Article pubs.acs.org/cm
Highly Elastic and Tough Interpenetrating Polymer NetworkStructured Hybrid Hydrogels for Cyclic Mechanical LoadingEnhanced Tissue Engineering Oju Jeon,† Jung-Youn Shin,† Robyn Marks,† Mitchell Hopkins,† Tae-Hee Kim,§ Hong-Hyun Park,† and Eben Alsberg*,†,$ †
Departments of Biomedical Engineering and $Orthopaedic Surgery, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, United States § Hathaway Brown School, 19600 North Park Boulevard, Shaker Heights, Ohio 44122, United States S Supporting Information *
ABSTRACT: Although hydrogels are extensively investigated as biomaterials due to their ability to mimic cellular microenvironments, they are often limited by their poor physical properties in response to mechanical loads, including weak gel strength, brittleness, and permanent deformation. Recently, interpenetrating polymer network (IPN) hydrogels have gained substantial attention for their use in investigating changes in encapsulated cell behaviors under mechanical stimulation. However, despite recent success in developing highly elastic IPN-structured hydrogels, it remains a great technical challenge to endow them with biocompatibility and biodegradability due to use of toxic chemicals, nonbiodegradable prepolymers, and harsh reaction conditions. In this study, we report on the synthesis and formation of highly elastic and tough IPN-structured hydrogels based on alginate and gelatin, which are biocompatible and biodegradable. Mechanical stimulation enhanced the proliferation and osteogenic differentiation of encapsulated human mesenchymal stem cells in the IPN-structured hydrogels. These new biocompatible, biodegradable, and tough elastomeric hydrogels provide an exciting platform for studying stem cell behaviors such as proliferation and differentiation under mechanical stimulation and may broaden the applications of hydrogels in the fields of tissue engineering and regenerative medicine.
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INTRODUCTION Tissue engineers aim to drive the formation of functional replacement tissues and organs, often by encapsulating cells with regenerative potential within a biomaterial matrix, presenting specific signals to control their behavior. Hydrogel biomaterials are in high demand for biomedical applications such as tissue engineering scaffolds, drug delivery vehicles, and wound healing dressings1−4 because they possess many physical and biochemical properties that are similar to natural extracellular matrix. The behavior of many cell populations can be modulated during development,5 homeostasis,6 and healing processes7 and in vitro via exposure to mechanical stimulation in the form of compressive, tensile, shear, and/or hydrostatic stress. It is for this reason that a great deal of research has been conducted focusing on understanding the role of mechanical stimulation applied to cells encapsulated in hydrogels to regulate their behaviors such as proliferation and differentiation and create functional tissue engineered constructs.8−12 The application specifically of cyclic compressive stress has been shown to increase stem cell proliferation13,14 and osteogenic15,16 and chondrogenic differentiation17,18 in hydrogels. However, currently, biocompatible, biodegradable hydrogels either permanently deform and/or break under repeated compressive loading; thus, there is a critical need for © 2017 American Chemical Society
highly elastic, strong hydrogels to progress this mechanostimulation strategy in the tissue engineering field. Over the past few decades, intense efforts have been devoted to the engineering of highly elastic and tough hydrogels, including microsphere composite hydrogels,19 nanocomposite hydrogels,20,21 and interpenetrating polymer network (IPN)structured hydrogels.22 Among them, IPN-structured hydrogels have attracted significant interest because of their simplicity of synthesis, impressive mechanical properties, high transparency, and their capacity to respond rapidly to external mechanical stimuli.23,24 These characteristics of IPN-structured hydrogels are desirable for investigating the role of mechanics on encapsulated cell behavior,25,26 but despite recent success in developing IPN-structured elastomeric hydrogels, it remains a great technical challenge to make them cytocompatible and biodegradable due to the use of toxic chemicals and nonbiodegradable prepolymers and involvement of harsh reactions.22,25,27 Alginate is one of the most versatile natural materials known to form hydrogels and has shown great promise as a biomaterial Received: July 17, 2017 Revised: September 6, 2017 Published: September 6, 2017 8425
DOI: 10.1021/acs.chemmater.7b02995 Chem. Mater. 2017, 29, 8425−8432
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Figure 1. Molecular structure and microstructure of highly elastomeric IPN-structured hybrid hydrogels formed by a triple network.
