Fabrication and characterization of electrospun polycaprolactone and gelatin composite cuffs for tissue engineered blood vessels.

Sewing cuffs incorporated within tissue-engineered blood vessels (TEBVs) enable graft anastomosis in vivo, and secure TEBVs to bioreactors in vitro. Alternative approaches to cuff design are required to achieve cuff integration with scaffold-free TEBVs during tissue maturation. To create porous materials that promote tissue integration, we used electrospinning to fabricate cuffs from polycaprolactone (PCL), PCL blended with gelatin, and PCL coated with gelatin, and evaluated cuff mechanical properties, porosity, and cellular attachment and infiltration. Gelatin blending significantly decreased cuff ultimate tensile stress and failure strain over PCL alone, but no significant differences were observed in elastic modulus or failure load. Interestingly, gelatin incorporation by blending or coating did not produce significant differences in cellular attachment or pore size. We then created tissue tubes by fusing self-assembled smooth muscle cell rings together with electrospun cuffs on either end. After 7 days, rings and cuffs fused seamlessly, and the resulting tubes were harvested for pull-to-failure tests to measure the strength of cuff-tissue integration. Tubes with gelatin-coated PCL cuffs failed more frequently at the cuff-tissue interface compared to PCL and PCL:gelatin blended groups. This work demonstrates that electrospun cuffs integrated successfully with scaffold-free TEBVs, and that the addition of gelatin did not significantly improve cuff integration over PCL alone for this application. Electrospun cuffs may aid cannulation for dynamic culture and testing of tubular constructs during engineered tissue maturation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 817-826, 2018.

Anti-Fas conjugated hyaluronic acid microsphere gels for neural stem cell delivery.

Central nervous system (CNS) injuries and diseases result in neuronal damage and loss of function. Transplantation of neural stem cells (NSCs) has been shown to improve locomotor function after transplantation. However, due to the immune and inflammatory response at the injury site, the survival rate of the engrafted cells is low. Engrafted cell viability has been shown to increase when transplanted within a hydrogel. Hyaluronic acid (HA) hydrogels have natural anti-inflammatory properties and the backbone can be modified to introduce bioactive agents, such as anti-Fas, which we have previously shown to promote NSC survival while suppressing immune cell activity in bulk hydrogels in vitro. Although bulk HA hydrogels have shown to promote stem cell survival, microsphere gels for NSC encapsulation and delivery may have additional advantages. In this study, a flow-focusing microfluidic device was used to fabricate either vinyl sulfone-modified HA (VS-HA) or anti-Fas-conjugated HA (anti-Fas HA) microsphere gels encapsulated with NSCs. The majority of encapsulated NSCs remained viable for at least 24 h in the VS-HA and anti-Fas HA microsphere gels. Moreover, T-cells cultured in suspension with the anti-Fas HA microsphere gels had reduced viability after contact with the microsphere gels compared to the media control and soluble anti-Fas conditions. This approach can be adapted to encapsulate various cell types for therapeutic strategies in other physiological systems in order to increase survival by reducing the immune response. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 608-618, 2017.

Aligned Nanotopography Promotes a Migratory State in Glioblastoma Multiforme Tumor Cells.

Glioblastoma multiforme (GBM) is an aggressive, Grade IV astrocytoma with a poor survival rate, primarily due to the GBM tumor cells migrating away from the primary tumor site along the nanotopography of white matter tracts and blood vessels. It is unclear whether this nanotopography influences the biomechanical properties (i.e. cytoskeletal stiffness) of GBM tumor cells. Although GBM tumor cells have an innate propensity to migrate, we believe this capability is enhanced due to the influence of nanotopography on the tumor cells' biomechanical properties. In this study, we used an aligned nanofiber film that mimics the nanotopography in the tumor microenvironment to investigate the mechanical properties of GBM tumor cells in vitro. The data demonstrate that the cytoskeletal stiffness, cell traction stress, and focal adhesion area were significantly lower in the GBM tumor cells compared to healthy astrocytes. Moreover, the cytoskeletal stiffness was significantly reduced when cultured on aligned nanofiber films compared to smooth and randomly aligned nanofiber films. Gene expression analysis showed that tumor cells cultured on the aligned nanotopography upregulated key migratory genes and downregulated key proliferative genes. Therefore, our data suggest that the migratory potential is elevated when GBM tumor cells are migrating along aligned nanotopographical substrates.

