Functional properties of chondrocytes and articular cartilage using optical imaging to scanning probe microscopy

Abstract

Mature chondrocytes in adult articular cartilage vary in number, size, and shape, depending on their depth in the tissue, location in the joint, and source species. Chondrocytes are the primary structural, functional, and metabolic unit in articular cartilage, the loss of which will induce fatigue to the extracellular matrix (ECM), eventually leading to failure of the cartilage and impairment of the joint as a whole. This brief review focuses on the functional and biomechanical studies of chondrocytes and articular cartilage, using microscopic imaging from optical microscopies to scanning probe microscopy. Three topics are covered in this review, including the functional studies of chondrons by optical imaging (unpolarized and polarized light and infrared light, two-photon excitation microscopy), the probing of chondrocytes and cartilage directly using microscale measurement techniques, and different imaging approaches that can measure chondrocyte mechanics and chondrocyte biological signaling under in situ and in vivo environments. Technical advancement in chondrocyte research during recent years has enabled new ways to study the biomechanical and functional properties of these cells and cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:620-631, 2018.

Keywords: articular cartilage; biomechanics; chondrocyte; functional study; imaging.

Fig 1

Three transmission electron micrographs of thin sections through rat growth-plate cartilage, where the histological processes were different. In (A), most of the proteoglycans (PGs) have been extracted, which causes the skeleton of collagenous fibrils to be revealed with great clarity. In (B), the loss of PGs has been prevented. Owing to the PG precipitation and consequent condensation, the matrix has a coarse, granular appearance. In (C), the PGs are preserved in situ and in their native, expanded state. The fine ultrastructural details of the collagenous fibrils are clearly visible. Bars = 1.5 μm (A, B) and 420 nm (C). (Reproduced with permission from Hunziker et al. Microsc Res Tech 28, 505–519, 1994)

Fig 2

Fluorescence confocal images of cartilage tissue blocks (left) immunolabeled for type-VI collagen at 0%, 10%, 30%, and 50% compressions. Typical shapes of chondrons in each zone under compression (right). Chondrons exhibited significant changes in height, shape, and volume in each zone, depending upon the applied compressive strain levels. (Reproduced with permission from Choi et al. J Biomech 40(12): 2596–2603, 2007)

Fig 3

The quantitative angle and retardation images of two chondrocyte clusters: (A) a cluster of two cells and (B) a cluster of four cells. The vector maps show graphically the average orientation of collagen fibrils forming “cocoons” around the cell clusters (meshed areas). (Reproduced with permission from Mittelstaedt et al. Connect Tissue Res 52(6): 512–522, 2011)

Fig 4

Porcine cartilage-bone explant prior to loading (left panel) and after cyclical loading (right panel). After 7 hours of loading (sinusoidal wave of 40% nominal tissue strain at 4Hz), surface fibrillation became apparent and the sample was subjected to second harmonic generation analysis of internal collagen fibril network disruption. There are several collagen fibril disruptions visible by eye and two of those are highlighted in white.

Fig 5

In situ testing of cartilage PCM/ECM. (A) Targeted indentation of tissue sections can be directed using (B) phase contrast and/or (C) fluorescence imaging. These techniques can be implemented in great variety to localize mechanical testing to regions where specific biomolecules are, or are not, present. (Reproduced with permission from Wilusz RE and Guilak F. J Mech Behav Biomed Mater 38: 183–97, 2014)

Fig 6

High-resolution force scanning image of sectioned, mouse femoral cartilage. Chondrocytes would be located in the bowl-like, dark blue regions, with the PCM immediately surrounding these voids. Location-specific moduli revealed a compliance gradient from where the cell membrane would be through the PCM (green/yellow) to the ECM (light/dark red). The force scanning mechanical property mapping technique is faster than standard approaches but requires relatively smooth materials for optimal outcomes. 3D image generated using data from Darling EM (Nanotechnology 22(17): 175707, 2011)

Fig 7

Normalized chondrocyte volume changes (means ± SD; n=5) as a function of time, from knee joints of live mice subjected to loading through controlled muscular contractions with an average pressure of 1.9 MPa that was held for 8 s and then removed. (A) Exemplar reconstructions of a single cell at various time points during loading and unloading of the knee joint. (B) Normalized and averaged volume changes of chondrocytes from five mice knee joints. Chondrocytes immediately lost 18–25% of their volume, which was only fully recovered after about seven minutes following load removal. (Reproduced with permission from Abusara et al. J Biomech 44:930–934, 2011)