ARTICLE pubs.acs.org/Biomac
Highly Extensible, Tough, and Elastomeric Nanocomposite Hydrogels from Poly(ethylene glycol) and Hydroxyapatite Nanoparticles Akhilesh K. Gaharwar, Sandhya A. Dammu, Jamie M. Canter, Chia-Jung Wu, and Gudrun Schmidt* Purdue University, Weldon School of Biomedical Engineering, West Lafayette, Indiana, United States ABSTRACT: Unique combinations of hard and soft components found in biological tissues have inspired researchers to design and develop synthetic nanocomposite gels and hydrogels with elastomeric properties. These elastic materials can potentially be used as synthetic mimics for diverse tissue engineering applications. Here we present a set of elastomeric nanocomposite hydrogels made from poly(ethylene glycol) (PEG) and hydroxyapatite nanoparticles (nHAp). The aqueous nanocomposite PEG-nHAp precursor solutions can be injected and then covalently cross-linked via photopolymerization. The resulting PEG-nHAp hydrogels have interconnected pore sizes ranging from 100 to 300 nm. They have higher extensibilities, fracture stresses, compressive strengths, and toughness when compared with conventional PEO hydrogels. The enhanced mechanical properties are a result of polymer nanoparticle interactions that interfere with the permanent cross-linking of PEG during photopolymerization. The effect of nHAp concentration and temperature on hydrogel swelling kinetics was evaluated under physiological conditions. An increase in nHAp concentration decreased the hydrogel saturated swelling degree. The combination of PEG and nHAp nanoparticles significantly improved the physical and chemical hydrogel properties as well as some biological characteristics such as osteoblast cell adhesion. Further development of these elastomeric materials can potentially lead to use as a matrix for drug delivery and tissue repair especially for orthopedic applications.
’ INTRODUCTION Injectable biomaterials and precursor solutions that can be crosslinked to form rigid biomaterials are highly desirable in tissue engineering because they allow for applying minimum invasive therapies.15 The development of injectable biomaterials for drug delivery and tissue engineering applications is mainly focused on in situ hydrogels that can take the anatomical shape of the defect site and avoid the need of prefabricated patient-specific scaffolds.59 The porous and interconnected structures of hydrogels support the diffusion of nutrients and wastes in and out of the hydrogel.1012 Such hydrogels usually provide a 3D microenvironment to cells, and the entrapped drugs within the hydrogel network stimulate specific cellular responses that may ultimately lead to functional tissues formation.1115 However, main drawbacks of using injectable hydrogels are weak mechanical properties, which limit their applications to drug delivery and soft tissue repair.8,9,16 Several new approaches using interpenetrating networks and nanocomposite hydrogels have been proposed to overcome some of the above-mentioned limitations. For example, Brigham et al. fabricated semi-interpenetrating (IPN) networks composed of collagen and hyaluronic acid (HA) with robust mechanical structure and cell-adhesive properties.17 These networks consist of molecularly entangled polymer chains of two different polymers. The synergistic interactions of two different polymer networks allow for the design of unique property combinations. The formation of semi-INP networks r 2011 American Chemical Society
was important in obtaining mechanically stiff hydrogels that can support cell adhesion and spreading.17 In a different study, Suri et al. reported on the fabrication of photo-cross-linkable collagen and HA IPNs with control over mechanical and structural properties.18 These materials were used to fabricate cell-laden microwells, microchannels, and other microstructures using a two-step photo-cross-linking process.17,18 Other approaches to formulate hydrogel networks with excellent mechanical properties use multifunctional nanoparticles for reinforcement.1921 Enhanced interactions between organic and inorganic components may result in mechanical property combinations that cannot be achieved by either material alone.2224 An advantage of using nanoparticles is to incorporate bioactive properties. For example, Gaharwar et al. developed a range of silicate cross-linked poly(ethylene oxide) (PEO) nanocomposites with bioactivity and controlled cell adhesion properties.23,2527 This study found that the addition of silicate nanoparticles to PEO promoted cell adhesion, spreading, and proliferation as well as an increase in alkaline phosphatase activity.26 Other studies of nanocomposite hydrogels that describe mechanical properties as well as cell growth include research on materials made of poly(Nisopropylacrylamide) and synthetic silicate nanoparticles.19,28 The Received: January 5, 2011 Revised: February 23, 2011 Published: March 17, 2011 1641
