Multivalent Host–Guest Hydrogels as Fatigue-Resistant 3D Matrix for

Sep 20, 2017 - However, hydrogels usually swell under physiological conditions and exhibit inevitable fatigue during excessive mechanical loadings. He...
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Cite This: Chem. Mater. 2017, 29, 8604-8610

Multivalent Host−Guest Hydrogels as Fatigue-Resistant 3D Matrix for Excessive Mechanical Stimulation of Encapsulated Cells Kongchang Wei,†,‡ Xiaoyu Chen,† Rui Li,† Qian Feng,†,¶ and Liming Bian*,†,§,∥,⊥ †

Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, Hong Kong, China Shun Hing Institute of Advanced Engineering and §Shenzhen Research Institute, The Chinese University of Hong Kong, Shatin, Hong Kong, China ∥ Centre for Novel Biomaterials, The Chinese University of Hong Kong, Shatin, Hong Kong, China ⊥ China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou 310000, China ¶ State and Local Joint Engineering Laboratory for Vascular Implants, Key Laboratory for Biorheological Science and Technology of Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400030, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: Fatigue resistance of hydrogels is critical to their applications in load-bearing sites of soft tissues that are usually subjected to continuous loadings, such as joint cartilage. However, hydrogels usually swell under physiological conditions and exhibit inevitable fatigue during excessive mechanical loadings. Here we show that hydrogels cross-linked by multivalent host−guest interactions can effectively dissipate a large fraction of the loading energy (>50%) under excessive compressions (over 80% strain, 1000 cycles) despite their high water contents (95%) under physiological conditions. No fatigue is observed in such highly swollen hydrogels during continuous cyclic compressions. We demonstrate that such hydrogels can be used as 3D cell carriers for excessive mechanical stimulation of the encapsulated stem cells, making them promising soft biomaterials for tissue engineering.

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INTRODUCTION Fatigue resistance is essential for deformable soft biomaterials used for load-bearing tissues, such as articular cartilage, that may need to function continuously for an extended period. As highly hydrated polymeric networks, hydrogels share many attributes with soft tissues and are promising biomaterials for tissue engineering.1−5 However, the use of hydrogels as 3D cell carriers for repairing load-bearing tissues has been severely limited by their mechanical weakness, especially under physiological conditions because of the swelling effect.6,7 To improve the mechanical strength, several types of tough hydrogels have been invented,8−15 including “nonswellable” hydrogels showing retained mechanical robustness with suppressed swelling under physiological conditions.16 No hysteresis or fatigue were found in such “nonswellable” hydrogels. However, most of the other deformable hydrogels reinforced by chemical or physical sacrificial bonds usually exhibit inevitable hysteresis during cyclic loadings even though they can be as tough as cartilage before swelling.12,13,17−24 The recovery of the hysteresis is usually slow and sometimes needs external stimulation, such as heat,12,24 leading to their inevitable fatigue during continuous cyclic loadings without resting.12,18,21,24 Such slow recovery of the hysteresis limits the © 2017 American Chemical Society

application of deformable hydrogels as soft-tissue mimetics to support excessive mechanical stimulation of the encapsulated cells. Multivalent noncovalent interactions have been commonly found in many biological structures and inspired scientists to develop advanced supramolecular hydrogels.25−29 Herein, to address the above-mentioned challenge, we report the facile preparation and excellent fatigue resistance of highly swollen hydrogels cross-linked by multivalent β-cyclodextrin/adamantane (βCD/AD) host−guest interactions. Under physiological conditions, the highly swollen hydrogels can withstand up to 90% compressive strains and rapidly recover spontaneously because of the fast binding kinetics of the host−guest complexes,30,31 giving rise to the excellent deformability and superior fatigue resistance. We also demonstrate that such hydrogels can therefore be used as a 3D matrix for excessive mechanical stimulations of encapsulated stem cells under physiological conditions. This may offer a reliable platform Received: May 29, 2017 Revised: September 19, 2017 Published: September 20, 2017 8604

