Influence of in Vitro Hydrolytic Degradation on the Morphology and

Biomacromolecules 2004, 5, 358-370

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Influence of in Vitro Hydrolytic Degradation on the Morphology and Crystallization Behavior of Poly(p-dioxanone) Marcos A. Sabino,† Julio Albuerne,† Alejandro J. Mu¨ller,*,† Josee´ Brisson,‡ and Robert E. Prud’homme§ Grupo de Polı´meros USB, Departamento de Ciencia de los Materiales, Universidad Simo´ n Bolı´var, Apartado 89000, Caracas 1080-A, Venezuela, Chemistry Department, Laval University, Que´ bec, Canada G1K 7P4, and Chemistry Department, Montreal University, Montre´ al, Canada H3C 3J7 Received September 19, 2003; Revised Manuscript Received November 21, 2003

We have studied the hydrolytic degradation of high molecular weight poly(p-dioxanone), PPDX, sutures. The samples were degraded either in distilled water or in a phosphate buffer at 37 °C, and the starting viscosity-average molecular weight was 130 kg/mol. The hydrolytic degradation of PPDX occurs in an approximate two stage process where the amorphous regions of the sample are attacked faster than the crystalline regions of the sample. The changes experienced by the samples as degradation proceeded were successfully monitored by viscosimetry, differential scanning calorimetry (DSC), weight loss, pH changes, and scanning electron microscopy (SEM). Polarized optical microscopy (POM) observations performed on PPDX films revealed that PPDX crystallizes in spherulites whose detailed morphology depends on the supercooling employed during isothermal crystallization. Changes in the spherulitic morphology as molecular weight is reduced are only pronounced when the molecular weight is equal or lower than 8 kg/mol. The dependence of lamellar thickness as a function of isothermal crystallization temperature was examined by atomic force microscopy (AFM) in thin films of PPDX together with melting point data obtained by DSC. Through the use of the Thomson-Gibbs equation, we obtained a value of 166 erg/cm2 for the fold surface free energy of PPDX. This value is in the same range as those obtained previously for similar linear polyesters. The lamellar thickness, as well as the melting point, was found to have a small decreasing dependence with the molecular weight of the samples. Introduction Poly(p-dioxanone) or PPDX is a synthetic poly(ester-ether) that exhibits appropriate molecular structure and properties for biomedical applications. Typical applications include suture material, pins for fracture fixation, surgical clips and fasteners.1-2 The chemical structure of PPDX is

A review on the synthesis, characterization, applications, and properties of PPDX and of copolymers based on PPDX has been recently published.1 It is well established that PPDX can be degraded by hydrolysis generating low molecular weight hydroxy-acid species that can be metabolized or bioabsorbed by the human body.1-2 It has been postulated that the hydrolysis process in semicrystalline polyesters occurs in two stages. The attack of the less dense amorphous regions where diffusion of the hydrolysis medium is easier is much faster than the attack on the crystalline regions; these differences in hydrolytic * To whom correspondence should be addressed. E-mail: [email protected]. † Universidad Simo ´ n Bolı´var. ‡ Laval University. § Montreal University.

degradation kinetics generate approximately a two-stage process. Even though such a two-stage process has been postulated for several biodegradable copolyesters such as for instance poly(D,L-lactic-co-glycolic acid) (PLGA),3 poly (D,Llactide-co-1,3-trimethylene carbonate),4 and poly(glycolideco-lactide) (tradename Vicryl)5 or even more recently for PPDX,6-8 a detailed description of morphological changes occurring during hydrolysis is still needed. The present work is based on our previous investigations into the morphology, nucleation, crystallization kinetics, and properties of neat and hydrolytically degraded PPDX.7,9-13 New results on the morphology of neat and degraded PPDX have been gathered by atomic force microscopy (AFM) and compared with observations performed by polarized optical microscopy (POM). The hydrolytic degradation process has been followed by viscometry and weight loss measurements and the depletion of the crystalline regions of the samples by differential scanning calorimetry (DSC). These results together with scanning electron microscopy (SEM) evidence allow us to draw a clearer morphological picture of how the hydrolytic degradation process in PPDX evolves with degradation time. Additionally, the variation of PPDX crystallization kinetics has been studied by determining spherulitic growth rates as a function of molecular weight,

10.1021/bm034367i CCC: $27.50 © 2004 American Chemical Society Published on Web 01/08/2004

Morphology and Crystallization Behavior of PPDX

Figure 1. Percentage of PPDX130 weight retention after exposure to the indicated hydrolysis media at 37 °C as a function of time.

