Strong Bioinspired Polymer Hydrogel with Tunable Stiffness and

Letter pubs.acs.org/macroletters

Strong Bioinspired Polymer Hydrogel with Tunable Stiffness and Toughness for Mimicking the Extracellular Matrix Teng Su,*,†,‡,⊥ Yi Liu,§,⊥ Hongjian He,† Jia Li,§ Yanan Lv,† Lili Zhang,§ Yao Sun,*,§ and Chunpu Hu*,∥ †

Department of Chemistry, Advanced Research Institute, Tongji University, Shanghai 200092, China Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695-7115, United States § Shanghai Engineering Research Center of Tooth Restoration and Regeneration, School of Stomatology, Laboratory of Oral Biomedical Science and Translational Medicine, Tongji University, Shanghai 200072, China ∥ Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237, China ‡

S Supporting Information *

ABSTRACT: Inspired by the delicate architecture of hyaline articular cartilage, we report on a biomimetic polymer hydrogel that incorporates strong intermolecular hydrogen bonding between urethane−urethane linkages as well as urethane−ester linkages. The resultant hydrogel, containing ≈75% water, can endure a compressive stress up to 56 MPa with a strain of 98%, and exhibit tunable compressive modulus (0.19−1.38 MPa), as well as toughness (3629−28290 J m−2) within a wide range. The tensile strength and elastic modulus reach as high as 0.56 and 5.5 MPa, respectively. The high stiffness and toughness enable the gel to withstand cyclic compressive loadings without fracturing. Moreover, our hydrogel mimics the extracellular matrices of cartilage and bone tissues and provides biochemical and physical cues that support the three-dimensional proliferation of chondrocytes and osteogenic differentiation of preosteoblasts.

B

Nature employs an intriguing strategy to create highperformance biomaterials with exquisite architecture. Hyaline articular cartilage is a delicate tissue comprising chondrocytes and the ECM that is elegantly organized by collagen (15−20%, by mass), proteoglycans (5−10%), and water (70−80%).22 Hydrophilic proteoglycan aggregates interweave between a dense network of type II collagen fibrils; this structure provides a shock-absorbing matrix that bestow upon articular cartilage a unique combination of high stiffness (compressive modulus 0.51−1.82 MPa), toughness (≈1000 J m−2) and lubrication.23,24 The highly organized and multiphasic nature of cartilage’s ECM is reminiscent of polyurethanes, a fascinating class of multiblock copolymers consisting of rigid hard segments and rubbery soft segments. In these structures, dense hydrogen-bonded assemblies readily form a microphaseseparated morphology that results in excellent mechanical properties. Recently, the fabrication of high-performance polyurethanes with elaborate architecture is receiving considerable attentions and increasingly demanded in frontier technological areas.25−29 However, the hydrogen bonds formed between urethane linkages in hard segments, which play a

one and cartilage support human bodies and protect the vital organs (e.g., brain, heart) from injury. The loss of their functions usually leads to restricted mobility and impaired quality of life. Since its conception by Langer and Vacanti in the mid-1980s, tissue engineering has seen the rapid progress that brings great hope for the regeneration of damaged tissues.1−6 Hydrogels are used intensively as cell scaffolds to mimic the extracellular matrix (ECM) of mammalian tissues. Seminal studies by many pioneering scientists have demonstrated successful cartilage and bone regeneration via the delicate manipulation of cells and their microenvironment (i.e., hydrogel matrix and biophysical and biochemical signals).7−11 However, most hydrogels suffer from low stiffness and tend to break under cyclic compressive loading, which precludes their practical applications to durable, load-bearing tissues. A biomaterial for cartilage substitution, for instance, requires high stiffness and toughness to resist deformation and bear load simultaneously. It remains a challenge for hydrogels to achieve a nice control of the mechanical strength, stiffness, toughness, fatigue resistance, and biological functionalities. Intense efforts are devoted to specially engineered tough hydrogels with exciting biophysical and biochemical properties, including double network hydrogels,12−14 ionic−covalent hybrid hydrogels,15,16 nanocomposite hydrogels,17−19 hydrogen bond crosslinked hydrogels,20 and tetra-PEG hydrogels.21 © XXXX American Chemical Society

