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Imaging Fibrillar Collagen with Optical Microscopy
Published in Jiro Nagatomi, Eno Essien Ebong, Mechanobiology Handbook, 2018
Tong Ye, Peng Chen, Yang Li, Xun Chen
Diseases or injury may modify the collagen structure significantly and cause compromise or loss of tissue function. SHG imaging can be a powerful method to assess the collagen structure and thereby detect change of tissue function or the progression of disease. The relevant collagen structural information can be obtained by quantitative analysis of SHG images. Several quantitative approaches have been reported. The ratio between forward and backward (F/B) SHG signals has been used to characterize collagen arrangement in tissue (Nadiarnykh et al. 2007; LaComb, Nadiarnykh, and Campagnola 2008b; Chaudhary et al. 2015; Houle et al. 2015). The F/B ratio correlates to the number of SHG photons propagating in the forward direction. The variation of the F/B ratio reflects the change of the scattering property, which usually associates with the fiber organization in the tissue. For example, Campagnola's group used the oim murine model and found that the F/B ratio could successfully feature the osteogenesis imperfecta (disease caused by mutations within the Col1A1 or Col1A2 genes) group with its high F/B ratio due to the reduced scattering from randomly aligned collagen fibers (Nadiarnykh et al. 2007). Fast Fourier transform (FFT) analysis is another method to quantify collagen structure (Matteini et al. 2009; Riccardo Cicchi et al. 2010; Tan et al. 2013; Mercatelli Raffaella et al. 2017). Randomly organized tissue collagen structure is expected to have more homogenously distributed frequency components in all directions in the Fourier frequency space. For example, tissue thermal treatment is involved in many clinical applications, such as cornea thickening and skin rejuvenation. However, under 50–60°C heat treatment, collagen within the tissue is gradually denatured and eventually causes the loss of tissue functions such as mechanical strength and optical transmission (Sun et al. 2006; Matteini et al. 2009). As shown in Figure 6.22, FFT analysis clearly indicates that the collagen fiber structure alters upon laser-induced heating; the frequency distribution can be further quantified by the aspect ratio, which is the ratio between the long and short axes of the ellipse of the frequency distribution (Matteini et al. 2009; Riccardo Cicchi et al. 2010). Quantification of the collagen structure has been extensively investigated; interested readers can refer to Cicchi et al. (2013) for a good overview.
Self-assembled monolayers of phosphonates promote primary chondrocyte adhesion to silicon dioxide and polyvinyl alcohol materials
Published in Journal of Biomaterials Science, Polymer Edition, 2019
Patrick E. Donnelly, Laurianne Imbert, Kirsty L. Culley, Russell F. Warren, Tony Chen, Suzanne A. Maher
There was an observed difference in chondrocyte morphology between chondrocytes cultured on 4-terminated SiO2 and PVA surfaces. These differences can be attributed to several factors: the studies were conducted on flat surfaces at low-seeding densities, and not three-dimensional gels, scaffolds, or in micromass that chondrocytes are typically cultured in [61–63] and as such, changes in morphology away from the more typical rounded shape are not surprising. The spindle-like morphology and the spreading of cells on the modified SiO2 surfaces suggested a fibroblast-like phenotypic shift of the cells respectively. To assess whether such a changes occurred, real-time quantitative RT-PCR (RT-qPCR) analysis was completed to assess the gene expression of chondrocytes cultured on standard tissue culture plastic (TCP), 4-terminated SiO2 and unmodified SiO2 surfaces after 3 days of culture. The genes analyzed (type I collagen (Col1a2), type II collagen (Col2a1), Aggrecan (Acan), Runx2, and Sox9) represent those that delineate the transition of normal chondrocytes to hypertrophic chondrocytes or dedifferentiation to fibroblast-like cells due to either their decrease or increased expression. We found no significant differences in chondrogenic or hypertrophic gene expression between chondrocytes cultured on TCP and 4-terminated SiO2 but a difference of the dedifferentiation marker Col1a2 between chondrocytes cultured on unmodified SiO2 and the other 2 conditions. These results suggest that bisphosphonic acid SAMP modification does not induce phenotypic change of chondrocytes any more than tissue culture plastic, and that the morphological differences are likely due to dedifferentiation of the chondrocytes into a more fibroblast-like cell when cultured on a cell-adherent stiff substrate [64,65]. This finding is further noted by the morphological differences noted between modified SiO2 and modified PVA – where chondrocytes cultured on SiO2 have a more fibroblastic morphology, that is not seen for chondrocytes cultured on modified PVA which has a much lower stiffness – PVA films have a Young’s modulus ranging from 1.50-3.75 GPa, while that of silicon ranges from 130 to 188 GPa [66,67].