Detection of Fluorescent Protein Mechanical Switching in Cellulo.

The ability of cells to sense and respond to mechanical forces is critical in many physiological and pathological processes. However, the mechanisms by which forces affect protein function inside cells remain unclear. Motivated by in vitro demonstrations of fluorescent proteins (FPs) undergoing reversible mechanical switching of fluorescence, we investigated if force-sensitive changes in FP function could be visualized in cells. Guided by a computational model of FP mechanical switching, we develop a formalism for its detection in Förster resonance energy transfer (FRET)-based biosensors and demonstrate its occurrence in cellulo in a synthetic actin-crosslinker and the mechanical linker protein vinculin. We find that in cellulo mechanical switching is reversible and altered by manipulation of cellular force generation as well as force-sensitive bond dynamics of the biosensor. Together, this work describes a new framework for assessing FP mechanical stability and provides a means of probing force-sensitive protein function inside cells. MOTIVATION: The ability of cells to sense mechanical forces is critical in developmental, physiological, and pathological processes. Cells sense mechanical cues via force-induced alterations in protein structure and function, but elucidation of the molecular mechanisms is hindered by the lack of approaches to directly probe the effect of forces on protein structure and function inside cells. Motivated by in vitro observations of reversible fluorescent protein mechanical switching, we developed an approach for detecting fluorescent protein mechanical switching in cellulo . This enables the visualization of force-sensitive protein function inside living cells.

Softening of the chronic hemi-section spinal cord injury scar parallels dysregulation of cellular and extracellular matrix content.

Regeneration following spinal cord injury (SCI) is challenging in part due to the modified tissue composition and organization of the resulting glial and fibrotic scar regions. Inhibitory cell types and biochemical cues present in the scar have received attention as therapeutic targets to promote regeneration. However, altered Young's modulus of the scar as a readout for potential impeding factors for regeneration are not as well-defined, especially in vivo. Although the decreased Young's modulus of surrounding tissue at acute stages post-injury is known, the causation and outcomes at chronic time points remain largely understudied and controversial, which motivates this work. This study assessed the glial and fibrotic scar tissue's Young's modulus and composition (scar morphometry, cell identity, extracellular matrix (ECM) makeup) that contribute to the tissue's stiffness. The spatial Young's modulus of a chronic (~18-wks, post-injury) hemi-section, including the glial and fibrotic regions, were significantly less than naïve tissue (~200 Pa; p < 0.0001). The chronic scar contained cystic cavities dispersed in areas of dense nuclei packing. Abundant CNS cell types such as astrocytes, oligodendrocytes, and neurons were dysregulated in the scar, while epithelial markers such as vimentin were upregulated. The key ECM components in the CNS, namely sulfated proteoglycans (sPGs), were significantly downregulated following injury with concomitant upregulation of unsulfated glycosaminoglycans (GAGs) and hyaluronic acid (HA), likely altering the foundational ECM network that contributes to tissue stiffness. Our results reveal the Young's modulus of the chronic SCI scar as well as quantification of contributing elastic components that can provide a foundation for future study into their role in tissue repair and regeneration.

Covalently Immobilizing Interferon-γ Drives Filopodia Production through Specific Receptor-Ligand Interactions Independently of Canonical Downstream Signaling.

Immobilizing a signaling protein to guide cell behavior has been employed in a wide variety of studies. This approach draws inspiration from biology, where specific, affinity-based interactions between membrane receptors and immobilized proteins in the extracellular matrix guide many developmental and homeostatic processes. Synthetic immobilization approaches, however, do not necessarily recapitulate the in vivo signaling system and potentially lead to artificial receptor-ligand interactions. To investigate the effects of one example of engineered receptor-ligand interactions, we focus on the immobilization of interferon-γ (IFN-γ), which has been used to drive differentiation of neural stem cells (NSCs). To isolate the effect of ligand immobilization, we transfected Cos-7 cells with only interferon-γ receptor 1 (IFNγR1), not IFNγR2, so that the cells could bind IFN-γ but were incapable of canonical signal transduction. We then exposed the cells to surfaces containing covalently immobilized IFN-γ and studied membrane morphology, receptor-ligand dynamics, and receptor activation. We found that exposing cells to immobilized but not soluble IFN-γ drove the formation of filopodia in both NSCs and Cos-7, showing that covalently immobilizing IFN-γ is enough to affect cell behavior, independently of canonical downstream signaling. Overall, this work suggests that synthetic growth factor immobilization can influence cell morphology beyond enhancing canonical cell responses through the prolonged signaling duration or spatial patterning enabled by protein immobilization. This suggests that differentiation of NSCs could be driven by canonical and non-canonical pathways when IFN-γ is covalently immobilized. This finding has broad implications for bioengineering approaches to guide cell behavior, as one ligand has the potential to impact multiple pathways even when cells lack the canonical signal transduction machinery.

