37/67-laminin receptor facilitates neural crest cell migration during enteric nervous system development.

Enteric nervous system (ENS) development is governed by interactions between neural crest cells (NCC) and the extracellular matrix (ECM). Hirschsprung disease (HSCR) results from incomplete NCC migration and failure to form an appropriate ENS. Prior studies implicate abnormal ECM in NCC migration failure. We performed a comparative microarray of the embryonic distal hindgut of wild-type and EdnrBNCC-/- mice that model HSCR and identified laminin-ß1 as upregulated in EdnrBNCC-/- colon. We identified decreased expression of 37/67 kDa laminin receptor (LAMR), which binds laminin-ß1, in human HSCR myenteric plexus and EdnrBNCC-/- NCC. Using a combination of in vitro gut slice cultures and ex vivo organ cultures, we determined the mechanistic role of LAMR in NCC migration. We found that enteric NCC express LAMR, which is downregulated in human and murine HSCR. Binding of LAMR by the laminin-ß1 analog YIGSR promotes NCC migration. Silencing of LAMR abrogated these effects. Finally, applying YIGSR to E13.5 EdnrBNCC-/- colon explants resulted in 80%-100% colonization of the hindgut. This study adds LAMR to the large list of receptors through which NCC interact with their environment during ENS development. These results should be used to inform ongoing integrative, regenerative medicine approaches to HSCR.

Manuka honey modulates the release profile of a dHL-60 neutrophil model under anti-inflammatory stimulation.

Manuka honey, a wound treatment used to eradicate bacteria, resolve inflammation, and promote wound healing, is a current focus in the tissue engineering community as a tissue template additive. However, Manuka honey's effect on neutrophils during the inflammation-resolving phase has yet to be examined. This study investigates the effect of 0.5% and 3% Manuka honey on the release of cytokines, chemokines, and matrix-degrading enzymes from a dHL-60 neutrophil model in the presence of anti-inflammatory stimuli (TGF-ß, IL-4, IL-4 +IL-13). We hypothesized that Manuka honey would reduce the output of pro-inflammatory signals and increase the release of anti-inflammatory signals. The results of this study indicate that 0.5% honey significantly increases the release of CXCL8/IL-8, CCL2/MCP-1, CCL4/MIP-1ß, CCL20/MIP-3α, IL-4, IL-1ra, and FGF-13 while reducing Proteinase 3 release in the anti-inflammatory-stimulated models. However, 3% honey significantly increased the release of TNF-α and CXCL8/IL-8 while reducing the release of all other analytes. We replicated a subset of the most notable findings in primary human neutrophils, and the consistent results indicate that the HL-60 data are relevant to the performance of primary cells. These findings demonstrate the variable effects of Manuka honey on the release of cytokines, chemokines, and matrix-degrading enzymes of this model of neutrophil anti-inflammatory activity. This study reinforces the importance of tailoring the concentration of Manuka honey in a wound or tissue template to elicit the desired effects during the inflammation-resolving phase of wound healing. Future in vivo investigation should be undertaken to translate these results to a physiologically-relevant wound environment.

Semi-interpenetrating network (sIPN) co-electrospun gelatin/insulin fiber formulation for transbuccal insulin delivery.

PURPOSE: This work was aimed at developing a semi-interpenetrating network (sIPN) co-electrospun gelatin/insulin fiber scaffold (GIF) formulation for transbuccal insulin delivery. METHODS: Gelatin was electrospun into fibers and converted into an sIPN following eosin Y-initiated polymerization of polyethylene glycol diacrylate (PEG-DA). The cytocompatibility, degradation rate and mechanical properties were examined in the resulting sIPNs with various ratios of PEG-DA to eosin Y to find a suitable formulation for transbuccal drug delivery. Insulin was co-electrospun with gelatin into fibers and converted into an sIPN-GIF using this suitable formulation. The in vitro release kinetics of insulin was evaluated using ELISA. The bioactivity of released insulin was analyzed in 3T3-L1 preadipocytes using Western blotting and Oil Red O staining. The transbuccal permeability of released insulin was determined using an in vitro porcine oral mucosa model. RESULTS: The sIPN-GF formulation of GF cross-linked by PEG-DA (1% w/v) with eosin Y (5% v/v) possessed no cytotoxic effect, a moderate degradation rate with degradation half-life of 49 min, and a significant enhancement in mechanical properties. This formulation was used to fabricate sIPN-GIF. Insulin release was extended up to 4 h by sIPN-GIF. The released insulin successfully triggered intracellular AKT phosphorylation and induced adipocyte differentiation in 3T3-L1 preadipocytes. The transbuccal permeability of released insulin was determined on the order of 10(-7) cm/s. CONCLUSIONS: Insulin can be fabricated into an sIPN-GIF formulation following co-electrospinning and cross-linking without losing bioactivity. It proved the potential of this new formulation for transbuccal insulin delivery.