dx.doi.org/10.1021/bm200027z | Biomacromolecules 2011, 12, 1641–1650
Biomacromolecules silicate nanoparticles usually act as covalently bound and multifunctional cross-linkers to the polymer, which leads to mechanical toughness, high tensile moduli, and rapid (de-) swelling in response to temperature changes.19 Cell adhesion on these nanocomposite hydrogels was controlled by changing the temperature from 37 to 25 °C, when a cell sheet could be detached from the surface.28 Most recently, Shin et al. showed that synergistic interactions between ferritin nanoparticles and a poly(vinyl alcohol) matrix resulted in mechanically strong nanocomposite hydrogel actuators that could be further developed into artificial muscles.29 In a different study, Tang et al. fabricated mechanically strong and temperature-sensitive hydrogels composed of N-isopropylacrylamide, 2-vinyl-4,6-diamino-1,3,5-triazine, and poly(ethylene glycol) (PEG).30 These hydrogels may anchor genes on the polymer surface and thus can be used as gene delivery matrix for the repair of load-bearing soft tissues.30 Olsson et al. fabricated highly porous (98%), flexible, and magnetically actuating aerogels (not hydrogels) from bacterial cellulose and magnetic nanoparticles that could be used in microfluidic devices and as electronic actuators.31 Wang et al. reported on the synthesis of highly hydrated nanocomposites from synthetic silicate and dendritic macromolecules to encapsulate proteins for biomedical use.21 Despite having a high water content (ca. 98%), Wang’s nanocomposites are mechanically robust while maintaining self-healing properties. In another study, Pek et al. proposed the use of thixotropic nanocomposite hydrogels made from PEG and silicate nanoparticles for 3D cell culture.20 The porosity in these hydrogels is due to the gel structure, which forms by aggregation of micrometer- and nano-sized aggregates that are themselves aggregates of nanoparticles. This study showed that reversible gelation characteristic of the nanocomposites could be used to entrap cells as well as biological factors. The differentiation of cells could be influenced by changing the matrix stiffness. Among the nanomaterials that have been used for orthopedic biomaterials design, hydroxyapatite nanoparticles (nHAp) is a good candidate because hydroxyapatite is biocompatible, osteoconductive, and already present in natural hard tissue.32 Hydroxyapatite nanoparticles are found to be nonimmunogenic and interact with the natural tissue without eliciting significant inflammatory responses.3336 A variety of nanocomposites have been fabricated from polymers and nHAp;3638 however, very few reports focused on the development of nanocomposite polymer hydrogels with tunable mechanical properties. We are interested in PEG-based nanocomposite hydrogels (PEG-nHAp) that can be formulated and covalently cross-linked to form highly elastic and tough materials. Photopolymerization of PEG is well known because this polymer has been used for numerous biomedical applications.3942 For example, one of the papers mentioning applications reported on the fabrication of photo-cross-linked and degradable PEG hydrogels with tunable mechanical properties.43 Temporal control over the biochemical properties of these hydrogels was used to influence the differentiation of encapsulated cells and to prevent the adhesion of thrombogenic and immunogenic proteins.44 In this work, we show how PEG-nHAp hydrogels can be formulated and injectable precursor solutions can be covalently cross-linked to form highly elastic and tough nanocomposite materials that are mechanically superior to conventional PEG hydrogels. We evaluate the effect of the nHAp on the structure and mechanical properties of the hydrogels and investigate why the addition of nHAp to PEG results in higher extensibility and toughness. Finally, we determine the hydrogel swelling kinetics
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in physiological environments. The new materials described have potential to be further developed as drug delivery matrixes and scaffolds for repair of tissue.
