Injectable neural hydrogel as in vivo therapeutic delivery vehicle.

Purpose: This study demonstrated in vivo delivery of a decellularized, injectable peripheral nerve (iPN) hydrogel and explored options for using iPN in combination with regenerative biomolecular therapies like stem cell secretome. Methods: Rat-derived iPN hydrogel solutions were combined with a dextran-dye before subcutaneous injection into adult Sprague Dawley rats. After injection, an in vivo imaging system (IVIS) was used to visualize hydrogels and quantify dextran-dye release over time. Poly(lactic-co-glycolic) acid (PLGA) was used to encapsulate the dextran-dye to prolong molecular release from the hydrogel scaffolds. Lastly, we investigated use of adipose-derived stem cell (ASC) secretome as a potential future combination strategy with iPN. ASC secretome was assessed for growth factor levels in response to media stimulation and was encapsulated in PLGA to determine loading efficiency. Results: Gelation of iPN hydrogels was successful upon subcutaneous injection. When combined with iPN, a 10 kDa dextran-dye was reduced to 54% its initial signal at 24 hours, while PLGA-encapsulated dextran-dye in iPN was only reduced to 78% by 24 hours. Modified media stimulation resulted in changes in ASC phenotype and dramatic upregulation of VEGF secretion. The PLGA encapsulation protocol was adapted for use with temperature sensitive biomolecules, however, considerations must be made with loading efficiency for cell secretome as the maximum efficiency was 28%. Conclusion: The results of this study demonstrated successful injection and subsequent gelation of our iPN hydrogel formulation in vivo. Biomolecular payloads can be encapsulated in PLGA to help prolong their release from the soft iPN hydrogels in future combination therapies. Lay Summary: We developed an injectable decellularized tissue scaffold from rat peripheral nerve tissue (called iPN), a potential minimally invasive therapeutic meant to fill lesion spaces after injury. This study was the first demonstration of iPN delivery to a living animal. The iPN solution was injected subcutaneously in a rat and properly formed a gelled material upon entering the body. Our results showed that encapsulating biomolecules in an FDA-approved polymer (PLGA) slowed the release of biomolecules from the iPN, which could allow therapeutics more time around the scaffold to help repair native tissue. Lastly, we investigated one potential avenue for combining iPN with other regenerative cues obtained from adipose-derived stem cells. Description of Future Works: Future work must focus on optimal loading conditions and release profiles from the iPN hydrogels. Next steps will be applying iPN in various combination therapies for spinal cord injury. We will focus efforts on developing a pro-regenerative secretome that directly promotes neurite extension and neural cell infiltration into iPN scaffolds upon transplantation in spinal cord.

Decellularized peripheral nerve as an injectable delivery vehicle for neural applications.

Damage to the nervous system can result in loss of sensory and motor function, paralysis, or even death. To facilitate neural regeneration and functional recovery, researchers have employed biomaterials strategies to address both peripheral and central nervous system injuries. Injectable hydrogels that recapitulate native nerve extracellular matrix are especially promising for neural tissue engineering because they offer more flexibility for minimally invasive applications and provide a growth-permissive substrate for neural cell types. Here, we explore the development of injectable hydrogels derived from decellularized rat peripheral nerves (referred to as "injectable peripheral nerve [iPN] hydrogels"), which are processed using a newly developed sodium deoxycholate and DNase (SDD) decellularization method. We assess the gelation kinetics, mechanical properties, cell bioactivity, and drug release kinetics of the iPN hydrogels. The iPN hydrogels thermally gel when exposed to 37°C in under 20 min and have mechanical properties similar to neural tissue. The hydrogels demonstrate in vitro biocompatibility through support of Schwann cell viability and metabolic activity. Additionally, iPN hydrogels promote greater astrocyte spreading compared to collagen I hydrogels. Finally, the iPN is a promising delivery vehicle of drug-loaded microparticles for a combinatorial approach to neural injury therapies.

Effects of Varied Stimulation Parameters on Adipose-Derived Stem Cell Response to Low-Level Electrical Fields.

