ARTICLE pubs.acs.org/Biomac
Porosity and Pore Size Regulate the Degradation Product Profile of Polylactide Karin Odelius,† Anders H€oglund,† Sanjeev Kumar,‡ Minna Hakkarainen,† Anup K. Ghosh,‡ Naresh Bhatnagar,§ and Ann-Christine Albertsson*,† †
Department of Fibre and Polymer Technology, Royal Institute of Technology, SE-100 44, Stockholm, Sweden Centre for Polymer Science and Engineering, Indian Institute of Technology, New Delhi 110016, India § Mechanical Engineering Department, Indian Institute of Technology Delhi, New Delhi 110016, India ‡
ABSTRACT: Porosity and pore size regulated the degradation rate and the release of low molar mass degradation products from porous polylactide (PLA) scaffolds. PLA scaffolds with porosities above 90% and different pore size ranges were subjected to hydrolytic degradation and compared to their solid analog. The solid film degraded fastest and the degradation rate of the porous structures decreased with decreasing pore size. Degradation products were detected earlier from the solid films compared to the porous structures as a result of the additional migration path within the porous structures. An intermediate degradation rate profile was observed when the pore size range was broadened. The morphology of the scaffolds changed during hydrolysis where the larger pore size scaffolds showed sharp pore edges and cavities on the scaffold surface. In the scaffolds with smaller pores, the pore size decreased during degradation and a solid surface was formed on the top of the scaffold. Porosity and pore size, thus, influenced the degradation and the release of degradation products that should be taken into consideration when designing porous scaffolds for tissue engineering.
’ INTRODUCTION In tissue engineering applications, the dimensions and shape of the porous scaffold are designed to mimic the tissue to be replaced, and it is well-known that different tissues require different optimum pore size ranges.1 It is believed that the ideal In Vivo degradation rate is equal or slightly less than the rate of tissue formation.2 It is therefore of great importance not only to determine the optimum pore size for tissue regeneration, but also for the degradation process because the degradation rate of the scaffolds can affect the cell vitality, cell growth, and even host response.3 In addition, the nature and formation profile of acidic low molar mass products formed during the degradation of aliphatic polyesters need to be assessed.4 An accumulation of acidic degradation products may evoke an unwanted inflammatory response at the implantation site of biodegradable polyesters.5,6 It is well-known that molar mass, crystallinity, hydrophilicity, sample size, and so on influence the degradation rate and degradation product profile.4 More recently, macromolecular design (triblock and multiblock) and polymer architectures (linear and cross-linked) were shown to be tools that significantly alter the degradation process and resulting degradation product patterns.7,8 Stereocomplexes have also been shown to produce shorter and more acidic degradation products, although the material had a much higher hydrolytic stability compared to the plain polymer.9 The degradation profile of polyesters can also be tuned by utilizing plasticizers of different architectures or hydrophilicities.10,11 Further, hydrophilic surface modification considerably influences the degradation process r 2011 American Chemical Society
and resulting degradation product patterns leading to rapid formation of water-soluble degradation products.12,13 Most studies on the degradation behavior of aliphatic polyesters have been performed on massive devices and films. It was shown early that hydrolytic degradation of aliphatic polyesters is autocatalyzed by carboxylic end groups generated by chain scissions of the ester bonds.14 Degradation products are formed both on the surface and within the bulk of the material, and in “thick” specimens the carboxylic groups may be trapped, resulting in heterogeneous degradation where the center of the specimen is degraded at a higher rate than the surface.15 This autocatalytic effect has also been observed in thin films and microspheres.16,17 It is well-known that the porosity greatly influences the rate of degradation, with a lower porosity leading to a faster degradation rate.18-20 However, the effect of pore size on the degradation process is still not well established. Some authors conclude that pore size determines the rate of hydrolysis,20 whereas other work state that pore size does not influence the degradation process.18 The increase in pore surface/volume ratio of foams prepared with salt in the range of 053 μm enhanced the release of degradation products, thus, diminishing the autocatalytic effect and resulting in slower foam degradation compared to foams with salt in the range of 106150 μm.21 Received: December 21, 2010 Revised: January 28, 2011 Published: February 23, 2011 1250
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Biomacromolecules In addition to the ambiguous results of the pore size effect on the degradation rate, little attention has been paid to the degradation products released from porous scaffolds during degradation. Several studies have investigated the changes in pH during degradation of porous scaffolds of various homo- and copolymers of aliphatic polyesters.16,22 This gives a good indication of when enough water-soluble products have been formed to influence the pH of the buffered solution. However, the release of longer oligomers does not initially influence the pH to the same extent as the monomeric hydroxy acids but are nevertheless important to assess due to the enhanced acidic effect upon prolonged degradation. The aim of this work was to regulate the degradation product profile and the degradation process of porous scaffolds and films of polylactide (PLA) by controlling the porosity and pore size of the scaffolds. Our hypothesis was that although the solid films and porous scaffolds are considered thin, their varied pore size and, hence, the thickness of the solid material between pores will affect the degradation rate. A smaller pore size will result in thinner pore walls, and consequently, the degradation rate will decrease. However, smaller pore sizes will also result in a larger number of pores and pore walls and the formed degradation products will therefore have a longer migration path through the scaffold to the surrounding medium. There could also be some isolated pores inside the scaffold that trap degradation products. In addition to designing porous scaffolds with the optimum pore size range for a given application, the influence of porosity and pore size on the degradation rate should also be taken into account.
