3.3.1 Sectioning of LR White embedded matrices for histology
Taking into account the desired orientation of the final tissue sections (sagittal or transverse), the resin embedded tissues were excised from the resin. The embedded tissue blocks were placed in a microtome (Ultracut E, Reichert-Jung, Depew, USA) sample holder and excess resin was trimmed from the face of the block with a sharp razor blade until the tissue was exposed. Glass knives were made (2178 Knifemaker II, LKB, Bromma, Sweden) and used to obtain a smooth flat surface of the embedded samples for sectioning. A new glass knife was used to obtain 2 µM sections of the samples. Cut sections were carefully placed onto a drop of distilled water on a microscope slide. The sections were semi-fixed to the slide by placing the slide onto a heating block (Dishwarmer 2, Photax, USA) for 24 hours. The slides were then stored in a slide container at RT until required (Partington, Mordan et al. 2013).
3.3.2 Histological analysis of LR White embedded matrices by toluidine blue staining
Toluidine blue, otherwise known as tolonium chloride, is an acidophilic metachromatic. Filtered toluidine blue was gently dropped onto the section and then immediately but gently washed off with distilled water. The sections were observed under an optical microscope (BX50, Olympus, UK) and imaged (Coolsnap-Pro cf color, Media Cybernetics, Bethesda, USA).
3.3.3 Histological analysis of LR White embedded matrices by haematoxylin and eosin (H&E) staining
The slides were immersed in Mayer’s haematoxylin for 1 hour at room temperature. The sections were then blued by being immersed in gently running tap-warm water for 20 minutes. The slides were then immersed in 5% eosin for 1 hour at room temperature. The sections were then rinsed very gently in static tap water. The sections were dried on the heating block for a few minutes then mounted in DPX Mountant (BDH Chemicals Ltd., Poole, UK). The sections were then observed under an optical microscope (BX50, Olympus, UK) and imaged (Coolsnap-Pro cf color,
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Media Cybernetics, Bethesda, USA). Five representative sections for each treatment stage were stained (Partington, Mordan et al. 2013).
3.3.4 Quantitative analysis of protein content
The quantitative protein assay used was the microplate procedure for the Pierce BCA Protein Assay Kit which utilises bicinchoninic acid (BCA) to detect the total amount of protein in a sample. The principle of the assay was based upon the ability of protein, in an alkaline solution, to reduce copper (II) ions or cupric ions (Cu2+) to copper (I) ion or cuprous ion (Cu+). The Cu+ ions form a purple coloured complex with BCA which could be quantitated with a spectrophotometer. Briefly, dilutions for a standard curve of BSA were prepared. The working reagent was prepared by combining Reagent A, containing the BCA, and Reagent B, containing cupric sulphate. The test samples were prepared using the tissue lysis component of the Qiagen DNeasy Blood and Tissue Kit. The working reagent was added to the BSA standards and the test samples in a 96 well plate, and then incubated at 37oC for 30 minutes. The plate was then cooled to room temperature and the absorbance measured at 562 nm using the Tecan Safire2™ (Tecan) and compared to a 20 - 2000 µg/mL standard curve. For each total protein quantitative test, the concentration of protein was normalized to the tissue weight and the mean ± SD was determined. Two-way ANOVA was used to find any statistically significant differences between native and decellularised trachea, as well as the corresponding non-decellularised control (NDC) (PBS treated) tissue (Partington, Mordan et al. 2013).
3.3.5 Quantitative analysis of soluble collagen content
The quantitative assay used to determine soluble collagen was the Biocolor Sircol™ Soluble Collagen Assay Kit, (Biocolor Ltd, Carrickfergus, UK) which was used according to the manufacturer’s instructions. The kit can be used to analyse cold acid and pepsin soluble collagen Types I to V that are produced in soft tissues and cartilages of the body, but is not suitable for covalently cross-linked collagen. The assay is a colorimetric assay which utilises the binding of Sirius Red in picric acid to the (Gly-X-Y) tri-peptide of the triple helix structure of collagen. Briefly, approximately 50 mg of tissue, frozen at -80oC since harvest, was taken and minced using razor blades. The tissue was digested overnight at 4ºC in pepsin (0.1 mg/ mL) (Sigma, Poole, UK) in 0.5M acetic acid (Sigma, Poole, UK). The acid solution was
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then neutralised. The sample was centrifuged to pellet any non-digested tissue and from the supernatant fraction the collagen was isolated and concentrated. The sample was then diluted appropriately; typically 1:20 in 0.5M acetic acid. The soluble collagen was then selectively bound and formed a complex with the Sirius Red in the dye reagent, which precipitated out of the solution. Following centrifugation, the bound soluble collagen-dye complex pelleted and the supernatant was removed. The soluble collagen-dye complex was then dissociated in an alkaline solution and the absorbance read at 555 nm using the Tecan Sunrise (Tecan) or the Tecan Safire2™ (Tecan) and compared to a 0 - 50 µg/mL standard curve. For each soluble collagen quantitative test, the concentration of soluble collagen was normalized to the lysate dilution, the tissue weight and the mean ± SD were determined. Two-way ANOVA was used to find any statistically significant differences between native and decellularised trachea, as well as the corresponding non-decellularised control (NDC) (PBS treated) tissue (Partington, Mordan et al. 2013).
3.3.6 Dynamic mechanical analysis (DMA) compression testing of tracheae tissue
Dynamic Mechanical Analysis (DMA) compression testing was used to evaluate the compressive properties of the cartilage rings in the tracheal wall. The PerkinElmer DMA 7E (PerkinElmer, Seer Green, UK) was used to mechanically interrogate and record, using the DMA 7E software (PerkinElmer, Seer Green, UK), in real time the tensile properties of each sample. For the tracheal wall testing, 5 mm x 5 mm (W x L) sections were punched out of a section of trachea tissue using a specimen cutting press (Model S1) (Wallace Instruments, Cambridge, UK). The adventitia and lamina propria were removed and the height of the tracheal wall section then measured before compression testing using callipers. The specimen was placed into position in the instrument and the measurements inputted into the software. The force was applied at 200 mN/minute up to 6000 mN and the results recorded in real time by the software. The Young’s (compressive) modulus (MPa) was determined for each analysis. All were plotted as the mean ± SD for each analysis. Two-way ANOVA was used to compare the decellularised (DC) scaffold findings with native and the corresponding non-decellularised control (NDC) (PBS treated) trachea findings over the decellularisation process (Partington, Mordan et al. 2013).
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