Interest in cellulose nanocrystal (CNC)-based hydrogels for drug delivery, tissue engineering, and other biomedical applications has rapidly expanded despite the minimal in vivo research reported to date. Herein, we assess both in vitro protein adsorption and cell adhesion as well as in vivo subcutaneous tissue responses and CNC biodistribution of injectable CNC-poly­(oligoethylene glycol methacrylate) (POEGMA) hydrogels. Hydrogels with different PEG side chain lengths, CNC loadings, and with or without in situ magnetic alignment of the CNCs are compared. CNC loading has a minimal impact on protein adsorption but significantly increases cell adhesion. In vivo, both CNC-only and CNC-POEGMA injections largely stay at their subcutaneous injection site over one month, with minimal bioaccumulation of CNCs in any typical clearance organ. CNC-POEGMA hydrogels exhibit mild acute and chronic inflammatory responses, although significant fibroblast penetration was observed with the magnetically aligned hydrogels. Collectively, these results suggest that CNC-POEGMA hydrogels offer promise in practical biomedical applications.

Decellularization efforts must balance the preservation of the extracellular matrix (ECM) components while eliminating the nucleic acid and cellular components. Following effective removal of nucleic acid and cell components, decellularized ECM (dECM) can be solubilized in an acidic environment with the assistance of various enzymes to develop biological scaffolds in different forms, such as sheets, tubular constructs, or three-dimensional (3D) hydrogels. Each organ or tissue that undergoes decellularization requires a distinct and optimized protocol to ensure that nucleic acids are removed, and the ECM components are preserved. The objective of this study was to optimize the decellularization process for dECM isolation from human lung tissues for downstream 2D and 3D cell culture systems. Following protocol optimization and dECM isolation, we performed experiments with a wide range of dECM concentrations to form human lung dECM hydrogels that were physically stable and biologically responsive. The dECM based-hydrogels supported the growth and proliferation of primary human lung fibroblast cells in 3D cultures. The dECM is also amenable to the coating of polyester membranes in Transwell™ Inserts to improve the cell adhesion, proliferation, and barrier function of primary human bronchial epithelial cells in 2D. In conclusion, we present a robust protocol for human lung decellularization, generation of dECM substrate material, and creation of hydrogels that support primary lung cell viability in 2D and 3D culture systems

Herein, we comprehensively investigate the internal morphology of fully injectable interpenetrating networks (IPNs) prepared via coextrusion of functionalized precursor polymer solutions based on thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and nonthermoresponsive poly(vinyl pyrrolidone) (PVP) by reactive mixing using kinetically orthogonal hydrazone and thiosuccinimide cross-linking mechanisms. Small-angle neutron scattering, probing both the full IPN as well as the individual constituent networks of the IPN using index-matching, suggests a partially mixed internal structure characterized by PNIPAM-rich domains entrapped in a clustered PVP-rich phase. This interpretation is supported by super-resolution fluorescence microscopy (direct stochastic optical reconstruction microscopy) measurements on the same gels on a different length scale, which show both the overall phase segregation typical of an IPN as well as moderate mixing of PNIPAM into the PVP-rich phase. Such a morphology is consistent with the kinetics of both gelation and phase separation in this in situ gelling system, in which gelation effectively traps a fraction of the PNIPAM in the PVP phase prior to full phase separation; by contrast, such interphase mixing is not observed in semi-IPN control hydrogels. This knowledge has significant potential for the design of an injectable hydrogel with internal morphologies optimized for particular biomedical applications.

Although two-dimensional hydrogel thin films have been applied across many biomedical applications, creating higher dimensionality structured hydrogel interfaces would enable potentially improved and more biomimetic hydrogel performance in biosensing, bioseparations, tissue engineering, drug delivery, and wound healing applications. Herein, we present a new and simple approach to control the structure of hydrogel thin films in 2.5D. Hybrid suspensions containing cellulose nanocrystals (CNCs) and aldehyde- or hydrazide-functionalized poly­(oligoethylene glycol methacrylate) (POEGMA) were spin-coated onto prestressed polystyrene substrates to form cross-linked hydrogel thin films. The films were then structured via thermal shrinking, with control over the direction of shrinking leading to the formation of biaxial, uniaxial, or hierarchical wrinkles. Notably, POEGMA-only hydrogel thin films (without CNCs) did not form uniform wrinkles due to partial dewetting from the substrate during shrinking. Topographical feature sizes of CNC–POEGMA films could be tuned across 2 orders of magnitude (from ∼300 nm to 20 μm) by varying the POEGMA concentration, the length of poly­(ethylene glycol) side chains in the polymer, and/or the overall film thickness. Furthermore, by employing adhesive masks during the spin-coating process, structured films with gradient wrinkle sizes can be fabricated. This precise control over both wrinkle size and wrinkle topography adds a level of functionality that to date has been lacking in conventional hydrogel networks.

Cellulose, the primary component of the plant cell wall, has fueled the wood, textile, pulp and paper industries for centuries, and has recently been used for the production of renewable nanomaterials. The tight crystalline packing of glucan chains within cellulose fibrils is responsible for its superior mechanical properties but renders this material recalcitrant to biochemical and chemical breakdown and limits its use as a green resource. The presence of nanoscale dislocations within cellulose fibrils has been postulated for decades and is thought to be responsible for the production and size of cellulose nanocrystals (CNCs) following acid hydrolysis. However, dislocations have never been directly visualized and their prevalence and size have remained elusive. In this study, we have used super-resolution (SR) fluorescence microscopy to directly visualize and measure alternating crystalline and disordered regions within individual fluorescently labelled bacterial cellulose fibrils. The measured size of the crystalline regions ranges from 40 – 400 nm and shows striking overlap with the length distribution of bacterial CNCs produced through sulfuric acid hydrolysis, supporting the fringed micellar model for the supramolecular structure of cellulose fibrils. The disordered regions were found to be 20 – 120 nm in length and show heterogeneous accessibility, which directs fibril cleavage during the initial stages of cellulose acid hydrolysis. Two-colour SR imaging of cellulose fibrils and bound exoglucanases (Cel7A), in combination with degree of crystallinity measurements suggest that these dislocations are nanoscale in size, and do not result in amorphous cellulose pockets large enough to accommodate enhanced enzyme binding. Through characterization of disordered regions in cellulose fibrils, we have gained insight into the role of cellulose nanostructure in its breakdown by chemical and enzymatic means.