’ EXPERIMENTAL SECTION Materials. The polylactides used in this study were commercial products from Nature Works Co. Ltd. U.S.A. (3051D, 96% L-LA and 4% D-LA). The materials were used as received and were chosen to obtain results comparable with previous work.11,12 Sodium chloride (NaCl; purity of 99.5%, Fischer chemicals, Germany) was used after separating the agglomerates by grinding in a mortar and sieving to a particle size of 0-90, 90-300, 300-500, and 90-500 μm. Chloroform (CHCl3; HPLC grade, Fischer Scientific), methanol (AR, BDH Prolabo), acetonitrile (J.T. Baker), dichloromethane (Fischer), triethylamine (Acros), hydrochloric acid (HCl; Fischer), D,L-2-hydroxyvaleric acid sodium salt (Prosynth LTD), and dichlorodimethylsilane (Aldrich) were used as received. Sample Preparation. Solid polymer films were prepared by dissolving PLA pellets in chloroform to a 5 wt % solution and subsequent solution casting on silanized glass molds. The solvent was evaporated, and the films were dried under vacuum for 1 week. Circular sample discs with a diameter of 10 mm and a thickness of approximately 250 μm were punched from the films. The porous scaffolds were prepared by a solvent casting and salt leaching technique, as described earlier.23 Briefly, PLA pellets were dissolved in CHCl3 to form 5 wt % homogeneous solutions and subsequently poured in silanized glass molds containing NaCl with a weight ratio of 1:10 (polymer/porogen). The mixture was slowly airdried under a lid for 5 days, followed by 5 days of drying without the lid. The composites were thereafter die-cut into small circular samples, 10 mm in diameter and approximately 2.5 mm in thickness, and immersed in deionized water to dissolve the salt particles. The water was changed after each 20 min period during 1 h and then once every hour for 5 h, followed by a water exchange 2-3 times every 24 h for four days. The porous scaffolds were thereafter dried under vacuum. 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
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AgCl, using a WPA UV-vis spectrophotometer version 1.6, after the instrument had been referenced with deionized water and AgNO3. SEM and gravimetric measurements were also used to confirm total salt leaching. The dimensions of the solid film were chosen to compare circular shapes of solid and porous discs with the same diameter and mass. Scaffold Properties. The porosity, P, of the scaffolds was determined by measuring the dimensions and mass of the scaffold and calculated according to eq 1: dp 100 ð1Þ P ¼ 1d where dp = (mp/Vp) is the scaffold density and d is the density of a nonporous film fabricated by the same procedure but without salt addition. The wall thicknesses were estimated using image analysis from SEM micrographs and given as average means from 30 measurements. Hydrolysis. The PLA samples were subjected to hydrolytic degradation in deionized water at 60 °C. Each specimen, irrespective of shape, had an approximate weight of 26 ( 1 mg and was placed in a vial containing 10 mL of water sealed with butyl/PTFE septum and aluminum lid and placed in an oven. At predetermined time intervals, between 1 and 182 days, triplicate samples of each material were withdrawn from the test environment, dried under vacuum, and subjected to the various analyses. In addition, the water-soluble degradation products in the sample solutions were analyzed after each hydrolysis time. Mass Loss. The course of degradation was followed by determining the remaining mass of the samples at the predetermined times. After withdrawing the samples from the hydrolysis medium, the solid samples were dried to constant weight under reduced pressure (0.5 10-3 mbar). The mass loss was determined by comparing the dry mass (md) at the specific time with the initial mass (m0) according to eq 2. m0 - md Δmd ¼ 100 ð2Þ m0