Exogenous electrical fields have been explored in regenerative medicine to increase cellular expression of pro-regenerative growth factors. Adipose-derived stem cells (ASCs) are attractive for regenerative applications, specifically for neural repair. Little is known about the relationship between low-level electrical stimulation (ES) and ASC regenerative potentiation. In this work, patterns of ASC expression and secretion of growth factors (i.e., secretome) were explored across a range of ES parameters. ASCs were stimulated with low-level stimulation (20 mV/mm) at varied pulse frequencies, durations, and with alternating versus direct current. Frequency and duration had the most significant effects on growth factor expression. While a range of stimulation frequencies (1, 20, 1000 Hz) applied intermittently (1 h × 3 days) induced upregulation of general wound healing factors, neural-specific factors were only increased at 1 Hz. Moreover, the most optimal expression of neural growth factors was achieved when ASCs were exposed to 1 Hz pulses continuously for 24 h. In evaluation of secretome, apparent inconsistencies were observed across biological replications. Nonetheless, ASC secretome (from 1 Hz, 24 h ES) caused significant increase in neurite extension compared to non-stimulated control. Overall, ASCs are sensitive to ES parameters at low field strengths, notably pulse frequency and stimulation duration.

Chondroitinase ABC/galectin-3 fusion proteins with hyaluronan-based hydrogels stabilize enzyme and provide targeted enzyme activity for neural applications.

Objective. Chondroitinase ABC (ChABC) has emerged as a promising therapeutic agent for central nervous system regeneration. Despite multiple beneficial outcomes for regeneration, translation of this enzyme is challenged by poor pharmacokinetics, localization, and stability.Approach. This study explored the function andin vitroapplication of engineered ChABC fused to galectin-3 (Gal3). Two previously developed ChABC-Gal3 oligomers (monomeric and trimeric) were evaluated for functionality and kinetics, then applied to anin vitrocellular outgrowth model using dorsal root ganglia (DRGs). The fusions were combined with two formulations of hyaluronan (HA)-based scaffolds to determine the extent of active enzyme release compared to wild type (WT) ChABC.Main Results. Monomeric and trimeric ChABC-Gal3 maintained digestive capabilities with kinetic properties that were substrate-dependent for chondroitin sulfates A, B, and C. The fusions had longer half-lives at 37 °C on the order of seven fold for monomer and twelve fold for trimer compared to WT. Both fusions were also effective at restoring DRG outgrowthin vitro. To create a combination approach, two triple-component hydrogels containing modified HA were formulated to match the mechanical properties of native spinal cord tissue and to support astrocyte viability (>80%) and adhesion. The hydrogels included collagen-I and laminin mixed with either 5 mg ml-1of glycidyl methacrylate HA or 3 mg ml-1Hystem. When combined with scaffolds, ChABC-Gal3 release time was lengthened compared to WT. Both fusions had measurable enzymatic activity for at least 10 d when incorporated in gels, compared to WT that lost activity after 1 d. These longer term release products from gels maintained adequate function to promote DRG outgrowth.Significance. Results of this study demonstrated cohesive benefits of two stabilized ChABC-Gal3 oligomers in combination with HA-based scaffolds for neural applications. Significant improvements to ChABC stability and release were achieved, meriting future studies of ChABC-Gal3/hydrogel combinations to target neural regeneration.

Development of novel apoptosis-assisted lung tissue decellularization methods.

Decellularized tissues hold great potential for both regenerative medicine and disease modeling applications. The acellular extracellular matrix (ECM)-enriched scaffolds can be recellularized with patient-derived cells prior to transplantation, or digested to create thermally-gelling ECM hydrogels for 3D cell culture. Current methods of decellularization clear cellular components using detergents, which can result in loss of ECM proteins and tissue architectural integrity. Recently, an alternative approach utilizing apoptosis to decellularize excised murine sciatic nerves resulted in superior ECM preservation, cell removal, and immune tolerance in vivo. However, this apoptosis-assisted decellularization approach has not been optimized for other tissues with a more complex geometry, such as lungs. To this end, we developed an apoptosis-assisted lung tissue decellularization method using a combination of camptothecin and sulfobetaine-10 (SB-10) to induce apoptosis and facilitate gentle and effective removal of cell debris, respectively. Importantly, combination of the two agents resulted in superior cell removal and ECM preservation compared to either of the treatments alone, presumably because of pulmonary surfactants. In addition, our method was superior in cell removal compared to a previously established detergent-based decellularization protocol. Furthermore, thermally-gelling lung ECM hydrogels supported high viability of rat lung epithelial cells for up to 2 weeks in culture. This work demonstrates that apoptosis-based lung tissue decellularization is a superior technique that warrants further utilization for both regenerative medicine and disease modeling purposes.

Three-Dimensional Bioprinted Hyaluronic Acid Hydrogel Test Beds for Assessing Neural Cell Responses to Competitive Growth Stimuli.