for tissue engineering.28,29 Although ionically cross-linked alginate hydrogels have been extensively investigated as biomaterial scaffolds to encapsulate stem cells, they are often limited under mechanical stimulation by their poor elastic properties.25,27 These alginate hydrogels can be permanently deformed and broken under mechanical stimulation due to their low elasticity and brittleness.27 Methacrylated gelatin (GelMA) also has been widely used in biomedical fields as a biomaterial because it has high biocompatibility and elasticity.30 However, drawbacks of GelMA include its poor mechanical strength and toughness and low biodegradability under physiological conditions.31 Here, IPN-structured hybrid hydrogels, which exhibited highly elastic properties, cytocompatibility, biodegradability, and toughness, based on an ionically cross-linked alginate and photo-cross-linked gelatin were engineered. The effect of alginate oxidation degree on IPN-structured hybrid hydrogel physical properties such as mechanical properties, swelling, and biodegradation was evaluated. Finally, it was determined whether mechanical stimulation could affect behaviors such as proliferation and differentiation of encapsulated stem cells.
are formed by photo-cross-linking of the methacrylate groups with low level UV light and a photoinitiator (Figure 1 and Figure S1b and e in Supporting Information). The completeness of GelMA photo-cross-linking was verified with 1H NMR. After photo-cross-linking, the disappearance of the vinyl methylene peaks in 1H NMR spectra (between 5.5 and 6 ppm)32 indicates complete reaction of the methacrylated groups (Figure S2 in Supporting Information). Alginate can be oxidized (OA) to form aldehyde groups in its polymer backbone, which enhances the rate of hydrolytic degradation of resulting hydrogels.33 Mixing the two macromers together results in imine bond formation via Schiff base reaction32 between the aldehyde groups of the OA and the amine groups of the GelMA (Figures S1c and f and Figure S2 in Supporting Information). As determined by 1H NMR, 54.9% of OA-2 aldehydes and 29.4% of OA-5 aldehydes reacted with amine groups of GelMA to form imine bonds (peak a, Figure S2 in Supporting Information). Homogeneity of the IPN-structured hybrid hydrogels at the microscale was confirmed with fluorescence microscopy (Figure S3 in Supporting Information). While ionically cross-linked alginate hydrogels were mechanically weak and had low elasticity27 (Movie S1 in Supporting Information), the IPN-structured hybrid hydrogels were tough, flexible, and highly elastic (Movie S2 in Supporting Information). When the elastic properties of hydrogels were evaluated by unconfined cyclic compression testing up to 50% strain, the nonoxidized alginate hydrogels (ALG) exhibited pronounced hysteresis and significant permanent deformation after each loading and unloading cycle (Figure 2a and Figure S4a in Supporting Information). The OA hydrogels were too weak to examine their elastic behavior (Figure 2b and Figure
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RESULTS AND DISCUSSION The overall strategy for the formation of the IPN-structured hydrogels is depicted in Figure 1. Alginate contains repeating units of 1,4-linked β-d-mannuronic acid (M unit) and α-lguluronic acid (G unit). The G units across different alginate macromers can form ionic cross-links with divalent cations such as Ca2+ in aqueous solution, resulting in a polymer network (Figure 1 and Figures S1a and d in Supporting Information). To generate a hydrogel with GelMA, which was chosen because of its high elasticity under deformation, cross-linked networks 8426
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Figure 2. Elasticity of hydrogels. Stress−strain hysteresis plots of (a) nonoxidized alginate (ALG, 1.25 w/v %), (b) 2% (theoretical) oxidized alginate (OA-2, 1.25 w/v %), (c) GelMA (10 w/v %), (d) ALG (1.25 w/v %)/GelMA (10 w/v %), (e) OA-2 (1.25 w/v %)/GelMA (10 w/v %), (f) 5% (theoretical) oxidized alginate (OA-5, 1.25 w/v %)/GelMA (10 w/v %), and (g) 10% (theoretical) oxidized alginate (OA-10, 1.25 w/v %)/GelMA (10 w/v %) hybrid hydrogels for 5 cycles of deformation. Strain increased by 10% increments with each successive cycle from 10% to 50%. Fatigue properties of IPN-structured hybrid hydrogels. Stress−strain hysteresis plots of (h) ALG/GelMA and OA-5/GelMA hybrid hydrogels during the 1st and 1000th cycle of loading and unloading and (i) a OA-5/GelMA hybrid hydrogel during the 10 000th and 20 000th cycles to a strain magnitude of 10%.