Solid-Phase Extraction (SPE). Hydrolysis products were extracted from the degradation medium by Solid-Phase Extraction (SPE). A total of 2 mL of the solution containing the degradation products was removed from each sample vial. A total of 200 μL of internal standard solution containing 1 mg/mL of D,L-2-hydroxyvaleric acid sodium salt was added, and the pH was lowered to 1 by addition of 37% HCl. SPE columns (ISOLUTE ENVþ 100 mg 1 mL, Biotage, Sweden) were used for the extractions. First, the columns were conditioned with 2 mL of methanol and equilibrated with 2 mL of acidified (pH = 1) solution medium. The 2 mL sample portion was subsequently allowed to pass through the column, after which the retained hydrolysis products were eluted with 0.5 mL of acetonitrile. A total of 1 μL of the eluted solution was removed and analyzed with gas chromatographymass spectrometry (GC-MS). SPE with subsequent GC-MS was chosen over other relevant analytical methodologies to determine lactic acid24 due to its proven selectivity for this application.25,26 Gas Chromatography-Mass Spectrometry (GC-MS). Chromatographic separation and mass spectrometric detection was performed using a ThermoFinnigan (San Jose, CA, U.S.A.) GCQ GCMS system. A Gerstel (M€ulheim and der Ruhr, Germany) MPS2 autosampler was used for injection of the samples. The GC was equipped with a WCOT CP-Wax 52 CB column (30 m 0.25 mm 0.25 μm) from Varian (Lake Forest, CA, U.S.A.). The GC was programmed to start at 40 °C, hold at this temperature for 1 min, and then increase the temperature by 10 °C/min to 250 °C, where it was held for 13 min. Helium of 99.99999% purity from Scangas (Stockholm, Sweden) was used as carrier gas at a constant average linear velocity of 1251
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Table 1. Polymer Properties before Hydrolysis Including Number-Average Molar Mass, Polydispersity Index, Glass-Transition Temperature, Melting Temperature, and Degree of Crystallinity
a c
sample name
denotation
Mna [g/mol]
PDIa
porosity
solid film porous scaffold 0-90 μm porous scaffold 90-300 μm porous scaffold 300-500 μm porous scaffold 90-500 μm
S P0/90 P90/300 P300/500 P90/500
109200 ( 1500 117100 ( 800 113200 ( 1500 117500 ( 200 104200 ( 3800
1.97 ( 0.01 1.86 ( 0.01 1.87 ( 0.01 1.86 ( 0.01 1.97 ( 0.08
91.2 ( 0.8 90.2 ( 1.6 91.8 ( 0.7 90.5 ( 1.0
wall thicknessb [μm] 4.7 ( 1.1 10.2 ( 3.0 17.7 ( 5.1 8.4 ( 3.9
Tmc [°C]
wcc [%]
Tmd [°C]
wcd [%]
Tgd [°C]
146.8 ( 1.9 147.7 ( 4.4 146.7 ( 0.4 146.6 ( 1.7 147.4 ( 0.6
24.6 ( 3.7 23.7 ( 1.5 20.0 ( 0.5 21.4 ( 2.7 19.9 ( 0.4
147.6 ( 1.9 147.0 ( 0.8 148.0 ( 1.7 147.9 ( 1.1 149.4 ( 1.4
0.4 ( 0.3 1.6 ( 0.8 0.6 ( 0.4 1.9 ( 1.1 1.5 ( 0.8
54.9 ( 4.9 53.6 ( 2.6 51.9 ( 2.9 50.7 ( 0.9 50.6 ( 1.3
Determined by THF-SEC calibrated with narrow molar mass polystyrene standards. b Estimated from SEM micrographs (30 measurements). Determined by DSC from the first heating scan. d Determined by DSC from the second heating scan.
