Sol–gel derived nanoscale bioactive glass (NBG) particles reinforced poly(ε-caprolactone) composites for bone tissue engineering

Materials Science and Engineering C 33 (2013) 1102–1108

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Sol–gel derived nanoscale bioactive glass (NBG) particles reinforced poly(ε-caprolactone) composites for bone tissue engineering Bo Lei a, Kwan-Ha Shin a, Da-Young Noh a, In-Hwan Jo a, Young-Hag Koh a,⁎, Hyoun-Ee Kim b, Sung Eun Kim c a b c

Department of Dental Laboratory Science and Engineering, Korea University, Seoul, 136-703, Republic of Korea Department of Materials Science and Engineering, Seoul National University, Seoul, 151-742, Republic of Korea Department of Orthopedic Surgery and Rare Diseases Institute, Korea University Medical College, Seoul, 152-703, Republic of Korea

a r t i c l e

i n f o

Article history: Received 8 April 2012 Received in revised form 5 November 2012 Accepted 29 November 2012 Available online 8 December 2012 Keywords: Bioactive glass Biodegradation Polymer Composite Hard tissue Mechanical properties

a b s t r a c t This study investigated the effect of the addition of sol–gel derived nanoscale bioactive glass (NBG) particles on the mechanical properties and biological performances of PCL polymer, in order to evaluate the potential applications of PCL/NBG composites for bone tissue regeneration. Regardless of the NBG contents (10, 20, and 30 wt.%), the NBG particles, which were synthesized through the sol–gel process using polyethylene glycol (PEG) polymer as a template, could be uniformly dispersed in the PCL matrix, while generating pores in the PCL/NBG composites. The elastic modulus of the PCL/NBG composites increased remarkably from 89 ± 11 MPa to 383 ± 50 MPa with increasing NBG content from 0 to 30 wt.%, while still showing good ultimate tensile strength in the range of 15–19 MPa. The hydrophilicity, water absorption and degradation behavior of the PCL/NBG composites were also enhanced by the addition of the NBG particles. Furthermore, the PCL/ NBG composite with a NBG content of 30 wt.% showed significantly enhanced in vitro bioactivity and cellular response compared to those of the pure PCL. © 2012 Elsevier B.V. All rights reserved.

1. Introduction In biomedical engineering, tissue engineering is one of the most popular approaches for repairing and regenerating the diseased or damaged tissues [1]. Biomaterials, which are indispensable for successful tissue regeneration, must provide special physicochemical, mechanical and biological properties for supporting not only the growth, proliferation and differentiation of cells but also the formation of new tissues [2]. Ideally, these materials should have similar subcellular micro-/ nano-scale features and mechanical properties matched to native tissues, as well as excellent bioactivity and controlled degradation rates [3]. For this goal, thus far, considerable effort has been made to combine flexible, biodegradable polymers and bioactive, stiff inorganic phases, because these organic/inorganic composites can mimic the architecture of natural tissues, e.g. bones [4–6]. As an organic phase, poly (ε-caprolactone) (PCL), approved by the Food and Drug Administration (FDA), has been extensively used for tissue regeneration owing to its excellent biocompatibility and biodegradable nature [7]. In addition, compared to other biodegradable polymers such as polylactic acid (PLA), poly (lactic-co-glycolic acid) (PLGA), chitosan and collagen, PCL possesses high mechanical toughness and easy processability, which makes it more suitable for bone tissue regeneration [8]. However, the pure PCL polymer still suffers from its ⁎ Corresponding author. Tel.: +82 29402844; fax: +82 29093502. E-mail address: [email protected] (Y.-H. Koh). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.11.039