DOI: 10.1021/acs.chemmater.7b02196 Chem. Mater. 2017, 29, 8604−8610

Article

Chemistry of Materials

Figure 1. (A) Structure of the host−guest macro-crosslinker (HGMC) formed with the host monomer Ac-βCD and the guest polymer ADxHA. (B) Schematic illustration of the hydrogel network with poly(N,N-dimethylacrylamide) (PDMAm) matrix (violet lines) cross-linked by the HGMCs. (C) Reversible host−guest complexation. (D, E) Successful injection of a star-shaped HGMC hydrogel object through a syringe (scale bar: 2 cm). (F) Compressive strain−stress curves of swollen HGMC hydrogels or chemical hydrogels cross-linked by methacrylated hyaluronic acid (Me37HA); the inset shows the Young’s modulus of the hydrogels (3 samples for each group). (G) Strain−stress curves of two compressive cycles of the HGMC hydrogels prepared with 1.0 wt % ADxHA (x = 15, 40, or 80); the inset shows the strain−stress curves of two compressive cycles of the chemical hydrogels cross-linked by Me37HA.

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for investigating the stem-cell behaviors responding to continuous large deformations in 3D environments. It is noteworthy that although reversible host−guest interactions have been widely used for the preparation of smart hydrogels, they usually act as discrete cross-linking points of the polymer networks, leading to the mechanical weakness of the resultant self-assembled hydrogels.32,33 Meanwhile, their contribution to fatigue resistance of host−guest-reinforced hydrogels has rarely been investigated.34,35 In our recent studies, we showed that supramolecular hydrogels of biopolymers, such as hyaluronic acid (HA) and gelatin, can be reinforced by multivalent host−guest interactions provided by biopolymer-derived host−guest macromers without using additional synthetic monomers (HGM hydrogels).36,37 In the current study, HA-derived host−guest macromers are formed by self-assembly of guest polymers (adamantane-functionalized HA, ADxHA) and host monomers (acrylated βCD, Ac-βCD) but act as multivalent crosslinkers of synthetic ductile polymers (Figure 1A). Therefore, they are termed “host−guest macrocrosslinkers” (HGMCs, Figure 1A). The hydrogels formed via the polymerization of the N,N-dimethylacrylamide (DMAm) monomer in the presence of the HGMCs are termed HGMC hydrogels (Figure 1B). Unlike most of the other host−guest supramolecular hydrogels that are not strong enough to form injectable freestanding objects, the HGMC hydrogels crosslinked by multivalent host−guest interactions along the HA backbones are much stronger and can even be injected as swollen objects (Figure 1C−E, Movie S1), thereby distinguishing our multivalent strategy from the other previously reported ones.

EXPERIMENTAL SECTION

Materials. Methacrylated hyaluronic acid (Me37HA), guest polymer ADxHA, and the acrylated β-cyclodextrin host monomer (Ac-βCD) were prepared as described elsewhere.36,37 N,N-dimethylacrylamide (DMAm, 99%; contains 500 ppm monomethyl ether hydroquinone as inhibitor) monomer from Sigma-Aldrich was used after passing through an alumina column. All other chemicals were purchased at the highest purity and were used as received. The human mesenchymal stem cells (hMSCs) were purchased from Lonza. Methods. Preparation of Hydrogel Precursor Solution. For the preparation of the HGMC hydrogels, guest polymer (ADxHA) and host monomer (Ac-βCD) were first dissolved in PBS buffer (pH = 7.4) and then mixed with photoinitiator 2-hydroxy-4′(2hydroxyethoxy)2-methylpropiophenone (I2959, final concentration: 0.05 wt %). For all experiments, the molar ratio between β-CD and AD was always kept as 1:1. N,N-dimethylacrylamide (DMAm, 1 M) monomer was then added into the solution. For the preparation of the chemical hydrogels, Me37HA was used as multivalent crosslinkers instead of the HGMCs. Preparation of Hydrogels for Compressive Test. For the preparation of the HGMC hydrogels, 50 μL of precursor solution containing ADxHA (n wt %), Ac-βCD (m wt %, equal molar ratio to AD), DMAm (1 M, 10.0 wt %), and I2959 (0.05 wt %) was loaded in the homemade mold of a plastic syringe (1.0 mL); a disklike hydrogel object was obtained after the solution was exposed to UV light (λ = 365 nm, 20 mW cm−2) for 30 min, and then swelled in PBS buffer at 37 °C for 40 h before being subjected to compressive studies. For the preparation of the chemical hydrogels, Me37HA was used as multivalent crosslinkers instead of the HGMCs. Preparation of hMSC-Laden Hydrogels. For the preparation of the HGMC hydrogels, 50 μL of precursor solution containing AD15HA (1.0 wt %), Ac-βCD (0.44 wt %), DMAm, (1M, 10.0 wt %), and I2959 (0.05 wt %) was used for suspending 1 million cells. After quick suspension of the cells, the cell-containing precursor solution was 8605

