Multi-scale structuring of cell-instructive cellulose nanocrystal composite hydrogel sheets via sequential electrospinning and thermal wrinkling.

Structured hydrogel sheets offer the potential to mimic the mechanics and morphology of native cell environments in vitro; however, controlling the morphology of such sheets across multiple length scales to give cells consistent multi-dimensional cues remains challenging. Here, we demonstrate a simple two-step process based on sequential electrospinning and thermal wrinkling to create nanocomposite poly(oligoethylene glycol methacrylate)/cellulose nanocrystal hydrogel sheets with a highly tunable multi-scale wrinkled (micro) and fibrous (nano) morphology. By varying the time of electrospinning, rotation speed of the collector, and geometry of the thermal wrinkling process, the hydrogel nanofiber density, fiber alignment, and wrinkle geometry (biaxial or uniaxial) can be independently controlled. Adhered C2C12 mouse myoblast muscle cells display a random orientation on biaxially wrinkled sheets but an extended morphology (directed preferentially along the wrinkles) on uniaxially wrinkled sheets. While the nanofiber orientation had a smaller effect on cell alignment, parallel nanofibers promoted improved cell alignment along the wrinkle direction while perpendicular nanofibers disrupted alignment. The highly tunable structures demonstrated are some of the most complex morphologies engineered into hydrogels to-date without requiring intensive micro/nanofabrication approaches and offer the potential to precisely regulate cell-substrate interactions in a "2.5D" environment (i.e. a surface with both micro- and nano-structured topographies) for in vitro cell screening or in vivo tissue regeneration. STATEMENT OF SIGNIFICANCE: While structured hydrogels can mimic the morphology of natural tissues, controlling this morphology over multiple length scales remains challenging. Furthermore, the incorporation of secondary morphologies within individual hydrogels via simple manufacturing techniques would represent a significant advancement in the field of structured biomaterials and an opportunity to study complex cell-biomaterial interactions. Herein, we leverage a two-step process based on electrospinning and thermal wrinkling to prepare structured hydrogels with microscale wrinkles and nanoscale fibers. Fiber orientation/density and wrinkle geometry can be independently controlled during the electrospinning and thermal wrinkling processes respectively, demonstrating the flexibility of this technique for creating well-defined multiscale hydrogel structures. Finally, we show that while wrinkle geometry is the major determinant of cell alignment, nanofiber orientation also plays a role in this process.

Injectable Anisotropic Nanocomposite Hydrogels Direct in Situ Growth and Alignment of Myotubes.

While injectable in situ cross-linking hydrogels have attracted increasing attention as minimally invasive tissue scaffolds and controlled delivery systems, their inherently disorganized and isotropic network structure limits their utility in engineering oriented biological tissues. Traditional methods to prepare anisotropic hydrogels are not easily translatable to injectable systems given the need for external equipment to direct anisotropic gel fabrication and/or the required use of temperatures or solvents incompatible with biological systems. Herein, we report a new class of injectable nanocomposite hydrogels based on hydrazone cross-linked poly(oligoethylene glycol methacrylate) and magnetically aligned cellulose nanocrystals (CNCs) capable of encapsulating skeletal muscle myoblasts and promoting their differentiation into highly oriented myotubes in situ. CNC alignment occurs on the same time scale as network gelation and remains fixed after the removal of the magnetic field, enabling concurrent CNC orientation and hydrogel injection. The aligned hydrogels show mechanical and swelling profiles that can be rationally modulated by the degree of CNC alignment and can direct myotube alignment both in two- and three-dimensions following coinjection of the myoblasts with the gel precursor components. As such, these hydrogels represent a critical advancement in anisotropic biomimetic scaffolds that can be generated noninvasively in vivo following simple injection.

Enhanced Mechanical Properties in Cellulose Nanocrystal-Poly(oligoethylene glycol methacrylate) Injectable Nanocomposite Hydrogels through Control of Physical and Chemical Cross-Linking.

While injectable hydrogels have several advantages in the context of biomedical use, their generally weak mechanical properties often limit their applications. Herein, we describe in situ-gelling nanocomposite hydrogels based on poly(oligoethylene glycol methacrylate) (POEGMA) and rigid rod-like cellulose nanocrystals (CNCs) that can overcome this challenge. By physically incorporating CNCs into hydrazone cross-linked POEGMA hydrogels, macroscopic properties including gelation rate, swelling kinetics, mechanical properties, and hydrogel stability can be readily tailored. Strong adsorption of aldehyde- and hydrazide-modified POEGMA precursor polymers onto the surface of CNCs promotes uniform dispersion of CNCs within the hydrogel, imparts physical cross-links throughout the network, and significantly improves mechanical strength overall, as demonstrated by quartz crystal microbalance gravimetry and rheometry. When POEGMA hydrogels containing mixtures of long and short ethylene oxide side chain precursor polymers were prepared, transmission electron microscopy reveals that phase segregation occurs with CNCs hypothesized to preferentially locate within the stronger adsorbing short side chain polymer domains. Incorporating as little as 5 wt % CNCs results in dramatic enhancements in mechanical properties (up to 35-fold increases in storage modulus) coupled with faster gelation rates, decreased swelling ratios, and increased stability versus hydrolysis. Furthermore, cell viability can be maintained within 3D culture using these hydrogels independent of the CNC content. These properties collectively make POEGMA-CNC nanocomposite hydrogels of potential interest for various biomedical applications including tissue engineering scaffolds for stiffer tissues or platforms for cell growth.