40 cm/s maintained by the Electronic Pressure Control (EPC) of the GC. The temperature of the injector was 250 °C. A splitless injection mode was used. The temperatures of the transfer line and ion source were 275 and 180 °C, respectively. The mass spectrometer was scanned in the range 35-400 m/z with a scan time of 0.43 s. Data were evaluated using the Xcalibur 1.2 software. The peak areas were calculated by integrating the total ion current (TIC). Electrospray Ionization Mass Spectrometry (ESI-MS). The water-soluble products were analyzed with a Finnigan LCQ ion trap mass spectrometer (Finnigan, San Jose, CA). Methanol (Fischer Scientific, super gradient) was added to the samples (2:1 v/v), and the solutions were continuously infused into the ESI ion source at a rate of 5 μL/min using the instrument syringe pump. The LCQ ion source was operating at 5 kV, and the capillary heater was set to 175 °C. Nitrogen was used as nebulizing gas, and helium was used as damping gas and collision gas in the mass analyzer. No cationizing agents were needed and positive ion mode was used for all analyses. Size Exclusion Chromatography (SEC). The molar mass changes of the PLA samples were analyzed with a Verotech PL-GPC 50 Plus system equipped with a PL-RI Detector and two PolarGel-M Organic (300 7.5 mm) columns from Varian. The samples were injected with a PL-AS RT autosampler for PL-GPC 50 Plus and THF was used as mobile phase (1 mL/min, 35 °C). Calibration was performed using narrow molar mass distribution polystyrene standards with a molar mass in the range of 162-400000 g/mol. Corrections for the flow rate fluctuations were made using toluene as an internal standard. CirrusTM GPC Software was used to process the data. Scanning Electron Microscopy (SEM). SEM images of the porous samples were taken using HITACHI TM-1000, having magnification from 20 to 10000 (digital zoom 2,4) using electron beam running at 15 kV. High vacuum was generated by a turbo-molecular pump (TMP) and the detector used worked on a highly sensitive BSE (back scattered electrons) technique. The samples were cryogenically broken using liquid nitrogen. The gold coating was not required because the instrument has a charge-up reduction mode to avoid the burning of the sample due to the accumulation of the charge by electron beam. Differential Scanning Calorimetry (DSC). The thermal properties were investigated using a DSC (Mettler Toledo DSC 820 module) under a nitrogen atmosphere. A total of 2-6 mg of the sample was placed in a 40 μL aluminum cap without a pin and sealed with a lid. Samples were heated under a nitrogen gas flow of 50 mL/min from 0 to 200 °C at a rate of 10 °C, held at 200 °C for 2 min, and thereafter cooled to 0 °C at a rate of 10 °C and held at the lowest temperature for 2 min. Finally, the samples were heated from 0 to 200 °C at a rate of 10 °C. Triplicate samples were analyzed at each time point. The melting temperatures were noted as the maximum value from the first and second heating scan, and the glass transition temperatures were taken as the midpoint of the glass transition. The approximate degree of crystallinity of the samples was calculated according to eq 3: wc ¼
ΔHf 100 ΔHf 0
ð3Þ
where wc is the degree of crystallinity, ΔHf is the heat of fusion of the sample, and ΔHf0 is the heat of fusion for a 100% crystalline polymer. The value used for ΔHf0 was 93 J/g.27
’ RESULTS AND DISCUSSION The influence of porosity, pore size, and wall thickness on the water-soluble product patterns and the degradation rate of PLAbased porous scaffolds were assessed during hydrolytic degradation at 60 °C in deionized water for up to 182 days. Four different scaffolds with the same initial porosity (>90%) and different pore sizes were fabricated using a solvent casting and porogen leaching technique as previously described and compared to a solid analogue (Table 1).23 The sieved size range of the particles was used to denote the pore size, although some salt particles may have agglomerated, which could affect the pore size range to some extent. It is, however, generally accepted that the average pore size is roughly equal to the porogen size.28 The sample names were given according to the pore size range of the respective porous scaffold. Degradation Products. The migrated oligomeric and monomeric degradation products after different hydrolysis times were analyzed by electrospray ionization-mass spectrometry (ESI-MS) and gas chromatography-mass spectrometry (GCMS), respectively. The expected oligomeric degradation products were lactic acid oligomers terminated with hydroxyl and carboxyl end groups, and the expected monomeric degradation product was lactic acid. For solid PLA film, water-soluble lactic acid oligomers were detected by ESI-MS after 14 days of degradation at 60 °C, Figure 1. The oligomers were observed as one series of peaks with a mass-to-mass peak increase of 72 Da, corresponding to the molar mass of the LA repeating unit. These peaks appear at m/z = (1 þ n 72 þ 17 þ 23), that is, lactic acid oligomers terminated with hydroxyl and carboxyl end groups forming adducts with one sodium ion each (general chemical structure in Figure 1). The oligomeric degradation product profile had a Gaussian-shaped curve with its maximum at m/z = 329 (tetramer of lactic acid). For the porous structures, no water-soluble lactic acid oligomers were observed after 14 days with the exception of small amounts of the lactic acid dimer at m/z = 185. After 28 days of hydrolysis of the porous structures, however, similar Gaussian-shaped oligomeric degradation profiles were recorded, as seen in Figure 2. At this stage, the oligomeric degradation product profile of the solid film had changed from Gaussian-shaped to an almost linearly declining curve with increasing molar mass of the oligomers. Hence, the degradation process proceeded with further degradation of the released oligomers resulting in the 1252
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Figure 1. Positive ESI-MS spectra of water-soluble degradation products of (a) solid PLA film after 14 days and of (b) P300/500 after 28 days of hydrolytic degradation at 60 °C in the mass range m/z 150-600.
