Optical clearing in collagen- and proteoglycan-rich osteochondral tissues

Abstract

Objective Recent developments in optical clearing and microscopy technology have enabled the imaging of intact tissues at the millimeter scale to characterize cells via fluorescence labeling. While these techniques have facilitated the three-dimensional cellular characterization within brain and heart, study of dense connective tissues of the musculoskeletal system have been largely unexplored. Here, we quantify how optical clearing impacted the cell and tissue morphology of collagen-, proteoglycan-, and mineral-rich cartilage and bone from the articulating knee joint. Methods Water-based fructose solutions were used for optical clearing of bovine osteochondral tissues, followed by imaging with transmission and confocal microscopy. To confirm preservation of tissue structure during the clearing process, samples were mechanically tested in unconfined compression and visualized by cryoSEM. Results Optical clearing enhanced light transmission through cartilage, but not subchondral bone regions. Fluorescent staining and immunolabeling was preserved through sample preparations, enabling imaging to cartilage depths 5 times deeper than previously reported, limited only by the working distance of the microscope objective. Chondrocyte volume remained unchanged in response to, and upon the reversal, of clearing. Equilibrium modulus increased in cleared samples, and was attributed to exchange of interstitial fluid with the more viscous fructose solution, but returned to control levels upon unclearing. In addition, cryoSEM-based analysis of cartilage showed no ultrastructural changes. Conclusion We anticipate large-scale microscopy of diverse connective tissues will enable the study of intact, three-dimensional interfaces (e.g. osteochondral) and cellular connectivity as a function of development, disease, and regeneration, which have been previously hindered by specimen opacity.

Mechanisms and Microenvironment Investigation of Cellularized High Density Gradient Collagen Matrices via Densification

“…Aggrecan expression appears locally near cells, but does not appear to be present in the interstitium between chondrocytes. Type VI collagen expression appears to form a shell around the cell membrane, similar to that seen in native cartilage [25] and potentially indicative of the formation of a pericellular matrix and the formation of chondrons.…”

Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

“…On the other hand, researchers are still searching for optical clearing methods that permit the imaging of the cells without damaging or killing them, which will impact on the realization of timecourse experiments (Calve, Ready, Huppenbauer, Main, & Neu, 2015;Neu, Novak, Gilliland, Marshall, & Calve, 2015). In fact, all the clearing methods described so far damage the cells (e.g., cells fixation and organic solvents utilization; Calve et al, 2015).…”

Advances in Ex Situ Tissue Optical Clearing

“…Bone is a heterogeneous tissue that contains solid mineral (along with soft, marrow components) and connective tissue, both of which are characterized by relatively high RIs of 1.55 and 1.47, respectively. So far, few chemically distinct TOC methods were assessed for their clearing utility, namely, SeeDB, CUBIC decalcification, dehydrating methods, Bone CLARITY, and tuned Sca l eS formula for clearing of the organ of Corti ( Figure ). In complementary studies performed by Neu and Calve on osteochondral tissue, 2.5‐mm thick murine and bovine samples of ligament and meniscus were treated with SeeDB.…”