Chondroitin sulfate, dermatan sulfate, and hyaluronic acid differentially modify the biophysical properties of collagen-based hydrogels.

Fibrillar collagens and glycosaminoglycans (GAGs) are structural biomolecules that are natively abundant to the extracellular matrix (ECM). Prior studies have quantified the effects of GAGs on the bulk mechanical properties of the ECM. However, there remains a lack of experimental studies on how GAGs alter other biophysical properties of the ECM, including ones that operate at the length scales of individual cells such as mass transport efficiency and matrix microstructure. This study focuses on the GAG molecules chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronic acid (HA). CS and DS are stereoisomers while HA is the only non-sulfated GAG. We characterized and decoupled the effects of these GAG molecules on the stiffness, transport, and matrix microarchitecture properties of type I collagen hydrogels using mechanical indentation testing, microfluidics, and confocal reflectance imaging, respectively. We complement these biophysical measurements with turbidity assays to profile collagen aggregate formation. Surprisingly, only HA enhanced the ECM indentation modulus, while all three GAGs had no effect on hydraulic permeability. Strikingly, we show that CS, DS, and HA differentially regulate the matrix microarchitecture of hydrogels due to their alterations to the kinetics of collagen self-assembly. In addition to providing information on how GAGs define key physical properties of the ECM, this work shows new ways in which stiffness measurements, microfluidics, microscopy, and turbidity kinetics can be used complementarily to reveal details of collagen self-assembly and structure. STATEMENT OF SIGNIFICANCE: Collagen and glycosaminoglycans (GAGs) are integral to the structure, function, and bioactivity of the extracellular matrix (ECM). Despite widespread interest in collagen-GAG composite hydrogels, there is a lack of quantitative understanding of how different GAGs alter the biophysical properties of the ECM across tissue, cellular, and subcellular length scales. Here we show using mechanical, microfluidic, microscopy, and analytical methods and measurements that the GAG molecules chondroitin sulfate, dermatan sulfate, and hyaluronic acid differentially regulate the mechanical, transport, and microstructural properties of hydrogels due to their alterations to the kinetics of collagen self-assembly. As such, these results will inform improved design and utilization of collagen-based scaffolds of tailored composition, mechanical properties, molecular availability due to mass transport, and microarchitecture.

Synthetic hydrogel mimics of the nuclear pore complex for the study of nucleocytoplasmic transport defects in C9orf72 ALS/FTD.

Dipeptide repeats (DPRs) associated with C9orf72 repeat expansions perturb nucleocytoplasmic transport and are implicated in the pathogenesis of amyotrophic lateral sclerosis. We present a synthetic hydrogel platform that can be used to analyze aspects of the molecular interaction of dipeptide repeats and the phenylalanine-glycine (FG) phase of the nuclear pore complex (NPC). Hydrogel scaffolds composed of acrylamide and copolymerized with FG monomers are first formed to recapitulate key FG interactions found in the NPC. With labeled probes, we find evidence that toxic arginine-rich DPRs (poly-GR and poly-PR), but not the non-toxic poly-GP, target NPC hydrogel mimics and block selective entry of a key nuclear transport receptor, importin beta (Impß). The ease with which these synthetic hydrogel mimics can be adjusted/altered makes them an invaluable tool to dissect complex molecular interactions that underlie cellular transport processes and their perturbation in disease.

Synthetic hydrogel mimics of the nuclear pore complex display selectivity dependent on FG-repeat concentration and electrostatics.

Synthetic hydrogels were utilized to explore influence of both charge and phenylalanine-glycine (FG) repeat concentration on translocation of select proteins. Hydrogels studied represent a biomimetic platform of the nuclear pore complex (NPC) found in eukaryotic cells. Polyacrylamide/phenylalanine-serine-phenylalanine-glycine (FSFG) peptide copolymers have previously demonstrated similar selectivity to native NPCs. Entry of a nuclear transport receptor (Impß) into hydrogels was monitored with fluorescence microscopy and observed to be greater within gels that contained larger concentrations of FG peptide. Low-resolution structural studies of gels demonstrated changes in morphology and porous network dimensions as FG-repeat concentration was varied. Copolymerization of charged acrylates within the polyacrylamide/FSFG matrix was performed to produce charged hydrogels. Enhanced entry of Impß, which is negatively charged, was observed in positively charged hydrogels, whereas entry was greatly diminished in negatively charged gels. Synthetic NPC mimics provide a useful testbed for further investigation of nucleocytoplasmic transport and may illuminate new routes for biomimetic separations.

Applications of nanopipettes in the analytical sciences.

In this review, we describe measurements and applications of interest to the analytical community that makes use of simple nanopipettes. Fabricated by applying heat during the separation of a glass capillary, nanopipettes provide a route for nanoscale studies of ion transport and for development of chemical and biochemical sensors. When mounted on a translation stage, nanopipettes also enable unique modes of imaging and material deposition. These facets of nanopipette research, as well as some of the unique properties of nanopipettes, will be discussed.