Imaging, Spectroscopic, Mechanical and Biocompatibility Studies of Electrospun Tecoflex® EG 80A Nanofibers and Composites Thereof Containing Multiwalled Carbon Nanotubes.

The present study discusses the design, development and characterization of electrospun Tecoflex® EG 80A class of polyurethane nanofibers and the incorporation of multiwalled carbon nanotubes (MWCNTs) to these materials. Scanning electron microscopy results confirmed the presence of polymer nanofibers, which showed a decrease in fiber diameter at 0.5% wt. and 1% wt. MWCNTs loadings, while transmission electron microscopy showed evidence of the MWCNTs embedded within the polymer matrix. The fourier transform infrared spectroscopy and Raman spectroscopy were used to elucidate the polymer-MWCNTs intermolecular interactions, indicating that the C-N and N-H bonds in polyurethanes are responsible for the interactions with MWCNTs. Furthermore, tensile testing indicated an increase in the Young's modulus of the nanofibers as the MWCNTs concentration was increased. Finally, NIH 3T3 fibroblasts were seeded on the obtained nanofibers, demonstrating cell biocompatibility and proliferation. Therefore, the results indicate the successful formation of polyurethane nanofibers with enhanced mechanical properties, and demonstrate their biocompatibility, suggesting their potential application in biomedical areas.

Neutrophil Activity and Extracellular Matrix Degradation: Drivers of Lung Tissue Destruction in Fatal COVID-19 Cases and Implications for Long COVID.

Pulmonary fibrosis, severe alveolitis, and the inability to restore alveolar epithelial architecture are primary causes of respiratory failure in fatal COVID-19 cases. However, the factors contributing to abnormal fibrosis in critically ill COVID-19 patients remain unclear. This study analyzed the histopathology of lung specimens from eight COVID-19 and six non-COVID-19 postmortems. We assessed the distribution and changes in extracellular matrix (ECM) proteins, including elastin and collagen, in lung alveoli through morphometric analyses. Our findings reveal the significant degradation of elastin fibers along the thin alveolar walls of the lung parenchyma, a process that precedes the onset of interstitial collagen deposition and widespread intra-alveolar fibrosis. Lungs with collapsed alveoli and organized fibrotic regions showed extensive fragmentation of elastin fibers, accompanied by alveolar epithelial cell death. Immunoblotting of lung autopsy tissue extracts confirmed elastin degradation. Importantly, we found that the loss of elastin was strongly correlated with the induction of neutrophil elastase (NE), a potent protease that degrades ECM. This study affirms the critical role of neutrophils and neutrophil enzymes in the pathogenesis of COVID-19. Consistently, we observed increased staining for peptidyl arginine deiminase, a marker for neutrophil extracellular trap release, and myeloperoxidase, an enzyme-generating reactive oxygen radical, indicating active neutrophil involvement in lung pathology. These findings place neutrophils and elastin degradation at the center of impaired alveolar function and argue that elastolysis and alveolitis trigger abnormal ECM repair and fibrosis in fatal COVID-19 cases. Importantly, this study has implications for severe COVID-19 complications, including long COVID and other chronic inflammatory and fibrotic disorders.

Electrospun blends of gelatin and gelatin-dendrimer conjugates as a wound-dressing and drug-delivery platform.

