Injectable Anisotropic Hydrogels

De France, Kevin J. (McMaster University)
Yager, Kevin G. (Brookhaven National Laboratory)
Chan, Katelyn J.W. (McMaster University)
Corbett, Brandon (McMaster University)
Cranston, Emily D. (McMaster University)
Hoare, Todd (McMaster University)

Introduction

While injectable hydrogels have several advantages in the context of experimental medicine and tissue engineering, their relatively weak mechanical properties and isotropic network structure often limit their practical applications or translation to clinical use. By physically incorporating rigid high aspect ratio cellulose nanocrystals (CNCs) into hydrazone crosslinked poly (oligoethylene glycol methacrylate) (POEGMA) hydrogels, we have shown an ability to significantly enhance the mechanical performance of these injectable gels. Furthermore, by utilizing the diamagnetic susceptibility of CNCs, their in situ alignment may be controlled via the application of an external magnetic field, leading to the first example of an injectable hydrogel with nanoscale anisotropy. Herein, we leverage our capacity to control the kinetics of both CNC alignment and POEGMA gelation to develop a new nanocomposite injectable hydrogel platform with readily tailorable anisotropic properties that can better mimic native muscle tissue microenvironments and direct the orientation of differentiated skeletal muscle myotubes in situ.

Materials and Methods

CNCs were extracted through traditional sulfuric acid-mediated hydrolysis of cotton. POEGMA copolymers were synthesized via free radical polymerization to contain either hydrazide or aldehyde functionality. Hydrogels were prepared by coextruding the aldehyde and hydrazide copolymer solutions from a double barrel syringe. CNCs were incorporated at different loading concentrations (0.2 – 1.65 wt%) in equal amounts in both barrels. Mixtures were then extruded into molds and allowed to gel as is, or in between a rare earth magnet (up to 1.2 T) to facilitate CNC alignment. The effect of exposure to a magnetic field was investigated through small angle X-ray scattering (SAXS). Cell growth experiments were conducted using C2C12 mouse myoblasts plated on top of or encapsulated within both aligned and non-aligned nanocomposite hydrogels. Growth, differentiation, and staining experiments were all performed according to manufacturer recommended protocols. Image analysis was conducted in Matlab following previously reported protocols for quantifying alignment.

Results

CNCs were mixed at various concentrations (0.2 – 1.65 wt%) with solutions of both hydrazide and aldehyde-functionalized POEGMA and were subsequently loaded into either side of a double barrel syringe and extruded in the presence or absence of a magnetic field to form hydrogels. If no magnetic field is applied, the resulting hydrogel appears isotropic (i.e., no preferential CNC orientation is observed via SAXS). However, exposure of the in situ gelling nanocomposite hydrogel to a magnetic field (0.56 - 1.2 T) induces quantifiable CNC orientation/anisotropy. CNC alignment also induces changes in hydrogel physical properties including swelling and mechanical strength. Magnetically aligned hydrogels swell significantly more than their isotropic counterparts, attributed to increased planar alignment of CNCs in the direction perpendicular to the applied magnetic field. Furthermore,CNC alignment results in anisotropic mechanical responses of the hydrogels (decreased shear modulus and increased compressive modulus) relative to the direction of CNC alignment. Finally, following C2C12 myoblast growth and differentiation on these nanocomposite hydrogels, significant differences in myotube alignment were observed between aligned and unaligned gels.

Discussion and Conclusion

In summary, we have developed an injectable nanocomposite hydrogel platform with tunable anisotropic properties facilitated by the application of an external magnetic field. CNCs were shown to align in situ on the order of minutes within the chemically crosslinked POEGMA hydrogel matrix, whereby CNC alignment remained "frozen" upon complete hydrogel gelation. We anticipate that this injectable hydrogel platform will be particularly relevant for engineering oriented tissues (e.g., muscle, cartilage, and nerve) by remotely inducing the required anisotropy in the scaffold using a safe and fully non-invasive magnetic field stimulus following simple injection, thus avoiding the need for surgical procedures.


Schematic representation of injectable CNC-POEGMA nanocomposite hydrogels. After reactive coextrusion, the mixture was placed within a magnetic field to align CNCs inside the POEGMA hydrogel network. Alignment is demonstrated via small angle x-ray scattering.

Small angle x-ray scattering linecuts for 1.65 wt% CNC gels (A) not exposed to a magnetic field during gelation and (B) exposed to a 1.2 T magnetic field. TEM images of the respective hydrogels (C, D) are shown, along with a plot of the orientational order parameter (E) and kinetics of alignment (F)

(A) C2C12 myoblasts cultured on TCPS control, unaligned, and aligned hydrogels for 48 hours. (B) Differentiated myotubes on TCPS, unaligned, and aligned hydrogels after culture for subsequent eight days. (C) Image analysis on myotube F-actin filaments, and (D) histograms of their orientational order

Acknowledgements

Funding from the Natural Sciences and Engineering Research Council of Canada (Discovery Grants RGPIN 356609 and 402329) as well as NSERC CREATE-IDEM (Integrated Design of Extracellular Matrices, grant 398058) is gratefully acknowledged.

References

1. De France, K. J.; Yager, K. G.; Chan, K. J. W.; Corbett, B.; Cranston, E. D.; Hoare, T. Nano Letters 2017, 17 (10), 6487–6495.

2. De France, K. J.; Chan, K. J. W.; Cranston, E. D.; Hoare, T. Biomacromolecules 2016, 17 (2), 649–660.

3. De France, K. J.; Yager, K. G.; Hoare, T.; Cranston, E. D. Langmuir 2016, 32 (30), 7564–7571.

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