’ MATERIALS AND METHODS Materials. PEG with a molecular weight Mw of 35 000 g/mol (Dalton) was purchased from Fluka Analytical (Sigma-Aldrich). The hydroxyl groups of PEG were acrylated by a five-fold molar excess of acryloyl chloride and a five-fold molar excess of triethylamine in 100 mL of dichloromethane under nitrogen. The reaction was stirred at room temperature for 24 h. The resulting solution was then filtered to remove any precipitates. Afterward PEG-diacrylate (PEGDA) was precipitated out from the filtrate by pouring in cold diethyl ether. The white PEG diacrylate precipitate was isolated and subsequently dried under vacuum for 1 day. The acrylation degree of PEGDA was >80%, as determined by 400 Hz 1H NMR on a Bruker ARX 400 spectrometer and calculated by the ratio of acryl protons of PEG diacrylate (CH2, δ = 5.86.4) to CH2O (δ = 3.63) of PEG. Hydroxyapatite nanoparticles (nHAp) (Ca10(PO4)6(OH)2) with an average size of 2070 nm and a specific surface area of 110 m2/g were purchased from Berkeley Advance Biomaterials (Berkeley, CA) (BABI-HAP-N20-A). The photoinitiator, IRGACURE 2959, was purchased from Ciba AG (Basel, Switzerland). Preparation of nHAp-PEG Nanocomposite Hydrogels. The nanocomposite hydrogels were prepared at ambient temperature (Figure 1). First, the nHAp was dispersed in the initiator solution (0.4% IRGACURE 2959 dissolved in deionized water) and dissolved by vortexing for 10 min and sonicating for 20 min more. It is important to utilize both vortexing and sonication to obtain a uniform distribution of nanoparticles. Then, the PEG diacrylate was added to the nHAp solution and was allowed to mix for 20 min. Finally, the mixture was centrifuged for 1 min at 200 rpm to remove any air bubbles. We did not observe any segregation or phase separation after centrifugation. The composite solution was injected into a mold and then photo-cross-linked for 10 min using a high-intensity UV lamp (B-100AP, Ultra-Violet Products, Upland, CA, wavelength used: 365 nm).The distance between the sample and the UV lamp was kept at 15 mm. The composition of the nanocomposite hydrogels is listed in Figure 1a (by mass fraction). Different types of sample shapes were prepared for the material characterizations. For tensile testing, dumb-bell-shaped specimens were prepared (5 mm in length, 3 mm wide, and 1 mm thick). For the rheological studies, cylindrical samples with a diameter of 20 mm and a height of 1 mm were used. For compression testing, cylindrical samples with 5 mm diameter and 10 mm height were prepared. Cryo-Scanning Electron Microscopy (Cryo-SEM). The microstructure of hydrogels was evaluated using a FEI NOVA nanoSEM (Hillsboro, OR) using 5 kV accelerating voltage. First, hydrogel samples were plunged in a liquid nitrogen slush, then transferred to GATAN Alto 2500 Cryo Units (Warrendale, PA) and cooled to 130 °C. The samples were fractured using a cold scalpel; then, the fractured surfaces were sublimated at 90 °C for 8 min, followed by sputter coating with platinum at 130 °C for 90 s. Afterward, the samples were transferred to a cryo-stage (140 °C) for imaging. Raman Spectroscopy. The Raman system (LabRam, Jobin-Yvon Horiba, Edison, NJ) used consists of a microscope (Olympus BX41), a stigmatic Raman spectrometer, a computer-controlled x-y stage for sample positioning, and a laser source (633 nm heliumneon laser). We prepared the samples by drying the nanocomposite hydrogel films under vacuum for 2 days. Two scans with a detector signal accumulation time of 60 s/ scan provided a good signal-to-noise ratio of Raman bands. Other parameters included: confocal hole size of 1100 μm, a slit width of 250 μm, and a 600 g/mm grating. The spectral data were baseline-corrected by a 5° polynomial fit. The instrument was calibrated using a silicon sample. 1642
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Biomacromolecules
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Figure 1. Preparation of PEG-nHAp nanocomposite hydrogels. (a) Hydrogel solution is prepared by combining nHAp (x = 0, 5, 10, 15%) and PEG diacrylate (20%) in an initiator solution. The solution was uniformly mixed using an ultrasonicator. Afterward, the solution was injected in a mold and crosslinked using UV light. (b) Composition of nanocomposite hydrogels. (c) Different shapes that can be formed from the nanocomposite hydrogels. Dogbone-shaped samples were used for the tensile testing, cylindrical samples were used for compression testing, and thin circular samples were used for rheological testing. The nanocomposite hydrogel can also be fabricated into thin fibers that can be stretched to extreme elongations even after been knotted.