In this work, we report a new nanofiber construct based on electrospun blends of gelatin and gelatin-dendrimer conjugates. Highly branched star-shaped polyamidoamine (PAMAM) dendrimer G3.5 was covalently conjugated to gelatin via EDC/NHS chemistry. Blends of gelatin and gelatin-dendrimer conjugates mixed with various loading levels of silver acetate (0, 0.83, 1.65, and 3.30% w/w) were successfully electrospun into nanofiber constructs (NCs). The NCs were further converted into semi-interpenetrating networks (sIPNs) with photoreactive polyethylene glycol diacrylate (Mn = 575 g mol(-1)) (PEG DA575). They were characterized in terms of fiber morphology, diameter, pore size, permeability, degradation, and mechanical properties. The resulting sIPN NCs retained nanofiber morphology, possessed similar fiber diameters to counterpart NCs, and gained improved structural stability. The sIPN NCs also showed good swelling capacity owing to porous structures and were permeable to aqueous solutions. Silver-containing sIPN NCs allowed sustained silver release and showed antimicrobial activity against two common types of pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. Incorporation of dendrimers into the gelatin nanofibers through covalent conjugation not only expands drug loading capacity of nanofiber constructs but also provides tremendous flexibility for developing multifunctional electrospun dressing materials.

Mitochondria as a therapeutic: a potential new frontier in driving the shift from tissue repair to regeneration.

Fibrosis, or scar tissue development, is associated with numerous pathologies and is often considered a worst-case scenario in terms of wound healing or the implantation of a biomaterial. All that remains is a disorganized, densely packed and poorly vascularized bundle of connective tissue, which was once functional tissue. This creates a significant obstacle to the restoration of tissue function or integration with any biomaterial. Therefore, it is of paramount importance in tissue engineering and regenerative medicine to emphasize regeneration, the successful recovery of native tissue function, as opposed to repair, the replacement of the native tissue (often with scar tissue). A technique dubbed 'mitochondrial transplantation' is a burgeoning field of research that shows promise in in vitro, in vivo and various clinical applications in preventing cell death, reducing inflammation, restoring cell metabolism and proper oxidative balance, among other reported benefits. However, there is currently a lack of research regarding the potential for mitochondrial therapies within tissue engineering and regenerative biomaterials. Thus, this review explores these promising findings and outlines the potential for mitochondrial transplantation-based therapies as a new frontier of scientific research with respect to driving regeneration in wound healing and host-biomaterial interactions, the current successes of mitochondrial transplantation that warrant this potential and the critical questions and remaining obstacles that remain in the field.

Near-field electrospinning of polydioxanone small diameter vascular graft scaffolds.

The ideal "off the shelf" tissue engineering, small-diameter (SD) vascular graft hinges on designing a scaffold to act as a template that facilitates transmural ingrowth of capillaries to regenerate an endothelized neointimal surface. Towards this goal, we explored two types of near-field electrospun (NFES) polydioxanone (PDO) architectures, as SD vascular graft scaffolds. The first architecture type consisted of a 200 × 200 µm and 500 × 500 µm grid geometry with random fiber infill, while the second architecture consisted of aligned fibers written in a 45°/45° and 20°/70° offset from the long axis written, both on a 4 mm diameter cylindrical mandrel. These vascular graft scaffolds were evaluated for their effective pore size, mechanical properties, and platelet-material interactions compared to traditionally electrospun (TES) scaffolds and Gore-Tex® vascular grafts. It was found that effective pore size, given by 9.9 and 97 µm microsphere filtration through the scaffold wall for NFES scaffolds, was significantly more permeable compared to TES scaffolds and Gore-Tex® vascular grafts. Furthermore, ultimate tensile strength, percent elongation, suture retention, burst pressure, and Young's modulus were all tailorable compared to TES scaffold characterization. Lastly, platelet adhesion was attenuated on NFES scaffolds compared to TES scaffold which approximates the low level of platelet adhesion measured on Gore-Tex®, with all samples showing minimal platelet activation given by P-selectin surface expression. Together, these results suggest a highly tailorable process for the creation of the next generation of small-diameter vascular grafts.

Neutrophil Extracellular Traps: Inflammation and Biomaterial Preconditioning for Tissue Engineering.

