Article pubs.acs.org/Macromolecules
Robust and Degradable Hydrogels from Poly(ethylene glycol) and Semi-Interpenetrating Collagen Charles W. Peak, Saumya Nagar, Ryan D. Watts, and Gudrun Schmidt* Weldon School of Biomedical Engineering, Purdue University, 206 South Martin Jischke Drive, West Lafayette, Indiana 47907, United States S Supporting Information *
ABSTRACT: Poly(ethylene glycol) (PEG) and interpenetrating collagen can be used to synthesize hydrogels that are elastomeric-like, can withstand reversible loadings, degrade, and are bioactive. Here we present the synthesis of a hydrogel system made of PEG modified with lactide and acrylate end groups and then photo cross-linked in the presence of type I collagen. The hydrogel precursor solutions are low viscous and the cross-linked hydrogels form elastomeric-like polymer networks. Mechanical properties of the hydrogels were found to depend predominantly on PEG concentration and less on collagen. This is possibly due to a balance of molecular interactions that reinforce and weaken the network structure. Hydrogel degradation times were strongly dependent on temperature. The experimental results from this project show how to generate robust, and degradable hydrogels containing bioactive collagen. The data show promise and show the versatility of making biotechnologically relevant soft materials from a few components.
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INTRODUCTION Robust and tough polymer hydrogels with tunable physical properties can be used to prepare a variety of gadgets and devices for technological and biomedical use. Applications are ranging from robust hydrogels for single use disposable devices to flexible hydrogels for sensors and actuators. Tough hydrogels have been used for microfluidics, “organ on a chip” devices, and food packaging as well as bioactive cell scaffold matrixes.1−6 The combination of synthetic polymers such as poly(ethylene glycol) (PEG) and interpenetrating natural polymers build hydrogels that can withstand physiological loadings, are potentailly degradable, and can support cell growth.7−10 When collagen is chosen as a natural polymer component, bioactivity is added to the hydrogel. Since collagen alone cannot yet be used for making robust and tough hydrogels, the synthesis of interpenetrating hydrogels offers ways to enhance mechanical and maintain biological properties at the same time. Collagen is abundant within tissues, and well studied for many pharmaceutical and biomedical applications. This natural polymer can be utilized to add bioactivity to synthetic matrices. Type I collagen is recognized by integrins on cell surfaces, allowing cells to adhere where present.2,9,11 Physical crosslinking of collagen usually occurs in collagen at 37 °C and at neutral pH in aqueous environments. This process is called fibrillogenesis. Although collagen is responsible for tensile strength in tissue, synthetic collagen hydrogels have very weak mechanical properties and reproducibility can be difficult because of batch to batch variations among other factors. However, when combined with synthetic polymers, mechanical strong hydrogels can be generated.12,13 © XXXX American Chemical Society
The interconnected porous microstructure of hydrogels formed by the combination of PEG and bioactive collagen allows diffusion of molecules.11,12,14−16 Physical cross-linking such as hydrogen bonding, dipole and ionic interactions among others keep the hydrogel from flowing. In aqueous solutions, these reversible interactions may take place between water molecules and adjacent PEG polymer chains, between different collagen molecules and between collagen and PEG chains. Covalent cross-linking of polymers such as PEG is usually irreversible and can be used to enhance mechanical properties, reinforce hydrogels, reduce swelling and prevent dissolution.17−19 Some parameters that can be varied are the polymer architecture (linear versus branched), the cross-link density, molecular weight and type of functional groups. Cross-linking via polymerization of end functionalized PEG can be initiated with light, temperature, pH, or solvent.11,17,18,20−22 For photo polymerization, ideally the samples should be transparent or translucent. The time of the light exposure, distance of the light source to the sample and the concentration of initiator will influence the cross-linking density.12,21,23 Cross-linking of collagen can been done with glutaraldehyde or carbodiimide, however this type of cross-linking chemistry will reduce collagen bioactivity, thus hamper the properties that made collagen advantageous in the first place.12,13,24−27 Therefore, combining PEG with “unmodified“ collagen as semi-interReceived: May 12, 2014 Revised: August 21, 2014
