromolecules 2006, 7, 1489– 1495. (5) Odelius, K.; Albertsson, A.-C. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 1249–1264. (6) Odelius, K.; Plikk, P.; Albertsson, A.-C. Biomacromolecules 2005, 6, 2718–2725. (7) Stridsberg, K.; Albertsson, A.-C. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 1774–1784. (8) Gogolewski, S.; Pennings, A. J. Colloid Polym. Sci. 1983, 261, 477– 84. (9) Mooney, D. J.; Baldwin, D. F.; Suh, N. P.; Vacanti, J. P.; Langer, R. Biomaterials 1996, 17, 1417–1422. (10) Schugens, C.; Maquet, V.; Grandfils, C.; Jerome, R.; Teyssie, P. Polymer 1996, 37, 1027–38. (11) Hou, Q.; Grijpma, D. W.; Feijen, J. J. Biomed. Mater. Res., Part B 2003, 67B, 732–740. (12) Wang, M.; Chen, L. J.; Ni, J.; Weng, J.; Yue, C. Y. J. Mater. Sci.: Mater. Med. 2001, 12, 855–60. (13) Vogt, S.; Larcher, Y.; Beer, B.; Wilke, I.; Schnabelrauch, M. Eur. Cell. Mater. 2002, 4, 30–8. (14) Zhang, R.; Ma, P. X. J. Biomed. Mater. Res. 2000, 52, 430–438. (15) Stjerndahl, A.; Wistrand, A. F.; Albertsson, A. C.; Ba¨ckesjo¨, C. M.; Lindgren, U. J. Biomed. Mater. Res. 2008, accepted for publication. (16) Stjerndahl, A.; Wistrand, A. F.; Albertsson, A.-C. Biomacromolecules 2007, 8, 937–940. (17) Bornstein, J.; La Liberte, B. R.; Andrews, T. M.; Montermoso, J. C. J. Org. Chem. 1959, 24, 886–7. (18) Stridsberg, K.; Albertsson, A.-C. J. Polym. Sci., Part A: Polym. Chem. 1999, 37, 3407–3417. (19) Dobrzynski, P.; Kasperczyk, J. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 3184–3201. (20) Draney, D. R.; Jarrett, P. K. Polym Prepr. (Am. Chem. Soc., DiV. Polym. Chem.) 1990, 31, 137–8. (21) Kricheldorf, H. R.; Stricker, A. Macromol. Chem. Phys. 1999, 200, 1726–1733. (22) Fischer, E. W.; Sterzel, H. J.; Wegner, G. Kolloid Z. Z. Polym. 1973, 251, 980–90.
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