relatively low elastic modulus, hydrophobic nature and limited bioactivity, which limits its wider applications for bone tissue regeneration [9]. Thus far, a variety of inorganic phases have been examined for use as a reinforcement for improving the mechanical properties and biological performances of biodegradable polymers [10–12]. More recently, bioactive glasses (BGs) have drawn increasing interest on account of their excellent bone-forming bioactivity and biodegradation ability, as well as the positive biological effects of their reaction products on osteoblastic responses [13–17]. These materials, which can be readily produced through the melting of oxides containing silica, calcium, phosphate, and sodium, have been utilized as a means of reinforcement for a range of biodegradable polymers [18–20]. More recently, sol–gel derived nanoscale bioactive glass (NBG) particles have been proven to provide enhanced biological response owing to their unique mesoporous structure [20–22], making them to be a very promising reinforcement for biodegradable polymers [18,23]. For example, it was demonstrated that NBG in the form of nanofiber could improve apatite-forming bioactivity and osteoblastic activity, as well as increasing mechanical properties, particularly owing to their extremely large surface area and high length-to-diameter ratio [24]. However, little attention has been paid to evaluate the potential of three-dimensional (3-D) particles with a well-defined morphology, which would be highly beneficial to improve the mechanical properties of polymers [25]. Therefore, in this study, we synthesized NBG particles with a tightly controlled morphology through the sol–gel template method using polyethylene glycol (PEG) polymer as a template and investigated

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their utility as a reinforcement for improving the mechanical properties and biological performances of PCL polymer. The effect of the addition of the NBG particles on the microstructural evolution, mechanical properties, hydrophilicity, degradation behavior, apatite-forming bioactivity and cellular response of the PCL polymer was evaluated. The potential applications of PCL/NBG composites with various NBG contents (0, 10, 20 and 30 wt.%) for bone tissue regeneration are discussed.

2. Materials and method 2.1. Starting materials Tetraethyl orthosilicate (TEOS), triethylphosphate (TEP), calcium nitrate tetrahydrate (CN), polyethylene glycol (PEG, MW= 6000), poly (ε-caprolactone) (PCL, average molecular weight (Mn)= 80,000), dichloromethane (DCM), hydrochloride acid (HCL, 0.1 M) and ammonia hydroxide (28 wt.%) were all purchased from Sigma-Aldrich (SigmaAldrich, St. Louis, MO, USA).

2.2. NBG particles synthesis Nanoscale bioactive glass (NBG) particles with a mole composition of 60% SiO2, 36% CaO and 4% P2O5 were synthesized using the acidcatalyzed sol–gel template method, particularly with an assistance of PEG polymer as the template. Typically, 30 mL of TEOS, 3 mL g of TEP, 18.9 g of CN, 4.8 g of PEG, 16.5 mL of H2O and 7.2 mL of HCl were mixed together using magnetic stirring at room temperature for 2 h. Subsequently, 15 mL of 0.1 N anhydrous ammonia was added to the clear BG sol to induce gelation. After this stage, the BG gels were washed using ethanol for 60 min and then freeze-dried for 24 h, followed by heat-treatment at 600 °C for 2 h.

2.3. PCL/NBG composites production The conventional solvent-casting technique was employed to produce PCL/NBG composites. First, a predetermined content of the NBG particles (10 wt.%, 20 wt.% and 30 wt.% in relation to the PCL polymer) was dispersed into 10 mL of DCM via ultrasonic vibration for 5 min. Next, the PCL pellets were dissolved in the DCM solvent containing the NBG particles and then mixed using magnetic stirring for 10 h. The prepared mixtures were then poured into glass Petri dishes and kept at room temperature for 48 h to remove the DCE solvent, followed by drying in air for 24 h to completely remove any of the liquid phases. Finally, the PCL/NBG composites were dried in a vacuum for another 72 h and kept for further examinations.