Tissue injury initiates a tissue repair program, characterized by acute inflammation and recruitment of immune cells, dominated by neutrophils. Neutrophils prevent infection in the injured tissue through multiple effector functions, including the production of reactive oxygen species, the release of granules, the phagocytosis of invaders, and the extrusion of neutrophil extracellular traps (NETs). However, these canonical protective mechanisms can also have detrimental effects both in the context of infection and in response to sterile injuries. Of particular interest to biomaterials and tissue engineering is the release of NETs, which are extracellular structures composed of decondensed chromatin and various toxic nuclear and granular components. These structures and their dysregulated release can cause collateral tissue damage, uncontrolled inflammation, and fibrosis and prevent the neutrophil from exerting its prohealing functions. This review discusses our knowledge of NETs, including their composition and morphology, signaling pathways, inhibitors, and contribution to inflammatory pathologies, as well as their role in the resolution of inflammation. In addition, we summarize what is known about the release of NETs as a preconditioning event in the response to biomaterials and highlight future considerations to target the neutrophil response and enhance biomaterial-guided tissue repair and regeneration. Impact statement Neutrophil extracellular trap (NET) release is an active process programmed into the neutrophil's molecular machinery to prevent infection. However, the release of NETs on biomaterials appears to be a significant preconditioning event that influences the potential for tissue healing with largely detrimental consequences. Given their contribution to inflammatory pathologies, this review highlights the role of NETs in the response to biomaterials. Together, the studies discussed in this review suggest that biomaterials should be designed to regulate NET release to avoid maladaptive immune responses and improve the therapeutic potential of tissue-engineered biomaterials and their applications in the clinical setting.

Methods for Quantifying Neutrophil Extracellular Traps on Biomaterials.

Neutrophils rapidly accumulate at sites of inflammation, including biomaterial implantation sites, where they can modulate the microenvironment toward repair through a variety of functions, including superoxide generation, granule release, and extrusion of neutrophil extracellular traps (NETs). NETs are becoming increasing implicated as a central player in the host response to a biomaterial, and as such, there is a need for reliable in vitro methods to evaluate the relative degree of NETs and quantify NETs on the surface of biomaterials. Such methods should be relatively high throughput and minimize sampling bias. In this chapter, we describe two procedures, (1) fluorescent image analysis and (2) a NETs-based ELISA, both of which have been specifically optimized to quantify NETs generated from human neutrophils on electrospun polydioxanone templates. Both methods are valid and also compatible with tissue culture plastic, but have a variety of advantages and disadvantages. Therefore, both methods can be used to concomitantly study NETs on the surface of a biomaterial. Finally, while these methods were developed for electrospun templates in a 96-well cell culture plate, they may be easily adapted to a large scale and for other biomaterials, including but not limited to metallics, ceramics, and natural and synthetic polymers.

Near-Field Electrospinning and Melt Electrowriting of Biomedical Polymers-Progress and Limitations.

Near-field electrospinning (NFES) and melt electrowriting (MEW) are the process of extruding a fiber due to the force exerted by an electric field and collecting the fiber before bending instabilities occur. When paired with precise relative motion between the polymer source and the collector, a fiber can be directly written as dictated by preprogrammed geometry. As a result, this precise fiber control results in another dimension of scaffold tailorability for biomedical applications. In this review, biomedically relevant polymers that to date have manufactured fibers by NFES/MEW are explored and the present limitations in direct fiber writing of standardization in published setup details, fiber write throughput, and increased ease in the creation of complex scaffold geometries are discussed.

Mechanical characterization and neutrophil NETs response of a novel hybrid geometry polydioxanone near-field electrospun scaffold.

Near-field electrospinning (NFES) is a direct fiber writing sub-technique derived from traditional electrospinning (TES) by reducing the air gap distance to the magnitude of millimeters. In this paper, we demonstrate a NFES device designed from a commercial 3D printer to semi-stably write polydioxanone (PDO) microfibers. The print head was then programmed to translate in a stacking grid pattern, which resulted in a scaffold with highly aligned grid fibers that were intercalated with low density, random fibers. As the switching process can be considered random, increasing the grid size results in both a lower density of fibers in the center of each grid cell as well as a lower density of 'rebar-like' stacked fibers. These scaffolds resulted in tailorable as well as greater surface pore sizes as given by scanning electron micrographs and 3D permeability as indicated by fluorescent microsphere filtration compared to TES scaffolds of the same fiber diameter. Furthermore, ultimate tensile strength, percent elongation, yield stress, yield elongation, and Young's modulus were all tailorable compared to the static TES scaffold characterization. Lastly, the innate immune response of neutrophil extracellular traps was attenuated on NFES scaffolds compared to TES scaffolds. These results suggest that this novel NFES scaffold architecture of PDO can be highly tailored as a function of programming for a variety of biomedical and tissue engineering applications.