Figure 2. Oligomeric degradation product profiles for solid and porous PLA scaffolds recorded by ESI-MS after 28 days of hydrolytic degradation at 60 °C for (b) solid film, (0) 0-90 μm, (Δ) 90-300 μm, () 300-500 μm, and (O) 90-500 μm.
formation of larger amounts of shorter oligomer chains. This transition in oligomeric degradation product profile from Gaussian-shaped to progressively declining was also observed for the porous structures between 28 and 49 days of degradation (Figure 3). The oligomeric degradation product profiles after 49 days of hydrolysis were similar for all materials with the largest intensity detected for the lactic acid dimer and thereafter decreasing intensity with increasing molar mass of the oligomers. These oligomeric degradation product profiles were retained throughout the study, that is, up to 182 days of degradation. The influence of pore size on the release of degradation products was more prominent when determining the monomeric degradation products by GC-MS. The relative amount of the released lactic acid monomer for the different materials increased as a function of degradation time, Figure 4. Negligible amounts of lactic acid monomer were detected from the different materials during the first 7 days of degradation where after the amounts increased progressively with degradation time for all materials. Interesting differences between the materials were, however, found during the intermediate degradation time
Figure 3. Oligomeric degradation product profiles of solid and porous structures of PLA recorded by ESI-MS after 49 days of hydrolytic degradation at 60 °C for (b) solid film, (0) 0-90 μm, (Δ) 90-300 μm, () 300-500 μm, and (O) 90-500 μm.
points, that is, from 14 to 49 days (close-up of Figure 4). P90/300, P300/500, and P90/500 showed very similar curves and the two former were therefore excluded from the figure close-up for clarity reasons. The amount of extracted lactic acid from the solid film increased rapidly between 7 and 14 days whereas the corresponding amounts from the porous structures only increased slightly. A similar rapid increase in monomer release was observed for P90/ 500 between 14 and 28 days, but again only a slight increase for P0/90. This material instead showed a rapid increase between 28 and 49 days of hydrolysis and the amounts of extracted lactic acid were approximately the same for all three materials after 49 days. This also shows that the solid film degrades faster than the porous scaffolds and the smaller the pore sizes the slower the hydrolysis (cf. Mass Loss and Molar Mass Changes). Mass Loss and Molar Mass Changes. The degradation process of the remaining material was also followed by monitoring the mass loss and molar mass changes during hydrolysis, Figure 5. As expected, the remaining mass decreased with hydrolysis time for all materials, Figure 5. Initially, higher mass loss with 1253
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Figure 4. Relative amount of lactic acid monomer extracted from the water fractions of solid and porous structures of PLA as a function of degradation time at 60 °C. (b) solid film, (0) 0-90 μm, (Δ) 90-300 μm, () 300-500 μm, and (O) 90-500 μm.