Modulation of MicroRNAs 34a and 21 Affects Viability, Senescence, and Invasion in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is an aggressive and invasive brain tumor. Current interventional strategies have been minimally successful. Three key characteristics of GBMs are (1) enhanced resistance to apoptosis, (2) increased proliferation rate, and (3) increased invasion potential, making them difficult to treat. MicroRNAs (miRs) have demonstrated beneficial therapeutic intervention; particularly miRs 34a and 21, which have been implicated in regulation of apoptosis, senescence, and invasion of GBM tumor cells. MiR21 is anti-apoptotic and pro-proliferative, whereas miR34a is proapoptotic and an anti-invasive regulator in tumor cells. Our study investigates the effects of modulating both miR34a and miR21, in addition to comparing the two individual treatments. Using targeted cationic liposomes that bind to the epidermal growth factor receptor (EGFR), we delivered miR34a and/or anti-sense oligonucleotide to miR21 (ASO21) to GBM tumor cell lines, U87MG and A172, in vitro. Our data demonstrate that co-delivery of miR34a and ASO21 results in enhanced reduction in viability and invasion, while increasing senescence in vitro. Additionally, there were significant decreases in pro-invasion and -proliferation gene markers, as well as an increase in pro-apoptotic markers. In vivo results demonstrate that the combination of miR34a and ASO21 reduced tumor volume and proliferation of the A172 tumor cells. Accumulation of rhodamine encapsulated EGFR-targeted cationic liposomes was observed throughout the primary tumor bed after systemic injection. To our knowledge, we are the first to modulate multiple miRs, while using a targeted cationic liposomal delivery for miR-based therapy. These results demonstrate a potential clinically relevant, miR therapeutic strategy for GBM.

Tunable, bioactive protein conjugated hyaluronic acid hydrogel for neural engineering applications.

Millions of Americans suffer from nervous system injuries. Hydrogels have been investigated to (1) bridge nerve gaps; (2) act as scaffolds for bioactive molecule delivery or cell transplantation; and/or (3) promote axonal outgrowth. In this study, we use a rapid, one-step Michael addition click chemistry reaction to fabricate a hyaluronic acid (HA) scaffold for neural repair. Briefly, some of the primary hydroxyl groups on the HA backbone were modified with vinyl sulfone functional groups for (1) conjugation of thiol based bioactive molecules and (2) hydrogel crosslinking, which was confirmed by proton nuclear magnetic resonance (1H-NMR) and Fourier transform infrared spectroscopy (FTIR). The degree of crosslinking creates a mechanically tunable hydrogel. Rheology confirmed that the storage modulus was within the order of magnitude to that of nervous tissue. Primary human dermal fibroblasts and primary mouse neural stem cells (NSCs) seeded in the HA hydrogel were viable and proliferative, thus demonstrating that the HA hydrogel is suitable as a scaffold for cell transplantation. The range of pore size demonstrated that the scaffold supports cell migration and neurite extension. Neurite outgrowth of cultured whole embryonic day 9 chick dorsal root ganglions signifies that the hydrogel supports axonal outgrowth. Reduction in immune and inflammatory cell viability was observed in the anti-Fas conjugated HA hydrogel, whereas the NSCs maintained viability in the anti-Fas HA hydrogel. Therefore, this one-step, rapid, controllable reaction is an efficient method for fabrication of tunable, biomolecule conjugated hydrogels for neural engineering applications.

Aqueous phase synthesis of highly luminescent, nitrogen-doped carbon dots and their application as bioimaging agents.

Carbon dots are promising scaffolds for multifunctional therapeutic systems because of their fluorescence property, good biocompatibility, and low toxicity. In this work, we prepared nitrogen-doped carbon dots through an aqueous phase strategy using folic acid as precursor. The carbon dots possess many attractive features including uniform dispersion with size about 9 nm, bright photoluminescence, high photoluminescence quantum yield of 23%, and low toxicity, making them excellent imaging probes for biomedical applications.

Central but not systemic administration of XPro1595 is therapeutic following moderate spinal cord injury in mice.