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2.5. Mechanical properties testing For the tensile strength tests, the PCL/NBG composites with a thickness of ~65± 8 μm were cut into rectangular strips, ~40 mm in length and 10 mm in width. The specimens were loaded at a cross-head speed of 10 mm/min using a screw-driven load frame (Oriental Testing Machine Co, Korea). The ultimate tensile strength, elastic modulus and elongation at fracture were determined from the stress versus strain responses of the specimens monitored during the tensile strength tests. Four samples were tested for each composite to obtain the mean and standard deviation. 2.6. Hydrophilicity and in vitro degradation tests The hydrophilicity of the PCL/NBG composites was evaluated using water contact angle measurements using an OCA15 contact angle analyzer (Dataphysics Co, Germany). A distilled water droplet size of ~2 μL from a syringe was placed carefully onto the surface of the composites at room temperature. After a period of 20 s, the contact angle was recorded. The mean value and standard deviation (SD) were calculated through testing at five different positions on the same sample. For evaluating the degradation behavior of the PCL/NBG composites, the water adsorption (WA %) and weight loss (WL %) were measured by immersing them at 37 °C in a simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma (i.e., 142 mM Na+, 5 mM K +, 2.5 mM Ca2+, 1.5 mM Mg2+, 147.8 mM Cl−, 4.2 mM HCO3−, 1 mM HPO42−, 0.5 mM SO42−) [26]. Three specimens for each composite were weighed (m1) and then immersed in SBF at 37 °C for various time periods of 1, 3, 7, 14, and 28 days. Thereafter, the specimens were extracted and weighed (m2), where the surface water was adsorbed gently onto them. The weight of the specimens (m3) was measured after drying at 40 °C for 24 h. The water adsorption (WA %) and weight loss (WL %) of the specimens were calculated by considering the m1, m2 and m3. 2.7. Apatite-forming bioactivity test The in vitro apatite-forming bioactivity of the PCL/NBG composites was tested using the simulated body fluid (SBF) at 37 °C for 7 days according to a method reported in the literature [26]. Briefly, the composites with dimensions of 5 mm × 5 mm were immersed in the SBF with the initial pH of 7.40 and placed inside an incubator at a controlled temperature of 37 °C and maintained at this temperature for 7 days. The composites were then extracted and washed three times using ethanol absolute and deionized water, and then finally dried at 37 °C for 24 h. The formation of the apatite layer on the surface of the composites was examined using FE-SEM, ATR-FTIR and X-ray diffraction (XRD, MXP18A-HF, MAC Science, Tokyo, Japan).

2.4. Microstructure and chemical analyses

2.8. In vitro cellular response test

Field emission scanning electron microscope (FE-SEM) (JSM6701F, JEOL, Japan) was used to characterize the surface morphology and microstructure of the NBG particles. The size distribution of the NBG particles was then determined by counting the number of particles > 100 using software image-Pro Plus 6.0 (Media Cybernetics, Inc. USA). The surface morphology and microstructure of the PCL/NBG composites produced with various NBG contents (0 wt.%, 10 wt.%, 20 wt.% and 30 wt.%) were examined by field emission scanning electron microscope (FE-SEM). The chemical compositions of the PCL/NBG composites were characterized using attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR, Nicolet 6700, Thermo Sci., USA). The porosity of the PCL/NBG composites was calculated by considering their apparent density, which could be calculated by measuring their dimensions and mass, and the theoretical densities of the PCL (1.145 g/cm3) and NBG (2.71 g/cm3).

The in vitro cellular response of the pure PCL and PCL/NBG composite with a NBG content of 30 wt.% was evaluated using a pre-osteoblast cell line (MC3T3-E1; ATCC, CRL-2593, Rockville, MD, USA). The MC3T3-E1 cells were cultured in Dublecco's modified eagle medium (DMEM: Welgene Co., Ltd., Seoul, Korea) supplemented with 10% fetal bovine serum (FBS), 1% penicillin–streptomycin, 10 mM β-glycerophosphate (sigma) and 10 μg mL−1 ascorbic acid. Prior to the cell seeding, the samples were soaked in 70% ethanol for 20 min and then in phosphate buffer solution (PBS) for 20 min, followed by washing 3 times by PBS. Subsequently, the cells were plated at a density of 2× 104 cells/mL and cultured in a humidified incubator in an atmosphere containing 5% CO2 at 37 °C for 1 and 3 days. The morphologies of the attached cells on the pure PCL and PCL/NBG composite with a NBG content of 30 wt.% after 1 day and 3 days of culturing were examined by FE-SEM. Prior to these observations, the

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Fig. 1. (A) Typical SEM image and (B) particle size distribution of the NBG particles synthesized through the sol–gel process using a PEG template.

