In vitro degradation of hydroxyapatite nanoparticle-coated biodegradable microspheres

Polymer Degradation and Stability 98 (2013) 377e386

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In vitro degradation of hydroxyapatite nanoparticle-coated biodegradable microspheres Syuji Fujii a, *, Yuki Miyanari a, Taiki Nishimura a, Yuichi Yokoyama a, Sho Hamasaki a, Masahiro Okada b, Tsutomu Furuzono c, Shojiro Matsuda d, Hideki Takamori d, Yoshinobu Nakamura a a

Department of Applied Chemistry, Faculty of Engineering, Osaka Institute of Technology, 5-16-1 Ohmiya, Asahi-ku, Osaka 535-8585, Japan Department of Biomaterials, Osaka Dental University, 8-1 Kuzuha-Hanazono, Hirakata, Osaka 573-1121, Japan Department of Biomedical Engineering, School of Biology-Oriented Science and Technology, Kinki University, 930 Nishi-Mitani, Kinokawa, Wakayama 649-6493, Japan d Research & Development Department, GUNZE Limited, 1 Ishiburo, Inokurashinmachi, Ayabe, Kyoto 623-8512, Japan b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 May 2012 Received in revised form 22 August 2012 Accepted 2 September 2012 Available online 7 September 2012

In vitro degradation behavior of hydroxyapatite (HAp) nanoparticle-coated poly(L-lactide-co-ε-caprolactone) (PLCL) microspheres, which were fabricated via ‘Pickering emulsion’ route, has been examined in terms of weight, molecular weight, thermal property, and morphological changes through incubation in a phosphate buffered saline up to 48 weeks. Gel permeation chromatography, scanning electron microscopy (SEM) and gravimetrical method were employed to characterize their degradation profiles. It was found that molecular weight of PLCL decreased rapidly after immersing the microspheres in the buffered saline and, on the other hand, the weight of the microspheres started to decrease after 16 weeks. These results support bulk degradation for the HAp-coated PLCL microspheres. During the degradation, the enthalpy of melting increased progressively, which should be due to the increase of crystallinity because of chain rearrangement of amorphous region induced by water uptake and the preferential degradation of amorphous regions. The microspheres retained spherical morphology at least for 48 weeks and the HAp nanoparticles detached from the microsphere surface, which was confirmed by SEM study. The microspheres were easy to be broken by external pressure when the molecular weight became below 35,000 g/mol after 24-week incubation. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: In vitro degradation Poly(L-lactide-co-ε-caprolactone) Microsphere Morphology Molecular weight

1. Introduction Biodegradable polymers have been used in the fields of orthopedic and reconstructive surgery [1] and tissue engineering [2], because it is not necessary to remove the polymeric materials after healing. There are strong needs for synthesis of biodegradable polymer in a form of microsphere [3], because they have found their applications as carriers for controlled drug delivery system [4] and 3D porous scaffold and cell carrier for tissue engineering [5].

Abbreviations: HAp, hydroxyapatite; PLCL, poly(L-lactide-co-ε-caprolactone); PLLA, poly(L-lactic acid); HAp-PLCL, hydroxyapatite nanoparticle-coated poly(Llactide-co-ε-caprolactone); PBS, phosphate buffered saline; SEM, scanning electron microscopy; GPC, gel permeation chromatography; OM, optical microscopy; TG, thermogravimetric; GC, gas chromatography; DSC, differential scanning calorimetry; Mw, weight-average molecular weight; Mn, number-average molecular weight; Dv, volume-average diameter; CH2Cl2, dichloromethane; BMNC, bone marrow mononuclear cell; GFP, green fluorescent protein. * Corresponding author. Tel.: þ81 6 6954 4274; fax: þ81 6957 2135. E-mail address: [email protected] (S. Fujii). 0141-3910/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.polymdegradstab.2012.09.003