Figure 5. Remaining mass as a function of hydrolysis time at 60 °C for (b) solid film, (0) 0-90 μm, (Δ) 90-300 μm, () 300-500 μm, and (O) 90-500 μm.
increasing pore size was observed. The solid films exhibited the fastest mass loss rate and P90/500 portrayed an intermediate degradation rate as compared to the other porous scaffolds. The mass loss differences leveled off with increasing degradation time correlating with almost no remaining number-average molar mass of the samples. The faster mass loss and lower average molar mass for the nonporous films compared to the porous samples are explained by an autocatalytic effect where more slowly diffusing degradation products catalyze further hydrolysis in the solid films. Faster mass loss was also observed with increasing pore size, because the pore wall thickness increases with increasing pore size.28 An autocatalytic effect has earlier been detected even for very thin films.16 The hydrolytic degradation of aliphatic polyester has been shown to be autocatalyzed by formed degradation products.14 It has also been shown that the degradation products may be trapped in thick specimens resulting in heterogeneous degradation.15 This would result in a delayed release of degradation products. However, degradation products were detected earlier from the solid films compared to the porous scaffolds. This is due to the additional migration path in the porous structures prior to release to the surrounding medium. Once formed, the degradation products first
Figure 6. Remaining number-average molar mass of the (b) solid film, (0) 0-90 μm, (Δ) 90-300 μm, () 300-500 μm, and (O) 90-500 μm as a function of hydrolysis time at 60 °C.
diffuse to the surface of the material and are thereafter released into the degradation medium. For porous scaffolds, the degradation products also need to travel through the pores prior to release from the structure. Although the diffusion distance is longer in the thicker solid films, there is no additional migration path to consider and degradation products are therefore detected earlier. In parallel, for larger pore sizes (thicker pore walls, Table 1), the migration rate is higher compared to smaller pore size scaffolds due to the facilitated migration path within the structure. Although the porosities were above 90% and the pores were relatively interconnected, there were still a large number of pore walls and isolated pores where degradation products could stay trapped (cf. Figure 9). Degradation products might therefore be trapped in the pores and need to migrate through several pores and past several pore walls before they are released to the surrounding medium. The molar mass changes for the solid and porous PLA materials during hydrolysis were determined by SEC, Figure 6. In general, the molar mass decreased rapidly with hydrolysis time. This is in accordance with previous results on degradation of PLA in deionized water at 37 °C, where a substantial drop in molar mass was seen already after 1 week of degradation.11 Moreover, comparing the remaining molar mass to the 1254
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Figure 7. Size exclusion chromatograms of solid films and porous scaffolds with a pore size of 0-90 μm before and after hydrolysis at 60 °C for 7-49 days.
remaining mass shows that the mass loss was considerably slower.29 This is due to the hydrophobic nature of PLA. The degradation products formed during hydrolysis are not watersoluble until they have a molar mass of ∼1000 g/mol and therefore remain in the polymer bulk.12 In addition, the same pore size trend can be observed for the changes in molar mass as was seen for the mass loss. The smaller the pore size, the slower the hydrolysis, with the solid film being by far the fastest degrading material. Once again, an intermediate behavior was observed for P90/500 compared to the other porous scaffolds. This very rapid loss of molar mass is expected at the elevated temperature used in this study. Split peaks were sometimes observed in the chromatograms, and the most prominent peak was selected in those cases. This is quite often found for semicrystalline aliphatic polyesters because the amorphous regions are degraded faster than the crystalline parts, in addition to a possible autocatalyzed degradation giving rise to fractions of different molar mass. This also results in an increase in polydispersity index with degradation time, Figure 7. Interestingly, split peaks were observed earlier for the solid samples, after approximately 2 weeks of hydrolysis, while an additional two weeks passed before the porous scaffolds showed the same behavior. Split peaks of semicrystalline polyesters originate from the faster degradation of the amorphous regions.27 For the solid films, this could occur earlier compared to the porous scaffolds due to the accumulation of degradation products, resulting in an autocatalytic effect. Morphology. The heterogeneous degradation of the solid film was seen from micrographs of the cross-section of the film, Figure 8. The cross-section of the solid film prior to degradation was smooth and homogeneous. It was evident that the faster degradation of amorphous regions resulted in the formation of crystalline structures throughout the bulk of the material after 2 weeks. This was also observed from the crystallinity, which increased from approximately 25-80% after 2 weeks of degradation (cf. Figure 11). However, the material structure close to the surface deviates from the bulk. This supports the theory of an autocatalytic process with a faster degradation inside the solid films as a contributing factor to the faster degradation of the solid films. Moreover, the film thickness did not change during degradation and the films retained their structure until fragmentation occurred.
