Glycosaminoglycans Modulate Long-Range Mechanical Communication Between Cells in Collagen Networks

Cells can sense and respond to mechanical forces in fibrous extracellular matrices (ECM) over distances much greater than their size. This phenomenon, termed long-range force transmission, is enabled by the realignment (buckling) of collagen fibers along directions where the forces are tensile (compressive). However, whether other key structural components of the ECM, in particular glycosaminoglycans (GAGs), can affect the efficiency of cellular force transmission remains unclear. Here we developed a theoretical model of force transmission in collagen networks with interpenetrating GAGs, capturing the competition between tension-driven collagen-fiber alignment and the swelling pressure induced by GAGs. Using this model, we show that the swelling pressure provided by GAGs increases the stiffness of the collagen network by stretching the fibers in an isotropic manner. We found that the GAG-induced swelling pressure can help collagen fibers resist buckling as the cells exert contractile forces. This mechanism impedes the alignment of collagen fibers and decreases long-range cellular mechanical communication. We experimentally validated the theoretical predictions by comparing collagen fiber alignment between cellular spheroids cultured on collagen gels versus collagen-GAG co-gels. We found significantly less alignment of collagen in collagen-GAG co-gels, consistent with the prediction that GAGs can prevent collagen fiber alignment. The roles of GAGs in modulating force transmission uncovered in this work can be extended to understand pathological processes such as the formation of fibrotic scars and cancer metastasis, where cells communicate in the presence of abnormally high concentrations of GAGs.Statement of significanceGlycosaminoglycans (GAGs) are carbohydrates that are expressed ubiquitously in the human body and are among the key macromolecules that influence development, homeostasis, and pathology of native tissues. Abnormal accumulation of GAGs has been observed in metabolic disorders, solid tumors, and fibrotic tissues. Here we theoretically and experimentally show that tissue swelling caused by the highly polar nature of GAGs significantly affects the mechanical interactions between resident cells by altering the organization and alignment of the collagenous extracellular matrix. The roles of GAGs in modulating cellular force transmission revealed here can guide the design of biomaterial scaffolds in regenerative medicine and provides insights on the role of cell-cell communication in tumor progression and fibrosis.

Abstract P351: Grayscale Texture Feature Angular Second Moment Correlates With Collagen Alignment in Carotid Artery Plaques

Introduction: Grayscale (GS) texture features that examine homogeneity and echogenicity have been used to identify vulnerable plaques with in vivo ultrasound imaging have been shown to correlate with plaque tissue composition. However, the relationship of collagen fiber organization to GS texture features extracted from in vivo images is a novel idea to provide additional information about plaque structure. We hypothesize that collagen fiber alignment is clinically relevant to identify vulnerable plaques. The objective of this feasibility study was to use multiscale imaging (in vivo ultrasound and high resolution optical microscopy) to determine how GS texture features are related to plaque collagen structure. Methods: Participants (n=6) scheduled for clinically indicated carotid endarterectomy underwent in vivo carotid ultrasound imaging with texture feature extraction (spatial gray level dependence matrices method for calculating angular second moment [SGLDM-ASM] and grayscale median value [GSM]). Plaque specimens were sent to histopathology and stained with H&E. The collagen fibers in the fibrous cap of the plaque histopathology slides were imaged with liquid crystal based polarization microscopy and quantified using an established software tool (CurveAlign). Correlations between collagen alignment coefficient (range 0-1, 1 represents perfectly aligned fibers) and the texture feature SGLDM-ASM (a measure of homogeneity, higher values are more homogenous) and GSM (a measure of echogenicity higher values are more echogenic) were examined. Results: Participants were mean (SD) 72.5 (6.1) years of age, had 71.67 (8.16) percent stenosis. The mean SGLDM-ASM was 0.0017 (0.0023), the mean SD GSM was 73.13 (30.98). SGLDM-ASM was significantly correlated to collagen alignment (r=0.83; p=0.028). There was no significant correlation detected between GSM and collagen alignment (r=-0.43;p=0.38). Conclusion: Results of this study indicate the potential role for using high resolution optical microscopy with ultrasound to characterize collagen fiber alignment in plaques with measures of homogeneity. Future studies are needed to see how multiscale imaging can be used to inform in vivo imaging for identification of vulnerable plaque features.