Biomacromolecules, Vol. 5, No. 2, 2004 359

Figure 2. Variation of solution pH during hydrolysis of PPDX130 in the indicated hydrolysis media at 37 °C as a function of time.

in a wide molecular weight range comprising 230 kg/mol to less than 5 kg/mol. Experimental Section Materials and Hydrolytic Degradation Method. Poly(p-dioxanone) monofilaments produced by Johnson & Johnson were employed. Two types of commercial samples were used: Undyed Pins of 1.3 mm diameter (Orthosorb) and dyed monofilaments of 0.486 mm diameter (PDS-II sutures by Ethicon). The sutures contained approximately 0.2 wt % of D&C Violet no.2 dye to aid visualization during surgery.14 The dye had no appreciable effect on the morphology, nucleation, and crystallization properties of PPDX since tests were performed with suture samples whose dye was extracted and no differences were found upon comparison with dyed samples. Similar conclusions have been obtained in the literature.14,15 To follow the hydrolytic degradation of PPDX, a series of 0.486 mm diameter monofilament samples (PDS-II sutures) were introduced in test tubes in a sterile environment containing either distilled water or a 0.2 M buffer phosphate solution (based on Na2HPO4 and KH2PO4) at pH ) 7.44 and 37 °C. Each test tube contained 16 mL of buffer solution, and 3 short segments of PDS-II suture of approximately 100 mg each were immersed in the solution. Samples were previously dry and weighed on a Mettler analytical balance. The hydrolysis was followed by weight loss and pH changes (with an Orion pH-meter) as a function of time (Figures 1 through 8 refer to results obtained employing directly commercial suture material). Each datum point in Figure 1 reports the mean weight loss of the 3 samples contained in each test tube, and no standard deviation is given since the number of samples is too low; however, the weight values were found to differ from each other in less than 3%. Average molecular weights were determined by capillary viscosity measurements using an Ubbelohde viscosimeter and solutions of PPDX samples in phenol/1,1,2,2 tetrachloro ethane (2:3 w/w) at 25 °C. The extrapolations of Huggins and Kraemer were employed to obtain intrinsic viscosities. The values of the Mark-Houwink constants K ) 79 × 10-3 cm3 g-1 and a ) 0.69 were employed to calculate the viscosity-average molecular weight, Mv. These approximate values of the Mark-Houwink constants were obtained by applying the group contribution treatment of van Krevelen.16

Figure 3. Changes in viscosity average molecular weight (Mv) during hydrolysis of PPDX130 at the indicated hydrolysis media at 37 °C as a function of time. The horizontal line indicates the approximate value of the weight average molecular weight of reference sample PPDX5.

Capillary viscometry experiments on the neat PPDX pins yielded a value of Mv ) 230 kg/mol, whereas the PDS-II monofilaments resulted in Mv ) 130 kg/mol. Because intrinsic viscosity determinations needed a substantial amount of material, only one determination per data point in Figure 3 was performed. For comparison purposes, we have also employed a low molecular weight PPDX standard sample generously provided by Prof. Philippe Dubois. The preparation of this type of PPDX sample has been previously described.17 Because its molecular weight was very low, the sample was able to dissolve in chloroform at 40 °C, and size exclusion chromatography (SEC) experiments were performed to determine its molecular weight and its distribution. The weight average molecular weight was 5.48 kg/mol and Mw/Mn ) 1.24. We were not able to obtain SEC traces of high molecular weight PPDX monofilaments in view of their poor solubility in the solvents commonly employed to perform SEC at low temperatures, and this is why we have employed capillary viscometry to follow the evolution of Mv as a function of degradation time since a suitable solvent mixture was found (see above). Therefore, we do not know the polydispersity of the commercial PPDX materials but we can infer that they are probably larger than 2. For instance, Abuzaina et al.18 employed a PPDX dyed resin (prepared by a tin catalyzed ring-opening bulk polymerization) and report an approximate Mw of 82 kg/mol and polydispersity index of 2.16. We will refer to the original samples as PPDX,230 PPDX,130 and PPDX,5 where the superscripts indicate their approximate

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Mv values (for the first two samples) or Mw value (for PPDX5) in kg/mol. It should be stressed, to avoid confusion, that the only sample where the Mw value is available is PPDX,5 and for the rest of the samples, only the Mv value is available. The hydrolytic degradation experiments were performed only to PPDX130 monofilaments. PPDX5 and PPDX230 were employed for comparison purposes. PPDX samples are extremely sensitive to humidity and even small traces of water can initiate hydrolytic degradation. Therefore, all samples were thoroughly dried in a vacuum oven at 30 °C and stored in a desiccator under vacuum at 25 °C at all times. Differential Scanning Calorimetry. Thermal analysis was performed with a Perkin-Elmer DSC-7 apparatus under an ultrahigh-purity nitrogen atmosphere. The equipment was calibrated with indium and tin standards. Sample weight was kept constant at 5.0 mg, and heating and cooling scans were recorded at 10 °C/min. The crystallinity, Xc, of a polymeric material can be expressed as the ratio of the heat of fusion ∆Hf of the sample and that of the 100% crystalline material ∆H°f Xc )

