In situ observation of cartilage matrix based on two-photon fluorescence microscopy.

Articular cartilage lesions remain a major challenge for clinicians and researchers. Several techniques, such as histological scoring, magnetic resonance imaging, and tissue section staining, are available for detecting cartilage degeneration and lesions and evaluating cartilage repairs. Nevertheless, these methods are complex and have numerous influencing factors, which may present obstacles to efficient communication between studies. In this study, we developed a fluorescence observation system that integrated a two-photon laser scanning confocal microscope (TPLSCM) with the second-harmonic generation (SHG) of a cartilage matrix. The observation system enabled the detection of autofluorescence emitted by the cartilage matrix without species specificity, facilitating both qualitative and quantitative analyses of the cartilage matrix. Notably, this observation could be applied three-dimensionally to a fresh specimen in situ up to a depth of 300 μm, obviating the need for traditional histological fixation, slicing, or staining. Furthermore, using this observation system, we reconstructed a three-dimensional (3D) image and a 3D model of the cartilage matrix. The utilization of the 3D fluorescence model may serve as a dependable option for the fabrication of cartilage matrix biomimetic scaffolds in future studies.