hydrogels to a high number of moderate strain cycles may be more relevant to their use in tissue engineering strategies.34 To further investigate the long-term resilience and fatigue properties of the IPN-structured elastomeric hybrid hydrogels, unconfined cyclic compression tests at 10% strain for up to 20 000 cycles were performed. The IPN-structured elastomeric hybrid hydrogels exhibited extraordinarily high resistance to fatigue (Figures 2h and i). ALG/GelMA (red line) and OA-5/ GelMA (yellow line) hybrid hydrogels exhibited negligible hysteresis and fully recovered immediately after 1000 cycles of loading and unloading. In addition, OA-5/GelMA fully recovered its original thickness even after 10 000 and 20 000 cycles of loading and unloading (Figure 2i). These results indicate that these IPN-structured hybrid hydrogels have great potential as biomaterials for biomedical applications that require full recovery from large strains and/or long-term cyclic compression.
S4b in Supporting Information). The GelMA hydrogels also showed pronounced hysteresis but fully recovered their original thickness after unloading (Figure 2c). Despite the high elasticity of GelMA hydrogels, their poor ability to withstand loads limits their use in applications that required mechanical strength.31 Although the IPN-structured hybrid hydrogels showed pronounced hysteresis as well, unlike the alginate hydrogels, they all fully recovered their original thickness immediately after each unloading. Additionally, mechanical properties of the IPN-structured hybrid hydrogels were greatly enhanced compared to hydrogels comprised only of a single biopolymer, alginate, or GelMA hydrogel (Figures 2a−g and Figures S4a−g in Supporting Information). The highly elastic nature of the IPN-structured hybrid hydrogels was further confirmed by cyclic tensile testing (Figure S5 in Supporting Information). While the IPN-structured elastomeric hybrid hydrogels responded with excellent shape recovery to physiologically extreme strain levels, fatigue resistance of the 8427
DOI: 10.1021/acs.chemmater.7b02995 Chem. Mater. 2017, 29, 8425−8432
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Figure 3. Hydrogel physical property changes over time. (a) Swelling (N = 3), (b) degradation (N = 3), and (c) modulus (N = 3) of IPN-structured elastomeric hybrid hydrogels. Stress−strain curves of (d) ALG/GelMA, (e) OA-2/GelMA, and (f) OA-5/GelMA hybrid hydrogels over time. *p < 0.05 compared with the other groups at a specific time point. **p < 0.05 compared with the other time points within a specific group.
time was determined as a measure of degradation (Figure 3b). Compared to the ALG/GelMA hybrid hydrogels, OA/GelMA hybrid hydrogels exhibited faster degradation. Due to the higher oxidation degree of alginate in OA-5/GelMA hybrid hydrogels, the degradation rate of OA-5/GelMA hybrid hydrogels was slightly faster than that of the OA-2/GelMA hybrid hydrogels. Because hydrogel degradation can affect the mechanical properties of materials,37,38 unconfined cyclic compression tests (two cycles) were performed over time to evaluate potential changes in the elasticity of the IPN-structured hybrid hydrogels. The elastic modulus of IPN-structured hybrid hydrogels decreased during the course of degradation (Figure 3c). As the oxidation level of alginate increased, the modulus decreased at day 7. After 21 days, the moduli of OA/GelMA hybrid
The swelling ratio and degradation profile of hydrogels are strongly dependent on the chemical properties of the polymer components, the network morphology, and the interaction between the polymer and the solvent.35,36 Because swelling and degradation can alter the elasticity of materials over time,37,38 the effect of oxidation level on swelling ratio changes, and degradation profiles of IPN-structured hybrid hydrogels in Dulbecco’s modified Eagle’s medium (DMEM) was investigated for 42 days. There was no significant difference in swelling ratio among three different hydrogels at day 1 (Figure 3a). After seven days of incubation, the swelling ratios of OA/ GelMA hybrid hydrogels were significantly higher than that of ALG/GelMA hybrid hydrogels. However, there was no significant difference in swelling after 42 days of incubation. The mass loss (%) of IPN-structured hybrid hydrogels over 8428