BACKGROUND: Glial cell activation and overproduction of inflammatory mediators in the central nervous system (CNS) have been implicated in acute traumatic injuries to the CNS, including spinal cord injury (SCI). Elevated levels of the proinflammatory cytokine tumor necrosis factor (TNF), which exists in both a soluble (sol) and a transmembrane (tm) form, have been found in the lesioned cord early after injury. The contribution of solTNF versus tmTNF to the development of the lesion is, however, still unclear. METHODS: We tested the effect of systemically or centrally blocking solTNF alone, using XPro1595, versus using the drug etanercept to block both solTNF and tmTNF compared to a placebo vehicle following moderate SCI in mice. Functional outcomes were evaluated using the Basso Mouse Scale, rung walk test, and thermal hyperalgesia analysis. The inflammatory response in the lesioned cord was investigated using immunohistochemistry and western blotting analyses. RESULTS: We found that peripheral administration of anti-TNF therapies had no discernable effect on locomotor performances after SCI. In contrast, central administration of XPro1595 resulted in improved locomotor function, decreased anxiety-related behavior, and reduced damage to the lesioned spinal cord, whereas central administration of etanercept had no therapeutic effects. Improvements in XPro1595-treated mice were accompanied by increases in Toll-like receptor 4 and TNF receptor 2 (TNFR2) protein levels and changes in Iba1 protein expression in microglia/macrophages 7 and 28 days after SCI. CONCLUSIONS: These studies suggest that, by selectively blocking solTNF, XPro1595 is neuroprotective when applied directly to the lesioned cord. This protection may be mediated via alteration of the inflammatory environment without suppression of the neuroprotective effects of tmTNF signaling through TNFR2.

Guiding intracortical brain tumour cells to an extracortical cytotoxic hydrogel using aligned polymeric nanofibres.

Glioblastoma multiforme is an aggressive, invasive brain tumour with a poor survival rate. Available treatments are ineffective and some tumours remain inoperable because of their size or location. The tumours are known to invade and migrate along white matter tracts and blood vessels. Here, we exploit this characteristic of glioblastoma multiforme by engineering aligned polycaprolactone (PCL)-based nanofibres for tumour cells to invade and, hence, guide cells away from the primary tumour site to an extracortical location. This extracortial sink is a cyclopamine drug-conjugated, collagen-based hydrogel. When aligned PCL-nanofibre films in a PCL/polyurethane carrier conduit were inserted in the vicinity of an intracortical human U87MG glioblastoma xenograft, a significant number of human glioblastoma cells migrated along the aligned nanofibre films and underwent apoptosis in the extracortical hydrogel. Tumour volume in the brain was significantly lower following insertion of aligned nanofibre implants compared with the application of smooth fibres or no implants.

Photocrosslinkable chitosan based hydrogels for neural tissue engineering.

Hydrogel based scaffolds for neural tissue engineering can provide appropriate physico-chemical and mechanical properties to support neurite extension and facilitate transplantation of cells by acting as 'cell delivery vehicles'. Specifically, in situ gelling systems such as photocrosslinkable hydrogels can potentially conformally fill irregular neural tissue defects and serve as stem cell delivery systems. Here, we report the development of a novel chitosan based photocrosslinkable hydrogel system with tunable mechanical properties and degradation rates. A two-step synthesis of amino-ethyl methacrylate derivitized, degradable, photocrosslinkable chitosan hydrogels is described. When human mesenchymal stem cells were cultured in photocrosslinkable chitosan hydrogels, negligible cytotoxicity was observed. Photocrosslinkable chitosan hydrogels facilitated enhanced neurite differentiation from primary cortical neurons and enhanced neurite extension from dorsal root ganglia (DRG) as compared to agarose based hydrogels with similar storage moduli. Neural stem cells (NSCs) cultured within photocrosslinkable chitosan hydrogels facilitated differentiation into tubulin positive neurons and astrocytes. These data demonstrate the potential of photocrosslinked chitosan hydrogels, and contribute to an increasing repertoire of hydrogels designed for neural tissue engineering.