Human neutrophil FcγRIIIb regulates neutrophil extracellular trap release in response to electrospun polydioxanone biomaterials.

During the acute inflammatory response, the release of neutrophil extracellular traps (NETs) is a pro-inflammatory, preconditioning event on a biomaterial surface. Therefore, regulation of NET release through biomaterial design is one strategy to enhance biomaterial-guided in situ tissue regeneration. In this study, IgG adsorption on electrospun polydioxanone biomaterials with differing fiber sizes was explored as a regulator of in vitro human neutrophil NET release. The propensity to release NETs was increased and decreased by modulating adsorbed IgG, suggesting a functional link between IgG and NET formation. Fiber-size dependent NET release was reduced by blocking FcγRIIIb, but not FcγRI, FcγRIIa, or Mac-1 (CD11b/CD18), indicating a specific receptor mediated neutrophil response. Inhibition of transforming growth factor-ß-activated kinase 1 (TAK1), which is activated downstream of FcγRIIIb, significantly reduced the release of NETs in a fiber size-independent manner. These results indicate that in vitro electrospun biomaterial-induced NET release is largely regulated by IgG adsorption, engagement of FcγRIIIb, and signaling through TAK1. Modulation of this pathway may have beneficial therapeutic effects for regulating neutrophil-mediated inflammation by avoiding the adverse effects of NETs and increasing the potential for in situ tissue regeneration. STATEMENT OF SIGNIFICANCE: Electrospun biomaterials have great potential for in situ tissue engineering because of their versatility and biomimetic properties. However, understanding how to design the biomaterial to regulate acute inflammation, dominated by neutrophils, remains a great challenge for successful tissue integration and regeneration. In this work, we demonstrate for the first time how protein adsorption on the biomaterial surface and engagement of a specific neutrophil receptor induces intracellular signals that regulate the pro-inflammatory release of neutrophil extracellular traps (NETs). Given the deleterious effects of NETs during the acute inflammatory response to a biomaterial, our work highlights the importance of considering biomaterial-neutrophil interactions on degradable and non-degradable biomaterials to achieve the desired biological outcome.

Neutrophils in Biomaterial-Guided Tissue Regeneration: Matrix Reprogramming for Angiogenesis.

Biomaterial-guided in situ tissue regeneration uses biomaterials to stimulate and guide the body's endogenous, regenerative processes to drive functional tissue repair and regeneration. To be successful, cell migration into the biomaterials is essential, which requires angiogenesis to maintain cell viability. Neutrophils, the first cells responding to an implanted biomaterial, are now known to play an integral part in angiogenesis in multiple tissues and exhibit considerable potential for driving angiogenesis in the context of tissue regeneration. In terms of biomaterial-guided in situ tissue regeneration, harnessing the proangiogenic potential of the neutrophil through its robust secretion of matrix metalloproteinase 9 (MMP-9) may provide a mechanism to improve biomaterial performance by initiating matrix reprogramming. This review will discuss neutrophils as matrix reprogrammers and what is currently known about their ability to create a microenvironment that is more conducive for angiogenesis and tissue regeneration through the secretion of MMP-9. It will first review a set of ground-breaking studies in tumor biology and then present an overview of what is currently known about neutrophils and MMP-9 in biomaterial vascularization. Finally, it will conclude with potential strategies and considerations to engage neutrophils in biomaterial-guided angiogenesis and in situ tissue regeneration. Impact statement This review draws attention to a highly neglected topic in tissue engineering, the role of neutrophils in biomaterial-guided tissue regeneration and angiogenesis. Moreover, it highlights their abundant secretion of matrix metalloproteinase 9 (MMP-9) for matrix reprogramming, a topic with great potential yet to be vetted in the literature. It presents strategies and considerations for designing the next generation of immunomodulatory biomaterials. While there is literature discussing the overall role of neutrophils in angiogenesis, there are a limited number of review articles focused on this highly relevant topic in the context of biomaterial integration and tissue regeneration, making this a necessary and impactful article.

Electrospun Polydioxanone Loaded With Chloroquine Modulates Template-Induced NET Release and Inflammatory Responses From Human Neutrophils.