∆Hf ∆H°f

(1)

Crystalline and amorphous materials usually do not hydrolyze at the same rate. We have followed the treatment of Joziasse et al.19 that considers the mass of crystalline material as a function of degradation time, mc(t), can be expressed as mc(t) ) Xc(t) m(t) )

( ) ( )

∆Hf(t) ∆Hf(t) m(t) ) m0 (1 - ml(t)) ∆H°f ∆H°f (2)

where m0 is the initial mass, m(t) the remaining mass at time t, and ml(t) the mass loss at time t (i.e., ml(t) ) 1 - m(t)/ m0). The amount of crystalline material relative to the initial value mc(0) can therefore be considered a function of time, such that19 mc(t) mc(0)

)

( ) ∆Hf(t)

∆Hf(0)

(1 - ml(t))

(3)

where ∆Hf(0) is the heat of fusion of the original material. Equation 3 can be used to monitor the changes experienced by the material during hydrolytic degradation. A constant value of mc(t)/mc(0) with time indicates that the crystalline part of the material is not being modified by hydrolysis, so that only the amorphous regions experience degradation. A value of mc(t)/mc(0) that increases with time indicates that crystallinity is increasing with hydrolysis time, and finally, if the ratio decreases with time, the crystalline regions are being depleted by hydrolytic degradation.19 When calculating crystallinity values from DSC enthalpies, we were careful to take into account if any cold crystallization process developed during the scan. The error in measuring enthalpies depends on the quality of the baseline calibration. In our case, the errors were estimated to be below 10%.

Scanning Electron Microscopy. The surface of PPDX monofilaments was observed after they had been removed from the hydrolysis medium and dried. Drying was performed in a vacuum oven at 25 °C for 7 days or until constant weight was achieved. We have assumed that the drying procedure has not significantly altered the surface morphology of the monofilaments. The samples were gold coated in a Balzars unit (SCD 030), and a Phillips SEM 505 instrument was employed for the observations of the sample surfaces. Low voltages were used to guarantee minimum radiation damage to the samples. AFM and POM Measurements. Thin PPDX films (before and after degradation) were prepared by spin-coating from a 2.5% (g/mL) solutions of PPDX in phenol/1,1,2,2 tetrachloro ethane (2:3 w/w) at a rotation speed of 3000 rpm during 20 s using a Headway Research Inc. EC101 apparatus. The final film thickness was on the order of 250-300 nm. Thicker films of thicknesses ranging from 600 to 900 nm were prepared by solution casting onto microscope slides for POM observations. A Zeiss Axioscop polarizing microscope and a Linkam hot stage, temperature controller, and cooling unit were used to follow the growth of the spherulites. Microphotographs were recorded with a Toshiba HVD27 3CCD camera. A λ/4 plate was used to facilitate the observations of the extinction patterns produced. The samples were melted at 140 °C for 5 min under a nitrogen atmosphere and then cooled at a rate of 50 °C/min to the crystallization temperature [which was held constant to within (0.1 °C)]. Bright field and phase contrast modes were used to make morphological observations, during and after isothermal crystallization. In some cases, the spin casted thin films were crystallized at specific Tc with the Linkam hot-stage under POM, and then they were quenched to room temperature and immediately used for AFM observations. We also performed POM observations in films prepared by hot melting small amounts of PPDX onto glass slides, and no influence of the method of preparation on the morphology and/or the growth rate kinetics was encountered. The only difference was that it is easier to obtain thinner films by the solution casting technique. A Nanoscope III, Dimension 3100, atomic force microscope (AFM) (Digital Instrument) was used to examine the surface topology of the thin films prepared. The instrument was operated in tapping mode with a J-5137JVH tip for observations at 100 µm resolution, whereas observations at 10 and 1 µm were performed with a E-4974EV tip. A useful review on the AFM technique and its application to biomaterials has been recently published by Jandt.20 Results and Discussion Hydrolytic Degradation Process of PPDX. A simple indication of the progress of hydrolytic degradation can be obtained by determining the weight loss experienced by the samples during exposure to the hydrolysis medium. Figure 1 shows the percentage of retained weight by the sample as a function of degradation time. After 10 weeks at 37 °C, the PPDX samples lost approximately 25% of their weight when the hydrolysis medium was phosphate buffer, whereas those in distilled water lost almost 50% weight.