Although biodegradable polymers found various applications, there is a disadvantage to be overcome: low cell adhesion to the polymers because of their hydrophobic surface character [6]. In order to improve the cell adhesion properties, hydroxyapatite (HAp) has been incorporated as either a filler or a coating agent (or both) either into or onto a biodegradable polymer matrix [7]. HAp is the main mineral of bones and teeth, and artificially synthesized HAp has been extensively used in a variety of applications such as biomaterials, ion exchangers, adsorbents, and catalysts [8]. Recently, we succeeded in synthesis of HAp nanoparticle-coated biodegradable microspheres by evaporation of the oil from the dichloromethane (CH2Cl2) solution of biodegradable polymer-inwater emulsions (Pickering emulsion method) and evaluate their cell adhesion properties [9]. In our previous study, the HAp nanoparticles were employed as a particulate emulsifier in order to prevent flocculation of emulsion droplets/microspheres as well as to improve cell adhesion properties of the microspheres obtained. The synthetic method has an advantage: neither molecular surfactant nor polymeric stabilizer was used, which is usually used to fabricate/stabilize the microspheres in media and has possibilities to cause allergy-like

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reactions and carcinogenicity [10] at the same time. It has been confirmed that the HAp-coated biodegradable microspheres can work as an effective injectable scaffold, which enhances cell-based therapeutic angiogenesis with bone marrow mononuclear cells (BMNCs) [11]. When BMNCs derived from enhanced green fluorescent protein (GFP) transgenic mice were injected into ischemic muscle, the GFP level in ischemic tissues was approximately 5-fold higher in the case of co-injection with HAp nanoparticle-coated poly(L-lactic acid) (PLLA) microspheres than in the case of BMNC alone or co-injection of BMNC with uncoated PLLA microspheres 1 week after injection. The HAp nanoparticle-coated PLLA microspheres were, thus, very effective as an injectable scaffold for cell-based therapeutic angiogenesis. For this purpose, it is ideal that the microspheres retain their spherical biodegradable polymer core/HAp nanoparticles shell morphology until they finish their roles as a scaffold and then are degraded into small fragments/molecules, which can be discharged out of body. Bearing this situation in mind, we felt that it is crucial to evaluate degradability of the HAp-coated biodegradable microspheres. There have been lots of reports on fabrication of biodegradable polymer microspheres, but there is little work on the characterization of their degradation behaviors. This omission is quite surprising, since the biodegradable polymer microspheres are widely used in medical field. In this study, in vitro degradation behavior of HAp nanoparticlecoated poly(L-lactide-co-ε-caprolactone) (PLCL) microspheres (HApPLCL microspheres) with several tens micrometer diameter has been examined in terms of weight, molecular weight, thermal property, and morphological changes through incubation in a phosphate buffered saline up to 48 weeks. 2. Materials and methods

1.1 g of PLCL in 18.9 g of CH2Cl2 in a capped bottle with overnight shaking to obtain a transparent solution. Then 0.2 g of HAp nanoparticles were dispersed in 200 mL of Milli-Q water in an ultrasonic bath for 10 min, after which the dispersion of HAp nanoparticles was added to the 5.5 wt% PLCL solution and a double layered mixture was obtained (the upper layer was the water phase and the lower one was the oil phase). Then the mixture was homogenized using an IKA Ultra-Turrax T-10 basic (Staufen, Germany) with a speed of 20,450 rpm for 3 min at room temperature. After that, the CH2Cl2 solvent remaining in the Pickering emulsion was removed by evaporation at room temperature for 1 day. The HAp-PLCL microspheres obtained were purified by several sedimentatione redispersion cycles after ultrasonic irradiation (38 kHz; 120 W; 5 min) in ethanol, with each successive supernatant solution being decanted and replaced with ethanol until no excess free HAp nanoparticles was observed by scanning electron microscopy. The HAp-PLCL microspheres sedimented quickly as compared with the free HAp nanoparticles dispersed in ethanol media. Finally, the HApPLCL microspheres were classified by testing sieves with different apertures of 25, 75 and 150 mm (Iida manufacture Co. Ltd., Osaka, Japan) and vacuum dried for 24 h to remove the residual ethanol. 2.4. Microsphere characterization 2.4.1. Optical microscopy (OM) A drop of the diluted emulsion or the HAp-PLCL particles was placed on a microscope glass slide and viewed using an optical microscope (Shimadzu Motic BA200) fitted with a digital camera (Shimadzu Moticam 2000). This technique was used to estimate mean droplet/microsphere sizes and morphologies in aqueous media (n ¼ 300).

