Journal: Bioengineering

Article Title: Pre-Clinical Cell Therapeutic Approaches for Repair of Volumetric Muscle Loss

doi: 10.3390/bioengineering7030097

Figure Lengend Snippet: Animal studies of cell-based therapies in VML treatment.

Article Snippet: Microvessel fragment (MVF) construct with myoblasts (MVF + Myoblasts) , Live/Dead assay demonstrates high viability of microvessels and seeded myoblasts and immunofluorescent staining shows microvessel networks increase more in MVF-Myoblast constructs than in MVF-only constructs. , 12 mm biopsy punch in biceps femoris muscle of Sprague Dawley rats. , Collagen hydrogel. , MVF-Myoblast constructs did not show muscle regeneration at both 2 weeks and 8 weeks post-implantation. , [ ] .

Techniques: In Vitro, Animal Model, In Vivo, Immunohistochemical staining, Imaging, Ex Vivo, Functional Assay, Construct, Expressing, Injection, Muscles, Immunofluorescence, Staining

Journal: Frontiers in Physiology

Article Title: Stromal Cells Promote Neovascular Invasion Across Tissue Interfaces

doi: 10.3389/fphys.2020.01026

Figure Lengend Snippet: The core-in-field (CIF) tissue boundary model. (A) Schematic of the CIF model showing the preformed “core” sitting on a thin bed of gelled collagen surrounded by an additional “field” of gelled collagen. (B) Top-view of a phase microscopy image of a microvessel-free and cell-free CIF construct. (C) SEM image of a cross-section view of a CIF construct. (D) Higher-magnification of the interface and peri-interface region of a CIF construct. (E) Second harmonic generation image of the native collagen fibril structure comprising the interface and adjacent regions. (F) Fibril densities of the three regions in the CIF construct based on a validated method of measurement from SHG images. Bars are mean ± SD, N = 15, one-way ANOVA with Holm-Sidak post hoc analysis. * P < 0.05. In all cases, arrows indicate the interface between the core and field.

Article Snippet: Microvessel fragments were isolated from adult Sprague-Dawley rats by limited collagenase digestion and sequential filtration to remove single cells and retain only MVs ( ).

Techniques: Microscopy, Construct

Journal: Frontiers in Physiology

Article Title: Stromal Cells Promote Neovascular Invasion Across Tissue Interfaces

doi: 10.3389/fphys.2020.01026

Figure Lengend Snippet: Simulations of neovessel growth in core-in-field models. (A) Top-view image of the interface of a CIF construct with microvessels growing along the tissue interface. White arrows point to the interface, black arrows point to microvessel being deflected along the interface. (B) Close-up of cut-view of cylindrical CIF geometry used in AngioFE. The core, interface, and field are colored in green, purple, and blue, respectively. Arrows indicate circumferential direction in the interface. (C) Simulations of vessel growth and behavior in CIF constructs with different interface densities and fibril organization. Visual results of the simulations for three different conditions of initial interface density (3 or 5 mg/mL) and fibril organization (anisotropic or isotropic) after 10 days of simulated culture. Cores are in light green, interfaces are in pink, fields are clear, and vessels are in red. (D) Log plot of predicted neovessel invasion across the interface for the three different simulated conditions, with an initial collagen concentration of either 3 or 4 mg/mL in the core. All groups are compared to the observed experimental value for microvessel-only constructs (white). Bars are mean ± SD, N = 4 for experiments, 10 for simulations. One-way ANOVA with Holm-Sidak post hoc analysis. *** P < 0.001 compared to experiments. There was an effect of interfacial matrix density on crossing regardless of core density. Further, simulated fibrils were more highly aligned than what was observed experimentally. (E) Validation of simulation mechanics determined by predicted final density after microvascular growth. Comparison of day 10 experimental (SHG) collagen densities after microvessel growth and contraction alongside predicted day 10 collagen densities from simulations. Simulations had an initial density in the core of 3 or 4 mg/mL. The initial interface and field densities were 5 and 3 mg/mL, respectively with isotropic fibrils for all simulations. The final densities predicted for each region by simulations are not different from those measured experimentally. One-way ANOVA performed on each region (core, interface, and field). Bars are mean ± SD. N = 15 (SHG) or N = 10 (simulation). P > 0.05.

Article Snippet: Microvessel fragments were isolated from adult Sprague-Dawley rats by limited collagenase digestion and sequential filtration to remove single cells and retain only MVs ( ).

Techniques: Construct, Concentration Assay, Biomarker Discovery, Comparison

Journal: Frontiers in Physiology

Article Title: Stromal Cells Promote Neovascular Invasion Across Tissue Interfaces

doi: 10.3389/fphys.2020.01026

Figure Lengend Snippet: Stromal vascular fraction cells promote neovessel crossing. (A) Without SVF cells, microvessels grow up to and then along the interface. (B) Inclusion of SVF cells results in neovessels crossing the interface to invade the field region. (C) SVF cells resulted in significant increases in neovessel crossing events, particularly when added to the field region. (D) Both cell number and spatial positioning affect crossing events. Bars are mean ± SEM, N = 4 (C) or N = 3 (D) . One-way ANOVA with Newman–Keuls (C) or Tukey (D) post hoc analysis. * P < 0.05 compared to all other or specified groups. White arrows point to interface.

Article Snippet: Microvessel fragments were isolated from adult Sprague-Dawley rats by limited collagenase digestion and sequential filtration to remove single cells and retain only MVs ( ).