Similar results have been reported previously for PPDX6-8 and for other aliphatic polyesters.3-5 For instance, the hydrolytic degradation of a 50:50 PLGA sample (Mw ) 37 kg/mol), under phosphate buffer conditions leads to 75% weight loss in a 70 days period. Another factor that influences the relative stability of the PPDX is the molecular weight and the shape and orientation of the material. In this case, we have employed oriented monofilaments of approximately Mv ) 130 kg/mol. In a previous work,7 suture monofilaments of Mv ) 184 kg/mol and a diameter of 0.486 mm were exposed to a 0.2 M phosphate buffer at 37 °C for 10 weeks and the samples only lost 3% weight. The difference with respect to the 25% weight loss obtained in the present work under similar conditions may be related to the molecular weight differences, because their crystallinity degree was approximately the same. More recently, Ooi and Cameron8 also found a weight loss of 3% after 80 days hydrolysis of PDS-II sutures (of diameter 0.357 mm). However, they did not determine the molecular weight of the material they employed. The changes in pH as degradation proceeds have been plotted in Figure 2. The pH for the buffer solution decreased from 7.1 to 5.6 during the 10 week degradation period, a similar result was obtained previously.7 This indicates that the hydrolysis is producing low molecular weight molecules that have acidic character5,21-22 as one can expect from hydrolysis of ester linkages according to the following general scheme:

This indicates that the buffer capacity of the medium has clearly been exceeded, because no regeneration of the buffer in the hydrolysis medium was performed. The buffer concentration was chosen to simulate human body conditions, unfortunately, the buffer was not regenerated during the hydrolysis period, and it should have been performed also in order to properly simulate in vivo conditions. If a regeneration of the buffer had been performed, the degradation rate could have been slower. When distilled water is used as a hydrolysis medium, there is no buffer to balance the pH, and the acidity of the medium is higher accelerating the hydrolysis of the polymer molecules. The viscosity average molecular weight was determined as a function of degradation time and is plotted in Figure 3. The weight average molecular weight of our PPDX5 sample that was obtained by SEC is shown in this plot as a reference value. The molecular weight of samples degraded for more than 10 weeks could not be measured because a small amount of sample was left and also the viscosities were too low and beyond the resolution of the Ubbelohde viscometer employed. If we consider the data shown in Figures 1-3, they reflect the way the hydrolytic degradation is proceeding, and it is clear that the initial degradation rate seems to be slower than that obtained from week 4 onward in the case of the buffer solution and even earlier in the case of distilled water. This may be related to the typical two-stage process that is


Figure 4. Changes experienced by the heat of fusion of PPDX130 samples determined during the first DSC heating scans performed after exposure to the indicated hydrolysis media at 37 °C as a function of time. The approximate Mv values of the samples that were degraded in the phosphate buffer solution (according to Figure 3) have been included in the top horizontal axis and the values corresponding to sample PPDX5 are plotted with reference to that axis.

common in hydrolytic degradation of polyesters. It is wellknown that initially the attack is faster on the amorphous regions because they are less dense, whereas the crystalline regions are more resistant to water diffusion. Therefore, although both regions are being attacked from the beginning of the experiment, the rate of attack on the amorphous regions (and the concomitant mass loss for instance) is faster, whereas that on the crystalline regions is slower. The overlap of these two types of kinetics as a function of time generates the apparent two-stage process. After week 7, the mechanical stability of the samples degraded in distilled water was lost and they were easily broken and seemed to crumble when manipulated with tweezers. The same happened with those degraded in the buffer solution after week 9. This is another indication that connecting tie chains within the amorphous regions as well as entanglements have been probably lost by hydrolysis after such time. Figure 4 shows the variations in sample crystallinity as hydrolytic degradation proceeded for PPDX sutures. The monofilament samples were removed from the hydrolysis medium, dried, weighted, and encapsulated in DSC pans after which they were heated in the DSC at 10 °C/min and the values of their heat of fusion recorded and plotted in Figure 4. Because no cold crystallization occurred during this first heating, the enthalpy of fusion was employed to calculate the value of crystallinity reported (the value corresponding to the enthalpy of fusion of a 100% PPDX sample employed for the calculations was that reported by Ishikiriyama et al.23). The reference values corresponding to PPDX5 are also plotted in this figure, and they are the values corresponding to 0.5 × 104 g/mol (5 kg/mol) in the upper x-axis label (notice that a trend line has been passed through the data points corresponding to PPDX130 and its derived degraded products only, i.e., not encompassing the 0.5 × 104 g/mol data points). A peculiar behavior is observed because the degree of crystallinity increases very rapidly at first with degradation time and then its rate of increase slows after week 7

