Murine models provide microvascular insights into the 3-D network disarray seen in retinopathy and cardiovascular diseases. Light-sheet fluorescence microscopy (LSFM) has emerged to capture retinal vasculature in 3-D, allowing for assessment of the progression of retinopathy and the potential to screen new therapeutic targets in mice. We hereby coupled LSFM, also known as selective plane illumination microscopy, with topological quantification, to characterize the retinal vascular plexuses undergoing preferential obliteration.
Electrospinning is a method in which materials in solution are formed into nano‐ and micro‐sized continuous fibers. Recent interest in this technique stems from both the topical nature of nanoscale material fabrication and the considerable potential for use of these nanoscale fibres in a range of applications including, amongst others, a range of biomedical applications processes such as drug delivery and the use of scaffolds to provide a framework for tissue regeneration in both soft and hard tissue applications systems. The objectives of this review are to describe the theory behind the technique, examine the effect of changing the process parameters on fiber morphology, and discuss the application and impact of electrospinning on the fields of vascular, neural, bone, cartilage, and tendon/ligament tissue engineering.
The development of small-diameter vascular grafts that can meet the long-term patency required for implementation in clinical practice presents a key challenge to the research field. Although techniques such as the braiding of scaffolds can offer a tunable platform for fabricating vascular grafts, the effects of braided silk fiber skeletons on the porosity, remodeling, and patency in vivo have not been thoroughly investigated. Here, we used finite element analysis of simulated deformation and compliance to design vascular grafts comprised of braided silk fiber skeletons with three different degrees of porosity. Following the synthesis of low-, medium-, and high-porosity silk fiber skeletons, we coated them with hemocompatible sulfated silk fibroin sponges and then evaluated the mechanical and biological functions of the resultant silk tubes with different porosities. Our data showed that high-porosity grafts exhibited higher elastic moduli and compliance but lower suture retention strength, which contrasted with low-porosity grafts. Medium-porosity grafts offered a favorable balance of mechanical properties. Short-term in vivo implantation in rats indicated that porosity served as an effective means to regulate blood leakage, cell infiltration, and neointima formation. High-porosity grafts were susceptible to blood leakage, while low-porosity grafts hindered graft cellularization and tended to induce intimal hyperplasia. Medium-porosity grafts closely mimicked the biomechanical behaviors of native blood vessels and facilitated vascular smooth muscle layer regeneration and polarization of infiltrated macrophages to the M2 phenotype. Due to their superior performance and lack of occlusion, the medium-porosity vascular grafts were evaluated in long-term (24-months) in vivo implantation. The medium-porosity grafts regenerated the vascular smooth muscle cell layers and collagen extracellular matrix, which were circumferentially aligned and resembled the native artery. Furthermore, the formed neoarteries pulsed synchronously with the adjacent native artery and demonstrated contractile function. Overall, our study underscores the importance of braided silk fiber skeleton porosity on long-term vascular graft performance and will help to guide the design of next-generation vascular grafts.
The Staged Electron Laser Acceleration — Laser Wakefield (STELLA‐LW) experiment is investigating two new methods for laser wakefield acceleration (LWFA) using the TW CO2 laser available at the Brookhaven National Laboratory Accelerator Test Facility. The first is seeded self‐modulated LWFA where an ultrashort electron bunch (seed) precedes the laser pulse to generate a wakefield that the laser pulse subsequently amplifies. The second is pseudo‐resonant LWFA where nonlinear pulse steepening of the laser pulse occurs in the plasma allowing the laser pulse to generate significant wakefields. The status of these experiments is reviewed. Evidence of wakefield generation caused by the seed bunches has been obtained as well as preliminary energy gain measurements of a witness bunch following the seeds. Comparison with a 1‐D linear model for the wakefield generation appears to agree with the data.