Sustained delivery of activated Rho GTPases and BDNF promotes axon growth in CSPG-rich regions following spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) often results in permanent functional loss. This physical trauma leads to secondary events, such as the deposition of inhibitory chondroitin sulfate proteoglycan (CSPG) within astroglial scar tissue at the lesion. METHODOLOGY/PRINCIPAL FINDINGS: We examined whether local delivery of constitutively active (CA) Rho GTPases, Cdc42 and Rac1 to the lesion site alleviated CSPG-mediated inhibition of regenerating axons. A dorsal over-hemisection lesion was created in the rat spinal cord and the resulting cavity was conformally filled with an in situ gelling hydrogel combined with lipid microtubes that slowly released constitutively active (CA) Cdc42, Rac1, or Brain-derived neurotrophic factor (BDNF). Treatment with BDNF, CA-Cdc42, or CA-Rac1 reduced the number of GFAP-positive astrocytes, as well as CSPG deposition, at the interface of the implanted hydrogel and host tissue. Neurofilament 160kDa positively stained axons traversed the glial scar extensively, entering the hydrogel-filled cavity in the treatments with BDNF and CA-Rho GTPases. The treated animals had a higher percentage of axons from the corticospinal tract that traversed the CSPG-rich regions located proximal to the lesion site. CONCLUSION: Local delivery of CA-Cdc42, CA-Rac1, and BDNF may have a significant therapeutic role in overcoming CSPG-mediated regenerative failure after SCI.

In situ gelling hydrogels for conformal repair of spinal cord defects, and local delivery of BDNF after spinal cord injury.

Permanent functional loss usually occurs after injury to the spinal cord. Currently, a clinical strategy to promote regeneration in the injured spinal cord does not exist. It has become evident that in order to promote regeneration, a growth permissive substrate at the injury site is critical. In this study, we report the utilization of an agarose scaffold that gels in situ, conformally filling an irregular, dorsal over-hemisection spinal cord defect in adult rats. Besides being growth permissive, the scaffolds also serve as carriers of trophic factors when embedded with BDNF releasing microtubules. We report that our thermo-reversible scaffolds are capable of supporting 3D neurite extension in vivo and are effective carriers of drug delivery vehicles for sustained local delivery of trophic factors. We demonstrate that BDNF encourages neurite growth into the scaffolds, and reduces further the minimal inflammatory response agarose gels generate in vivo as evidenced by quantitative analysis of the extent of NF-160 kDA positive neurons and axons, GFAP positive reactive astrocytes, and CS-56 positive chondroitin sulfate proteoglycan at the interface of the scaffold and host spinal cord. We suggest that these thermo-reversible scaffolds have great potential to serve as growth permissive 3D scaffolds, and to present neurotrophic factors and potentially anti-scar agents to the injury site and enhance regeneration after spinal cord injury.

Modulation of Rho GTPase activity alleviates chondroitin sulfate proteoglycan-dependent inhibition of neurite extension.

The central nervous system (CNS) fails to regenerate after injury. A glial scar forms at the injury site, contributing to regenerative failure partly resulting from the chondroitin sulfate proteoglycans (CSPGs) in the glial scar. The family of Rho GTPases, which includes Cdc42, Rac1, and RhoA, is involved in growth cone dynamics. Although the response of neural cells to the inactivation of Rho when contacting myelin-related substrates, or CSPG, has been investigated, Rac1's and Cdc42's abilities to modulate CSPG-dependent inhibition have yet to be explored. In this study, a stripe assay was utilized to examine the effects of modulating all three Rho GTPases on neurite extension across inhibitory CSPG lanes. Alternating laminin (LN) and CSPG lanes were created and NG108-15 cells and E9 chick dorsal root ganglia (DRGs) were cultured on the lanes. By using the protein delivery agent Chariot, the neuronal response to exposure of constitutively active (CA) and dominant negative (DN) mutants of the Rho GTPases, along with the bacterial toxin C3, was determined by quantifying the percentage ratio of neurites crossing the CSPG lanes. CA-Cdc42, CA-Rac1, and C3 transferase significantly increased the number of neurites crossing into the CSPG lanes compared with the negative controls for both the NG108-15 cells and the E9 chick DRGs. We also show that these mutant proteins require the delivery vehicle, Chariot, to enter the neurons and affect neurite extension. Therefore, activation of Cdc42 and Rac, as well as inhibition of Rho, helps overcome the CSPG-dependent inhibition of neurite extension.