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approximately. Because the PPDX sample employed had a rather high initial molecular weight, we interpret these results by proposing that the initial attack on the amorphous zones depletes many entanglements and tie chains between crystalline regions that reduce conformational hindrances, and because the temperature at which the hydrolysis is being carried out (37 °C) is much higher than the Tg (originally at approximately -10 °C) of PPDX, the material can crystallize further. This very clear trend of crystallinity increase was not so evident in the results obtained by Ooi and Cameron8 during the hydrolysis of similar PDS-II samples during 80 days. Nevertheless, they did find a small decrease in lamellar thickness after 25 days of degradation which they argued could be consistent with main-chain scission enabling fragments of undegraded chains to reorganize and form new thinner crystals. Lin et al.6 also found an increase of crystallinity with degradation in PPDX commercial suture material by DSC measurements. It is interesting to note that they employed a suture PPDX fiber marketed as PDS which was the predecessor product of PDS-II suture, the material employed in this work. PDS-II is reported to have a different fiber morphology because it has a skin-core morphology that the PDS suture does not possess.8 Similar changes in sample morphology has been described by Zong et al. and Fu et al. in their studies of in vitro hydrolytic degradation of poly(glycolide) and poly(glycolideco-lactide).24-26 They have referred to the increase in crystallinity with time as “cleavage-induced crystallization”. They have also found experimental evidences to suggest that there is a decrease in sample polydispersity with degradation time and that there is a greater chance that higher molecular weight chains, such as tie molecules, undergo degradation.24-26 The increase in crystallinity with degradation time may have a limit, because after most amorphous regions have been depleted, the crystallinity of the sample tends to increase more slowly with time (see Figure 4). It is not surprising that the samples become fragile and break after 7 weeks hydrolytic degradation because an important amount of amorphous zones have been depleted and the crystallinity degree has been increased. Scanning electron microscopy was employed to reveal the overall morphological features of the sample surfaces as degradation proceeded. Figure 5 shows low magnification pictures of PPDX monofilaments surfaces after 6 weeks exposure to the hydrolysis medium. The sample that was degraded in water already shows a series of micro-cracks that are located roughly perpendicular and sometimes parallel to the fiber axis (in a kind of grid pattern) indicating water penetration and hydrolytic degradation to specific parts of the fiber that could correspond to inter-lamellar amorphous regions. After 8 weeks of hydrolytic degradation, SEM observations at a higher magnification (pictures not shown here) showed that the degradation occurs in regions that are mostly perpendicular to the fiber axis. Some fiber debris was observed indicating that material is being progressively removed from the monofilament surfaces. After 10 weeks, Figure 6 demonstrates that the material subjected to distilled water hydrolysis appears much more heavily attacked, at the

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Figure 5. SEM micrograph of a PPDX130 monofilament after (a) 6 weeks exposure to a 0.2 M phosphate buffer solution at 37 °C and (b) 6 weeks exposure to distilled water. The length of the white bar is 1 mm.

Figure 6. SEM micrograph of a PPDX130 monofilament after (a) 12 weeks exposure to a 0.2 M phosphate buffer solution at 37 °C and (b) 12 weeks exposure to distilled water. The length of the white bar is 0.1 mm. The white arrows indicate the fiber axis directions.

same magnification, than that immersed in the phosphate buffer solution. The fiber axis in Figure 6 has been indicated with white arrows. Figure 6 shows that the hydrolytic attack on the amorphous zones seems to be occurring layer by layer in a kind of zigzag pattern that is consistent with the “Swiss cheese” model proposed by Prevosek and reviewed by Williams.27 The


Figure 7. Variation of relative crystalline fraction (eq 3) as a function of hydrolysis time in the indicated media at 37 °C. The approximate Mv values of the samples that were degraded in the phosphate buffer solution (according to Figure 3) have been included in the top horizontal axis.

missing material corresponds to hydrolyzed amorphous regions whose molecules have been degraded to low molecular weight material that is now soluble in the hydrolysis medium. This is consistent with the weight loss of the sample (Figure 1) and with the decreasing pH values (Figure 2). We have employed eq 3 to calculate the relative amount of crystalline material present as a function of degradation time as indicated in the Experimental Section. Figure 7 shows how the relative amount of crystalline material evolves with hydrolytic degradation. Figure 7 shows that as a result of the “cleavage induced crystallization” the rate of increase in the crystalline regions is higher than the rate of depletion of the crystalline regions during the first 4 weeks of hydrolysis for the PPDX samples in the buffer solution and during the first 2 weeks for the samples in water. The downturn in the curves of Figure 7 could be taken as indicative of the time at which the crystalline regions in the sample are being attacked although a much clearer indication would be when the mass ratio plotted in Figure 7 falls below 1. Using the first criterion, the crystalline regions of the samples exposed to the buffer solution start to be attacked at a time greater than 4 weeks and greater than 2 weeks for those in water. From all of the results gathered in this section, an approximate picture of the detailed hydrolytic degradation of PPDX can be proposed. A tentative schematic representation of such process is provided in Figure 8. Two major initial routes are depicted: (I) kinetically favored hydrolysis on the amorphous regions of PPDX and (II) diffusion hindered attack on the crystalline lamellae. Both routes can be present from the start of the hydrolysis experiment. However, route (I) occurs faster than route (II) at least during the first few weeks of degradation depending on the media employed (water or buffer solution). In route (I), the amorphous regions are first hydrolyzed, and new crystals are formed at the expense of the shorter chains in the amorphous regions (i.e., the cleavage-induced crystallization24). As degradation time increases, greater attack on the amorphous regions leads to the fragmentation of groups of lamellae (like in a) whose inter-lamellar amorphous regions are finally depleted by hydrolysis and eventually dissolve in the hydrolysis medium