2.1. Materials Unless otherwise stated, all materials were guaranteed reagent grade and used as received from Nacalai Tesque Inc. Calcium nitrate (Ca(NO3)2$4H2O), diammonium hydrogen phosphate ((NH4)2HPO4), 25% ammonia aqueous solution and 60% HNO3 were used as received. CH2Cl2 was purchased from SigmaeAldrich, Inc. Milli-Q water (Millipore Corp., MA, USA) with a specific resistance of 18.2  106 U cm was used for the synthesis of the HAp nanoparticles and for the preparation of emulsion. Phosphate buffered saline, Dulbecco’s PBS() (Ca, Mg Not-inc.), was used as received. PLCL used in this study was synthesized by ring-opening polymerization. The PLCL (Llactide/ε-caprolactone, 75/25 molar ratio) has a weight-average molecular weight of 460,000 g/mol, a melting point of 156.1  C and a density of 1.283 g/cm3 (determined by helium pycnometry using a AccuPyc II 1340 instrument). The pristine PLCL bulk sample has irregular shape with sizes between 0.3 and 5 mm (a number average size, 2.2 mm). The needle (NN-2238R: 22 gauge, inner and outer diameters of 480 mm and 700 mm, respectively) and syringe (SS-OIT: 1 mL) were used as received from Terumo Co., Tokyo, Japan. 2.2. Hydroxyapatite nanoparticles synthesis The HAp nanoparticles were prepared by a wet chemical process with Ca(NO3)2 aqueous solution and (NH4)2HPO4 aqueous solution (pH, 12, room temperature), followed by calcination at 800  C for 1 h with an anti-sintering agent, poly(acrylic acid, calcium salt) and removal of the agent with water after calcination [12]. 2.3. Fabrication of HAp nanoparticle-stabilized emulsion and HApPLCL microspheres HAp-PCL microspheres were prepared according to the previous study [9c]. First, a CH2Cl2 solution of PLCL was prepared by dissolving

2.4.2. Thermogravimetric (TG) analysis The loading amount of the HAp nanoparticles on the PLCL microspheres was determined by TG measurement of the HAp-PLCL microspheres after purification. The instrument used for the thermogravimetric analysis was a TG/DTA6300 (SII NanoTechnology Inc., Chiba, Japan). The dried HAp-PLCL microspheres after purification were heated from 25 to 500  C at a scanning rate of 10  C/min under nitrogen. The organic PLCL component was completely pyrolyzed under these conditions, but HAp nanoparticles remained. 2.4.3. Elemental microanalysis The HAp nanoparticle loading of the HAp-PLCL microspheres were determined by comparing the carbon contents determined by CHN elemental microanalysis (MT-5 CHN Corder, Yanaco New Science Inc., Kyoto, Japan) with that of pristine PLCL bulk sample. 2.4.4. Gas chromatography (GC) measurement The residual CH2Cl2 in the aqueous dispersion of the emulsion droplets/PLCL microspheres was determined with a GC (GC-8A, Shimadzu Corp., Kyoto, Japan) using a packed glass column (TSG-1 15% Shincarbon A 60/80; 3.2 mm ID  3.1 m; Shimadzu Corp.) at a column temperature of 120  C. The retention time of CH2Cl2 was approximately 1.9 min. The GC signal was quantified on a C-R8A chromatopac integrator (Shimadzu Corp.). 2.5. Degradation behavior characterizations 2.5.1. Experimental design The dried HAp-PLCL microspheres (0.200 g) were placed in glass vials (50 mL), each containing 30 mL of PBS (pH ¼ 7.3). Before starting degradation experiments, it was confirmed that all the microspheres dispersed/sedimented in the PBS and no bubbles attached to the samples. The samples were stored with lids on the

