Mueller Matrix Analysis of Collagen and Gelatin Containing Samples Towards More Objective Skin Tissue Diagnostics

Abstract

Electrospun polycaprolactone:gelatin (PCL:GT) fibre scaffolds are widely employed in the field of tissue implants. Here, the orientation of fibres plays an important role in regard to implantation due to the impact on the mechanical properties. Likewise, the orientation of collagen fibres in skin tissue is relevant for dermatology. State-of-the-art fibre orientation measurement methods like electron microscopy are time consuming and destructive. In this work, we demonstrate polarimetry as a non-invasive approach and evaluate its potential by measuring the Mueller matrix (MM) of gelatin and collagen containing samples as simple skin tissue phantoms. We demonstrate that it is possible to determine the orientation of PCL:GT fibre scaffolds within one MM measurement. Furthermore, we determine the structural orientation in collagen film samples. Currently, the diagnosis of skin diseases is often performed by image analysis or histopathology respectively, which are either subjective or invasive. The method presented, here, provides an interesting alternative approach for such investigations. Our findings indicate that the orientation of collagen fibres within skin lesions might be detectable by MM measurements in the future, which is of interest for skin diagnostics, and will be further investigated during the next step.

Keywords: Mueller matrix; Raman spectroscopy; collagen; electrospinning; fibre alignment; gelatin; tissue engineering.

Figure 1

Overview of the experimental setup and adjusted process parameters. The electrospinning device used is composed of a grounded and rotating drum collector, high voltage supply, cannula, syringe and syringe pump. The adjusted parameters—the needle tip to collector distance, applied voltage, total process time, relative collector velocity, diameter and width of the drum collector, flow rate, inner cannula diameter, setup type, polymer and solvent as well as the mass ration of the used blend—are displayed.

Figure 2

Raman spectra of pure PCL, PCL:GT 1.2 m/s, PCL:GT 10.7 m/s and collagen film. PCL is displayed in black, PCL:GT 1.2 m/s in dark grey, PCL:GT 10.7 m/s in light grey and collagen film in green. The vertical lines represent characteristic wavenumbers: phenylalanine (1010 cm^–1; dotted dark blue), PCL (1110 cm^–1; solid dark blue and 1725 cm^–1; solid blue), amide (II: 1220 cm^–1; dashed dark blue, III: 1280 cm^–1; dashed blue and I: 1667 cm^–1; dashed light blue) and lysine (1450 cm^–1; small dashed dark blue), with the solid lines marking the PCL-typical bands and the dashed and dotted lines marking the protein typical bands.

Figure 3

Boxplots of the degree of fibre orientation in ° for each of the 13 relative collector velocities. The resulting boxplots, including outliers, showed a trend of decreasing IQR with increasing relative collector velocity. This trend continued until 8.3 and 9.1 m/s, after which it reversed. Correspondingly, the dispersion followed a similar trend and decreased with increasing relative collector velocity. Based on the QQ-plots, indicating normal distribution, and the recorded values are independent, parametric tests were conducted. Statistical significances for all groups were investigated via one-way repeated measure ANOVAs. Mean differences between the group for 1.2 m/s and the others were analyzsed by the Dunnett post-hoc test and labelled as follows: * (p < 0.05), ** (p < 0.01) and *** (p < 0.001).

Figure 4

Boxplots of degree of fibre orientation in ° for 1.2 and 8.3 m/s relative collector velocity. The individually measured values are presented on the left side of each boxplot. The depicted boxplots show the results for the lowest relative collector velocity (1.2 m/s), which is defined as “control” for the Dunnett post-hoc test, and the results for 8.3 m/s without outliers. Both data sets show a distribution within a similar range, but for 8.3 m/s the majority of values is located around 0°. A much wider distribution for 1.2 m/s can be observed. The comparison of both data sets shows a decreased IQR and dispersion with increased relative collector velocity. The depicted IQR indicates normally distributed values.

Figure 5

SEM images of the transparent collagen film. (a) shows a magnification of a factor of 35. Here the perforation of the film can be clearly seen. (b) shows a higher magnification of a factor of 50,000. The black arrow points in the unrolling direction of the collagen film. Probably during a rolling process, the film was stretched in this direction, which could have led to the structures visible here.

Figure 6

Average values of the MM images for relative collector velocity 8.3 m/s (n = 3; error bars: min. and max.) for different angles of the sample measured in transmission with 532 nm. The fibres are oriented horizontally at 0°.

Figure 7

Stokes vector of the fast axis of retardation for different sample orientation. The vectors are averaged over the given sample size of three and each angle was measured twice as the sample was rotated by 370°.

Figure 8

MM results for rotation of the transparent collagen film measured in (a) transmission and (b) reflection (n = 5; ±SD). Measurement was performed with 532 nm light source.

Figure 9

Stokes vector of the fast axis of retardation for different sample orientation of the transparent collagen film in transmission (a) and reflection (b). The vectors are averaged over a given sample size of three and each angle was measured twice because the sample was rotated by 370°.

Figure 10

Correlation between polarizance P (resulting from MM measurements) and absolute orientation (measured SEM-based) of the PCL/GT fibre scaffolds. The green circles represent the mean values for the polarizance P with error bars (min. and max.) whereas the blue squares show the results for the absolute orientation with error bars (±SD).