Figure 8. Schematic model of morphological transformation during hydrolytic degradation of PPDX. Route (I) refers to the preferential attack on the amorphous regions, whereas route (II) indicates how the crystalline regions may be attacked (see text).

which is indicated in Figure 8 by the final (e) stage. Figure 8 shows in route (II) the hydrolysis of the crystalline regions of the sample. The process starts hypothetically by hydrolysis of the chain folds located on the basal plane of the lamellae (b). Such chain fold depletion leads to lamellar cleavage (c), a process that continues until smaller crystal fragments are produced (d), until eventually, individual chains can be extracted from the crystals and dissolved in the hydrolysis medium (e). Characterization of Hydrolytically Degraded PPDX. Figure 9a shows DSC cooling scans after heating the samples to 140 °C in order to erase thermal history. Subsequent DSC heating scans are shown in Figure 9b. Neat samples, PPDX230, PPDX130, and PPDX5 are also included in Figure 9. The effect of molecular weight of the material can be appreciated in these Figures and in Table 1 where the relevant enthalpies and temperatures extracted from Figure 9 are reported. The higher molecular weight neat materials PPDX230 and PPDX130 behave in a very similar way. Their enthalpy of crystallization and melting are almost identical. There is a difference in the value of the peak crystallization temperature during cooling, a fact that probably reflect differences in nucleating densities between these two commercial materials but their final peak melting temperatures are very similar (106.8 vs 106.4 °C). Despite the large variations in molecular weight experienced by the samples during hydrolytic degradation in buffer solution, their thermal properties were not so sensitive to these molecular weight changes. After hydrolytic degradation of PPDX130 in phosphate buffer, five samples with different degradation times were selected for DSC studies: 2, 4, 6, 8, and 12 weeks. For the first 4 samples, the Mv values were measured and are reported in Figure 3, and these values are used as superscripts (in kg/mol) to identify the samples, i.e., PPDX121, PPDX101, PPDX78, and PPDX.48 In the case of the sample with 12 weeks degradation time in the buffer solution, we do not have a measured value for its molecular weight. Nevertheless, an arbitrary value of 7.5 kg/mol was employed and an integer value of 8 was used as superscript. The relative much narrower width of the crystallization exotherm and melting endotherm of PPDX5 as compared to

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Figure 9. DSC thermal characterization of PPDX samples of different molecular weights. The numbers on each scan indicate PPDX average molecular weights in kg/mol. (a) DSC cooling scans (at 10 °C/min) after erasing the samples thermal history by heating at 140 °C for 3 min. (b) Subsequent DSC heating scans (at 10 °C/min) after the cooling runs shown in part (a). Relevant temperatures and enthalpies are listed in Table 1.

PPDX130 or PPDX230 is probably due to its narrower molecular weight distribution. Figure 10 shows how the glass transition temperatures (Tg), the peak crystallization temperature during cooling, and the onset and peak melting temperatures vary as a function of hydrolytic degradation time or approximate values of average molecular weight. The values corresponding to PPDX230 and PPDX5 were included for comparison purposes at the top of the figure, where the Mv values are included as a label in the top x axis. The tendency of Tg to decrease as degradation time increases is noteworthy because it is consistent with the molecular weight reduction detected by viscometry (Figure 3). After 6 weeks degradation, the value of Tg could not be detected because it was out of our measurement temperature range. Figure 10 shows that Tc tends to decrease slightly with molecular weight (from 43.8 °C for PPDX230 up to 46.5 °C for PPDX5), a trend reflecting greater nucleation efficiency as the molecular weight decreases. Both onset and peak Tm values decrease slowly as degradation time increases. It should be noticed that the decrease in Tm is more pronounced for samples with 8 weeks of degradation or more, i.e., when the hydrolytic degradation was in its advanced stage and the crystalline regions of the sample were attacked. A peculiar behavior is observed during the cooling scans, because the enthalpy of crystallization initially decreases sharply with hydrolytic degradation time (from week 0, PPDX130, to week 2, PPDX121, in Table 1) and then increases

