Unraveling the Supramolecular Structure and Nanoscale Dislocations of Bacterial Cellulose Ribbons Using Correlative Super-Resolution Light and Electron Microscopy.

Cellulose is a structural linear polysaccharide that is naturally produced by plants and bacteria, making it the most abundant biopolymer on Earth. The hierarchical structure of cellulose from the nano- to microscale is intimately linked to its biosynthesis and the ability to process this sustainable resource for materials applications. Despite this, the morphology of bacterial cellulose microfibrils and their assembly into higher order structures, as well as the structural origins of the alternating crystalline and disordered supramolecular structure of cellulose, have remained elusive. In this work, we employed high-resolution transmission electron and atomic force microscopies to study the morphology of bacterial cellulose ribbons at different levels of its structural hierarchy and provide direct visualization of nanometer-wide microfibrils. The non-persistent twisting of cellulose ribbons was characterized in detail, and we found that twists are associated with nanostructural defects at the bundle and microfibril levels. To investigate the structural origins of the persistent disordered regions that are present along cellulose ribbons, we employed a correlative super-resolution light and electron microscopy workflow and observed that the disordered regions that can be seen in super-resolution fluorescence microscopy largely correlated with the ribbon twisting observed in electron microscopy. Unraveling the hierarchical assembly of bacterial cellulose and the ultrastructural basis of its disordered regions provides insights into its biosynthesis and susceptibility to hydrolysis. These findings are important to understand the cell-directed assembly of cellulose, develop new cellulose-based nanomaterials, and develop more efficient biomass conversion strategies.

The CaT stretcher: An open-source system for delivering uniaxial strain to cells and tissues (CaT).

Integration of mechanical cues in conventional 2D or 3D cell culture platforms is an important consideration for in vivo and ex vivo models of lung health and disease. Available commercial and published custom-made devices are frequently limited in breadth of applications, scalability, and customization. Herein we present a technical report on an open-source, cell and tissue (CaT) stretcher, with modularity for different in vitro and ex vivo systems, that includes the following features: 1) Programmability for modeling different breathing patterns, 2) scalability to support low to high-throughput experimentation, and 3) modularity for submerged cell culture, organ-on-chips, hydrogels, and live tissues. The strategy for connecting the experimental cell or tissue samples to the stretching device were designed to ensure that traditional biomedical outcome measurements including, but not limited to microscopy, soluble mediator measurement, and gene and protein expression remained possible. Lastly, to increase the uptake of the device within the community, the system was built with economically feasible and available components. To accommodate diverse in vitro and ex vivo model systems we developed a variety of chips made of compliant polydimethylsiloxane (PDMS) and optimized coating strategies to increase cell adherence and viability during stretch. The CaT stretcher was validated for studying mechanotransduction pathways in lung cells and tissues, with an increase in alpha smooth muscle actin protein following stretch for 24 h observed in independent submerged monolayer, 3D hydrogel, and live lung tissue experiments. We anticipate that the open-source CaT stretcher design will increase accessibility to studies of the dynamic lung microenvironment through direct implementation by other research groups or custom iterations on our designs.

Recent Microscopy Advances and the Applications to Huntington's Disease Research.

Huntingtin is a 3144 amino acid protein defined as a scaffold protein with many intracellular locations that suggest functions in these compartments. Expansion of the CAG DNA tract in the huntingtin first exon is the cause of Huntington's disease. An important tool in understanding the biological functions of huntingtin is molecular imaging at the single-cell level by microscopy and nanoscopy. The evolution of these technologies has accelerated since the Nobel Prize in Chemistry was awarded in 2014 for super-resolution nanoscopy. We are in a new era of light imaging at the single-cell level, not just for protein location, but also for protein conformation and biochemical function. Large-scale microscopy-based screening is also being accelerated by a coincident development of machine-based learning that offers a framework for truly unbiased data acquisition and analysis at very large scales. This review will summarize the newest technologies in light, electron, and atomic force microscopy in the context of unique challenges with huntingtin cell biology and biochemistry.

Efficient Labeling of Nanocellulose for High-Resolution Fluorescence Microscopy Applications.

The visualization of naturally derived cellulose nanofibrils (CNFs) and nanocrystals (CNCs) within nanocomposite materials is key to the development of packaging materials, tissue culture scaffolds, and emulsifying agents, among many other applications. In this work, we develop a versatile and efficient two-step approach based on triazine and azide-alkyne click-chemistry to fluorescently label nanocelluloses with a variety of commercially available dyes. We show that this method can be used to label bacterial cellulose fibrils, plant-derived CNFs, carboxymethylated CNFs, and CNCs with Cy5 and fluorescein derivatives to high degrees of labeling using minimal amounts of dye while preserving their native morphology and crystalline structure. The ability to tune the labeling density with this method allowed us to prepare optimized samples that were used to visualize nanostructural features of cellulose through super-resolution microscopy. The efficiency, cost-effectiveness, and versatility of this method make it ideal for labeling nanocelluloses and imaging them through advanced microscopy techniques for a broad range of applications.

A Robust Protocol for Decellularized Human Lung Bioink Generation Amenable to 2D and 3D Lung Cell Culture.

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.

Tuning the Nanotopography and Chemical Functionality of 3D Printed Scaffolds through Cellulose Nanocrystal Coatings.

In nature, cells exist in three-dimensional (3D) microenvironments with topography, stiffness, surface chemistry, and biological factors that strongly dictate their phenotype and behavior. The cellular microenvironment is an organized structure or scaffold that, together with the cells that live within it, make up living tissue. To mimic these systems and understand how the different properties of a scaffold, such as adhesion, proliferation, or function, influence cell behavior, we need to be able to fabricate cellular microenvironments with tunable properties. In this work, the nanotopography and functionality of scaffolds for cell culture were modified by coating 3D printed materials (DS3000 and poly(ethylene glycol)diacrylate, PEG-DA) with cellulose nanocrystals (CNCs). This general approach was demonstrated on a variety of structures designed to incorporate macro- and microscale features fabricated using photopolymerization and 3D printing techniques. Atomic force microscopy was used to characterize the CNC coatings and showed the ability to tune their density and in turn the surface nanoroughness from isolated nanoparticles to dense surface coverage. The ability to tune the density of CNCs on 3D printed structures could be leveraged to control the attachment and morphology of prostate cancer cells. It was also possible to introduce functionalization onto the surface of these scaffolds, either by directly coating them with CNCs grafted with the functionality of interest or with a more general approach of functionalizing the CNCs after coating using biotin-streptavidin coupling. The ability to carefully tune the nanostructure and functionalization of different 3D-printable materials is a step forward to creating in vitro scaffolds that mimic the nanoscale features of natural microenvironments, which are key to understanding their impact on cells and developing artificial tissues.

2.5D Hierarchical Structuring of Nanocomposite Hydrogel Films Containing Cellulose Nanocrystals.

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.

Tissue Response and Biodistribution of Injectable Cellulose Nanocrystal Composite Hydrogels.

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.

Nanostructure of Fully Injectable Hydrazone-Thiosuccinimide Interpenetrating Polymer Network Hydrogels Assessed by Small-Angle Neutron Scattering and dSTORM Single-Molecule Fluorescence Microscopy.

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.