The clustering and morphology of chondrocytes in normal and mildly degenerate human femoral head cartilage studied by confocal laser scanning microscopy

Abstract

Chondrocytes are the major cell type present in hyaline cartilage and they play a crucial role in maintaining the mechanical resilience of the tissue through a balance of the synthesis and breakdown of extracellular matrix macromolecules. Histological assessment of cartilage suggests that articular chondrocytes in situ typically occur singly and demonstrate a rounded/elliptical morphology. However, there are suggestions that their grouping and fine shape is more complex and that these change with cartilage degeneration as occurs in osteoarthritis. In the present study we have used confocal laser scanning microscopy and fluorescently labelled in situ human chondrocytes and advanced imaging software to visualise chondrocyte clustering and detailed morphology within grade-0 (non-degenerate) and grade-1 (mildly degenerate) cartilage from human femoral heads. Graded human cartilage explants were incubated with 5-chloromethylfluorescein diacetate and propidium iodide to identify the morphology and viability, respectively, of in situ chondrocytes within superficial, mid- and deep zones. In grade-0 cartilage, the analysis of confocal microscope images showed that although the majority of chondrocytes were single and morphologically normal, clusters (i.e. three or more chondrocytes within the enclosed lacunar space) were occasionally observed in the superficial zone, and 15-25% of the cell population exhibited at least one cytoplasmic process of ~ 5 μm in length. With degeneration, cluster number increased (~ 50%) but not significantly; however, the number of cells/cluster (P < 0.001) and the percentage of cells forming clusters increased (P = 0.0013). In the superficial zone but not the mid- or deep zones, the volume of clusters and average volume of chondrocytes in clusters increased (P < 0.001 and P < 0.05, respectively). The percentage of chondrocytes with processes, the number of processes/cell and the length of processes/cell increased in the superficial zone of grade-1 cartilage (P = 0.0098, P = 0.02 and P < 0.001, respectively). Processes were categorised based on length (L0 - no cytoplasmic processes; L1 < 5 μm; 5 < L2 ≤ 10 μm; 10 < L3 ≤ 15 μm; L4 > 15 μm). With cartilage degeneration, for chondrocytes in all zones, there was a significant decrease (P = 0.015) in the percentage of chondrocytes with 'normal' morphology (i.e. L0), with no change in the percentage of cells with L1 processes; however, there were significant increases in the other categories. In grade-0 cartilage, chondrocyte clustering and morphological abnormalities occurred and with degeneration these were exacerbated, particularly in the superficial zone. Chondrocyte clustering and abnormal morphology are associated with aberrant matrix metabolism, suggesting that these early changes to chondrocyte properties may be associated with cartilage degeneration.

The Role of Chondrocyte Morphology and Volume in Controlling Phenotype—Implications for Osteoarthritis, Cartilage Repair, and Cartilage Engineering

“…Full-depth osteochondral explants from human femoral heads obtained from femoral neck fracture with ethical permission and patient consent, were incubated with CMFDA (5-chloromethyl-fluorescein diacetate) and propidium iodide (10 μM each), prepared for confocal scanning laser microscopy (CLSM) and imaged in the axial direction (i.e. viewed down onto the cartilage surface using a × 40 0.8 NA water-immersion lens) as described [27 •]. Chondrocytes are labelled green or red for living or dead cells, respectively.…”

“…Chondrocytes are labelled green or red for living or dead cells, respectively. Representative images of chondrocyte morphology are taken from images in Karim et al [27 •] The scale bar shown in (a) is the same for all panels …”

“…Approximately half of the chondrocytes within macroscopically normal, aged human tibial plateau cartilage have fine cytoplasmic processes usually extending beyond the pericellular matrix (PCM) or lacunar space into the inter-territorial matrix [ 8 ]. Studies on human femoral head cartilage also identified abnormal chondrocyte morphology and classified the cytoplasmic processes based on their length and number per chondrocyte [27 •]. In grade-0 (non-degenerate) cartilage, the majority of chondrocytes were single and morphologically normal, and topographically arranged as described [ 7 ].…”

p47phox siRNA-Loaded PLGA Nanoparticles Suppress ROS/Oxidative Stress-Induced Chondrocyte Damage in Osteoarthritis

“…Chondrocyte clustering is a histological sign of late-stage OA [1,37]; this type of clustering and its associated morphological abnormalities are particularly evident in the superficial zones of OA tissues. To explore whether p47phox was associated with ROS production and cartilage damage, samples of degenerating articular cartilage from patients with OA were assessed.…”

Osteochondral Live Slices as Physiological in-vitro Model of the Human Joint for High-throughput Applications

“…Since chondrocytes are of mesenchymal origin, it might be possible, that they leave their tissue speci c position and migrate to the periphery of the tissue, but in our setting CLSM showed highly conserved cell architecture inside the tissue (21). Cartilage is divided into three different cellular zones: The super cial zone (tightly packed with MSC like cells), the mid zone (pearl-bead-like alignment) and the deep zone (column-like chondrocyte alignment) (22) (23). The conservation of the cellular zones depends on the supply with nutrients and physiological cartilage infrastructure -minimizing cellular stress by malnutrition and/or hypoxia (24).…”