Subcutaneous priming of protein-functionalized chitosan scaffolds improves function following spinal cord injury.

Strategies using neural stem cells (NSCs) to aid regeneration following spinal cord injury (SCI) show much promise, but challenges remain regarding implementation and efficacy. In this work, we explored the use of an NSC-seeded scaffold consisting of covalently immobilized interferon-γ and rat NSCs within a hydrogel matrix (methacrylamide chitosan). We placed the scaffolds within the subcutaneous environment of rats, allowing them to incubate for 4 weeks in order to prime them for regeneration prior to being transplanted into a right lateral hemisection SCI model in the same animal. We found that subcutaneous priming reduced the lineage commitment of encapsulated NSCs, as observed by increased nestin expression and decreased NeuN expression. When combined with intracellular σ peptide administration (which reduces inhibition from the glial scar), subcutaneous maturation improved functional outcomes, which were assessed by BBB score and quantitative gait parameters (fore and hind limb duty factor imbalance, right and left paw placement accuracy). Although we did not observe any direct reconnection of the transplanted cells with the host tissue, we did observe neurofilament fibers extending from the host tissue into the scaffold. Importantly, the mechanism for improved functional outcomes is likely an increase in trophic support from subcutaneously maturing the scaffold, which is enhanced by the administration of ISP.

Subcutaneous priming of protein-functionalized chitosan scaffolds improves function following spinal cord injury.

Strategies using neural stem cells (NSCs) to aid regeneration following spinal cord injury (SCI) show much promise, but challenges remain regarding implementation and efficacy. In this work, we explored the use of an NSC-seeded scaffold consisting of covalently immobilized interferon-γ and rat NSCs within a hydrogel matrix (methacrylamide chitosan). We placed the scaffolds within the subcutaneous environment of rats, allowing them to incubate for 4 weeks in order to prime them for regeneration prior to being transplanted into a right lateral hemisection SCI model in the same animal. We found that subcutaneous priming reduced the lineage commitment of encapsulated NSCs, as observed by increased nestin expression and decreased NeuN expression. When combined with intracellular σ peptide administration (which reduces inhibition from the glial scar), subcutaneous maturation improved functional outcomes, which were assessed by BBB score and quantitative gait parameters (fore and hind limb duty factor imbalance, right and left paw placement accuracy). Although we did not observe any direct reconnection of the transplanted cells with the host tissue, we did observe neurofilament fibers extending from the host tissue into the scaffold. Importantly, the mechanism for improved functional outcomes is likely an increase in trophic support from subcutaneously maturing the scaffold, which is enhanced by the administration of ISP.

Concurrent Delivery of Soluble and Immobilized Proteins to Recruit and Differentiate Neural Stem Cells.