Received: September 14, 2016 Accepted: October 14, 2016

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DOI: 10.1021/acsmacrolett.6b00702 ACS Macro Lett. 2016, 5, 1217−1221

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ACS Macro Letters

Figure 1. Schematic description of the hydrogel prepared from ACU macromonomer, MPC, NIPAm, and gelatin, using NaCl particles as porogen. The hydrogel is synthesized utilizing a photoinduced copolymerization in DMSO under (i) UV irradiation combined with a subsequent (ii) porogen leaching process.

method, we can create interconnected macropores of different sizes in the hydrogel, which is beneficial for growing different types of cells. The resultant hydrogel, containing ≈75% water, can endure a compressive stress up to 56 MPa, with a strain of 98%, and exhibit tunable compressive modulus (0.19−1.38 MPa) as well as toughness (3629−28290 J m−2). The tensile strength and elastic modulus reach as high as 0.56 and 5.5 MPa, respectively. The high stiffness and toughness enable the gel to withstand cyclic compressive loadings and never break. Moreover, the cells cultured within the gel show high viability and significant proliferation. Our hydrogel could address the challenge to achieve a nice control of the mechanical strength, stiffness, toughness, fatigue resistance, and biological functionalities for mimicking the ECM of durable load-bearing mammalian tissues. We envision that these hydrogels might open up new possibilities for developing high-performance biomaterials for diverse applications. By the precise control over the stoichiometry of the reactants according to previous studies,33−35 we synthesized four types of ACU macromonomers with different hard segment (HS) contents, designated as ACU-27.4, ACU-33.6, ACU-37.4, and ACU-42.8, respectively (see Experimental Section, Figures S1− S4 and Table S1 in the Supporting Information for details). We noticed that the equilibrium water content (EWC) of the hydrogel decreases when we increase the concentration of ACU (Figures S5 and S6). Unexpectedly, after reaching equilibrium in water, our hydrogel does not undergo further swelling in various aqueous media for months even under physiological

pivotal role in determining the mechanical performance of polyurethane, are vulnerable to polar solvents, such as water. Consequently, the introduction of strong hydrophilic segments into polyurethane backbone, which is a strategy commonly employed when synthesizing hydrogels, leads to uncontrolled swelling in conjunction with reduced mechanical strength.30−32 Herein, we report on a bioinspired polymer hydrogel that can simultaneously achieve ultrahigh strength, stiffness, toughness, and fatigue resistance. Inspired by the multiphasic structures and dense hydrogen-bonded assemblies in the ECM of cartilage, we base our strategy for hydrogel fabrication on several key ideas (Figure 1): (1) The incorporation of a polymerizable acrylated polyurethane (ACU) macromonomer that contains a hydrophobic poly(hexamethylene carbonate) (PHC) as soft segment. Polycarbonate segments can both repel water and form hydrogen bonds with urethane linkages, thus, creating a nonpolar environment for the stabilization of hydrogen bonding between urethane linkages in hard segments against aqueous media. Therefore, the strong intermolecular hydrogen bonding between urethane−urethane linkages as well as urethane−ester linkages could be maintained in hydrogel. (2) The introduction of the highly hydrophilic 2-methacryloyloxyethyl phosphorylcholine (MPC) units can compensate for the hydrophobicity of the hydrogel network. (3) To facilitate the functionalization of gel network with bioactive molecules (e.g., gelatin), N-isopropylacrylamide (NIPAm) is introduced owing to the ability of its polymer to immobilize proteins via noncovalent interactions. (4) By utilizing a porogen leaching 1218