samples were fixed with 2.5% glutaraldehyde for 30 min and then dehydrated for 5 min in graded ethanol (70, 90 and 100% ethanol in sequence), followed by drying at ambient condition. The sample was coated with a thin layer of Pt for the SEM observations. 2.9. Statistics analysis The significant differences between the data were analyzed using a one-way ANOVA test based on the software Origin Lab 8.0 (Microcal Co, USA). The p value of b0.05 (*) was considered to be significant. 3. Results and discussion 3.1. Characteristics of NBG particles Nanoscale bioactive glass (NBG) particles with a narrow particle size distribution were successfully synthesized through the acid-catalyzed

sol–gel template method using PEG polymer as a template, as shown in Fig. 1(A) and (B). The synthesized NBG particles showed wellcontrolled particle morphology without any noticeable agglomerations (Fig. 1(A)). The sizes of the NBG particles were in the range of 50– 150 nm with a mean diameter of 95 nm (Fig. 1(B)). This suggests that the NBG particles synthesized in this study could be effectively used as a reinforcement for improving the mechanical properties and biological performances of PCL polymer. 3.2. Microstructure of PCL/NBG composites The typical SEM images of PCL/NBG composites with various NBG contents (0 wt.%, 10 wt.%, 20 wt.% and 30 wt.%), which were produced using the conventional solvent casting method, are shown in Fig. 2(A)– (L). Basically, regardless of the NBG content, all the produced composites showed good dispersion of the NBG particles in the PCL matrix, which was attributed to the use of the well-defined nanoscale particles

Fig. 2. SEM images showing the top surface ((A)–(D)) and cross-section ((E)–(L)) of the PCL/NBG composites with various NBG contents of 0 wt.% ((A),(E), (I)), 10 wt.% ((B),(F),(J)), 20 wt.% ((C),(G),(K)) and 30 wt.% ((D),(H),(L)).

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(Fig. 2(D), (H), (L)) still showed pores. However, the number and size of the pores decreased with increasing NBG content, which was presumably attributed to an increase in interfacial interaction between the NBG particle and PCL matrix [28]. The porosities of the PCL/NBG composites with the NBG contents of 0 wt.%, 10 wt.%, 20 wt.% and 30 wt.% were 3± 2 vol.%, 19 ±2 vol.%, 11 ±2 vol.% and 8± 5 vol.%, respectively. 3.3. Chemical structure of PCL/NBG composites

Fig. 3. ATR-FTIR spectra of (A) the pure PCL, (B) the PCL/NBG composite with a NBG content of 10 wt.% and (C) the PCL/NBG composite with a NBG content of 30 wt.%.

without severe agglomeration and ultrasonification for dispersing the NBG particles in DCM solvent. However, the morphology of the composites was strongly affected by the content of the NBG particles. The pure PCL revealed a relatively smooth top surface (Fig. 2(A)) and a dense microstructure throughout the sample (Fig. 2(E), (I)). This was primarily attributed to the use of a hydrophobic DCM as a solvent for dissolving the PCL polymer. On the other hand, when a NBG content of 10 wt.% was added, the PCL/NBG composite showed a quite different microstructure, i.e., a number of pores were formed on the top surface and inside the composite (Fig. 2(B), (F)). This change was due to a decrease in the hydrophobicity of the PCL polymer by the presence of hydrophilic NBG particles [20,27]. In addition, the PCL/NBG composites with the NBG contents of 20 wt.% (Fig. 2(C), (G), (K)) and 30 wt.%

The chemical structure of the PCL/NBG composites was evaluated using ATR-FTIR. The typical ATR-FTIR spectrums of the PCL/NBG composites with the NBG contents (0 wt.%, 10 wt.% and 30 wt.%) are shown in Fig. 3(A)–(C). The pure PCL showed the typical characteristic bands of PCL polymer. That is, three dominant adsorption bands at ~1720 cm−1, 1180 cm−1 and 1105 cm−1 corresponding to the representative of C_O, C\O\C and C\O bonds of PCL polymer were observed (Fig. 3(A)) [29]. On the other hand, when the NBG particles were added, an additional adsorption band appeared at ~795 cm−1 [30], which was attributed to the Si\O\Si bond in the sol–gel derived NBG particles (Fig. 3(B), (C)). 3.4. Mechanical properties The mechanical properties of the PCL/NBG composites with various NBG contents (0 wt.%, 10 wt.%, 20 wt.% and 30 wt.%) were evaluated using tensile strength tests. The stress versus strain response of the PCL/NBG composites was strongly affected by the content of the NBG particles, as show in Fig. 4(A)–(D). The pure PCL and PCL–NBB composite with a NBG content of 10 wt.% exhibited a typical characteristic of ductile thermoplastic polymers, i.e., ductile fracture with a high extent of elongation, as shown in Fig. 4(A) and (B). On the other hand, the PCL/NBG composites with the NBG contents of 20 wt.% and 30 wt.%