Hyaluronic acid (HA) is an abundant extracellular matrix (ECM) component in soft tissues throughout the body and has found wide adoption in tissue engineering. This study focuses on the optimization of methacrylated HA (MeHA) for three-dimensional (3D) bioprinting to create in vitro test beds that incorporate regeneration-promoting growth factors in neural repair processes. To evaluate MeHA as a potential bioink, rheological studies were performed with PC-12 cells to demonstrate shear thinning properties maintained when printing with and without cells. Next, an extrusion-based Cellink BIO X 3D printer was used to bioprint various MeHA solutions combined with collagen-I to determine which formulation was the most optimal for creating 3D features. Results indicated that MeHA (10 mg/mL) with collagen-I (3 mg/mL) was most suitable. As Schwann cells (SCs) are a critical component of neural repair and regeneration, SC adhesion assessment via integrin ß1 immunostaining indicated that the bioink candidate adequately supported SC adhesion and migration when compared to Col-I, a highly cell-adhesive ECM component. MeHA/collagen-I bioink was adapted for neural specific applications by printing with the neural growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF). These test beds were conducive for SC infiltration and presented differential migration responses. Finally, a two-chamber in vitro test bed design was created to study competitive biochemical cues. Dorsal root ganglia were seeded in test beds and demonstrated directional neurite extension (measured by ß-III tubulin and GAP43 immunostaining) in response to NGF and GDNF. Overall, the selected MeHA/collagen-I bioink was bioprintable, improved cell viability compared to molded controls, and was conducive for cell adhesion, growth factor sequestration, and neural cell infiltration. MeHA is a suitable bioink candidate for extrusion-based bioprinting and will be useful in future development of spatially complex test beds to advance in vitro models as an alternative to common in vivo tests for neural repair applications.

Mechano-stimulation initiated by extracellular adhesion and cationic conductance pathways influence astrocyte activation.

Traumatic brain injury (TBI) represents a major cause of long-term disability worldwide. Primary damage to brain tissue leads to complex secondary injury mechanisms involving inflammation, oxidative stress and cellular activation/reactivity. The molecular pathways that exacerbate brain cell dysfunction after injury are not well understood and provide challenges to developing TBI therapeutics. This study aimed to delineate mechanisms of astrocyte activation induced by mechano-stimulation, specifically involving extracellular adhesion and cationic transduction. An in vitro model was employed to investigate 2D and 3D cultures of primary astrocytes, in which cells were exposed to a single high-rate overpressure known to cause upregulation of structural and proliferative markers within 72 h of exposure. An inhibitor of focal adhesion kinase (FAK) phosphorylation, TAE226, was used to demonstrate a relationship between extracellular adhesion perturbations and structural reactivity in the novel 3D model. TAE226 mitigated upregulation of glial fibrillary acidic protein in 3D cultures by 72 h post-exposure. Alternatively, incubation with gadolinium (a cationic channel blocker) during overpressure, demonstrated a role for cationic transduction in reducing the increased levels of proliferating cell nuclear antigen that occur at 24 h post-stimulation. Furthermore, early changes in mitochondrial polarization at 15 min and in endogenous ATP levels at 4-6 h occur post-overpressure and may be linked to later changes in cell phenotype. By 24 h, there was evidence of increased amine metabolism and increased nicotinamide adenine dinucleotide phosphate oxidase (NOX4) production. The overproduction of NOX4 was counteracted by gadolinium during overpressure exposure. Altogether, the results of this study indicated that both extracellular adhesion (via FAK activation) and cationic conductance (via ion channels) contribute to early patterns of astrocyte activation following overpressure stimulation. Mechano-stimulation pathways are linked to bioenergetic and metabolic disruptions in astrocytes that influence downstream oxidative stress, aberrant proliferative capacity and structural reactivity.

Extracellular Matrix Disparities in an Nkx2-5 Mutant Mouse Model of Congenital Heart Disease.

