Evaluating the effects of glutaraldehyde concentration and incubation time on the structural integrity of the human pericardium

Figures

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  FIGURE 1. Schematic representation of the image analysis pipeline. The process begins with the raw image input, which undergoes grayscale conversion followed by contrast enhancement. A fringe filter is then applied, after which thresholding and skeletonization are performed. Both the raw image and the skeletonized data are subsequently used for post-processing and quantitative analysis.

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  FIGURE 2. Quantification of collagen waviness and schematic representation of treatment effects. (A) Measurement of Euclidean distance in a wavy structure. The straight-line distance between endpoints A and B represents the Euclidean distance [L0], while the actual path length [Lf] follows the undulating contour of the structure. The difference between these lengths reflects the degree of waviness [W], with increased undulation resulting in a longer path length relative to the straight-line distance. (B) Schematic illustration of the hypothesized effect of glutaraldehyde (GA) treatment on collagen architecture. From left to right, increasing GA concentration and incubation time (indicated by the arrow) are associated with progressive collagen fiber bundling and reduced waviness. Collagen fibers are depicted in red, transitioning from loosely organized, wavy structures to more aligned, compact bundles, representing a reduction in crimp and increased structural organization.

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  FIGURE 3. Pericardial processing with glutaraldehyde (GA) at different incubation times. (A) Representative image of human pericardium prior to treatment. Black arrows indicate the initial width (80 mm) and height (40 mm). The dotted lines indicate the cutting direction used for sample preparation, based on specimen geometry rather than predefined collagen fiber orientation. Half of the sample was allocated for histological analysis and the other half for tensile testing. Untreated tissue served as the baseline control (0% GA, 0 min incubation) for all subsequent analyses. (B) Pericardial tissue sections treated with 2.5% GA at different incubation times (5, 20, and 90 min, from left to right), stained with Picrosirius red and imaged using confocal microscopy. Images represent different regions of the same pericardial specimen for each condition, rather than the same region imaged over time. White arrows indicate collagen fiber alignment. Scale bar: 100 μm.

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  FIGURE 4. Fiber Orientation Analysis of glutaraldehyde (GA) treated human pericardium. (A) Interaction plot of fiber orientation [degree°] in dependence of different Glutaraldehyde (GA) concentrations (Control (Ctrl)…grey and marked with x; 0.1%…blue and marked with ●; 0.6%…green and marked with ■; 1.0%.red marked with ◆ and 2.5%…pink marked with ▲) and incubation times (5, 20 and 90 min). A consistent increase in the mean orientation across all concentrations, with no systematic differences between the concentration groups is shown. Each data point represents the mean of six biological replicates (patient samples), the error bars show the standard deviation.

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  FIGURE 5. Effect of treatment time and concentration on maximum strain in tensile testing. (A) Representative stress–strain curve from uniaxial tensile testing. Stress (σ) is expressed in MPa and strain (ε) in %. Blue dots indicate measured data points. The elastin-dominated phase is highlighted in orange with a dotted line representing the linear fit in the low-strain region, while the collagen-dominated phase is highlighted in green with a dotted line representing the linear fit in the high-strain region. The grey dot marks the transition point (εtrans) between the two phases, and the red dot indicates the maximum stress point (σmax). (B) Barplot showing the maximum strain (Emax) across different Glutaraldehyde (GA) concentrations. Each bar represents the mean from all patients treated for 5 min (red), 20 min (green), or 90 min (blue) at the respective GA concentrations. Error bars indicate variability (standard deviation) across patients (n = 3). (C) Interaction plot showing the effects of treatment time and concentration (Control (Ctrl)…grey and marked with x; 0.1%…blue and marked with ●; 0.6%…green and marked with ■; 1.0%.red marked with ◆ and 2.5%…pink marked with ▲) on maximum…

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  FIGURE 6. Effect of treatment time and concentration on transition strain in tensile testing. (A) Barplot showing the transition strain (εtrans) across different Glutaraldehyde (GA) concentrations. Each bar represents the mean from all patients treated for 5 min (red), 20 min (green), or 90 min (blue) at the respective GA concentrations. Error bars indicate variability (standard deviation) across patients (n = 3). (B) Interaction plot showing the effects of treatment time and concentration (Control (Ctrl)…grey and marked with x; 0.1%…blue and marked with ●; 0.6%…green and marked with ■; 1.0%.red marked with ◆ and 2.5%…pink marked with ▲) on transition strain. Each data point represents the mean of three biological replicates (patient samples), the error bars show the standard deviation. (C–E) Linear regression plots showing the relationship between GA concentration and transition strain (εtrans) for each incubation time: (C) 5 min (red), (D) 20 min (green), (E) 90 min (blue). In each plot, the x-axis represents increasing GA concentrations, and the y-axis shows the corresponding transient strain. Trend lines illustrate the linear relationship for each time point.

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  FIGURE 7. Within-person repeated-measures correlations among structural and mechanical parameters. Heatmap of within-person correlations (rmcorr) between structural and mechanical variables across all treatment conditions. Each cell represents the repeated-measures correlation coefficient (r) between two variables, accounting for non-independence of observations within the same sample. The color scale ranges from −1.0 (blue, strong negative correlation) to +1.0 (red, strong positive correlation). Asterisks (*) indicate statistically significant correlations (p < 0.05).