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In Vivo Assessment
Published in Karen J.L. Burg, Didier Dréau, Timothy Burg, Engineering 3D Tissue Test Systems, 2017
Maria Yanez, Scott Collins, Thomas Boland
Frozen tissues were placed on Leica cryocut 1800 maintained at −30°C and sectioned to 7–20 μm. The sections were collected on poly-l-Lysine-coated slides for improved adherence. Tissue samples were stained with hematoxylin and eosin (H&E) staining and Masson's trichrome staining. Tissue sections for immunohistochemistry were hydrated using successive concentrations of ethanol starting from 100%, 95%, 70%, 50% and finishing with distilled water. For antigen unmasking, the samples were boiled for 15 min in an antigen retrieval solution consisting of 10 mM sodium citrate, 0.05% Tween 20 at pH 6.0, cooled on a bench top for 30 min, and rinsed with 1× PBS. Cells were permeabilized with 100% methanol at −20°C for 6 min to provide access to the antibody. Tissue samples were blocked for 1 h at room temperature in 5% normal goat serum. Samples were incubated for 1 h at room temperature with FITC, labeled mouse monoclonal CD34 antibody in PBS (1:100). Unbound antibodies were removed by washing the samples three times with PBS 10 min each. During the final rinse, the cells were incubated with DAPI. Samples were mounted with DAKO mounting media and covered with a coverslip. Imaging was performed with a Nikon Confocal microscope using a 10× objective.
Biological-Derived Biomaterials for Stem Cell Culture and Differentiation
Published in Gilson Khang, Handbook of Intelligent Scaffolds for Tissue Engineering and Regenerative Medicine, 2017
Creation of a bioartificial lung requires engineering of viable and functional lung architecture to enable ventilation, perfusion, and gas exchange. Petersen et al. harvested lungs from adult Fischer 344 rats and treated the vascular and airway compartments with a solution containing CHAPS, a zwitterionic detergent, in a phosphate buffer at 1 M salt concentration. They maintained the vascular perfusion pressure below 20 mmHg for 2–3 h.111 The cells and nuclear material were removed during the decellularization process, but the alveolar septal architecture remained undisturbed. A DNA quantification assay confirmed that approximately 99% of the DNA was removed by the decellularization process. Price et al. used lungs from 8–12-week-old mice for decellularization.112 Infusion of decellularization solutions via both the trachea and the vascular route (though the right ventricle) resulted in more complete decellularization than either route alone. Masson’s trichrome staining demonstrated that the decellularized lungs were acellular and that the pulmonary matrix, including collagen and elastin components, was intact. Immunostaining for laminin, which is important in alveolar epithelial cell differentiation, showed that the deposition of this matrix component was also relatively unaffected by the decellularization process.
Biocomposites as Implantable Biomaterials
Published in Yaser Dahman, Biomaterials Science and Technology, 2019
In-vivo studies were conducted by attaching the MWC scaffold onto rabbits’ femoral defects. The experiment was conducted using a total of 24 rabbits. 12 rabbits were used for MWC and WP scaffolding. Then, of the 4 rabbits were used for the MWC and WP scaffolding every 4 weeks (at 4 weeks, 8 weeks, and 12 weeks after the initial testing of 12 rabbits). A radial defect of 6 mm was created in the left thighbone of the rabbits through median incision. The defects were then fitted with the scaffolds. New bone formation was observed under a high power microscope. Samples were stained with Masson trichrome and observed under microscope. Masson trichrome staining proves the in-vivo effect of the MWC scaffold on new bone creation and degradation. Statistical study of histological sections was used to quantify newly formed bone. The same image was passed through analytical image software to find the new bone vs mature bone ratio. Synchrotron-microcomputed tomography (SRmCT) analysis was conducted using monochromatic light to detect bone defect repair after scaffold implantation. Samples were decalcified, washed, and dehydrated in alcohol for 30 days at room temperature. The results showed that defects were restored in line, without any complexity. The effectiveness and speed of bone defect repair was greater than that of WP. Histological elevation showed that the new bone vs mature bone ratio was higher in MWC scaffold. By 12 weeks, the MWC and WP scaffold degraded but the WP scaffold degraded faster than the MWC scaffold. Immunohistochemical analysis using XRD showed that stained MWC scaffold had a higher intensity than WP scaffold indicating better osteogenic properties. Histomorphometry analysis based on Masson trichrome staining suggested that the MWC scaffold stimulates effective new bone tissue growth with faster and improved bone regeneration and remodelling. So, Masson trichrome staining through immunohistochemical analysis showed that bone regeneration of the MWC scaffold is effective, improved, and faster, showing better osteogenic properties in the MWC scaffold (Xia et al., 2016).
Preparation of hybrid meniscal constructs using hydrogels and acellular matrices
Published in Journal of Biomaterials Science, Polymer Edition, 2023
Gizem Zihna, Bengisu Topuz, Gülçin Günal, Halil Murat Aydin
Hematoxylin & Eosin (H&E) and DAPI staining were used to confirm the removal of cell nuclei and the integrity of ECM. Posterior regions of meniscus tissues were fixed in 10% (v/v) formalin and subsequently dehydrated by graded series of ethanol and xylene. Then, tissues were embedded in paraffin blocks and sectioned at 5-micron thickness via microtome. The cross-sections were stained with H&E and DAPI. Moreover, Masson’s trichrome staining was performed to observe collagen fibers in native and decellularized tissues. Proteoglycans were visualized with Safranin-O/Fast Green staining. All staining was imaged with a light microscope (Leica, Germany).
Collagen-coated silk fibroin nanofibers with antioxidants for enhanced wound healing
Published in Journal of Biomaterials Science, Polymer Edition, 2023
Sowmya Selvaraj, Chandrasekar Inbasekar, Suryalakshmi Pandurangan, Nishad Fathima Nishter
Collagen formation is one of the key indicators in tissue engineering and it is necessary for the structure and function of healthy skin. Masson’s trichrome staining was performed to examine the amount of collagen deposition in wounds (indicated by blue color) and the results are shown in Figure 7(b). Matured collagen bundles were observed in wounds treated with Col SF- KH and Col SF-SH nanofibers on day 16 and it was not observed in the other two groups. Thus, Col SF-KH nanofibers showed enhanced wound healing, which could be due to its anti-oxidant and anti-inflammatory properties.