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The effect of pore size on the degradation rate of the porous scaffolds was also evaluated by SEM, Figure 9. Prior to degradation, the scaffolds were regular structures with homogeneously distributed pores. The shape and structure of the pores was influenced, but not completely determined, by the size range of the sieved salt particles used (Figure 9; first column a,d,g). The initial porosities of the scaffolds were above 90%, with the majority of the pores being in the size range of 0-90, 90-300, 300-500, and 90-500 μm, respectively. No significant changes in the surface morphology were seen after 14 days of hydrolysis for the porous scaffolds with the larger pores, that is, P90/500 and P300/500. After 28 days of hydrolysis, however, the scaffolds displayed sharp pore edges (third column in Figure 9). In aqueous media, semicrystalline aliphatic polyesters degrade in two steps. First, water penetrates the lessorganized amorphous regions, degrading these by hydrolytic chain scission of the ester bonds. When most of these regions are degraded, the hydrolysis is also initiated in the crystalline regions, moving from the edges toward the core.4 Hence, these sharp edges indicate that degradation has occurred predominantly in the amorphous regions, with much of the crystalline regions remaining. Cavities were also found on the scaffold surface after 28 days of hydrolysis, most obvious for P300/500, where a loss of structure had occurred. This is in line with the higher mass loss of the materials and indicates that a loss of scaffold material possibly by fragmentation of the scaffold has occurred during the hydrolysis process. Fragmentation is an undesirable process and should be taken into consideration when considering these pore size ranges for tissue engineering applications. In contrast, smaller pores were formed in P90/300 after 14 days and a solid surface on the top of the scaffold after 28 days. Comparable observations have been found for amorphous PLGA with a pore size range of 180-250 μm, where the pores gradually became flattened, and after 8 weeks, the surfaces were similar to that of a solid film.30 In addition, it has also been reported that the number and size of the surface pores of PLGA with a pore size range of 180-280 μm decreased with degradation time, and some fiber-like connections appeared. Most pores had vanished after 24 weeks, and the remaining pores were smaller than initially.22 For P0/90, which portray the slowest rate of hydrolysis, no apparent change in the surface morphology was observed during the first 14 days of degradation. The scaffolds were too brittle to handle after 28 days of hydrolysis time and could not be evaluated by SEM (has also been observed by others18). Although the slowest degradation rate was observed for the scaffolds with smaller pore sizes and thinnest pore walls, their physical integrity is not maintained during the same hydrolysis time span as the scaffolds with larger pore sizes. This is most likely due to the thinner pore walls in the scaffolds with smaller pore sizes. Moreover, all scaffolds collapsed and lost their dimensional stability after 49 days of hydrolysis. Thermal Properties. The changes in thermal properties during hydrolysis were evaluated with DSC in two consecutive steps. The specimens were first subjected to a first heating scan, which reflected the thermal effects related to the morphological changes from aging. A second run was carried out after cooling the samples, allowing for a comparison between the bulk properties and the physical changes caused by aging. All samples had approximately the same melting temperature prior to degradation (cf. Table 1). The melting temperature increased with approximately 4 °C for all samples already during the first day of hydrolysis, where 1255
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Figure 8. Scanning electron micrographs of the cross-section of solid PLA film hydrolyzed for (a) 0 and (b) 14 days in deionized water at 60 °C.
Figure 9. Scanning electron micrographs of the surface of porous PLA scaffolds hydrolyzed in deionized water at 60 °C, with an initial pore size range of 90-300 μm (first row a-c), pore size range of 300-500 μm (second row d-f), and pore size range of 90-500 μm (third row g-i) hydrolyzed for 0 days (first column a,d,g), 14 days (second column b,e,h), and 28 days (third column c,f,i).
after the Tm was virtually unchanged during the first 14 days of degradation. After this time point, the Tm started to decrease rapidly (Figure 10). The initial elevation jump in Tm is believed to be caused by an annealing effect occurring when the materials were placed in 60 °C and the continued increase over the first two weeks is due to the continued increase in the crystal thickness during hydrolysis.31 At longer degradation times, the melting temperatures started to decrease because of the formation of shorter polymer chains. During the full duration of the study, the lowest decrease in melting temperature was, similar to mass loss and changes in molar mass, observed for the porous scaffolds. In parallel to the decrease in Tm, an increase in the degree of crystallinity, wc, was also observed (Figure 11).