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dx.doi.org/10.1021/ma500972y | Macromolecules XXXX, XXX, XXX−XXX
Macromolecules
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use. The design and synthesis of such materials as well as their property optimization processes are then guided by the commercial application of the end product. An example of semi-IPN hydrogels made from two natural polymers are those reported by Brigham et al. This group photopolymerized solutions of collagen and methacrylated hyaluronic acid12 into robust hydrogels with improved mechanical strength and high cell viability.12 The synthesis of another collagen containing interpenetrating hydrogel was published by Sargeant et al.27 These hydrogels were made degradable by using PEG succinimidyl glutarate in combination with type I collagen. The choice of these polymers allowed for controlling mechanical, degradation, and biological properties.27 We have developed a simple procedure that can be used to generate tough hydrogels from few components and that can be easily scaled up. Our materials can potentially be used for making disposable devices or as flexible hydrogels for sensors and actuators. Other possible applications are seen in microfluidics or as materials for food packaging or bioactive cell scaffold matrixes. Currently, we have no specific application for our hydrogels in mind but instead offer a protocol that can be easily tailored toward the requirements of many different applications. The use of simple and few components in synthesis is very desirable and important in designing soft materials that need to be FDA approved. Below we present a robust and degradable biomimetic hydrogel made from PEG−lactide endfunctionalized with diacrylate (PEG−LA−DA) and type I collagen. In our pervious work we have developed nondegradable hydrogel versions from PEGDA and collagen.47 In this study, we focus on synthesizing an interpenetrating and degradable system with mechanical and degradation properties.
penetrating network would solve some of the above mentioned problems related to the crosslinking of collagen. PEG based hydrogels5,7,28,29 have been evaluated for materials used in disposable devices, sensors and actuators, and microfluidics and as scaffolds for tissue repair and for sealants.30−34 Advantages of using PEG include its bioinert nature, biocompatibility, hydrophilicity, low protein adsorption, and versatility when being chemically modified.5,7,28,29 PEG is widely available and most often used in its linear form. Linear PEG macromolecules have hydroxyl end groups that, using the appropriate chemistry, can be modified with acrylates, amines, maleimide, and aldehyde functionalities thereby changing the chemical, physical, and biological properties.35,36 PEG− diacrylate (PEGDA) is a very common form that has been used to form cross-linked rigid hydrogels.11,17,37−39 Cells can be encapsulated into these PEGDA networks;40 and the PEG can be functionalized e.g. with lactides to allow for cell adhesion, proliferation,15,41 and degradability.42 In addition to composition, the ratio between covalent cross-linking of acrylate end groups and physical cross-linking of collagen to collagen and collagen to PEG, will influence the viscoelastic properties of hydrogels. Alternatively, collagen can be covalently attached to PEG chains directly, and this conjugate can then be used to prepare hydrogels. Scott et al. have investigated PEGDA and collagen conjugate networks.43 The authors found that the conjugation of collagen to the PEG chains allowed for better distribution of collagen molecules within the hydrogel. PEG hydrogel networks were synthesized at 3, 4, and 5 wt % PEGDA with covalently attached collagen added at concentrations ranging from 0.1 to 100 ug/mL. With an increase in collagen, it was found that there was a decrease in the mechanical stiffness of the gel.43 In a different study, the authors prepared hydrogels from PEG−dimethacrylate and poly(lactic acid) (PLA) to be used for encapsulation of chondrocytes.30,44,45 The cross-link density of the hydrogel was found to affect the compressive modulus. High extracellular matrix production was observed when the system was intermediately and densely cross-linked.30 This PEG/PLA hydrogel system appears to be semi-interpenetrating. Other semi-interpenetrating hydrogel networks made from synthetic PEGDA and natural hyaluronic acid have been reported.46 The addition of only small amounts of hyaluronic acid into the PEGDA network