Techniques:

Journal: Frontiers in Physiology

Article Title: Stromal Cells Promote Neovascular Invasion Across Tissue Interfaces

doi: 10.3389/fphys.2020.01026

Figure Lengend Snippet: Stromal vascular fraction cells do not disrupt gross fibril structure of the interface. (A) Collagen fibril densities at and near the interface as measured by SHG imaging. Acellular CIF constructs are compared to microvessels cultured in CIF constructs (MV) and microvessels and SVF cells cultured for 10 days (MV + SVF). Bars are mean ± SD. N = 22, 15, and 10 for acellular, MV, and MV + SVF, respectively. Separate one-way ANOVA for densities in the core, interface, or field, with P > 0.05 in all cases. (B–D) SEM images of CIF constructs containing either microvessels (MV) or microvessels and SVF cells (MV + SVF). Arrows indicate the interface between the core and field. (D) is a higher magnification of the area in panel (C) highlighted by the dashed box. Arrows point to interface.

Article Snippet: Microvessel fragments were isolated from adult Sprague-Dawley rats by limited collagenase digestion and sequential filtration to remove single cells and retain only MVs ( ).

Techniques: Imaging, Construct, Cell Culture

Journal: Frontiers in Physiology

Article Title: Stromal Cells Promote Neovascular Invasion Across Tissue Interfaces

doi: 10.3389/fphys.2020.01026

Figure Lengend Snippet: Stromal cell migration from the core to the field. (A,B) Confocal image stacks of CIF constructs formed with microvessels (red, rhodamine labeled Lectin stain) and SVF cells (green, GFP+) in the core after 10 days of culture. (C,D) Images of Hoechst stained samples without (C) or with (D) SVF incorporated into the core region. (E) Graph of Hoechst stained cells counted in the field region after 10 days of culture, with or without initial SVF incorporation in the core region. The circle, triangle, and box each represent a different experiment, with the line representing the mean of the experiments. Each experiment was statistically evaluated individually using a student’s t -test. P < 0.05 within the circle experiment and square experiment, but not the triangle experiment. In (A–D) , white arrows point to microvessels, black arrows point to SVF. White dashed line indicates the interface.

Article Snippet: Microvessel fragments were isolated from adult Sprague-Dawley rats by limited collagenase digestion and sequential filtration to remove single cells and retain only MVs ( ).

Techniques: Migration, Construct, Labeling, Staining

Journal: Frontiers in Physiology

Article Title: Stromal Cells Promote Neovascular Invasion Across Tissue Interfaces

doi: 10.3389/fphys.2020.01026

Figure Lengend Snippet: Effect of VEGF-A on neovessel invasion. Effect of a VEGF trap on (A) interface crossings and (B) vessel density, compared SVF alone and a control IgG chimera protein. (C) Normalized neovessel invasion and (D) vessel density in CIF constructs containing microvessels alone or microvessels with recombinant VEGF-A 165 added to the media (+VEGF). One Way ANOVA with Newman–Keuls test (A,B) or Student’s t -test (C,D) . Bars are mean ± SEM, N = 5. * P < 0.05 compared to all other groups.

Article Snippet: Microvessel fragments were isolated from adult Sprague-Dawley rats by limited collagenase digestion and sequential filtration to remove single cells and retain only MVs ( ).

Techniques: Control, Construct, Recombinant

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Rapid Perfusion and Network Remodeling in a Microvascular Construct After Implantation

doi: 10.1161/01.atv.0000124103.86943.1e

Figure Lengend Snippet: Figure 4. Smooth muscle coverage of vessel elements changes during vascularization. Confocal image stacks of double-stained vessel elements (greenMHC and red-actin) were used to generate vessel and perivascular volumes. Single-image planes of (a) a freshly isolated microvessel fragment (day 0), (b) a day 8 cultured fragment, and (c) day 28 postimplantation branched vessel element. d, Percentage of vessel elements covered by -actin–positive perivascular cells measured at different time points in the model. Coverage was assessed during culturing of microvascular constructs (day 0c–day 10c) and during implanta- tion of constructs precultured for 8 days before implantation (day14i–day 28i).

Article Snippet: Rat fat microvessel fragments (RFMFs) were isolated from epididymal fat pads of retired breeder male Sprague-Dawley rats.

Techniques: Staining, Isolation, Cell Culture, Construct

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Rapid Perfusion and Network Remodeling in a Microvascular Construct After Implantation

doi: 10.1161/01.atv.0000124103.86943.1e

Figure Lengend Snippet: Figure 5. Microvascular constructs assembled from rat-derived or human-derived freshly isolated microvessel fragments (no preculturing, see Methods) also form a vascular bed on implan- tation. a, En bloc fluorescence immunostaining for all cells (anti- MHC antibody) reveals that vessels within the construct form tree-like structures. b, Ink perfusion of the host and a superficial vessel connection (arrowheads) between host and construct (arrows indicate host muscle/construct boundary). Immunostain- ing of human-derived, day 15 implants for von Willebrand factor (c) or -actin (d) indicates that vessels (arrows) are differentiated and mature. Representative sections from the same human- derived implant stained with the human-specific lectin, UEA1 labels only vessels (arrows in e) within the construct, whereas the rodent-specific vascular marker GS-1 labels vessels within only the surrounding host mouse tissue (arrows in f). The dashed lines indicate the boundary between the implant and underlying host tissue.

Article Snippet: Rat fat microvessel fragments (RFMFs) were isolated from epididymal fat pads of retired breeder male Sprague-Dawley rats.

Techniques: Construct, Derivative Assay, Isolation, Immunostaining, Staining, Marker