Insufficient endogenous neural stem cell (NSC) migration to injury sites and incomplete replenishment of neurons complicates recovery following central nervous system (CNS) injury. Such insufficient migration can be addressed by delivering soluble chemotactic factors, such as stromal cell-derived factor 1-α (SDF-1α), to sites of injury. However, simply enhancing NSC migration is likely to result in insufficient regeneration, as the cells need to be given additional signals. Immobilized proteins, such as interferon-γ (IFN-γ) can encourage neurogenic differentiation of NSCs. Here, we combined both protein delivery paradigms: soluble SDF-1α delivery to enhance NSC migration alongside covalently tethered IFN-γ to differentiate the recruited NSCs into neurons. To slow the release of soluble SDF-1α, we copolymerized methacrylated heparin with methacrylamide chitosan (MAC), to which we tethered IFN-γ. We found that this hydrogel system could result in soft hydrogels with a ratio of up to 70:30 MAC/heparin by mass, which enabled the continuous release of SDF-1α over a period of 2 weeks. The hydrogels recruited NSCs in vitro over 2 weeks, proportional to their release rate: the 70:30 heparin gels recruited a consistent number of NSCs at each time point, while the formulations with less heparin recruited NSCs at only early time points. After remaining in contact with the hydrogels for 8 days, NSCs successfully differentiated into neurons. CNS regeneration is a complex challenge, and this system provides a foundation to address multiple aspects of that challenge.

Automated Gait Analysis Detects Improvements after Intracellular σ Peptide Administration in a Rat Hemisection Model of Spinal Cord Injury.

A promising treatment strategy for spinal cord injury (SCI) is to reduce inhibition from chondroitin sulfate proteoglycans (CSPGs). For example, administering intracellular σ peptide (ISP) can improve the ability of axons to cross inhibitory CSPGs and improve function in rodent models of SCI. To translate such treatments into the clinic, we need robust and sensitive methods for studying rodent models. In this study, we applied a newly developed suite of quantitative gait analysis tools: gait analysis instrumentation and technology optimized for rodents (GAITOR), which consists of an arena and open-source software (AGATHA: automated gait analysis through hues and areas). We showed that GAITOR can be used to detect subtle functional improvements (measured by hindlimb duty factor imbalance) in rats following ISP administration in a T10 hemisection injury model. We demonstrated that SCI-specific parameters (right paw placement accuracy and phase dispersion) can be easily added to GAITOR to track recovery. We confirmed the gait observations via retrograde tracer uptake. We concluded that GAITOR is a powerful tool for measuring recovery after moderate/mild SCI, and could be used to replace expensive/inflexible commercially-available gait analysis techniques.

Molecular Tension Sensors: Moving Beyond Force.

Nearly all cellular processes are sensitive to mechanical inputs, and this plays a major role in diverse physiological processes. Mechanical stimuli are thought to be primarily detected through force-induced changes in protein structure. Approximately a decade ago, molecular tension sensors were created to measure forces across proteins within cells. Since then, an impressive assortment of sensors has been created and provided key insights into mechanotransduction, but comparisons of measurements between various sensors are challenging. In this review, we discuss the different types of molecular tension sensors, provide a system of classification based on their molecular-scale mechanical properties, and highlight how new applications of these sensors are enabling measurements beyond the magnitude of tensile load. We suggest that an expanded understanding of the functionality of these sensors, as well as integration with other techniques, will lead to consensus amongst measurements as well as critical insights into the underlying mechanisms of mechanotransduction.

The Open Source GAITOR Suite for Rodent Gait Analysis.

Locomotive changes are often associated with disease or injury, and these changes can be quantified through gait analysis. Gait analysis has been applied to preclinical studies, providing quantitative behavioural assessment with a reasonable clinical analogue. However, available gait analysis technology for small animals is somewhat limited. Furthermore, technological and analytical challenges can limit the effectiveness of preclinical gait analysis. The Gait Analysis Instrumentation and Technology Optimized for Rodents (GAITOR) Suite is designed to increase the accessibility of preclinical gait analysis to researchers, facilitating hardware and software customization for broad applications. Here, the GAITOR Suite's utility is demonstrated in 4 models: a monoiodoacetate (MIA) injection model of joint pain, a sciatic nerve injury model, an elbow joint contracture model, and a spinal cord injury model. The GAITOR Suite identified unique compensatory gait patterns in each model, demonstrating the software's utility for detecting gait changes in rodent models of highly disparate injuries and diseases. Robust gait analysis may improve preclinical model selection, disease sequelae assessment, and evaluation of potential therapeutics. Our group has provided the GAITOR Suite as an open resource to the research community at www.GAITOR.org , aiming to promote and improve the implementation of gait analysis in preclinical rodent models.