DOI: 10.1021/acs.chemmater.7b02196 Chem. Mater. 2017, 29, 8604−8610

Article

Chemistry of Materials

= 139.3 ± 5.3 kPa for x = 80), but less deformable (Figure 1F and inset). Moreover, the ductile HGMC hydrogel (x = 15) can recover the original shape immediately after the large deformation, as demonstrated by the successful injection of a star-shaped hydrogel object through a small hole of the syringe (Figure 1D,E, Figure S3a,b, and Movie S1). Such ductility is also demonstrated by the ribbon-shaped HGMC hydrogel in the knotted state (Figure S3c) and the disk-shaped HGMC hydrogel resisting the slicing with a cutter (Figure S3d and Movie S2). Such excellent deformability and the rapid recovery of the original shape can be attributed to the effective energy dissipation mediated by the reversible and fast host−guest interactions. Unlike the overlapped stress−strain curves of the chemical hydrogels (Me37HA, Figure 1G inset), significant hysteresis was observed when the HGMC hydrogels were under cyclic compressions, indicating substantial energy dissipation during the compression of the HGMC hydrogels (Figure 1G).39 Moreover, the spontaneous recovery of the hysteresis is fast as made evident by the nearly identical hysteresis loops between two different cycles (Figure 1G). These results indicate that the reversible host−guest crosslinking is important to both the ductility and fast recovery of the HGMC hydrogels. Effective Energy Dissipation, Fast Spontaneous Recovery and Fatigue Resistance of HGMC Hydrogels. Hereafter, HGMC hydrogels with 1.0 wt % ADxHA (x = 15, n = 1.0) will be used for further investigations because of their aforementioned extremely good ductility and high EWC (94.5 wt %) unless otherwise specified. Rheological measurements confirm that the HGMC hydrogels are extremely stable and ductile, yet still maintain the reversible feature of the host− guest cross-linking. Frequency-sweep reveals that no “gel−sol” transition is observed with the frequency as low as 10−3 Hz (Figure 2A), whereas most βCD-AD-based polymeric hydrogels show a clear “gel−sol” transition within the frequency range 0.01−10 Hz.40−42 Such excellent hydrogel stability is attributed to the multivalent effect of host−guest interactions. Amplitude-sweep shows that the swollen HGMC hydrogels are weakened by swelling but are still highly ductile, as revealed by the broad linear viscoelastic region (Figure S4). Meanwhile, the step-strain time-sweep measurement reveals the rapid and full recovery of the HGMC hydrogel structure following large deformations due to the intrinsic reversible nature of the host− guest interactions (Figure 2B). Such a combination of multivalently enhanced network stability, ductility, and rapid recovery of the hydrogels gives rise to the unique macroscopic properties. The representative stress−strain curves of the HGMC hydrogels under increasing compressive strains are presented with offset and magnification for clarity (Figure 2C). Significant hysteresis was observed when the HGMC hydrogels were compressed to a strain above 30% in PBS buffer, indicating substantial energy dissipation during the compression of the HGMC hydrogels.39 The energy-dissipating efficiency (the ratio of the area of the hysteresis loop to the area beneath the loading curve, Figure S5) depends on the loading strains (Figure 2D in PBS), indicating that more host−guest dissociations occur at higher strains. The energy dissipating efficiency is as high as 55% when the HGMC hydrogels are compressed to the strain of 80% (Figure 2D). Furthermore, the strength and the energy-dissipating efficiency were both significantly reduced when the HGMC hydrogels were tested in PBS buffer containing competitive guest molecules (Figure