The implantation of a biomaterial quickly initiates a tissue repair program initially characterized by a neutrophil influx. During the acute inflammatory response, neutrophils release neutrophil extracellular traps (NETs) and secrete soluble signals to modulate the tissue environment. In this work, we evaluated chloroquine diphosphate, an antimalarial with immunomodulatory and antithrombotic effects, as an electrospun biomaterial additive to regulate neutrophil-mediated inflammation. Electrospinning of polydioxanone was optimized for rapid chloroquine elution within 1 h, and acute neutrophil-biomaterial interactions were evaluated in vitro with fresh human peripheral blood neutrophils at 3 and 6 h before quantifying the release of NETs and secretion of inflammatory and regenerative factors. Our results indicate that chloroquine suppresses NET release in a biomaterial surface area-dependent manner at the early time point, whereas it modulates signal secretion at both early and late time points. More specifically, chloroquine elution down-regulates interleukin 8 (IL-8) and matrix metalloproteinase nine secretion while up-regulating hepatocyte growth factor, vascular endothelial growth factor A, and IL-22 secretion, suggesting a potential shift toward a resolving neutrophil phenotype. Our novel repurposing of chloroquine as a biomaterial additive may therefore have synergistic, immunomodulatory effects that are advantageous for biomaterial-guided in situ tissue regeneration applications.

Patients with COVID-19: in the dark-NETs of neutrophils.

SARS-CoV-2 infection poses a major threat to the lungs and multiple other organs, occasionally causing death. Until effective vaccines are developed to curb the pandemic, it is paramount to define the mechanisms and develop protective therapies to prevent organ dysfunction in patients with COVID-19. Individuals that develop severe manifestations have signs of dysregulated innate and adaptive immune responses. Emerging evidence implicates neutrophils and the disbalance between neutrophil extracellular trap (NET) formation and degradation plays a central role in the pathophysiology of inflammation, coagulopathy, organ damage, and immunothrombosis that characterize severe cases of COVID-19. Here, we discuss the evidence supporting a role for NETs in COVID-19 manifestations and present putative mechanisms, by which NETs promote tissue injury and immunothrombosis. We present therapeutic strategies, which have been successful in the treatment of immunο-inflammatory disorders and which target dysregulated NET formation or degradation, as potential approaches that may benefit patients with severe COVID-19.

Manuka Honey Reduces NETosis on an Electrospun Template Within a Therapeutic Window.

Manuka honey, a topical wound treatment used to eradicate bacteria, resolve inflammation, and promote wound healing, is a focus in the tissue engineering community as a tissue template additive. However, its effect on neutrophil extracellular trap formation (NETosis) on a tissue engineering template has yet to be examined. As NETosis has been implicated in chronic inflammation and fibrosis, the reduction in this response within the wound environment is of interest. In this study, Manuka honey was incorporated into electrospun templates with large (1.7-2.2 µm) and small (0.25-0.5 µm) diameter fibers at concentrations of 0.1%, 1%, and 10%. Template pore sizes and honey release profiles were quantified, and the effect on the NETosis response of seeded human neutrophils was examined through fluorescence imaging and myeloperoxidase (MPO) analysis. The incorporation of 0.1% and 1% Manuka honey decreased NETosis on the template surface at both 3 and 6 h, while 10% honey exacerbated the NETosis response. Additionally, 0.1% and 1% Manuka honey reduced the MMP-9 release of the neutrophils at both timepoints. These data indicate a therapeutic window for Manuka honey incorporation into tissue engineering templates for the reduction in NETosis. Future in vivo experimentation should be conducted to translate these results to a physiological wound environment.

An atorvastatin calcium and poly(L-lactide-co-caprolactone) core-shell nanofiber-covered stent to treat aneurysms and promote reendothelialization.