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vials in a 37  C environment on standing for various periods up to 48 weeks. At the end of each time point, the samples were taken out from PBS by filtering using filter paper (No. 42: diameter, 110 mm; Particle retention in liquid, 2.5 mm: Whatman). In order to remove ions and water-soluble PLCL oligomers/monomer, the sample on the filter paper was rinsed with water (100 mL) and then were air-dried overnight and vacuum-dried for 84 h. The weight, weight-average molecular weight (Mw), number-average molecular weight (Mn) and surface morphology were recorded on three samples, namely pristine PLCL bulk sample and the HAp-PLCL microspheres with 25e 75 mm and 75e150 mm diameters. Weight decrease % data were expressed as percentages relative to the initial values. 2.5.2. Gel permeation chromatography (GPC) The molecular weights of the pristine PLCL bulk sample and the microspheres during the degradation were determined by GPC using a Shimadzu LC-10AT VP pump with a Shimadzu RID-10A refractive index detector. For the determination of molecular weights of the raw polymer and microspheres, the following conditions were adopted: a Shimadzu 802c, 804c, and 806c columns were used; chloroform was the mobile phase at a flow rate of 1 mL/min. The microspheres were dissolved in chloroform, filtered and then injected into GPC column. Weight-average molecular weights were calculated from the GPC curve using a series of polystyrene standards. 2.5.3. Differential scanning calorimetry (DSC) The thermal property of the samples was studied using DSC-60 (Shimadzu, Kyoto, Japan) under a nitrogen atmosphere. DSC was calibrated by using indium as standards. The measurements were performed from room temperature to 200  C at a heating rate of 10  C/min under a nitrogen atmosphere to observe the endothermic peaks of calorimetric transition. The melting temperature was recorded as Tm. 2.5.4. Scanning electron microscopy (SEM) Scanning electron microscopy (SEM; Keyence VE-8800, 12 kV) studies were conducted with gold sputter-coated (Elionix SC-701 Quick Coater) dried samples. Dried samples were placed on an aluminum stub and sputter coated with gold in order to minimize sample-charging problems. This technique was used to measure mean sizes of the HAp nanoparticles and microspheres in dried state (n ¼ 300), and the resulting data were presented as mean size  standard deviation. Surface coverage of the microspheres with HAp nanoparticles was estimated using magnified printed SEM images gravimetrically. 2.5.5. Microsphere diameter and its distribution A Malvern Mastersizer 2000 instrument equipped with a small volume Hydro 2000SM sample dispersion unit (ca. 100 mL including flow cell and tubing), a HeNe laser operating at 633 nm and solid-state blue laser source operating at 466 nm were used to size the HAp-PLCL microspheres. The stirring rate was adjusted to 2000 rpm in order to disperse the microspheres well in the aqueous media. The microsphere diameter was taken to be the volumeaverage diameter (Dv), which is mathematically expressed as Dv ¼ P P 4 Di Ni = D3i Ni (Di, the diameters of individual microspheres; Ni, the number of microspheres corresponding to the diameters). 3. Results and discussions 3.1. Fabrication and characterization of HAp-PLCL microspheres HAp nanoparticle-stabilized CH2Cl2 solution of PLCL emulsion droplets were prepared in aqueous media in the absence of any