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Figure 4. IPN-structured elastomeric hydrogels are cytocompatible, and mechanical stimulation on hMSC encapsulated IPN-structured elastomeric hybrid hydrogels enhances hMSC osteogenesis. Live/Dead staining of encapsulated hMSCs in IPN-structured elastomeric hybrid ALG/GelMA (a and c), OA-2/GelMA (b and e), and OA-5/GelMA (c and f) hydrogels at day 0 (a−c) and day 14 (d−f). The scale bars indicate 100 μm. (g) Quantification of DNA content of IPN-structured elastomeric hybrid ALG/GelMA, OA-2/GelMA, and OA-5/GelMA hydrogel/hMSC constructs over time (N = 3). *p < 0.05 compared with the other groups at a specific time point. Quantification of (h) DNA and (i) ALP/DNA (N = 4) in hMSCs encapsulated within hydrogels after 28 days of culture in osteogenic differentiation media. *p < 0.05 compared with stimulation group at a specific time point. **p > 0.05 and ***p > 0.05 compared with day 7 and day 28, respectively, within a specific group; otherwise, p < 0.05. Mineralization of cell−hydrogel constructs analyzed by Alizarin red staining of (j) bulk (upper disks: stimulation group and lower disks: no stimulation group; scale bar indicates 10 mm) and sectioned (k) stimulation and (l) no stimulation constructs (scale bars indicate 200 μm). (m) Quantification of calcium content (N = 4) in the constructs. *p < 0.05 compared with stimulation group at a specific time point. **p > 0.05 compared with day 7 within a specific group; otherwise, p < 0.05.
hydrogels were significantly lower than that of ALG/GelMA hybrid hydrogels. Although the stiffness of OA/GelMA IPNstructured hybrid hydrogels rapidly changed during degradation, they exhibited excellent resilience (Figures 3e and f). ALG/GelMA IPN-structured hybrid hydrogels also maintained their high elasticity even after three weeks of degradation (Figure 3d). All IPN-structured hybrid hydrogels recovered
from applied compressive strain (50%) to their original thickness as evidenced by representative cyclic loading/ relaxation curves taken over the course of 21 days. This result indicates that the all IPN-structured elastomeric alginates/ GelMA hybrid hydrogels are biomaterials that retain their high elasticity in response to large strains during degradation for at least 21 days. 8429
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entiation by measuring alkaline phosphatase (ALP) activity, an early osteogenic differentiation marker. Compared to the no stimulation group, the ALP activity of encapsulated hMSCs in the stimulation group was significantly higher at early time points (days 3 and 7); however, there was no significant difference between groups at day 14 (Figure 4i). It is possible that ALP activity peeked in the stimulation group between 14 and 28 days, as this osteogenic differentiation marker was higher in the no stimulation group at the latter time point. Because mineralization of tissue engineered bone constructs is the definitive marker of stem cell osteogenic differentiation, calcium deposition in the hMSC/hydrogel constructs was visualized by Alizarin red S staining and quantified. Compared to the no stimulation group, more intense Alizarin red staining was observed in the stimulation group at day 28 (Figures 4j−l). Calcium deposition increased over time in both groups and was significantly higher at days 7, 14, and 28 in the stimulation group compared to the no stimulation group (Figure 4m), confirming the day 28 Alizarin red S staining results. These findings demonstrate that mechanical stimulation of the IPNstructured elastomeric hybrid hydrogels enhances osteogenic differentiation of encapsulated stem cells and resulted in bonelike mineralization of the extracellular environment.