The increase in wc is once again coupled to the initial degradation of the more susceptible amorphous regions which leads to an increase in the overall wc during hydrolysis of semicrystalline polyesters. The higher mobility of the shorter chains allows for a reorientation of the crystalline regions and subsequently an increase in wc. This process is substantially faster for the solid films due to overall faster degradation rate as observed by SEC and gravimetrical measurements. Bimodal melting peaks were found in the second heating scan in the DSC chromatograms recorded after longer degradation times and the most prominent peak was selected in those cases. This splitting of the melting peak is caused by the fractions of different molar mass as explained earlier (cf. mass loss and molar mass changes). Correlating to the split peaks found in the SEC evaluation, the DSC bimodal peaks were observed earlier for the 1256
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Figure 10. Melting temperature determined from the first heating scan as a function of hydrolysis time at 60 °C for (b) solid film, (0) 0-90 μm, (Δ) 90-300 μm, () 300-500 μm, and (O) 90-500 μm.
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Figure 12. Degree of crystallinity determined from the second heating scan as a function of hydrolysis time at 60 °C for (b) solid film, (0) 0-90 μm, (Δ) 90-300 μm, () 300-500 μm, and (O) 90-500 μm.
showed lower Tgs compared to the porous scaffolds due to the faster degradation rate in these materials. The Tg values recorded for the different porous structures were relatively similar although P0/90 showed a slightly slower decrease in Tg with degradation time. The Tg values were taken from the second heating scan due to undefined peaks in the first heating scan.
Figure 11. Degree of crystallinity determined from the first heating scan as a function of hydrolysis time at 60 °C for (b) solid film, (0) 0-90 μm, (Δ) 90-300 μm, () 300-500 μm, and (O) 90-500 μm.
solid samples, that is, after approximately 1 week of hydrolysis while an additional week passed before the porous scaffolds showed the same behavior. This is explained by the faster degradation of the solid films as compared to the porous scaffolds. No peak splitting was apparent after the first heating scan. The second run reveals information on the molar mass dependence of Tm and wc, because the heating destroyed the initial crystallites and recrystallization involves degraded chains. Comparing the degree of crystallinity of the first and second heating scans shows that the wc of the second heating scan is always lower. This is caused by the shorter crystallization time upon the rapid heating in the second scan. It is also observed that the curve shapes are different, where the wc is constant up to 91 days of degradation in the first heating scan while it decreases in the second heating scan. This indicates that the initially formed crystallites maintain their integrity and are much less affected by degradation as compared to the amorphous regions (Figures 11 and 12). The glass transition temperature, Tg, of all samples decreased with increasing degradation time (data not shown). This is also due to the formation of shorter chains with higher mobility. Similar to the observations in the other analyses, the solid films
’ CONCLUSIONS The degradation product profiles and the degradation rate of solid and porous polylactide (PLA) were controlled by the sample porosity and pore size and hence the pore wall thickness. Solid films and larger pore size scaffolds degraded faster due to autocatalyzed degradation with trapped degradation products. Degradation products were nevertheless detected from the solid films after shorter degradation times due to the additional migration path within the porous structures. Possibly degradation products are also trapped inside isolated pores in the scaffold. Pore size and pore size range also influenced the degradation process; the smaller the pore size the slower the hydrolysis. In addition, a broad pore size range resulted in an intermediate degradation behavior compared to small or large pore size scaffolds. The porous structures maintained their morphology during the early stages of degradation but with time the larger pore size scaffolds portrayed sharp pore edges and cavities on the scaffold surface. The size of the pores in the smaller pore size scaffolds decreased during degradation with a subsequent formation of a solid surface on the top of the scaffold. Eventually, all scaffolds collapsed and lost their dimensional stability. The effect of porosity and pore size on the degradation and the degradation product profiles should be taken into account when designing porous scaffolds for a specific application. ’ AUTHOR INFORMATION Corresponding Author
*Tel.: þ46-8-790 82 74. Fax: þ46-8-20 84 77. E-mail: aila@ polymer.kth.se.
’ ACKNOWLEDGMENT The authors gratefully acknowledge Professor Indra K. Varma for valuable scientific discussions and the ERC Advanced Grant, PARADIGM, (Grant Agreement No. 246776) and STINT (the 1257
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Swedish Foundation for International Cooperation in Research and Higher Education) for their financial support of this work.
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