High-Performance Polarization Microscopy Reveals Structural Remodeling in Rat Calcaneal Tendons Cultivated In Vitro

Abstract

Collagenous tissues exhibit anisotropic optical properties such as birefringence and linear dichroism (LD) as a result of their structurally oriented supraorganization from the nanometer level to the collagen bundle scale. Changes in macromolecular order and in aggregational states can be evaluated in tendon collagen bundles using polarization microscopy. Because there are no reports on the status of the macromolecular organization in tendon explants, the objective of this work was to evaluate the birefringence and LD characteristics of collagen bundles in rat calcaneal tendons cultivated in vitro on substrates that differ in their mechanical stiffness (plastic vs. glass) while accompanying the expected occurrence of cell migration from these structures. Tendon explants from adult male Wistar rats were cultivated for 8 and 12 days on borosilicate glass coverslips (n = 3) and on nonpyrogenic polystyrene plastic dishes (n = 4) and were compared with tendons not cultivated in vitro (n = 3). Birefringence was investigated in unstained tendon sections using high-performance polarization microscopy and image analysis. LD was studied under polarized light in tendon sections stained with the dichroic dyes Ponceau SS and toluidine blue at pH 4.0 to evaluate the orientation of proteins and acid glycosaminoglycans (GAG) macromolecules, respectively. Structural remodeling characterized by the reduction in the macromolecular orientation, aggregation and alignment of collagen bundles, based on decreased average gray values concerned with birefringence intensity, LD and morphological changes, was detected especially in the tendon explants cultivated on the plastic substrate. These changes may have facilitated cell migration from the lateral regions of the explants to the substrates, an event that was observed earlier and more intensely upon tissue cultivation on the plastic substrate. The axial alignment of the migrating cells relative to the explant, which occurred with increased cultivation times, may be due to the mechanosensitive nature of the tenocytes. Collagen fibers possibly played a role as a signal source to cells, a hypothesis that requires further investigation, including studies on the dynamics of cell membrane receptors and cytoskeletal organization, and collagen shearing electrical properties.

Keywords: optical anisotropy; polarization microscopy; tendon explants; tissue culture.

Figure 1

Birefringence images and respective average gray evaluations obtained for sections of control and cultivated rat calcaneal tendons. (A). Images representative of control tendon sections are shown before (a,c,e) and after (b,d,f) birefringence compensation. The intensity variations in birefringence brilliance levels are caused by variations in collagen fiber orientation with respect to the crossed polarizers. When the long axes of tendons were positioned parallel to the polarizer’s PPL, the crimp structure becomes identifiable (c,d). Interference colors obtained when using the DIC-PLM system are depicted for sections of control tendons (e,f) and for tendons cultivated on glass coverslips (h,j). Images (h,j), obtained with the DIC-PLM system, correspond to images g and i, respectively, which were obtained with ordinary polarization microscopy. Structural changes in collagen bundles were visualized with increasing time of tissue cultivation (g,h—8 days; i,j—12 days). Scale bars equal 50 µm. (B). Polydispersed distributions of birefringence average gray values expressed in pixels corresponding to optical retardations for collagen bundles of tendon sections immersed in water. The higher frequencies of average gray values that were detected in control tendons shift to smaller values in tendons cultivated for 8 and 12 days on plastic dishes and in tendons cultivated for 12 days on glass coverslips. The long axes of the tendons were positioned at 45° with respect to the PPL.

Figure 2

Positive LD in Ponceau SS-stained tendon collagen bundles. (A) Images representative of stained collagen bundles in control tendons (a,b) and tendons cultivated on glass coverslips (c–h). The long axes of the tendons were positioned parallel (a,c,e,g) and perpendicular (b,d,f,h) to the PPL. Images (g,h) correspond to the extremity tendon zone distal to the region originally occupied by the enthesis in vivo. (B) Images representative of stained collagen bundles in tendons cultivated on plastic dishes (a–h). The long axes of the tendons were positioned parallel (a,c,e,g) and perpendicular (b,d,f,h) to the PPL. Images (c,d,g,h) were obtained at zones distal to the region originally occupied by the enthesis. A less intense LD phenomenon was observed in tendons cultivated for 12 days, especially at zones distal to the region originally occupied by the enthesis (g,h). Scale bars equal 50 µm (A(a–h)) and (B(e–h)) and 100 µm (B(a–d)).

Figure 3

Negative LD in TB-stained tendon collagen bundles. Images representative of stained control tendons (a,b) and tendons cultivated for 8 (c,d) and 12 (e,f) days on glass coverslips (c–f). The long axes of the tendons were positioned perpendicular (a,c,e) (violet color) and parallel (b,d,f) (blue color) to the PPL. Images e and f were obtained for zones distal to the region originally occupied by the enthesis in vivo. The LD phenomenon is maintained in cultivated tendons, although it exhibits slightly less intense colors in comparison with control tendons. Scale bars equal 50 µm.

Figure 4

Tenocyte distributions in tendons (T) cultivated in vitro as observed with inverted light microscope. Images representative of the distribution of tenocytes that migrated from the lateral regions of tendons (a–d, arrows) to the plastic substrate after a 6-day period of tendon cultivation (a,b) and that acquired a preferential orientation parallel to the long tendon axis 24 h later (c–e). Details of dense tenocyte distributions oriented parallel to the long axis of the tendon are indicated by an arrow (e). The number of the parallel aligned rows after a 12-day period of tendon cultivation increased even more as seen in a preparation stained with toluidine blue at pH 4.0 (f). Scale bars equal 250 µm (a–d,f) and 100 µm (e).

Figure 5

Graphical representation summarizing the main results of the present study.