Microstructural and Mechanical Properties of the Anterolateral Ligament (ALL) of the Knee

Objectives: The anterolateral ligament (ALL) of the knee has recently emerged as a potential contributor to rotational stability of the knee, with growing interest in ALL reconstruction as a supplement to anterior cruciate ligament reconstruction. The prevalence of the ALL in the knee has varied in anatomic dissection and imaging studies, raising questions about its importance as a knee stabilizer. The purpose of this study was to assess the microstructural and mechanical properties of the anterolateral knee, to better understand the ALL structure compared to the surrounding anterolateral capsule (ALC) and lateral collateral ligament (LCL). A polarized light imaging technique was used to quantify collagen fiber alignment simultaneously with measurement of tensile mechanical properties. Our primary hypothesis was that there is no difference in the microstructural and mechanical properties between the ALL and ALC. Our secondary hypothesis was that the properties of the LCL are different from the ALL and ALC. Methods: Twenty-five knee specimens from sixteen donors (five males, eleven females; mean age 45.6 +/- 6.4; age range 35-59 years; mean BMI 26.5 +/- 8.4) were obtained as determined by a priori power analysis. The anatomic technique to dissect the anterolateral knee structures was performed as described previously. Three tissue samples (LCL, ALL, and ALC) were harvested (Fig. 1). The ALL was taken as a quadrilateral piece of tissue starting posterior/proximal from the lateral femoral epicondyle and ending at the lateral border of Gerdy’s tubercle. During gross dissection, the knee was assessed for the presence or absence of a distinct visible and palpable structure within the area defined as the ALL. Harvested samples were thinned to approximately 1-mm thick using a freezing-stage sliding microtome. Cross-sectional area was measured using a 3D laser scanning system. Four 0.8-mm diameter aluminum beads were attached to the sample surface to enable strain measurement. Mechanical testing was performed with preconditioning followed by both a stress-relaxation test and a quasi-static ramp to failure. Microstructural analysis was performed using transmitted circularly-polarized incident light and a high-resolution, division-of-focal-plane polarization camera. The average degree of linear polarization (AVG DoLP; i.e., mean strength of collagen alignment) and standard deviation of the angle of polarization (STD AoP; i.e., degree of variation in collagen angle orientation) were calculated for the region of interest of each sample. Statistical analysis was performed using Kruskal-Wallis test (assuming nonparametric data) with Dunn’s correction for multiple comparisons. Results: Mechanical analysis of elastic moduli for the toe- and linear-region of the stress-strain curves showed no difference between the ALL and ALC but were significantly higher for the LCL (p<0.0001; Fig. 2). Microstructural analysis of the ALL and ALC during quasi-static ramp to failure showed no difference in AVG DoLP and STD AoP values at all strain levels (Fig. 3). Larger DoLP values (i.e., stronger collagen fiber alignment) were observed for the LCL than both the ALL and ALC (p<0.0001). Larger STD AoP values (i.e., more variation in collagen orientation) were observed for the ALL and ALC compared to the LCL (p<0.0001; Fig. 3). When looking at correlations between mechanical and microstructural properties (Fig. 4), we found clustering of the LCL data points at high linear modulus and AVG DoLP while the ALL and ALC data points were clustered together. Similarly, we found clustering of the LCL at high linear modulus and low STD AoP while the ALL and ALC were clustered together. Only three of 25 knee specimens (12%) were observed to have a distinct, ligamentous structure in the region of the ALL. Interestingly, these distinct ALL samples (outlined in black on figures) showed relatively larger elastic moduli, higher AVG DoLP, and lower STD AoP (i.e., uniform and organized collagen alignment) across the stress-strain curve compared to samples harvested from knees without a distinct ALL. The distinct ALL tissues were also seen clustered near the LCL data points in the correlation plots. Conclusions: Overall, there were no differences in the mechanical and microstructural properties between the ALL and ALC, while the LCL demonstrated different properties compared to both the ALL and ALC. Both the ALC and ALL show significantly weaker collagen fiber alignment and more variation in the direction of collagen fiber alignment compared to the LCL. These findings suggest that the ALL has similar properties to capsule (i.e., ALC). However, when a distinct ALL was present at dissection (12%), the data indicates stronger and more uniform collagen alignment suggestive of more ligament-type qualities. Further research is needed to more precisely define the prevalence and properties of distinct ALLs in the knee.