The advent of light-sheet fluorescence microscopy (LSFM) has revolutionized the imaging of the cardiovascular system, allowing for visualizing the 3-D intact vascular network with high-spatiotemporal resolution and minimal photobleaching. We introduce the custom-built LSFM combined with simplified passive CLARITY (SPC) method to study the vascular development in murine retina bypassing the flat-mount sample preparation. This imaging strategy enables the rapid acquisition of entire retina with a single scan to interrogate vascular development and study angiogenesis. For the sample preparation, we dissected the retina from C57BL/6 mice at P10 for clearing. The clearing method was modified based on the previous passive CLARITY method. In brief, mouse retinas were fixed in 4% Paraformaldehyde (wt/vol) for 2 hours and transferred to the Eppendorf filled with monomer solution (4% Acrylamide (wt/vol), 0.05% Bis-Acrylamide (wt/vol), and 0.25% VA-044 initiator (wt/vol) in PBS) and incubated overnight. After thorough incubation, we put the sample in 37 degrees water bath for hydrogel polymerization for 4 hours. The retinal were then placed into a clearing solution comprised of 4% w/v sodium dodecyl sulfate (SDS) and 1.25% w/v boric acid (pH 8.5). The samples were incubated at 37 °C until cleared (Figure 1B). Our clearing method allowed for the generation of tissue-hydrogel hybrids to support the intact bowl shape structure of the retina. After the SPC, the retinas were washed with PBS and incubated with Biotinylated GS I4B lectin in 1:20 dilution for overnight and secondary antibody Alexa-488 conjugated streptavidin with 1:100 dilution for 2 days to complete the staining on vasculature. Our in-house dual-sided illumination LSFM applied a continuous wave laser with triple wavelengths as the illumination source. The detection module was installed perpendicular to the illumination plane, and it was composed of a stereo microscope with a 1X magnification objective (NA = 0.25), a scientific CMOS and a set of filters (Figure 1A). The lateral resolution and axial resolution of this LSFM system are 3.25 μm and 5 μm respectively. The cleared retinas were immersed in the refractive index (RI) matching solution (RI: 1.46–1.4844) with 1% agarose solution was mounted in a Borosilicate glass tubing (RI = 1.47) to reduce refraction and reflection among various interfaces. We succeeded in imaging the 3-D vascular network (Figure 1C), demonstrating the feasibility of this strategy to provide a structural analysis of the microvascular development. In addition, the adapted SPC preserved the native structure of the retina comparing to the flat-mount. It further reduces the complexity for clearing by removing vacuum chamber and nitrogen tank. This advanced imaging strategy is not limited to the shallow primary plexus layer for morphometric measurements but providing an accessibility for investigating the deeper secondary vascular plexus in murine. Unlike traditional confocal or wide-field imaging of the flat-mount retina, LSFM allows for rapid scanning with high axial resolution and low photo-bleaching, enabling 3D spatial localization of the tissue structure and cellular events with multi-channels of fluorescence. Support or Funding Information NIH (5R01HL083015-10) Figure 1Open in figure viewerPowerPoint A. Schematic diagram of a dual-sided illumination for the light-sheet fluorescent microscope. B. P10 mouse retinas before SPC (left), after clearing was complete (right) at day 7 of the clearing. C. 3-D perception of Alexa-488 labeled retina samples in ROI displaying the 3D vascular network. Scale bar: (B) 1 mm (C) 200 μm for. M1-10: mirror; PH: pinhole; ND: neutral density filter; BE: beam expander; S: slit; CL1-2: cylindrical lens; L1-4: achromatic doublets; Ob1-2: objective This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A simple, one-step technology is developed to generate a hydroxyapatite (HA)-containing silk fibroin nanofibrous scaffold which has great potential as osteogenesis promoting scaffolds for constructing tissue-engineered periosteum.
Abstract Porous three‐dimensional (3D) silk fibroin (SF) scaffolds were widely applied for bone regeneration and showed excellent biocompatibility and biodegradability. Recently graphene was developed for bone scaffolds due to its osteogenic properties. Thus, we combine the SF and graphene to improve the osteogenic properties of SF scaffolds. In our study, we explored the incorporation of SF scaffolds with graphene to develop osteogenic scaffolds capable of accelerating bone formation. The 3D SF scaffolds were fabricated with different contents of graphene (0, 0.5, and 2%). Fluorescence images showed that the graphene nanosheets were homogeneously dispersed in the SF scaffolds. The addition of graphene affected the microarchitecture of the scaffolds. The G/SF scaffolds were cocultured with rat bone marrow‐derived mesenchymal stem cells (rBMSCs) for 21 days. The cell morphology and cell proliferation study suggested that 0 and 0.5% G/SF scaffolds displayed good cell proliferation. In addition, immunofluorescent staining (e.g., osteonectin, osteopontin, and osteocalcin) and ALP activities indicated that the osteogenic properties was more actively exhibited on 0.5% G/SF scaffolds compared with the other groups. Our results indicated that SF scaffolds incorporated with graphene could be an appropriate scaffold for bone tissue engineering.
A salt-leached porous silk fibroin carrier was fabricated to improve the handling properties of DBM powder and to support the attachment, proliferation and osteogenic differentiation of BMSCs.
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