Congenital heart disease (CHD) affects almost one percent of all live births. Despite diagnostic and surgical reparative advances, the causes and mechanisms of CHD are still primarily unknown. The extracellular matrix plays a large role in cell communication, function, and differentiation, and therefore likely plays a role in disease development and pathophysiology. Cell adhesion and gap junction proteins, such as integrins and connexins, are also essential to cellular communication and behavior, and could interact directly (integrins) or indirectly (connexins) with the extracellular matrix. In this work, we explore disparities in the expression and spatial patterning of extracellular matrix, adhesion, and gap junction proteins between wild type and Nkx2-5 +/R52G mutant mice. Decellularization and proteomic analysis, Western blotting, histology, immunostaining, and mechanical assessment of embryonic and neonatal wild type and Nkx2-5 mutant mouse hearts were performed. An increased abundance of collagen IV, fibronectin, and integrin ß-1 was found in Nkx2-5 mutant neonatal mouse hearts, as well as increased expression of connexin 43 in embryonic mutant hearts. Furthermore, a ventricular noncompaction phenotype was observed in both embryonic and neonatal mutant hearts, as well as spatial disorganization of ECM proteins collagen IV and laminin in mutant hearts. Characterizing such properties in a mutant mouse model provides valuable information that can be applied to better understanding the mechanisms of congenital heart disease.

Novel Sodium Deoxycholate-Based Chemical Decellularization Method for Peripheral Nerve.

Decellularized peripheral nerve has been proven to be an effective clinical intervention for peripheral nerve repair and a preclinical cell carrier after spinal cord injury. However, there are currently a lack of decellularization methods for peripheral nerve that remove cells and maintain matrix similar to the previously established, clinically translated technique (the Hudson method) that relies on the discontinued Triton X-200 detergent. Therefore, the aim of this study was to optimize a novel chemical decellularization method for peripheral nerves based on the currently available anionic detergent sodium deoxycholate. Sprague Dawley rat sciatic nerves were isolated, frozen in buffered solution, and then subject to sequential washes in water, salt buffer, zwitterionic detergents sulfobetaines -10 and -16, and varying concentrations of sodium deoxycholate (SDC). To optimize DNA removal after SDC decellularization, nerves were subjected to deoxyribonuclease (DNase) incubation and salt buffer washes. Immunohistochemical results demonstrated that utilization of 3% SDC in the decellularization process preserved extracellular matrix (ECM) components and structure while facilitating significantly better removal of Schwann cells, axons, and myelin compared with the Hudson method. The addition of a 3-h DNase incubation to the 3% SDC decellularization process significantly removed cellular debris compared with the Hudson method. Proteomic analysis demonstrated that our novel decellularization method based on 3% SDC +3-h DNase used in conjunction with zwitterionic detergents, and salt buffers (new decellularization method using 3% SDC + 3-h DNase, zwitterionic detergents, and salt buffers [SDD method]) produced a similar proteomic profile compared with the Hudson method and had significantly fewer counts of cellular proteins. Finally, cytotoxicity analysis demonstrated that the SDD decellularized scaffolds do not contain significant cytotoxic residuals as eluted media supported metabolically active Schwann cells in vitro. Overall, this study demonstrates that SDD decellularization represents a novel alternative utilizing currently commercially available chemical reagents. Impact Statement Decellularized nerves are clinically relevant materials that can be used for a variety of regenerative applications such as peripheral nerve and spinal cord injury repair. However, discontinuation of key detergents used in a proven chemical decellularization process necessitates the optimization of an equivalent or better method. This research presents the field with a novel chemical decellularization method to replace the previous validated standard. Scaffolds generated from this method provide an extracellular matrix-rich material that can be used in a variety of in vitro applications to understand cellular behavior and in vivo applications to facilitate regeneration after neural injury.

Astrocyte Mechano-Activation by High-Rate Overpressure Involves Alterations in Structural and Junctional Proteins.

Primary blast neurotrauma represents a unique injury paradigm characterized by high-rate overpressure effects on brain tissue. One major hallmark of blast neurotrauma is glial reactivity, notably prolonged astrocyte activation. This cellular response has been mainly defined in primary blast neurotrauma by increased intermediate filament expression. Because the intermediate filament networks physically interface with transmembrane proteins for junctional support, it was hypothesized that cell junction regulation is altered in the reactive phenotype as well. This would have implications for downstream transcriptional regulation via signal transduction pathways like nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Therefore, a custom high-rate overpressure simulator was built for in vitro testing using mechanical conditions based on intracranial pressure measurements in a rat model of blast neurotrauma. Primary rat astrocytes were exposed to isolated high-rate mechanical stimulation to study cell junction dynamics in relation to their mechano-activation. First, a time course for "classical" features of reactivity was devised by evaluation of glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) expression. This was followed by gene and protein expression for both gap junction (connexins) and anchoring junction proteins (integrins and cadherins). Signal transduction analysis was carried out by nuclear localization of two molecules, NF-κB p65 and mitogen-activated protein kinase (MAPK) p38. Results indicated significant increases in connexin-43 expression and PCNA first at 24 h post-overpressure (p < 0.05), followed by structural reactivity (via increased GFAP, p < 0.05) corresponding to increased anchoring junction dynamics at 48 h post-overpressure (p < 0.05). Moreover, increased phosphorylation of focal adhesion kinase (FAK) was observed in addition to increased nuclear localization of both p65 and p38 (p < 0.05) during the period of structural reactivity. To evaluate the transcriptional activity of p65 in the nucleus, electrophoretic mobility shift assay was conducted for a binding site on the promoter region for intracellular adhesion molecule-1 (ICAM-1), an antagonist of tight junctions. A significant increase in the interaction of nuclear proteins with the NF-κB site on the ICAM-1 corresponded to increased gene and protein expression of ICAM-1 (p < 0.05). Altogether, these results indicate multiple targets and corresponding signaling pathways which involve cell junction dynamics in the mechano-activation of astrocytes following high-rate overpressure.