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once more, at the beginning rather slowly (weeks 4-6, samples PPDX101 through PPDX78) and then at a higher rate (weeks 8-12, PPDX48 and PPDX8), see Table 1. This is reflected in the DSC heating scans (Figure 9b), because PPDX tends to crystallize during heating in a noticeable cold crystallization exotherm at temperatures between 20 and 50 °C. Figure 10b shows how this cold crystallization exotherm, labeled (I), has an enthalpy of crystallization that first increases (from week 0 to week 2) and then stays more or less constant within the error of the measurements of enthalpy (4-6 weeks) and finally decreases at a high rate, see in Table 1 the values labeled ∆HcI. We have previously shown11 that PPDX easily undergoes partial melting and recrystallization during the scan and this is partly reflected in the cold exotherm labeled (II) in Figure 9b that appears just before a very broad melting range. Most crystals melting in the final endotherm have been recrystallized during the scan, and we have shown in ref 11 by performing melting experiments after isothermal crystallization that their final peak melting temperature is independent of crystallization conditions and only depends on the heating rate.11 Similar conclusions about recrystallization during the scan have been reached independently by Andjelic et al.14,15 and Pezzin et al.28 Therefore, in the present case, any variations in the peak melting temperature reflect a variation in the molecular weight of the sample as a result of hydrolytic degradation. The lack of exotherm labeled (I) in PPDX5 in Figure 9b indicates that the sample has crystallized all that was possible during the cooling performed in Figure 9a. However, these crystals formed during cooling at 10 °C/min are not stable enough and therefore are partially molten and recrystallized at higher temperatures; therefore, the sample exhibits the cold crystallization exotherm labeled (II) and a complex melting endotherm. The lower temperature melting peak reflects the melting of the rather more imperfect crystals that formed during cooling, whereas the higher temperature melting peak is due to the melting of the reorganized crystals during the scan.11 In all of the other samples, the double melting peak is not resolved, most probably because of the wide molecular weight distribution of the samples, but the melting peak is very broad, particularly in the low-temperature tail. Polarized Optical Microscopy (POM). The first report on the superstructural morphology of neat PPDX was published by Sabino et al.11 It revealed that PPDX230 when crystallized from the melt produced a range of superstructures. Representative examples of these can be seen in Figure 11. At the highest supercooling shown in Figure 11, the nucleation density is relatively high and well-defined small spherulites with Maltese-cross and banding extinction patterns were observed (e.g., Tc ) 65 °C). At intermediate supercoolings, the density of nuclei was substantially smaller and large spherulitic structures are easily seen, such as those grown at 70 and 85 °C (see Figure 11). Extensive observations revealed that the banding observed in these spherulites has two periodicities and corresponds to a double banding morphology.11 Because the banding spacing increases with crystallization temperature, the spherulites obtained of samples crystallized at Tc < 75 °C appeared as single banded


Morphology and Crystallization Behavior of PPDX Table 1. Thermal Properties Determined by DSC sample PPDX230 PPDX130 (0 weeks) PPDX121 (2 weeks) PPDX101 (4 weeks) PPDX78 (6 weeks) PPDX48 (8 weeks) PPDX8 (12 weeks) PPDX5

scan (10°C/min) cooling second heating cooling second heating cooling second heating cooling second heating cooling second heating cooling second heating cooling second heating cooling second heating

Tg (°C)

∆Hc (J g-1)

Tcpeak (°C)

Tconset (°C)

35.0

43.8

64.0

31.2

49.0

65.3

6.1

42.8

56.8

8.0

43.0

56.6

14.8

43.1

56.8

55.9

44.8

61.6

67.2

45.5

64.2

70.1

46.5

53.9

-9.6 -6.4 -11.8 -12.6 -14.5

Figure 10. Variation of relevant thermal transition temperatures as a function of hydrolysis time (in a 0.2 M phosphate buffer at 37 °C) and average molecular weight. The data for reference samples PPDX230 and PPDX5 are referred to the top horizontal axis.

because the mean banding spacing was below 5 µm.11 The presence of extinction patterns appearing as single or double banding in spherulites has been previously discussed.11,29-32 Double banding has been observed in several polyesters.31-34 In Figure 11, double banding is difficult to observe at the magnifications shown in the micrographs corresponding to PPDX230 (a higher magnification picture is available in ref 11). It can be better observed in an equivalent micrograph shown in Figure 11 and taken during isothermal crystallization of PPDX130 at 85 °C. One of the best examples of very clear double banded spherulites can be seen in Figure 11 for PPDX8 crystallized at 60 °C, thanks in part to the very good contrast provided by the different extinction colors that can be obtained using a λ/4 plate inserted between the crossed polars. Pezzin et al.28 reported the observation of single banded spherulites in isothermally crystallized PPDX (commercial PDS sutures, no molecular weight reported) at 65 °C. At this temperature, it is difficult to ascertain whether double banded spherulites are present because band spacing is too low. On the other hand, Andjelic et al.14 reported the observation of spherulites without banding in their PPDX resin (Mw ∼ 82 kg/mol).