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aggregate and strengthen the gel network (Figure 1). With the increase in ACU concentration, there are more hydrogenbonded hard segments that enhance the modulus of the network, while the polycarbonate segments as well as PMPC and PNIPAm segments act as soft ductile components that efficiently dissipate energy during compressive loading. Intriguingly, the stiffness and toughness of our hydrogel can be readily tuned by changing the chemical structure of the ACU macromonomer. We prepared two types of hydrogels (BP-M and BP-L gels) by using salt porogen of different sizes, small (180−300 μm) and large (425−600 μm), respectively. Both the compressive modulus and toughness display a maximum value at an intermediate HS content (Figure 2c,d and Table S3), which could be explained by the hydrogen bonding behavior of urethane linkages in the gel network (Figure S10 and Table S5). Our hydrogel exhibits excellent self-recovering ability. It can rapidly recover its mechanical strength after a large-amplitude oscillatory breakdown (Figures S11 and S12). It can also realize rubber-like recovery when subjected to a fatigue cyclic compression test at a large compressive strain (ε = 70%) with at least 50 successive loading−unloading cycles or sustain at least 500 successive loading−unloading cycles at a small strain (ε = 20%). It is notable that after unloading, these hydrogels are able to recover their original shapes within 2 h when reimmersed in deionized water (Figure S13). In our hydrogel, the interconnected macropores occur ubiquitously, with the size comparable to that of the salt porogen employed (Figure 3a). Thus, the pore size of the

conditions (Figure S7). These results indicate that strong intermolecular interactions and subtle hydrophilic−hydrophobic balance may exist in the hydrogel, which suppress the expansion of the network. This nonswellable property of our hydrogel is highly desirable for its application in biomedical areas.21,36−38 Compressive tests reveal that the introduction of ACU significantly enhances the mechanical properties of hydrogels. Amazingly, the hydrogel initially prepared with 12.0% ACU shows an astonishing compressive stress up to 56 MPa at 98% strain (Figure 2a). Moreover, both the compressive modulus

Figure 2. (a) Compressive stress−strain curves of the hydrogels initially prepared with different ACU macromonomer (ACU-37.4) concentrations, using 180−300 μm NaCl particles as porogen. (b) Compressive toughness and modulus as a function of the concentration of ACU-37.4 macromonomer. (c) Compressive modulus and (d) toughness of hydrogel vary with the HS content in ACU macromonomer. Error bars show standard deviation (n = 3).

and toughness of the hydrogel increase with increasing ACU concentration. The hydrogel with the maximum ACU concentration exhibits compressive toughness as high as 28290 ± 1280 J m−2, which is 28× the fracture toughness of articular cartilage (≈1000 J m−2),19 while it maintains a high modulus (1.38 ± 0.04 MPa) that is comparable to that of cartilage (0.51−1.82 MPa; Figure 2b and Table S2). The maximum tensile strength and modulus achieved for the hydrogel prepared with 12.0% ACU are 0.56 and 5.5 MPa, respectively, which are over 2.4× and 5.8× higher than those of the hydrogel prepared with 6.6% ACU, whereas the corresponding fracture strain only decreases a little (Figure S8). To the best of our knowledge, this is a rear report of a polymer hydrogel exhibiting excellent mechanical performance that even rivals natural elastomers.39 We interpret the simultaneous enhancement of stiffness and toughness as follows. In our hydrogel, the hydrophilic segments of PMPC and PNIPAm are covalently cross-linked with the hydrophobic polyurethane chains, thus, endowing the gel network with an intrinsic amphiphilic nature. When the gel is placed in an aqueous environment, the polyurethane chains could adopt a folded conformation and cluster together as a result of hydrophobic effect. This process is much like the formation of tertiary structures in natural proteins.40,41 The hydrogen bonds formed between urethane−urethane linkages as well as between urethane−ester linkages could be stabilized in the hydrophobic region where the polyurethane chains

Figure 3. (a) SEM image of BP-M hydrogel. (b) Confocal image (maximum intensity projection) of BP-M hydrogel after selectively staining the gelatin within the gel with 5-(2-aminoethylamino)-1naphthalenesulfonic acid sodium salt (EDANS). (c, d) Threedimensional confocal images of (c) MC3T3-E1 preosteoblasts cultured within BP-M hydrogel and (d) chondrocytes cultured within BP-L hydrogel for 7 days.

hydrogel can be facilely adjusted by varying the size of porogen. Hydrogels with an interconnected macroporous structure and tunable pore sizes are attractive for their practical application in tissue engineering.42,43 Moreover, the gelatin molecules bind effectively to the gel matrix through a noncovalent interaction44−46 and are distributed evenly on the surface of pore walls (Figures 3b and S14 and Table S3). The hydrogels prepared with or without gelatin exhibit similar compressive 1219