loaded in the homemade mold of the plastic syringe (1.0 mL); a disklike hydrogel object with hMSCs encapsulated inside was obtained after the solution was exposed to UV light (λ = 365 nm, 7 mW cm−2) for 8 min. The obtained cell-laden hydrogels were immediately washed with PBS buffer (supplemented with 0.01 wt % penicillin/ streptomycin) three times (every 5 min), and growth media (αminimum essential medium with 16.7% fetal bovine serum, 1% penicillin/streptomycin, and 1% L-glutamine) three times (every 10 min). For the preparation of the cell-laden chemical hydrogels, Me37HA was used as multivalent crosslinkers instead of the HGMCs. Unconfined Compressive Test. Unconfined compressive tests were performed on the MACH-1 Micromechanical system. Hydrogels without cells were tested in PBS buffer at 37 °C. Cell-laden hydrogels were tested in chondrogenic medium (DMEM, 1% (v/v) ITS+ Premix, 50 μg mL−1 L-proline, 0.1 μM dexamethasone, 0.9 mM sodium pyruvate, 50 μg mL−1 ascorbate, and antibiotics, supplemented with 10 ng mL−1 TGF-β3) at 37 °C. Culturing of hMSC-Laden Hydrogels. The cell-laden hydrogels were cultured in chondrogenic medium (DMEM, 1% (v/v) ITS+ Premix, 50 μg mL−1 L-proline, 0.1 μM dexamethasone, 0.9 mM sodium pyruvate, 50 μg mL−1 ascorbate, and antibiotics, supplemented with 10 ng mL−1 TGF-β3). The medium was refreshed every day. Confocal Microscopic Observation of Cell Morphologies. The cell-laden hydrogels were washed by PBS buffer and then incubated in PBS solution containing 0.1 vol % calcein AM for 20 min.51 The observations were under a Nikon Eclipse TI microscope (Nikon). ITC Experiment. The isothermal titration experiments were carried out on a MicroCal iTC200 isothermal titration calorimeter (ITC) at 25.00 ± 0.01 °C, using single injection mode titration.36 The PBS solution of guest polymer AD15HA (1.7 μM, containing 0.3 mM of HA disaccharide units) or pure PBS was incubated in the ITC sample cell (200 μL). The host monomer Ac-βCD is injected into the sample cell in one single injection (40 μL).

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RESULTS AND DISCUSSION The swelling and mechanical properties of the HGMC hydrogels can be tuned by adjusting the modification degree (x) and the concentration (n wt %) of ADxHA while keeping the DMAm concentration constant (1 M, 10 wt %) and the molar ratio between Ac-βCD and AD at unity. The HGMC hydrogels have high equilibrium water contents (EWCs, ranging from 75 to 95 wt %, Figure S1) after swelling to the equilibrium state under physiological conditions (37 °C, 40 h, in PBS buffer; Figure S1). The EWC value of the hydrogels decreases when x or n increases because of the higher crosslinking density. Hereafter, the HGMC hydrogels with 1.0 wt % ADxHA (n = 1.0) will be investigated at their equilibrium swollen states because of their high EWCs (>90 wt %, Figure S1). Tunable Mechanical Properties and Excellent Ductility of HGMC Hydrogels. For investigation of the contribution of the reversible host−guest cross-linking to the mechanical properties of the HGMC hydrogels, chemical hydrogels with a PDMAm matrix cross-linked by methacrylated HA (Me37HA, Figure S2) were also prepared and characterized.38 With reversible host−guest interactions as the sacrificial bonds, the HGMC hydrogels are more deformable under compression. The chemical hydrogels (Figure 1F inset; Me37HA, Young’s modulus E = 89.5 ± 3.1 kPa) fractured when the strain reached over 59%, while the HGMC hydrogels can withstand significantly larger deformations, and the failure strain depends on x (Figure 1F). With lower cross-linking density (x = 15, E = 51.8 ± 5.2 kPa), the HGMC hydrogels prepared with AD15HA containing 94.5 wt % water can even withstand a strain up to 90% (Figure 1F and Figure S1). With increasing x, the hydrogels become stiffer (E = 94.1 ± 3.6 kPa for x = 40, and E 8606