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molecular surfactant, followed by the evaporation of CH2Cl2 leading to the HAp-PLCL microspheres, as described previously [9c]. GC study indicated the residual CH2Cl2 in the aqueous dispersion of HAp-PLCL microspheres could not be detected after 24-h evaporation at 25  C, which indicates the concentration of CH2Cl2 was below a GC detection limit of 10 ppm. The international conference on harmonization of technical requirements for registration of pharmaceuticals for human use (ICH) has classified CH2Cl2 as a Class 2 solvent and described the concentration limit of 600 ppm in a product can be applicable under the assumption that the product mass of 10 g is administered daily [13]. The residual CH2Cl2 amount in the microspheres produced in this study is one order of magnitude lower than that limited in ICH. The microspheres sedimented in the aqueous medium within 1 min due to gravity on standing, however they could easily redisperse in the medium by hand shaking. It was confirmed that the microspheres stored in aqueous medium at 4  C, can redisperse into the aqueous medium as a single microsphere even after 48 weeks, which was confirmed by optical microscopy studies and laser diffraction microsphere size distribution analyses. It was also confirmed that dried microspheres, which were stored at 4  C in dry state for 48 weeks, can be redispersed into aqueous medium as a single microsphere. OM and SEM studies (Fig. 1) indicated productions of polydisperse HAp-PLCL microspheres and laser diffraction studies (Fig. 2 and Table 1) confirmed volume-average diameters of 59  18 mm and 115  28 mm after sieving, respectively, which were in relatively good accordance with those estimated from OM images. The SEM studies also confirmed that the microspheres possess rough surface morphologies in both (sub)micrometer and nanometer dimensions (Fig. 1 inset): (sub)micrometer-sized wrinkles and nanometer-sized roughness on the wrinkle. Similar morphologies were observed for the HAp-PLLA microsphere system [9a]. In the case of the bare PLCL microspheres prepared by removal of the HAp nanoparticles using HNO3, the wrinkled surface with no distinct nanomorphology was observed (Fig. S1). From these results, it is expected that the HAp nanoparticles adsorbed on the surface of the PLCL microspheres. The (sub)micrometer-sized wrinkles on the microsphere surface should be formed due to the evaporation of CH2Cl2 from the emulsion droplet accompanied by a decrease of the interfacial area between oil and water. This decrease of the oil-water interfacial area could also lead to desorption of the HAp nanoparticles from the interface. These desorbed HAp nanoparticles should be included in the free HAp particles and were removed by washing. TG analyses gave mean HAp contents of approximately 3 and 1% for 59 mm and 115 mmsized microspheres, respectively (Fig. S2), which indicates approximately 17% (for 59 mm) and 5% (for 115 mm) of HAp nanoparticles added were adsorbed on the PLCL microspheres. The HAp nanoparticle incorporation efficiency is relatively lower than that determined for the HAp-PLLA microsphere system [9a]. There are two possibilities explaining this result: (1) specific surfaces area of the HAp-PLCL microspheres (59 mm and 115 mm) are smaller than that of the HAp-PLLA microspheres (8 mm) and (2) the HAp-PLCL microspheres were washed with sonication, which led to desorption of excess HAp nanoparticles adsorbed on the microspheres (the HAp-PLLA microspheres were washed without sonication). The smaller microspheres (59 mm) had larger amount of HAp component comparing with the larger microspheres (115 mm), which is reasonable result considering specific surface area of the microspheres. The HAp amounts determined by elemental microanalyses were similar to those determined by thermogravimetric analyses (Table 1). Injection of the biodegradable microspheres into body is the final step in the drug/cell delivery process [14]. The HAp-PLCL

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Fig. 1. (a,b) Optical micrographs and (c,d) SEM images of HAp-PLCL microspheres. Aqueous dispersions were taken in (a) and (b). Diameters: (a,c) 25e75 mm and (b,d) 75e150 mm.

microspheres are dispersed in a liquid injection vehicle and are injected under the skin or intramuscularly [11,12]. Toward this application, it is crucial to investigate injectability of the aqueous dispersion of the microspheres through medical gauge slip-tip needles. The needle used in this study was 22 gauge (an inner diameter of 480 mm) and the aqueous dispersion of the HAp-PLCL microspheres (25e75 mm, 1 mL, 1.5 wt%) was passed through the needle by pushing syringe plunger, and passing percent of the microspheres was estimated gravimetrically. It was confirmed that the passing percent of approximately 70% after the first injection and approximately 90% after the third injection was achieved without blockage of the syringe with the microspheres. OM and laser diffraction studies indicated there was no change in dispersity,

shape and size distribution of the microspheres before and after injection experiment (Fig. S3). In the case of 75e125 mm-sized HApPLCL microspheres, the passing percent after the first and the third injections were measured to be approximately 40 and 50%, respectively, and the microspheres blocked the syringe. Therefore, the HAp-PLCL microspheres having 25e75 mm diameter were suitable as an injectable scaffold for drug/cell delivery system. 3.2. Degradation behaviors of the HAp-PLCL microspheres and pristine PLCL bulk powder No/little change in stiffness of the microspheres which should be related to molecular weight of PLCL is required for the microspheres as the scaffolds in order to enhance cell-based therapeutic angiogenesis with BMNCs, because the HAp-PLCL microspheres need to hold BMNCs without breakage at least for 1 week: 1 week is required for functional expression as the scaffold. After 1 week, it is

Table 1 Summary of volume-average microsphere diameters, elemental microanalytical data and HAp/PLCL weight ratio data of pristine PLCL bulk sample and HAp-PLCL microspheres. Sample name

Volume-average diametera/mm

C/%b

H/%b

PLCL:HApc (w/w) (CHN%)

PLCL:HAp (w/w) (TG)

Pristine PLCL bulk sample HAp-PLCL 25e75 mm microspheres HAp-PLCL 75e150 mm microspheres

e

52.5

6.2

e

e

59  18

51.2

6.1

98:2

97:3

115  28

51.9

6.1

99:1

99:1

a

Fig. 2. Laser diffraction microsphere size distribution curves obtained for HAp-PLCL microspheres dispersed in aqueous media. Diameters: (a) 25e75 mm and (b) 75e 150 mm.