Biomaterial strategies for limiting the impact of secondary events following spinal cord injury.

The nature of traumatic spinal cord injury (SCI) often involves limited recovery and long-term quality of life complications. The initial injury sets off a variety of secondary cascades, which result in an expanded lesion area. Ultimately, the native tissue fails to regenerate. As treatments are developed in the laboratory, the management of this secondary cascade is an important first step in achieving recovery of normal function. Current literature identifies four broad targets for intervention: inflammation, oxidative stress, disruption of the blood-spinal cord barrier, and formation of an inhibitory glial scar. Because of the complex and interconnected nature of these events, strategies that combine multiple therapies together show much promise. Specifically, approaches that rely on biomaterials to perform a variety of functions are generating intense research interest. In this review, we examine each target and discuss how biomaterials are currently used to address them. Overall, we show that there are an impressive amount of biomaterials and combinatorial treatments which show good promise for slowing secondary events and improving outcomes. If more emphasis is placed on growing our understanding of how materials can manage secondary events, treatments for SCI can be designed in an increasingly rational manner, ultimately improving their potential for translation to the clinic.

Covalent growth factor tethering to direct neural stem cell differentiation and self-organization.

Tethered growth factors offer exciting new possibilities for guiding stem cell behavior. However, many of the current methods present substantial drawbacks which can limit their application and confound results. In this work, we developed a new method for the site-specific covalent immobilization of azide-tagged growth factors and investigated its utility in a model system for guiding neural stem cell (NSC) behavior. An engineered interferon-γ (IFN-γ) fusion protein was tagged with an N-terminal azide group, and immobilized to two different dibenzocyclooctyne-functionalized biomimetic polysaccharides (chitosan and hyaluronan). We successfully immobilized azide-tagged IFN-γ under a wide variety of reaction conditions, both in solution and to bulk hydrogels. To understand the interplay between surface chemistry and protein immobilization, we cultured primary rat NSCs on both materials and showed pronounced biological effects. Expectedly, immobilized IFN-γ increased neuronal differentiation on both materials. Expression of other lineage markers varied depending on the material, suggesting that the interplay of surface chemistry and protein immobilization plays a large role in nuanced cell behavior. We also investigated the bioactivity of immobilized IFN-γ in a 3D environment in vivo and found that it sparked the robust formation of neural tube-like structures from encapsulated NSCs. These findings support a wide range of potential uses for this approach and provide further evidence that adult NSCs are capable of self-organization when exposed to the proper microenvironment. STATEMENT OF SIGNIFICANCE: For stem cells to be used effectively in regenerative medicine applications, they must be provided with the appropriate cues and microenvironment so that they integrate with existing tissue. This study explores a new method for guiding stem cell behavior: covalent growth factor tethering. We found that adding an N-terminal azide-tag to interferon-γ enabled stable and robust Cu-free 'click' immobilization under a variety of physiologic conditions. We showed that the tagged growth factors retained their bioactivity when immobilized and were able to guide neural stem cell lineage commitment in vitro. We also showed self-organization and neurulation from neural stem cells in vivo. This approach will provide another tool for the orchestration of the complex signaling events required to guide stem cell integration.

A Hydrogel Bridge Incorporating Immobilized Growth Factors and Neural Stem/Progenitor Cells to Treat Spinal Cord Injury.