significantly improved the elastic moduli of the hydrogel and enhanced spreading and proliferation of encapsulated human dermal fibroblast cells.46 The interpenetrating network (IPNs) or semi-IPN approach can be used to enhance the mechanical properties of hydrogels without hampering bioactivity of the polymer. Any combination of polymers can be used, but often a synthetic polymer and a natural polymer are combined, mixed, and then cross-linked into interpenetrating structures.10,13,14,26 The synthetic polymer may be chosen to generate a network structure with reproducible and controlled mechanical properties. The natural polymer component may provide specific biological properties that affect cellular activity. Among the natural polymers, hyaluronic acid, alginate, collagen, and chondrointin sulfate have been combined with PEG polymers for generating soft materials that satisfy requirements of some biomedical applications. Biodegradable moieties are usually introduced to tailor and fine-tune degradation times for these applications. Other functionalities can be introduced to adjust for biological requirements or mechanical properties. Most of such hydrogels are synthesized in smaller batches and for specific biomedical
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EXPERIMENTAL SECTION
Materials. Poly(ethylene glycol) (Mw = 3400 g·mol−1), lactide monomer, and tin(II) 2-ethylhexanoate were purchased from Fluka Analytical (Sigma-Aldrich, MO). Diethyl ether and dichloromethane were obtained from Mallinckrodt Baker (VWR International, PA). Triethylamine was purchased from EMD Chemicals Inc., and acryloyl chloride was obtained from Alfa Aesar. Irgacure 2959, 1-[4-(2Hydroxyethoxy)phenyl]-2-hydroxy-2-methyl-1-propane-1-one, was received from Ciba AG (Basel, Switzerland). Nutragen, from bovine type I collagen solution, was purchased from Advanced Biomatrix (San Diego, CA) at a stock concentration of 6.0 mg·mL−1. All chemicals were used as received without further processing. Synthesis of Hydrogels. The synthesis of PEG−lactide− diacrylate was done according to a modified procedure from literature.42 The addition of lactide to each of the PEG hydroxyl end groups was done via polymerization with tin(II) 2-ethylhexanoate as catalyst. Briefly, 10 g of PEG, 1.694 g of lactide, and 40 μL of tin(II) 2-ethylhexanoate were stirred under nitrogen, at 180 °C and for ca. 4 h. The resulting product, PEG−lactide (PEG−LA), was dissolved in dichloromethane and precipitated in ice-cold diethyl ether. The precipitate was dried and then purified three times by redissolving in dichloromethane. The dried PEG−LA was used for acrylation. In short, 10 g of PEG−LA was dissolved in 30 mL of dichloromethane containing 2.50 mL of acryloyl chloride. A dichloromethane solution with 12 molar excess of triethylamine was added dropwise to the PEG−LA solution. The reaction was stirred until completion at 0 °C and the product was precipitated in ice-cold diethyl ether and purified several times. The polymer (PEG−LA−DA) was dried in a vacuum. 1 H NMR (CDCl3) was used to verify the product on a Bruker ARX 400 Hz spectrometer. The acrylated product was synthesized and characterized according to an optimized procedure reported in literature.19,42,48 PEG−lactide diacrylate (PEG−LA−DA) was disB
dx.doi.org/10.1021/ma500972y | Macromolecules XXXX, XXX, XXX−XXX
Macromolecules
Article
Figure 1. Left: Schematic of hydrogel synthesis. A solution of PEG−LA−DA (with n = 77 repeat units and m = 1−2 lactide groups) is vigorously mixed with a type I collagen solution to create a precursor solution. The precursor solution is then photo polymerized into a robust hydrogel with geometries appropriate for mechnaical testing. Right: The table inserted shows hydrogel compositions according to PEG−LA−DA wt % and collagen wt %. Colors correspond to the collagen weight percent (orange 0%, green 0.25%, blue 0.50%, red 0.75%) used for graphing purposes (see Figure 2). Bottom right: Schematic representation of a hydrogel network structure made from covalently cross-linked PEG (red) and semiinterpenetrating collagen (black). solved in a photoinitiator solution (0.4% w/v Irgacure 2959 in 1X PBS) and then kept in an ice bath prior to photo cross-linking. Collagen solution was prepared according to a manufacturer’s procedure. Functionalized PEG−lactide−diacrylate solution (10, 15, 20 wt %) was added slowly to the neutralized collagen solution and mixed in an ice bath for 2 h. The resulting solutions were translucent or nearly opaque. The precursor solutions were injected into a glass or silicone mold (thickness = 1 mm) and photo-cross-linked for