Several elastomeric alginate hydrogels based on IPN structure have been developed.25,27,39 While these hydrogels exhibited high elasticity in response to both applied tension and compression, none of them possessed good cytocompatibility or permitted long-term survival of cells encapsulated within them. To verify that the IPN-structured elastomeric alginate/ GelMA hydrogels were cytocompatible and could be used as 3D matrixes for long-term culture of viable encapsulated cells, human bone marrow-derived mesenchymal stem cells (hMSCs) were encapsulated within them and cultured in serum containing media. High hMSC viability was observed throughout all groups for 14 days (Figures 4a−f), indicating that the mixing and photo-cross-linking process, the macromers, the IPN-structured elastomeric hydrogels themselves, and their degradation products are nontoxic to the cells. Quantifying DNA content of the cell-laden IPN-structured elastomeric hybrid hydrogels demonstrated that cells proliferated within the gels, reaching a more than two-fold increase of over day 0 values after 14 and 28 days for the OA/GelMA and ALG/GelMA conditions, respectively (Figure 4g). The hMSCs encapsulated in the OA/GelMA hydrogels exhibited a significantly faster growth rate with greater cell spreading up to 14 days (Figures 4e−g). A potential explanation may be that the greater swelling and faster degradation of IPN-structured OA/GelMA elastomeric hybrid hydrogels (Figures 2a and b) provided more space and weaker network formation, permitting increased cell spreading, migration, and proliferation.40,41 Additionally, these physical changes may allow for improved transport of oxygen and nutrients, which are essential for cell survival and proliferation. This finding also corroborates other reports where increasing the biodegradation rate of hydrogels enhanced the proliferation of encapsulated cells.33,42 Cells are subjected to a variety of physical stresses in vivo,43 and these mechanical stimuli play a key role in controlling cell behaviors. Therefore, mechanical stimulation of cells in biomaterials is an area of increasing interest for tissue engineering and regenerative medicine applications.44,45 When mechanical stress is applied to hydrogels, this stress is transmitted to encapsulated cells and transduced into biochemical signals that can alter cell gene expression and function.46 Dynamic cyclic compression, for example, has been demonstrated to induce osteogenic differentiation of hMSCs within hydrogels and modulate bone-specific extracellular matrix synthesis, resulting in improved mechanical properties of engineered bone.47−49 Due to the nonelastomeric nature of most cytocompatible hydrogels, they may not be able to completely recover from deformation and effectively transmit the defined mechanical stimulation to encapsulated cells. Because elastomeric materials have the potential to efficiently transmit mechanical stimulation to cells,25,50 hMSCs were encapsulated in IPN-structured elastomeric alginate/GelMA hybrid hydrogels. These hydrogel constructs were cultured in osteogenic differentiation media under dynamic cyclic compression to investigate the effect of mechanical stimulation on the osteogenic differentiation of stem cells in this system. The number of encapsulated hMSCs significantly increased over 14 days as a result of mechanical stimulation of the IPNstructured elastomeric hybrid hydrogels compared to the no stimulation group as measured by DNA content (Figure 4h). Cyclic compression may enhance the supply of nutrients and oxygen to the encapsulated hMSCs by perfusing media,51 resulting in the increased cell proliferation. hMSC/hydrogel constructs were then evaluated for hMSC osteogenic differ-
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CONCLUSIONS In this work, we engineered novel cytocompatible, biodegradable, and tough IPN-structured elastomeric hybrid hydrogels based on ionically cross-linked alginate and photo-cross-linked gelatin. While alginate-only hydrogels exhibited significant permanent deformation after unloading, the highly elastic tough IPN-structured hydrogels fully recovered their original thickness from large strains and long-term cyclic strain loading. The physical properties of IPN-structured hydrogels were controllable, and their high elasticity was preserved during degradation. Encapsulated hMSCs maintained long-term high viability in the IPN-structured hydrogels, and mechanical stimulation enhanced their proliferation and osteogenic differentiation. This hydrogel system is an exciting new platform for understanding the influence of mechanical stimulation on cell behavior as the biomaterial can fully recover from large strains and long-term cyclic loading, and it may broaden the application of this class of hydrogels in tissue engineering.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.7b02995.
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Experimental details and additional data (PDF) (ZIP) (XLSX)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Eben Alsberg: 0000-0002-3487-4625 Notes
The authors declare no competing financial interest. 8430
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ACKNOWLEDGMENTS The authors gratefully acknowledge funding from the National Institutes of Health’s National Institute of Arthritis and Musculoskeletal and Skin Diseases under award numbers R01 AR069564 (E.A.), R01AR066193 (E.A.), and T32AR007505 (O.J.). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.
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DOI: 10.1021/acs.chemmater.7b02995 Chem. Mater. 2017, 29, 8425−8432
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DOI: 10.1021/acs.chemmater.7b02995 Chem. Mater. 2017, 29, 8425−8432