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In summary, a bioinspired polymer hydrogel is prepared that incorporates strong intermolecular hydrogen bonding between urethane−urethane linkages as well as urethane−ester linkages. Our hydrogel can simultaneously achieve ultrahigh strength, stiffness, toughness, and fatigue resistance. The mechanical properties can be readily tuned within a wide range by varying the concentration and chemical composition of the acrylated polyurethane macromonomer. Moreover, the pore size of the hydrogel can be easily modulated by using a porogen leaching technique and adaptive to a variety of microstructuring requirements for cell culturing. The hydrogel supports the proliferation of chondrocytes and the osteogenic differentiation of preosteoblasts. Considering the tailorability of the macromolecular architecture of polyurethane and the vast range of hard and soft “building blocks” available,51,52 novel multifunctional hydrogels for the replacement of load-bearing tissues could be created with specific bioactive cues and controlled degradation profile that matches the tissue remodeling process. These studies are currently under way. Our hydrogel may serve as a facile and versatile platform for the development of highperformance biomaterials for diverse applications.

modulus and toughness (Figure S15), indicating that the incorporation of bioactive macromolecules, such as gelatin, does not negatively affect mechanical strength. Our hydrogels have excellent cytocompatibility as confirmed by the cell viability assay (Figures S16−S18). To evaluate the ability of the hydrogels to mimic the matrices of cartilage and bone tissues, we examined the proliferation of two cell lines, that is, chondrocytes and preosteoblasts (MC3T3-E1), within the hydrogels. It has been reported that large pores (400−540 μm) are favorable for chondrocyte proliferation,47,48 while smaller pores (120−325 μm) are suitable for osteoblast growth.49,50 We seeded the chondrocytes into BP-L gels and the MC3T3-E1 cells into BP-M gels, respectively. Live/dead staining analysis reveals that the MC3T3-E1 cells (Figure 3c) as well as the chondrocytes (Figure 3d) are viable and distributed evenly after 7 days of culture within the hydrogels. Real-time polymerase chain reaction (PCR) analysis provides useful information about the changes in tissue-specific gene expression. For the chondrocytes cultured within BP-L hydrogels, type II collagen (Col-II), a hyaline cartilage-specific marker, is significantly up-regulated after 14 days of culture; its expression levels in the two hydrogel (BP-L1 and BP-L3) groups are comparable to that in the control group, where the cells are cultured on standard tissue culture polystyrene plates. Aggrecan (AGG) has similar expression levels across all three groups on day 14 (Figure 4a). For the MC3T3-E1 cells

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.6b00702. Experimental details and additional figures and tables (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Author Contributions ⊥

These authors contributed equally to this work (T.S. and Y.L.).

Figure 4. Real-time PCR analysis for (a) proliferation of chondrocytes and (b) osteogenic differentiation of MC3T3-E1 preosteoblasts cultured within different hydrogels. The expressions of cartilagespecific genes (Col-II and AGG) in chondrocytes and the expressions of osteogenic genes (ALP, OSX, OPN, and BSP) in MC3T3-E1 cells were quantified at 3 and 14 days, respectively. GAPDH was used as a housekeeping gene. The data represent the mean ± standard deviation (n = 3), where *P < 0.05 and **P < 0.01.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 81571017 and 81470715), the Fundamental Research Funds for Central Universities, and the Recruitment Program of Global Experts.

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cultured within BP-M hydrogels, the expression of alkaline phosphatase (ALP), an early marker of osteogenic differentiation, increases markedly after 14 days of osteogenic induction (Figure 4b). Expressions of the bone differentiation markers osterix (OSX), osteopotin (OPN), and bone sialoprotein (BSP) are also evidently enhanced. Moreover, we notice that the MC3T3-E1 cells cultured under osteogenic condition within BP-M3 hydrogel for 14 days show a significant up-regulation in ALP, OSX, and OPN expression compared with those cultured in BP-M1 hydrogel. This result implies that high-stiffness hydrogel may favor the osteogenic differentiation of preosteoblasts. This finding is intriguing and merits further investigation. These results indicate that our hydrogels provide biochemical and physical cues that support cell proliferation and differentiation.

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DOI: 10.1021/acsmacrolett.6b00702 ACS Macro Lett. 2016, 5, 1217−1221