240 Chapter 9 calculation of the average lowest threshold for identifiable specific staining for all images. These 10 images were opened in FIJI 1.53F51 (LOCI, University of Wisconsin) [36] as 8-bit images and split into different channels that captured the different staining colors, using the appropriate vector for the thresholds of the staining using color deconvolution. An in-house built macro (Supplementary Document 1 for PSR Images and Supplementary Document 2 for AB Images) was used along with the FIJI SlideJ plugin (tile size: 20000) to calculate the outputs from all images. Results were saved as a text to tab file and opened in R studio 4.1.1 (Boston, MA, USA) to sort the percentage area of positively stained pixels and the average intensity of the positively stained pixels data of the different staining levels (Supplementary Figure 39) using an in-house built macro as previously described [37]. Briefly, the strength of the signal from each pixel was determined and categorized as “weak”, “moderate” or “strong” based on the level of the strength. The pixels that belong to each of these categories were then combined per image and used in the following steps. Area percentage and intensity values for each category per image were calculated in Microsoft Excel 2016 (Microsoft, Resmond, WA, USA). For these calculations, the sorted rows Area_ Colour (area) and RawIntDen_Colour (intensity) were used. Stained area (%) and intensity values (arbitrary units) of each category was calculated using equation (i) and equation (ii), respectively, as shown below. (i) calculated in Microsoft Excel 2016 (Microsoft, Resmond, WA, USA). For these calculations, the sorted rows Area_Colour (area) and RawIntDen_Colour (intensity) were used. Stained area (%) and intensity values (arbitrary units) of each category was calculated using equation (i) and equation (ii), respectively, as shown below. (i) Area (%)= Number of pixels meeting category criteria Total number of pixels ×100% (ii) Intensity (au)=255- Sum of intensity values in pixels meeting category criteria Total number of pixels Analysis of Fluorescence Microscopy Images For every sample, images were generated of 6 randomly selected areas and saved as TIF (LZW) files. Each image was analyzed using the TWOMBLI-plugin in FIJI 1.53F51 (LOCI), and the following parameters were examined: the area, total fiber length, end points, branchpoints, curvature (with curvature windows 20 and 50), percentage of high density matrix and alignment of fibers [38]. Mechanical testing with Low Load Compression Testing (LLCT) Stiffness values and viscoelastic relaxation properties of the hydrogels at day 7 and 14 were measured using a Low Load Compression Tester as previously described [32, 39]. The LLCT analysis was performed on three different randomly selected locations on each hydrogel using 20% fixed strain rate. The measurement locations had at least 1.5 mm distance between them and 0.5 mm from the edges to ensure robustness and representativeness of the measurements. The stress ((Equation (iii)) and strain (Equation (iv)) values were calculated as described below and the slope of the line was used to calculate Young’s modulus (E, stiffness) (Equation (v)) until the peak point for the highest measurement observed). Relaxation values were calculated starting from the time point at which highest stiffness was observed by using the formula (Equation (vi)). Time duration for reaching 100% relaxation was recorded as ‘Relaxation time’. Time duration for reaching 50% of the total relaxation was recorded as (ii) calculated in Microsoft Excel 2016 (Microsoft, Resmond, WA, USA). For these calculations, the sorted rows Area_Colour (area) and RawIntDen_Colour (intensity) were used. Stained area (%) and intensity values (arbitrary units) of each category was calculated using equation (i) and equation (ii), respectively, as shown below. (i) Area (%)= Number of pixels meeting category criteria Total number of pixels ×100% (ii) Intensity (au)=255- Sum of intensity values in pixels meeting category criteria Total number of pixels Analysis of Fluorescence Microscopy Images For every sample, images were generated of 6 randomly selected areas and saved as TIF (LZW) files. Each image was analyzed using the TWOMBLI-plugin in FIJI 1.53F51 (LOCI), and the following parameters were examined: the area, total fiber length, end points, branchpoints, curvature (with curvature windows 20 and 50), percentage of high density matrix and alignment of fibers [38]. Mechanical testing with Low Load Compression Testing (LLCT) Stiffness values and viscoelastic relaxation properties of the hydrogels at day 7 and 14 were measured using a Low Load Compression Tester as previously described [32, 39]. The LLCT analysis was performed on three different randomly selected locations on each hydrogel using 20% fixed strain rate. The measurement locations had at least 1.5 mm distance between them and 0.5 mm from the edges to ensure robustness and representativeness of the measurements. The stress ((Equation (iii)) and strain (Equation (iv)) values were calculated as described below and the slope of the line was used to calculate Young’s modulus (E, stiffness) (Equation (v)) until the peak point for the highest measurement observed). Relaxation values were calculated starting from the time point at which highest stiffness was observed by using the formula (Equation (vi)). Time duration for reaching 100% relaxation was recorded as ‘Relaxation time’. Time duration for reaching 50% of the total relaxation was recorded as Analysis of Fluorescence Microscopy Images For every sample, images were generated of 6 randomly selected areas and saved as TIF (LZW) files. Each image was analyzed using the TWOMBLI-plugin in FIJI 1.53F51 (LOCI), and the following parameters were examined: the area, total fiber length, end points, branchpoints, curvature (with curvature windows 20 and 50), percentage of high density matrix and alignment of fibers [38]. Mechanical testing with Low Load Compression Testing (LLCT) Stiffness values and viscoelastic relaxation properties of the hydrogels at day 7 and 14 were measured using a Low Load Compression Tester as previously described [32, 39]. The LLCT analysis was performed on three different randomly selected locations on each hydrogel using 20% fixed strain rate. The measurement locations had at least 1.5 mm distance between them and 0.5 mm from the edges to ensure robustness and representativeness of the measurements. The stress ((Equation (iii)) and strain (Equation (iv)) values were calculated as described below and the slope of the line
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