Fig. 4. Typical stress versus strain responses of the PCL/NBG composites with various NBG contents of (A) 0 wt.%, (B) 10 wt.%, (C) 20 wt.% and (D) 30 wt.%.

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Fig. 5. (A) Ultimate tensile strength (UTS) and (B) elastic modulus of the PCL/NBG composites as a function of the NBG content.

3.5. Hydrophilicity and degradation behavior

Table 1 Contact angle of the PCL/NBG composites as a function of the NBG content. NBG content [wt %] Contact angle [°]

0

10

20

30

75 ± 3

27 ± 3

41 ± 5

29 ± 8

displayed a considerable decrease in the strain at failure with negligible plastic deformation, as shown in Fig. 4(C) and (D). The ultimate tensile strength (UTS) and elastic modulus of the PCL/ NBG composites with various NBG contents (0 wt.%, 10 wt.%, 20 wt.% and 30 wt.%) are plotted as a function of the NBG content, as shown in Fig. 5(A) and (B), respectively. The addition of the NBG particles to the PCL polymer resulted in a decrease in the ultimate tensile strength (Fig. 5(A)), as is often the case with inorganic reinforced polymer composites [10,28,31]. However, the PCL/NBG composites with various NBG contents (10 wt.%, 20 wt.% and 30 wt.%) still showed high ultimate tensile strengths in the range of 15–19 MPa. On the other hand, the elastic modulus increased remarkably from 89 ± 11 MPa to 383 ± 50 MPa (Fig. 5(B)), owing to the presence of the stiff NBG particles in the PCL/NBG composites. These high mechanical properties could be attributed to good dispersion of the NBG particles that have a welldefined morphology with a narrow size distribution [31].

The hydrophilicity of the PCL/NBG composites with various NBG contents (0 wt.%, 10 wt.%, 20 wt.% and 30 wt.%) was evaluated by measuring their incident contact angles, as summarized in Table 1. The PCL/NBG composites with a range of NBG contents (10 wt.%, 20 wt.% and 30 wt.%) showed contact angles in the range of 29±9–37±2°, which was much lower than that (75 ±3°) of the pure PCL. This was attributed to the presence of the hydrophilic NBG particles in the PCL/ NBG composites. The increase in the hydrophilicity by means of the addition of the NBG particles to the PCL polymer led to the considerable increases in the water absorption and weight loss of the PCL/NBG composites, as shown in Fig. 6(A) and (B), respectively. In addition, the water adsorption and weight loss increased with increasing NBG content, which could be attributed to the favorable degradation of the NBG particles [32]. The PCL/NBG composite with a NBG content of 30 wt.% exhibited water adsorption of 20% and weight loss of 16% after soaking in the SBF for 28 days. It is well recognized that the pure PCL polymer has negligible degradation ability in the in vitro environment with no lipasetype enzymes as assistants [33]. This suggests that the weight loss of the PCL/NBG composites was mainly attributed to the degradation of the NBG particles. However, the degree of the weight loss decreased after 7 days, suggesting the extensive precipitation of apatite crystals on the PCL/NBG composites owing to the presence of the bioactive NBG particles [32].

Fig. 6. (A) Water adsorption and (B) weight loss of the PCL/NBG composites with various BGM contents (0 wt.%, 10 wt.%, 20 wt.% and 30 wt.%) as a function of the soaking time.