Determined by laser diffraction method. Measured by elemental microanalysis method. Determined by comparing C % of HAp-PLCL microspheres with that of PLCL bulk sample. b

c

S. Fujii et al. / Polymer Degradation and Stability 98 (2013) 377e386

preferable for the HAp-PLCL microspheres to be degraded into small fragments/molecules, which can be discharged out of body. 3.2.1. Weight loss The dynamics of weight loss for the HAp-PLCL microspheres (59 mm and 115 mm) were similar to that for PLCL bulk sample (Fig. 3). For all systems, the weight remained constant for one week. For the pristine PLCL bulk sample system, the weight remained almost constant for 16 weeks after incubation in PBS at 37  C, and then a gradual decrease was observed. Rate of weight loss was determined to be 0.9%/week. The HAp-PLCL microspheres (59 mm) maintained w99% of the initial weight during 16 weeks of degradation in PBS, started to lose weight after 16 weeks and their weight reduced to 80% by 48 weeks due to dissolution of water soluble oligomers/monomers of PLCL generated during the degradation. Rate of weight loss was determined to be 0.7%/week. Detachment of the HAp nanoparticles from the microspheres in one of factors for weight loss (see below), however this should be negligible because HAp loading amount is w2%, based on HAp-PLCL microspheres which is difficult to be detected gravimetrically in this study. The 115 mm-sized HAp-PLCL microspheres showed almost the same dynamics of weight loss with 59 mm-sized microspheres, which suggests there is little effect of microsphere size on the degradation kinetics. (Specific surface area ratio of 59 and 115 mm-sized microspheres was calculated to be 7.8/4.1, using the PLCL density of 1.283 g/cm3, which gives little effect on degradation rate.)

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3.2.2. Molecular weight loss Unlike the profile for mass loss, the molecular weight of the HAp-PLCL microspheres (59 mm and 115 mm) started to decrease immediately after placement in PBS and continued to decrease throughout the time course (Fig. 4). Significant loss of Mw was observed for the HAp-PLCL microspheres with diameters of 59 mm and 115 mm, with only 1.7 and 2.0% of the initial Mw remaining after 48 weeks of degradation, respectively (Fig. 4b, c). The profile of molecular weight loss obtained for the microspheres is almost the same with that for the pristine PLCL bulk sample: 1.7% of the initial Mw remained after 48 weeks. Molecular weight distribution (polydispersity, Mw/Mn) remained almost constant (2e3.5) during the degradation experiment for the HApPLCL microspheres and the pristine PLCL bulk sample. Interestingly, the GPC chromatograms of the degraded HAp-PLCL (59 mm) microspheres suggest that degradation proceeded heterogeneously (Fig. 5). The microspheres had a broad single peak at 413,000 before degradation and Mw decreased down to 32,000 by 24 weeks of degradation in PBS, resulting in a broad peak at 28,000 with a shoulder around 8000 (Fig. 5b). After 24 weeks, the shoulder became prominent with time. These results indicate the presence of both fast and slow degrading domains, corresponding to the amorphous and crystalline areas of PLCL (see below). Tsuji et al. also observed a similar shoulder/new peak during degradation of PLLA and indicated the degradation rate difference between amorphous and crystalline areas [15]. The original PLCL contains crystalline area and there is also a possibility that

Fig. 3. Percent of weight remaining for (a) pristine PLCL bulk sample and (b,c) HAp-PLCL microspheres (diameters: (b) 25e75 mm and (c) 75e150 mm) compared to week 0 value as a function of degradation time. The samples were incubated in PBS at 37  C.

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Fig. 4. Evolution of molecular weights and their polydispersities for (a) pristine PLCL bulk sample and (b,c) HAp-PLCL microspheres (diameters: (b) 25e75 mm and (c) 75e150 mm). The samples were incubated in PBS at 37  C.

crystalline is formed during degradation of PLCL [15a]. The molecular weight of the shoulder/new peak observed during the degradation of PLLA reported by Tsuji et al. [15] is almost the same with that observed for the HAp-PLCL microspheres (8000 g/

mol). The presence of shoulder for degraded samples suggests that the change in molecular weight distribution during degradation follows the same mechanism as the pristine PLCL bulk powder degradation.