Aneurysmal subarachnoid hemorrhage is a common complication caused by an intracranial aneurysm that can lead to hemorrhagic stroke, brain damage, and death. Knowing this clinical situation, the purpose of this study was to develop a controlled-release stent covered with a core-shell nanofiber mesh, fabricated by emulsion electrospinning, for the treatment of aneurysms. By encapsulating atorvastatin calcium (AtvCa) in the inner of poly (L-lactide-co-caprolactone) (PLCL) nanofibers, the release period of AtvCa was effectively extended. The morphology and inner structure of the core-shell nanofibers were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. The release of AtvCa from the nanofiber system continued for more than ten weeks without a significant initial burst release. The nanofiber mesh structure degraded gradually but maintained its fiber morphology before neovascularization. The results of this study further elucidated the reendothelialization mechanism of AtvCa by analyzing the nitric oxide (NO) expression from seeded HUVECs. The in vivo studies demonstrated that the PLCL-AtvCa covered stents were capable of separating the aneurysm dome from the blood circulation, leading to the abolishment of the aneurysm. Moreover, the AtvCa controlled release promoted the in vitro proliferation of HUVECs on the nanofiber meshes, and the PLCL-AtvCa covered stents induced in vivo neovascularization. STATEMENT OF SIGNIFICANCE: Intracranial aneurysms are pathological dilatations of blood vessels that have developed an abnormally weak wall structure, thus prone to rupture. Covered stents had been demonstrated to be a method for the treatment of intracranial aneurysm. We prepared a controlled-release stent covered with a core-shell nanofiber mesh, fabricated by emulsion electrospinning, which encapsulated atorvastatin calcium in the inner portion of nanofibers. The results of this study further elucidated the reendothelialization mechanism of AtvCa by analyzing the nitric oxide (NO) expression from seeded HUVECs. The generated AtvCa-load covered stents separated the aneurysm dome from the blood circulation, and keep long-term patency of the parent artery. But also induced neovascularization, thus provide further protection against recurrence of aneurysms after nanofiber meshes degradation.

Characterization of Polydioxanone in Near-Field Electrospinning.

Electrospinning is a popular method for creating random, non-woven fibrous templates for biomedical applications, and a subtype technique termed near-field electrospinning (NFES) was devised by reducing the air gap distance to millimeters. This decreased working distance paired with precise translational motion between the fiber source and collector allows for the direct writing of fibers. We demonstrate a near-field electrospinning device designed from a MakerFarm Prusa i3v three-dimensional (3D) printer to write polydioxanone (PDO) microfibers. PDO fiber diameters were characterized over the processing parameters: Air gap, polymer concentration, translational velocity, needle gauge, and applied voltage. Fiber crystallinity and individual fiber uniformity were evaluated for the polymer concentration and translational fiber deposition velocity. Fiber stacking was evaluated for the creation of 3D templates to guide the alignment of human gingival fibroblasts. The fiber diameters correlated positively with polymer concentration, applied voltage, and needle gauge; and inversely correlated with translational velocity and air gap distance. Individual fiber diameter variability decreases, and crystallinity increases with increasing translational fiber deposition velocity. These data resulted in the creation of tailored PDO 3D templates, which guided the alignment of primary human fibroblast cells. Together, these results suggest that NFES of PDO can be scaled to create precise geometries with tailored fiber diameters for biomedical applications.

Manuka Honey Modulates the Inflammatory Behavior of a dHL-60 Neutrophil Model under the Cytotoxic Limit.

Recent work has shown that Manuka honey, an increasingly popular wound additive with potent antibacterial properties, also has anti-inflammatory properties. However, little research has been done examining its effect on neutrophils. This study investigates the hypothesis that Manuka honey reduces neutrophil superoxide release and chemotaxis and reduces the activation of the inflammatory nuclear factor-κB (NF-κB) signaling pathway under honey's cytotoxic limit. A differentiated HL-60 cell line was used as a neutrophil model and cultured in various concentrations of Manuka honey for 3 and 24 hours to measure cytotoxicity via mitochondrial activity and visual trypan-exclusion count. Cytochrome C and Boyden chamber assays were used to measure the effect of Manuka honey on superoxide release and chemotaxis toward fMLP, respectively. Additionally, a Western blot for NF-κB inhibitor α (IκBα) was performed to measure Manuka honey's effect on the NF-κB pathway via IκBα phosphorylation. The results indicate a cytotoxic limit of 3-5% v/v. The presence of 1% honey decreased superoxide release at 24 hours. The 0.5, 1, and 3% honey concentrations reduced chemotaxis and IκBα phosphorylation in a dose-dependent fashion. These results suggest that Manuka honey significantly reduces neutrophil recruitment and inflammatory behavior in the wound site in a dose-dependent fashion under the cytotoxic limit.