DOI: 10.1021/acs.chemmater.7b02196 Chem. Mater. 2017, 29, 8604−8610

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Chemistry of Materials

physical interactions as sacrificial bonds of deformable hydrogels. It was reported that the βCD-AD association rate constant kon is approximately 108 M−1 s−1 and that the dissociation rate constant koff is approximately 2 × 103 s−1, corresponding to a measured equilibrium constant Keq = 4.6 × 104 M−1.30,31 Such fast binding kinetics enables very rapid equilibration compared to the time scale of mechanical testing (i.e., seconds).30 Accordingly, within a broad range of compression frequencies, the loading curves of the HGMC hydrogels exhibit negligible differences (Figure 2F). The HGMC hydrogels can still effectively dissipate the loading energy even when the large deformation (strain: 80%) is imposed in only 1 s (compression frequency: 0.5 Hz), suggesting that the HGMC hydrogels are promising soft materials for repairing load-bearing tissues that are usually subjected to loadings of a similar frequency. It is noteworthy that the unloading curves in the high-strain region (55−80%) shift to higher stress with increasing compression frequencies (Figure 2F, red arrow). Meanwhile, the opposite shift of the unloading curves and the negative compressive stress with increasing loading speeds in the low-strain region (18−55%) was observed (Figure 2F, blue arrow). Such a minor change in the unloading curves could be attributed to the slow dynamics of the flexible polymer chains under stress.45−47 When the compression frequency is higher than 0.025 Hz, the shape recovery of the hydrogel is slightly slower than the movement of the indenter, resulting in the appearance of the residual strain and negative stress, which however has no obvious effect on the following compression cycles and the restoration of the mechanical properties as made evident by the identical consecutive loading curves. Another important feature of such HGMC hydrogels is that their fatigue resistance remains uncompromised under continuous loadings with predescribed residual strains.39 This is also essential for soft biomaterials used for the load-bearing tissues, such as articular cartilage, that may need to function continuously for an extended period without resting. The HGMC hydrogels are compressed to a large deformation (peak strain εp = 80%) and then relaxed to a certain residual strain (εr) in the initial cycle (C0). Subsequently, four consecutive compression cycles (C1−C4) between this residual strain and the peak strain are carried out as demonstrated by the strain− time profile (Figure 3A, inset). For all tested residual strains (from 20% to 70%), the loading−unloading curves are perfectly overlapped without any reduction in the peak stresses, indicating the fast and full recovery of the hydrogel network and mechanical properties. Moreover, all the unloading curves with the varying residual strains follow an identical trace, indicating that the hysteresis recovery is strictly residual-strain dependent (Figure 3B, only C0-loading and C0-unloading curves are presented with the C1-loading curves of varied residual strains). Considering that the energy dissipation of the chemical hydrogels is negligible (Me37HA, Figure 1G inset), the reversible host−guest complexation can be quantitatively related to the strain variation during the loading−unloading cycles, showing more reassociation of host−guest complexes with increasing recovered strains (Figure 3C; Figure S7, the calculation of the reassociation ratio). To the best of our knowledge, such an excellent fatigue resistance of hydrogels under continuous nonresting conditions has never been reported before. Excessive Mechanical Stimulation of Stem Cells Encapsulated in the HGMC Hydrogel 3D Matrix. Mechanical loading is important for the proper development

