Abstract
Summary
In adult articular cartilage, the pericellular matrix (PCM) mediates chondrocyte-matrix-interactions and is associated with the spatial cellular organization. Immunofluorescence microscopy, multiphoton-induced autofluorescence and second harmonic generation (SHG) imaging, as well as point pattern analyses revealed that both PCM and spatial organization were absent in fetal chondrocytes.
Introduction
In adult articular cartilage, the pericellular matrix mediates the biomechanical, biophysical and biomechanical interactions between the chondrocyte and the extracellular matrix. The PCM is also associated with the spatial organization of human superficial chondrocytes, which are situated in four distinct patterns of strings, clusters, pairs or single cells. However, little is known about the PCM and the spatial organization during fetal development. In this study, we asked the question whether fetal chondrocytes display a spatial organization comparable to that of adult chondrocytes, and whether a PCM is present or absent in the early stages of fetal cartilage development.
Methods
Articular cartilage sections (100µm thickness) were prepared from the condyles of human fetal knee joints (pregnancy weeks 7–10) and from macroscopically intact condylar areas (knee joint replacement procedures). The samples were characterised by immunofluorescence microscopy and multiphoton-induced autofluorescence imaging combined with quantitative SHG signal profiling, a technique that allows quantifying the fibrillar collagen content. The spatial organization was analyzed by point pattern analyses as we described previously. For these analyses the Cartesian coordinates of each nucleus were determined by converting the immunofluorescence images into gray-scale images and finding the local gray-scale maxima with ImageJ (NIH).
Results
In adult superficial cartilage, the PCM surrounded single or groups of chondrocytes and defined their spatial organization of groups of cellular strings. PCM was characterised by a strong collagen VI staining signal and high collagen intensity as measured by SHG (156.7±12.4). In fetal cartilage, the condyles were characterised by a high cellular density, no recognizable spatial organization, and little amounts of extracellular matrix. No collagen VI was detected in the matrix surrounding the fetal chondrocytes. Furthermore, SHG imaging revealed that the fibrillar collagen intensity was significantly weaker (4.7±0.8; p<0.001) when compared to the adult tissues. During maturation from the fetal to the adult stage the cell density per volume decreased significantly (p<0.001). The cell distance from each fetal chondrocyte to its nearest neighbor was 15.70±0.12µm and significantly larger for adult cartilage (35.86±1.37µm; p<0.001). The level of spatial clustering as measured by the integral of the pair correlation function was significantly higher in the adult cartilage (p<0.001). Overall, a cellular grouping comparable to that of adult chondrocytes was not present in fetal cartilage. Together these parameters suggest that the spatial organization typical for adult condylar cartilage was not present in human fetal condylar cartilage.
Discussion/Conclusion
In human fetal articular cartilage the spatial organization typical for adult chondrocytes was not present. Instead, the chondrocytes were situated densely and in close proximity. This study determined for the first time that the collagenous components that are typical for the adult PCM were not present in fetal cartilage. In conclusion, the PCM and also the spatial organization of superficial human chondrocytes develop with cartilage maturation and thus are not inborn but instead acquired characteristics.