Fig. 5. GPC chromatograms obtained at various incubation times during in vitro degradation experiment in PBS at 37  C: (a) pristine PLCL bulk sample and (b) HAp-PLCL microspheres (25e75 mm).

S. Fujii et al. / Polymer Degradation and Stability 98 (2013) 377e386

Fig. 6. Enthalpy of melting obtained at various incubation times during in vitro degradation experiment in phosphate buffered saline (PBS) at 37  C: ( ) pristine PLCL bulk sample and ( ) HAp-coated PLCL microspheres (25–75 mm ).

3.2.3. Thermal analysis PLCL (L-lactide/ε-caprolactone, 75/25 M ratio) is known to be one of crystalline polymers and our DSC studies showed there are

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crystalline regions in the PLCL component of the HAp-PLCL microspheres. The enthalpy of melting (DH) for the HAp-PLCL microspheres and PLCL bulk sample is plotted as a function of degradation time, as shown in Fig. 6. Before the degradation, the HAp-PLCL microspheres and PLCL bulk sample melt at 148  C (DH ¼ 17.9 J/g) and 160  C (DH ¼ 28.8 J/g), respectively. Generally, degradation is known to start in the amorphous regions and then extend to the crystalline domains. The mass loss coming from the chain scission and dissolution of resulting water soluble oligomers/ monomers in amorphous domains apparently leads to an increase of crystallinity. For both the HAp-PLCL microspheres and PLCL bulk sample, crystallinity increased progressively after 16 weeks when the weight loss started. However, DH increased from 17.9 J/g to 27.4 J/g before the weight loss started (12 weeks) for the HAp-PLCL microspheres, as shown in Fig. 6. This may be due to the chain rearrangement of amorphous region induced by water uptake that results in the increase of crystallinity [16]: Tm increased from 148  C to 159  C, which should indicate the size increase of crystalline. Thus, the increase of crystallinity was more apparent than mass loss in the early degradation period. For the PLCL bulk sample, a little change was observed in DH until 16 weeks, which might indicate crystallization due to rearrangement of amorphous region was not prosperous comparing to the HAp-PLCL microspheres. 3.2.4. Microsphere morphology and diameter Biodegradable polymers can be classified into two groups on the basis of the mechanism by which they degrade: bulk degradation

Fig. 7. SEM images of HAp-coated PLCL microspheres (25e75 mm) degraded in PBS at 37  C: taken at different degradation times.

Fig. 8. SEM images of pristine PLCL bulk sample surface degraded in PBS at 37  C: taken at different degradation times.

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Fig. 9. Optical micrographs of the dried HAp-PLCL microspheres after loading external pressure: Incubation period (PBS, 37  C) (a) 0 week and (b) 48 weeks.

and surface degradation [17]. In the bulk degradation, a penetration rate of water into the biodegradable polymer matrix is faster than the rate of polymer degradation. This process is homogeneous and degradation occurs at a uniform rate throughout the polymer matrix. On the other hand, in surface degradation, the rate of water penetration into the polymer matrix is slower than the rate of polymer degradation. This process, therefore, is heterogeneous and