Figure 2. (A) Oscillatory frequency-sweep measurement reveals that the HGMC hydrogels are extremely stable, and no “gel−sol” transition is observed with the frequency as low as 10−3 Hz. (B) Rheological step-strain oscillatory time-sweep measurements displaying complete and rapid recovery of the hydrogel structure following the highmagnitude deformation (shear strain: 300%) of the HGMC hydrogels. (C) Representative stress−strain curves of the HGMC hydrogels under cyclic compressions with the strain rate of 1.3% s−1. For clarity, the stress−strain curves are offset relative to one another. Final strains are shown on the curves. Inset shows the details of the stress−strain curves with low strain levels. (D) Energy-dissipating ratio (the ratio of the area of the hysteresis loop to the area beneath the loading curve) of the hydrogels compressed to different final strains. (E) Representative loading−unloading curves under continuous 1000 cyclic compressions with a compression frequency of 0.05 Hz. (F) Loading−unloading curves obtained with varying compression frequencies.

2C, 80% + AD; and Figure 2D, in AD/PBS solution). Such responsiveness confirms that the host−guest dissociations are responsible for the energy dissipation. It is remarkable that the content of the HGMCs used for the preparation of the energy dissipating hydrogel is as low as 1.44 wt % (containing 1.0 wt % AD15HA and 0.44 wt % Ac-βCD), indicating that the βCD-AD host−guest complexes are ideal motifs for energy dissipation. The energy required for the rupture of the βCD-AD complexes in these HGMC hydrogels was estimated to be approximately 2.73 kcal mol−1 (ΔH, Figure S6),43 and this value is comparable to the energy needed to rupture the immunoglobulin domain of muscle proteins (4 kcal mol−1).44 Effective energy dissipation and the fast recovery of the resultant hysteresis is essential for fatigue resistance of highly deformable soft materials. The almost identical stress−strain curves for 1000 consecutive cycles of compressions demonstrate the excellent fatigue resistance of the HGMC hydrogels (Figure 2E, and Movie S3; for clarity, only the selected cycles are presented). It is noteworthy that the full recovery of the hysteresis is purely spontaneous without any external assistance such as high temperatures, and the recovery is fast with no need of any resting between cycles because of the fast βCD-AD binding kinetics. This has rarely been achieved by using other 8607

DOI: 10.1021/acs.chemmater.7b02196 Chem. Mater. 2017, 29, 8604−8610

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Chemistry of Materials

Figure 4. (A) Representative strain and stress curves during the cyclic compression of the cell-laden hydrogels. (B) Viability of cells encapsulated in the chemical hydrogels (Me37HA) subjected to continuous cyclic compressions with a strain of 40% or in the HGMC hydrogels (prepared with 1 wt % AD15HA) subjected to 650 continuous compressive cycles with a strain of 80% (as shown in panel A: 0.05 Hz, 4 h). The viability assay is conducted after certain days (1, 3, or 7 days) of culture following the compression (4 samples for each group were evaluated). Reconstructed 3D confocal microscopic image of living cells in (C) the noncompressed hydrogel, (D) the hydrogel being compressed to a strain of 80%, and (E) the hydrogel recovered from the compressed state after the removal of the compression. The scale bar is 50 μm in panels C−E.

Figure 3. Fatigue resistance of HGMC hydrogels under nonresting conditions. (A) Stress−strain curves of the hydrogels during consecutive loading−unloading with varying residual strains (εr) after being compressed to the peak strain (εp). The strain−time profile is demonstrated in the inset. (B) Stress−strain curves from panel A presented in the superimposed position [only the initial loading− unloading curves (C0-loading and C0-unloading) are presented with the C1-loading curves of varied residual strains]. (C) Reassociation ratio of the forced dissociated host−guest complexes depending on the recovered strain, εre (εre = εp − εr).

of musculoskeletal tissues.48 However, the compressive mechanical loadings that can be applied to cell-laden hydrogels have usually been limited within a small range of strain (