∆HcI (J g-1)

TcI (°C)

∆HcII (J g-1)

TcII (°C)

∆ Hm (J g-1)

Tmonset (°C)

Tmpeak (°C)

17.7

45.0

5.2

87.0

58.3

98.0

106.8

14.5

43.4

3.4

85.7

56.9

98.0

106.0

43.1

43.4

6.9

84.4

65.2

98.4

106.4

46.3

39.2

8.5

82.0

78.4

99.3

106.4

42.6

37.2

10.5

80.4

80.2

98.0

105.7

14.5

20.2

5.1

76.8

89.6

92.0

102.0

9.2

24.0

10.6

77.5

88.8

91.9

102.2

13.0

76.9

87.3

91.7

88.5

99.4

A change in the appearance of PPDX230 spherulites occurs when the crystallization is carried out at low supercoolings (Tc > 85 °C). The banding becomes progressively poorly coordinated and almost disappears; additionally, the Maltese cross also becomes progressively faint as Tc increases and is very difficult to observe at Tc ) 100 °C or higher. The spherulites take a “granular” appearance; an example is provided in Figure 11 for PPDX230 crystallized at 90 °C. The disappearance and/or distortion of banding upon increasing crystallization temperatures has been observed in several polyesters.34 It has been postulated that the regular twisting of lamellar structures as they grow radially within the spherulitic superstructures leads to the banding phenomenon.29-32 The reason behind this twisting is controvertial,29-32 but one possible explanation is that lamellae twist to relieve surface stresses as they grow. Higher temperatures lead to slower growth rates, and the lamellae formed are thicker and more perfect and may not need to relieve any stress buildup because this may not happen when the chains have ample time for conformational rearrangements at high Tc. Polymorphism was ruled out by wide-angle X-ray scattering (WAXS) measurements. Experiments performed on samples isothermally crystallized at 60, 70, 80, and 90 °C of PPDX230 yielded identical reflections whose angular positions did not change with Tc. The fading of the Maltese cross when the spherulites appear “granular” (like in PPDX230 crystallized at 90 °C) may be more difficult to explain. One possible explanation could be a growth regime change. The analysis of the growth rate data with the Lauritzen and Hoffman nucleation theory performed in a previous work on the same material yielded a regime III to II transition as the crystallization temperature increased. However, the transition temperature was much lower than that at which the spherulites turned granular.11 It should also be noted that Andjelic et al. performed spherulitic growth kinetics in their PPDX resin and did not find a regime growth change in a wider temperature range than that employed here and still report the morphological change of the spherulites to a granular morphology at higher crystallization temperatures.14 Additionally, DSC crystallization

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Sabino et al.

Figure 11. Polarized optical microscopy micrographs for isothermally crystallized PPDX samples of the indicated average molecular weights. The white scale bar size is 100 µm for all micrographs.

kinetics performed at low supercoolings indicated that, despite the granular appearance, the Avrami index was close to 4 indicating three-dimensional superstructures (i.e., spherulites) growing from sporadic nuclei.11 In other words, the granular superstructures are three-dimensional and should not be confused with axialites. Extensive observations indicate that, although much more difficult to see, there were always signs of a very faint Maltese cross superimposed on the apparent granular structures. Similar results on the disappearance of the Maltese cross at higher crystallization temperatures were reported by Andjelic et al.14 on their PPDX resin. The influence of hydrolytic degradation on the spherulitic morphology of PPDX was the subject of a previous brief communication by us.12 In Figure 11, we present a more complete set of data comprising a very wide molecular weight range since we have also included our neat PPDX materials (PPDX230, PPDX130, and PPDX5). An important general observation that can be drawn from Figure 11 is that a minimum molecular weight is needed for the material to display the characteristic regular double banding extinction pattern. For samples with average mo-

lecular weights of 5 kg/mol or lower, the banding observed was highly irregular. For PPDX130 and PPDX230, the morphology and its dependence with crystallization temperature were similar to each other. We have also performed observations with a PPDX180 and a PPDX290 and the results were equivalent. In the range 100-290 kg/mol, the morphology of PPDX and its temperature dependence are therefore almost equivalent. The only differences observed were the exact temperature at which the spherulites become “granular” which tends to increase as the molecular weight increases (see more on this effect below). Also, the measurements of radial growth yielded almost identical growth rate results for PPDX in the 100-230 kg/mol range. Noticeable changes in spherulitic morphology were detected after 6 weeks of hydrolytic degradation in phosphate buffer solutions. Figure 11 shows the spherulitic morphology for PPDX78 (degraded for 6 weeks), PPDX8 (degraded for 12 weeks), and PPDX

Influence of in Vitro Hydrolytic Degradation on the Morphology and

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