degradation reaction confined to the surface of the biodegradable polymer materials. The gross appearance of all the HAp-PLCL microspheres did not change so much over time during degradation. The microspheres can stably disperse in the PBS media and remained white color at least for 48 weeks. SEM studies confirmed that the microspheres retained their spherical morphologies with relatively smooth surfaces (in micrometer dimension) up to 48 weeks in PBS. Taking the mass loss and molecular weight loss results into account, these results indicate that PLCL degraded in bulk degradation mode [17]. Cracks with sizes ranging from a few to a few tens mm were formed for the microsphere samples placed in PBS over 24 weeks (Fig. 7 inset), which can be also observed in the pristine PLCL bulk sample (Fig. 8). OM studies indicated there was no clear crack on the microspheres in PBS, and the cracks observed in SEM images should be formed during drying. The absorbed water is believed to be bound to the PLCL polymer matrix because the HAp-PLCL microspheres became to have carboxylic acid group by degradation, which might cause the formation of cracks by drying. Interestingly, the microspheres placed in PBS over 48 weeks can be easily broken/deformed by external pressure (Fig. 9): cover glass (18 mm  18 mm, 0.1175 g) was placed on the dried HAp-PLCL microspheres and 1.00 kg scale weight was set on the cover glass for 1 min. On the other hand, original HAp-PLCL microspheres before placing in PBS kept their spherical morphology even after addition of the external pressure. These results indicate the microspheres with Mw of below 10,000 were deformable/breakable. Surface morphology change was investigated using SEM at higher magnification (Fig. 10 and Table 2). Percentage of HAp nanoparticle coverage on the PLCL microsphere was 90% before placement in PBS, which indicated 10% of PLCL underlying skin contacted directly with PBS, and remained intact during 1 week in PBS at 37  C. The coverage percentage started to decrease after 1 week from 90% down to 17% throughout the time course for 48 weeks soaked in PBS. We proposed possible mechanism for the HAp nanoparticles to detach from the surface of HAp-PLCL microspheres as shown below. PLCL molecules should be bonded with the HAp nanoparticles at microsphere surface via interaction between carbonyl/carboxyl groups of PLCL and Ca ion rich plane of HAp: the interaction points are multiple. With a proceed of degradation, the molecular weight of PLCL decreased and it should be expected that anchoring effect of the PLCL decreased and the HAp nanoparticles with PLCL oligomer on their surface can detach and disperse into the aqueous media. Laser diffraction studies indicated that the size of HAp-PLCL microspheres did not change during 1 week placing in PBS and

Fig. 10. Magnified SEM images of surface of HAp-PLCL microspheres (25e75 mm) degraded in PBS at 37  C: taken at different degradation times.

S. Fujii et al. / Polymer Degradation and Stability 98 (2013) 377e386 Table 2 Profile for volume-average diameters of the HAp-PLCL microspheres and surface coverage percentage of the microspheres with HAp during in vitro degradation. [2] Volume-average diameter of HAp-PLCL microspheres/mm 0 week 1 week 4 weeks 12 weeks 24 weeks 36 weeks 40 weeks 48 weeks

59 60 59 e e e 58 55

 18  18  18

 20  17

Surface coverage with HAp/% 90 90 84 69 48 33 e 17

slowly decreased from 59  18 mm to 55  17 mm after 1 week (Table 2), retaining their dispersion stability. The decrease in size should be due to the decrease of microsphere volume caused by dissolution of degraded PLCL components (oligomers and monomer) from the microspheres into aqueous media. Dispersion stability of the microspheres should be due to HAp nanoparticles and the carboxylic acid group generated during the degradation on the microsphere surface. 4. Conclusions In vitro degradation behavior of the HAp-PLCL microspheres has been studied in terms of weight, molecular weight, thermal property, and morphological change during an incubation period of up to 48 weeks. It was found that molecular weight decreased rapidly after immersing the microspheres in the buffer solution at 37  C and, on the other hand, the weight of the microspheres started to decrease after 16 weeks, which was the similar degradation profile with the pristine PLCL bulk sample. This result supports the bulk degradation for the HAp-PLCL microspheres. During the degradation, the enthalpy of melting increased progressively, which should be due to the increase of crystallinity because of chain rearrangement of amorphous region induced by water uptake and the preferential degradation of amorphous regions. The HAp nanoparticles detached from the microsphere surface during the degradation, which was confirmed by SEM study. The microspheres were fragile and easy to be broken by external pressure when the molecular weight became at and below 35,000 after 24 weeks incubation period. No/little change in weight of the HAp-PLCL microspheres and molecular weight of PLCL and surface morphology was observed after 1 week in PBS at 37  C, and the microspheres should be useful for the injectable scaffold [11].

[3]

[4]

[5]

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[7]

[8]

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Acknowledgments We are grateful to Dr. Shinya Fukumoto and Dr. Yohei Mima of Osaka City University for fruitful discussions. This work was supported by the R&D of Nano-devices for Practical Utilization of Nanotechnology of the New Energy and Industrial Technology Development Organization (NEDO), Japan. [10]

Appendix A. Supplementary material Supplementary material related to this article can be found at http://dx.doi.org/10.1016/j.polymdegradstab.2012.09.003.

[11]

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