Abstract
Background: Chondrocyte dedifferentiation is emerging as a major concern for the functional reconstruction of hyaline articular cartilage.
Objective: The aim of this study was to characterize morphological and molecular changes during chondrocyte dedifferentiation processes using confocal Raman microscopy (CRM).
Material and methods: Human chondrocytes were isolated from articular cartilage harvested from a patient's knee during surgery, following informed consent. Cells were enzymatically digested with collagenase, then expanded directly onto Calcium Fluoride (CaF2) slides in two-dimensional (2D) monolayer cultures. Evaluations using wide-field optical microscopy, as well as Raman spectral measurements at D7, D14, D21, and D28 were performed for each passage from P1 to P4.
Results: Analysis of the different passages showed morphological and biochemical changes associated with cell passages. The greater the number of passages, the more the cells adopted a fibroblastic morphology. Raman bands located at 1063, 1255, and 1665 cm-1 were essential for monitoring changes in the molecular composition of glycosaminoglycans (GAGs), type II collagen and type I collagen over the passages.
Conclusion: CRM is proving to be a powerful analytical tool capable of tracking in real time the morphological and biochemical changes occurring during chondrocyte dedifferentiation. The P2 passage could therefore be considered sufficient to obtain the cell density required for chondrocyte redifferentiation.
Keywords
Articular cartilage, Chondrocytes, Dedifferentiation, Functional reconstruction, Confocal Raman microscopy