The translation of cell-based therapies for ischemic tissue repair remains limited
The translation of cell-based therapies for ischemic tissue repair remains limited by several factors, including poor cell survival and limited target site retention. hind limb ischemia, an intramuscular injection of BMPACs within this bioactive peptide nanofiber matrix resulted in greater retention of cells, enhanced capillary density, increased limb perfusion, reduced necrosis/amputation, and maintained function of the ischemic limb compared to treatment with cells alone. This self-assembling, bioactive peptide nanofiber matrix showing an integrin-binding domain name of fibronectin enhances regenerative efficacy of cell-based strategies in ischemic tissue by enhancing cell survival, retention, and reparative functions. assays, a soluble concentration of 0.2 w/v% of PA nanofibers in media was used. All experiments were repeated at least three occasions. Cell viability was assessed using an MTS Assay (Promega) using 5103 cells/well in 96-well dishes. Cells were treated for 24 hours with the numerous PAs or media only, following which conversion of the MTS substrate was assessed after 4 hours in triplicate per condition by absorbance at 490 nm (background 650 nm). Apoptosis was evaluated by plating BMPACs in 4-chamber SIRT1 photo slides at 5104 cells/chamber and inducing apoptosis through treatment with H2O2 (50 M), following which cells were assayed by microscopy in 3 impartial high-power fields (20x) per condition after TUNEL staining using the Fluorescein In Situ Cell Death Detection Kit (Roche). For adhesion assays, cells were pre-treated with PA or media for 48 hours on temperature-sensitive cell culture dishes (Nunc) to preserve cell surface proteins. Pretreated cells were then added to 96-well dishes (2.5103/well) coated with collagen type-1, laminin, or vitronectin, in addition to an uncoated surface. After 1 hour, the surface was washed and adherent cells were fixed and stained with DAPI for quantification using fluorescent microscopy in 5 high-power fields at 20x. To assess tube formation, 3104 serum-starved cells were seeded per well in a 48-well plate coated with growth-factor-reduced Matrigel (BD Biosciences). The mean tube length and total number of tube-like forms were quantified by KN-93 Phosphate bright-field microscopy. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) RNA was isolated from cells with RNA STAT-60 (TEL-TEST Electronics Labs, Inc.) according to the manufacturers instructions. Total RNA was reverse transcribed with a Taqman cDNA Synthesis Kit (Applied Biosystems) and amplified using a Taqman KN-93 Phosphate 7500 analyzer (Applied Biosystems). The comparative manifestation of each mRNA was calculated by the comparative threshold cycle (CT) method and normalized to 18S manifestation. Immunoblotting Protein concentrations from cell lysate were decided by a Bradford assay, and equivalent protein amounts were loaded. Following standard SDS PAGE using 10% Tris-HCl SDS gels (Bio-Rad), phosphorylation of Akt and mitogen-activated protein kinase (MAPK) ERK 1/2 (p44/42) were detected using anti-Akt, anti-phospho-Akt, anti-p44/p42, and anti-phospho-p44/p42 antibodies (all 1:1000, Cell signaling). Membranes were developed via horseradish peroxidase-coupled secondary antibodies (1:2000, Bio-Rad) and enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate kit, Thermo Scientific). Protein phosphorylation levels are given as the ratio of phosphorylated to total protein. Scanning electron microscopy Scanning electron microscopy (SEM) was performed using a KN-93 Phosphate S4800 scanning electron microscope with a 3 kV accelerating voltage (Hitachi). Samples of cells on coverslips were fixed with glutaraldehyde, sequentially dehydrated in ethanol, crucial point dried, and coated with osmium tetraoxide. Animals For cell isolation and hind-limb ischemia (HLI) studies, 8-week aged male FVB/N wild type mice (Charles-River) were used. For HLI studies using hCD34+ cells, 8C10 week aged male BALB/c nude mice (Charles-River) were used. For bioluminescence studies, hemizygous male FVB/N-Tg(-Actin-luc)-Xen transgenic mice (Xenogen) with a altered firefly luciferase gene under the constitutive murine -Actin promoter were used. For hind limb ischemia studies, 15 mice per group were planned. Following triage of outliers and removal of animals with severe ischemia over the course of the study, each time point includes an average of between 7C13 mice per group. For comparable reasons, bioluminescence imaging studies included 4C10 animals per group per time point. All KN-93 Phosphate animal studies were approved by the Northwestern University or college Animal Care and Use Committee. Hind-limb ischemia, laser Doppler imaging, and functional limb assessment For the HLI model , mice were anesthetized with isoflurane. Using a dissecting microscope, the femoral nerve was separated from the ship package. The femoral artery was ligated and excised including all superficial and deep twigs. Immediately after the procedure, laser Doppler perfusion imaging (LDPI, MoorLDI-SIM System, Moor Devices) at a wavelength of 785 nm was performed to make sure ischemia, indicated by a ratio of ischemic/non-ischemic limb of 0.20. At postoperative day 3,.