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3.6. Apatite-forming bioactivity The in vitro bioactivity of the pure PCL and PCL/NBG composite with a NBG content of 30 wt.% was evaluated by soaking them in SBF for 7 days at 37 °C. The pure PCL did not exhibit any sign of the precipitation of apatite crystals on its surface (Fig. 7(A)), suggesting its poor bioactivity. On the other hand, the surface of the PCL/NBG composite became very porous after soaking in SBF for seven days due to a vigorous precipitation of apatite crystals (Fig. 7(B)). The precipitated apatite crystals showed a high aspect ratio with ~15–25 nm in width and ~100–200 nm in length (Fig. 7(C)). The crystalline structure of the apatite crystals precipitated on the PCL/NBG composites after 7 days in SBF at 37 °C was evaluated by XRD, as shown in Fig. 8(A) and (B). Before soaking in SBF, the composite showed only peaks at 2θ = 21° and 23°, which corresponded to those of PCL polymer [34] (Fig. 8(A)). On the other hand, crystalline peaks at 2θ = 26°, 32° and 41° were observed after soaking in SBF for 7 days, which corresponded well to those of hydroxyapatite (HA) crystalline phase (JCPDS 09-0432) (Fig. 8(B)) [35]. This suggests that the apatite

Fig. 8. XRD pattern of the PCL/NBG composite with a NBG content of 30 wt.% (A) before and (B) after soaking in SBF for 7 days.

crystals were formed vigorously on the surface of the PCL/NBG composite owing to the excellent apatite-forming bioactivity of the NBG particles. 3.7. In vitro cellular response The in vitro cellular response of the PCL/NBG composite with a NBG content of 30 wt.% was assessed by in vitro cell tests using MC3T3-E1 cells, in order to evaluate its potential applications in bone tissue engineering. For comparison purpose, the pure PCL was also tested. Basically, both the PCL and PCL/NBG composite showed good cellular response, where cells adhere and spread on their surfaces after 1 day of culturing (Fig. 9(A),(B)). However, the PCL/NBG composite showed better cell spreading with filopodia-like extension and branching. In addition, the surface of the PCL/NBG composite was almost covered with a cellular layer (Fig. 9(D)) after 3 days of culturing. These findings suggest that the hydrophilicity, surface roughness, degradation behavior and apatite-forming ability of the PCL could be significantly improved by the addition of the bioactive NBG particles, consequently resulting in a considerable improvement in cellular response [36,37]. However, a further study should be carried out to characterize the release of ions from the PCL/NBG composites, which would affect their cellular response in vitro and in vivo. 4. Conclusions We herein produced PCL/NBG composites with various NBG contents (0 wt.%, 10 wt.%, 20 wt.% and 30 wt.%) and evaluated their microstructural evolution, mechanical properties, biodegradation behavior and in vitro bioactivity. NBG particles synthesized through the sol–gel process using a PEG template showed a well-defined morphology with a narrow size distribution, which allowed them to be dispersed uniformly in the PCL matrix. This led to a considerable increase in the elastic modulus 89±11 MPa to 383±50 MPa with increasing NBG content from 0 wt.% to 30 wt.%. Furthermore, the addition of the NBG particles remarkably increased the hydrophilicity, water absorption and weight loss of the PCL/NBG composites. The apatite crystals with a high aspect ratio were vigorously formed on the surface of the PCL/NBG composite with a NBG content of 30 wt.% when immersed in SBF for seven days, suggesting its excellent in vitro bioactivity. These findings suggest that the PCL/NBG composite with a NBG content of 30 wt.% with reasonably high mechanical properties, excellent in vitro apatite-forming ability and good cellular response would have great potential for bone tissue regeneration. Acknowledgments

Fig. 7. SEM images of the pure PCL ((A)) and PCL/NBG composite with a NBG content of 30 wt.% ((B),(C)) after soaking in SBF for 7 days.

Contract grand sponsor: Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea; contract grant number: A110416.

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Fig. 9. SEM images of the MC3T3-E1 on the pure PCL ((A),(C)) and the PCL/NBG composite with a NBG content of 30 wt.% ((B),(D)) after 1 day ((A),(B)) and 3 days ((C),(D)) of culturing.

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