Tensile and Compression Experiments. Tensile and unconfined compressive tests were performed at room temperature on the asprepared samples using an ARES rheometer (TA Instruments) equipped with a 2000 gf transducer (n = 5). For tensile tests, a strain rate of 5 mm/s was applied to determine the mechanical properties of each sample. Elastic modulus, fracture stress, and ultimate strain were calculated from the stressstrain curves. For compression testing, a crosshead speed of 0.01 mm/sec was used. Hysteresis (or energy lost due to permanent deformation) was measured by subjecting the sample to a loading and unloading cycle. The compression limit was set to be 50% strain to protect the load cell. Rheological Experiments. Rheological properties of the nanocomposite hydrogels were measured using an AR2000 stress controlled rheometer (TA Instruments) (n = 3). Stress sweep and frequency sweep experiments were performed at 37 °C using a 20 mm parallel plate geometry and a gap of 500 μm. A solvent trap was used to minimize sample evaporation. For the stress sweep, the oscillatory stress was set ranging from 0.1 to 1000 Pa and the frequency at 1 Hz. For the frequency sweep, the frequency was set ranging from 0.01 to 100 Hz and the oscillatory stress at 10 Pa. All samples were tested immediately after covalent cross-linking with UV light. Swelling Kinetics. The swelling behavior of hydrogels was assessed in phosphate-buffered saline (PBS) at different temperatures (4, 25, and 37 °C) for 96 h (n = 5). As-prepared cylindrical samples with 5 mm diameter and 5 mm height were used for the swelling experiments. The swollen samples were removed from PBS and weighed after blotting off the excess water from the sample surface with a filter paper. The swelling degree was defined as the weight ratio of the net liquid uptake to the asprepared nanocomposite hydrogel hydration degree ¼
ðMt Mo Þ 100% Mo
where Mo is the initial weight and Mt is the wet weight of the nanocomposite hydrogels. In Vitro Cell Culture. MC3T3-E1 subclone 4 mouse preosteoblast cells (American Type Culture Collection) were seeded on fully hydrated hydrogels to determine cell adhesion properties. Cells were grown in MEM Alpha (Gibco) supplemented with 10% fetal bovine serum, 100 U/mL
penicillin, and 100 μg/mL streptomycin. Fully hydrated nanocomposite hydrogel films were cut into 1.5 cm diameter circular discs and sterilized under high-intensity UV radiation for 30 min. Before seeding the cells on the hydrogels, samples were allowed to hydrate in the media for 24 h. Cell adhesion was determined by seeding the hydrogels and control wells with the preosteoblast cells (15 000 cells/cm2) in ultralow attachment 24-well plates (Corning) (n = 3). Twenty-four hours post seeding, cells were fixed using 3.7% formaldehyde solution, and the cytoskeleton of the cells was labeled with Alexa Fluor 488 phalloidin fluorescent dye (Invitrogen). Fluorescence images were taken with an Olympus FV1000 confocal microscope with an excitation wavelength of 488 nm. Representative images are shown. Statistical Analysis. Data are presented as mean ( standard error of the mean values. Statistical analysis was performed using Minitab (version 16, Minitab) to determine the statistical differences. Statistical comparisons were performed with one-way analysis of variance (ANOVA) for an average of three to five replicates. After ANOVA was performed on the data set, Tukey’s method was used to test all pairwise mean comparisons. Statistical significance for all tests was set to be at a p value