Spinal cord injury (SCI) causes permanent, often complete disruption of central nervous system (CNS) function below the damaged region, leaving patients without the ability to regenerate lost tissue. To engineer new CNS tissue, a unique spinal cord bridge is created to deliver stem cells and guide their organization and development with site-specifically immobilized growth factors. In this study, this bridge is tested, consisting of adult neural stem/progenitor cells contained within a methacrylamide chitosan (MAC) hydrogel and protected by a chitosan conduit. Interferon-γ (IFN-γ) and platelet-derived growth factor-AA (PDGF-AA) are recombinantly produced and tagged with an N-terminal biotin. They are immobilized to streptavidin-functionalized MAC to induce either neuronal or oligodendrocytic lineages, respectively. These bridges are tested in a rat hemisection model of SCI between T8 and T9. After eight weeks treatments including chitosan conduits result in a significant reduction in lesion area and macrophage infiltration around the lesion site (p < 0.0001). Importantly, neither immobilized IFN-γ nor PDGF-AA increased macrophage infiltration. Retrograde tracing demonstrates improved neuronal regeneration through the use of immobilized growth factors. Immunohistochemistry staining demonstrates that immobilized growth factors are effective in differentiating encapsulated cells into their anticipated lineages within the hydrogel, while qualitatively reducing glial fibrillary acid protein expression.

Tunable Keratin Hydrogels for Controlled Erosion and Growth Factor Delivery.

Tunable erosion of polymeric materials is an important aspect of tissue engineering for reasons that include cell infiltration, controlled release of therapeutic agents, and ultimately to tissue healing. In general, the biological response to proteinaceous polymeric hydrogels is favorable (e.g., minimal inflammatory response). However, unlike synthetic polymers, achieving tunable erosion with natural materials is a challenge. Keratins are a class of intermediate filament proteins that can be obtained from several sources, including human hair, and have gained increasing levels of use in tissue engineering applications. An important characteristic of keratin proteins is the presence of a large number of cysteine residues. Two classes of keratins with different chemical properties can be obtained by varying the extraction techniques: (1) keratose by oxidative extraction and (2) kerateine by reductive extraction. Cysteine residues of keratose are "capped" by sulfonic acid and are unable to form covalent cross-links upon hydration, whereas cysteine residues of kerateine remain as sulfhydryl groups and spontaneously form covalent disulfide cross-links. Here, we describe a straightforward approach to fabricate keratin hydrogels with tunable rates of erosion by mixing keratose and kerateine. SEM imaging and mechanical testing of freeze-dried materials showed similar pore diameters and compressive moduli, respectively, for each keratose-kerateine mixture formulation (∼1200 kPa for freeze-dried materials and ∼1.5 kPa for hydrogels). However, the elastic modulus (G') determined by rheology varied in proportion with the keratose-kerateine ratios, as did the rate of hydrogel erosion and the release rate of thiol from the hydrogels. The variation in keratose-kerateine ratios also led to tunable control over release rates of recombinant human insulin-like growth factor 1.

Alkylation of human hair keratin for tunable hydrogel erosion and drug delivery in tissue engineering applications.

Polymeric biomaterials that provide a matrix for cell attachment and proliferation while achieving delivery of therapeutic agents are an important component of tissue engineering and regenerative medicine strategies. Keratins are a class of proteins that have received attention for numerous tissue engineering applications because, like other natural polymers, they promote favorable cell interactions and have non-toxic degradation products. Keratins can be extracted from various sources including human hair, and they are characterized by a high percentage of cysteine residues. Thiol groups on reductively extracted keratin (kerateine) form disulfide bonds, providing a more stable cross-linked hydrogel network than oxidatively extracted keratin (keratose) that cannot form disulfide crosslinks. We hypothesized that an iodoacetamide alkylation (or "capping") of cysteine thiol groups on the kerateine form of keratin could be used as a simple method to modulate the levels of disulfide crosslinking in keratin hydrogels, providing tunable rates of gel erosion and therapeutic agent release. After alkylation, the alkylated kerateines still formed hydrogels and the alkylation led to changes in the mechanical and visco-elastic properties of the materials consistent with loss of disulfide crosslinking. The alkylated kerateines did not lead to toxicity in MC3T3-E1 pre-osteoblasts. These cells adhered to keratin at levels comparable to fibronectin and greater than collagen. Alkylated kerateine gels eroded more rapidly than non-alkylated kerateine and this control over erosion led to tunable rates of delivery of rhBMP-2, rhIGF-1, and ciprofloxacin. These results demonstrate that alkylation of kerateine cysteine residues provides a cell-compatible approach to tune rates of hydrogel erosion and therapeutic agent release within the context of a naturally-derived polymeric system.