A Customizable Chamber for Measuring Cell Migration.

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between cells, the extracellular matrix, and soluble and non-soluble chemical factors (e.g., chemoattractants). Standard methods for measuring the migration of cells, such as the Boyden chamber assay, work by counting cells on either side of a divider. These techniques are easy to use; however, they offer little geometric modification for different applications. In contrast, microfluidic devices can be used to observe cell migration with customizable concentration gradients of soluble factors1,2. However, methods for making microfluidics based assays can be difficult to learn. Here, we describe an easy method for creating cell culture chambers to measure cell migration in response to chemical concentration gradients. Our cell migration chamber method can create different linear concentration gradients in order to study cell migration for a variety of applications. This method is relatively easy to use and is typically performed by undergraduate students. The microchannel chamber was created by placing an acrylic insert in the shape of the final microchannel chamber well into a Petri dish. After this, poly(dimethylsiloxane) (PDMS) was poured on top of the insert. The PDMS was allowed to harden and then the insert was removed. This allowed for the creation of wells in any desired shape or size. Cells may be subsequently added to the microchannel chamber, and soluble agents can be added to one of the wells by soaking an agarose block in the desired agent. The agarose block is added to one of the wells, and time-lapse images can be taken of the microchannel chamber in order to quantify cell migration. Variations to this method can be made for a given application, making this method highly customizable.

Role of Glia in Memory Deficits Following Traumatic Brain Injury: Biomarkers of Glia Dysfunction.

Historically, glial cells have been recognized as a structural component of the brain. However, it has become clear that glial cells are intimately involved in the complexities of neural networks and memory formations. Astrocytes, microglia, and oligodendrocytes have dynamic responsibilities which substantially impact neuronal function and activities. Moreover, the importance of glia following brain injury has come to the forefront in discussions to improve axonal regeneration and functional recovery. The numerous activities of glia following injury can either promote recovery or underlie the pathobiology of memory deficits. This review outlines the pathological states of glial cells which evolve from their positive supporting roles to those which disrupt synaptic function and neuroplasticity following injury. Evidence suggests that glial cells interact extensively with neurons both chemically and physically, reinforcing their role as pivotal for higher brain functions such as learning and memory. Collectively, this mini review surveys investigations of how glial dysfunction following brain injury can alter mechanisms of synaptic plasticity and how this may be related to an increased risk for persistent memory deficits. We also include recent findings, that demonstrate new molecular avenues for clinical biomarker discovery.

Two and Three-Dimensional in Vitro Models of Blast-Induced Neurotrauma.

Blast-induced neurotrauma (BINT) has become an increasingly significant concern in Veterans returning from warfare. Associated brain injury and cognitive deficits are difficult to diagnose as the nature of this injury is progressive. In order to better understand the mechanisms of BINT at the microscopic level, two- and three-dimensional models of astrocytes were studied. The 3-D model was developed using Matrigel® to embed the cells. Injury was induced by exposure to an overpressure of 20 psi (140 kPa) using a shock wave generator which simulates a free field blast profile. Cellular viability was measured by MTT assays conducted at 24 and 48 hours post-blast. Gene expression levels of glial-fibrillary acidic protein (GFAP), ß-actin, and vinculin were analyzed as potential structural biomarkers of injury at 48 and 72 hours post-blast. Results indicated that glial cells survived and became activated by 72 hours following exposure. Specifically, gene expression of GFAP was elevated in simulated blast cultures as compared to controls. Moreover, 2- and 3-D cultures were observed to have different time periods of activation. These activation markers may be useful when designing therapeutic targets to mitigate injury progression.