High-Resolution Fourier Transform Infrared Spectroscopy Characterization of the Cell Microenvironment in Healthy and Degenerated Meniscus

The pathogenesis of knee meniscus degeneration is poorly understood. The tissue matrix surrounding meniscal cells, i.e. , the cell microenvironment, is crucial for maintaining cell homeostasis and optimal cell-matrix interactions in a healthy meniscus. Collagen, a key component in the mechanoresponsivity of the meniscus, plays a significant role in this process and may change at different stages of meniscal degeneration. Therefore, we aimed to investigate whether the collagen content of the meniscus cell microenvironment is altered at different stages of meniscal degeneration. Human menisci (N = 11) were obtained from six deceased donors and seven total knee arthroplasty patients with ethical permissions (Dnrs 2015/39 & 2016/865, Lund University). Meniscus sections, including the posterior horn and mid-body, were fixed, dehydrated, and embedded in paraffin. Histological slices were stained with hematoxylin-eosin and Safranin-O–Fast Green for Pauli scoring (grades 1–4). Additional unstained slices were imaged using high-resolution Fourier transform infrared spectroscopy (pixel size: 1.1×1.1 µm 2 ; spectral range: 800–3800 cm −1 ; spectral resolution: 4 cm −1 ; scans per pixel: 128). Menisci were divided into five regions, and 5–10 cell microenvironments per region were analyzed (total n = 489). The area under the Amide I (1590–1720 cm −1 ) region was used to calculate the (estimated) collagen content maps for each slice. The collagen content was analyzed within a 5.5 µm wide region of interest (ROI) extending 3.3 µm from the cell edge. The ROI was aligned parallel to the meniscus surface, and data from both sides of the cell were averaged. Differences in collagen content across Pauli grades were compared using a linear mixed model, with Pauli grade and compartment as fixed variables, and meniscus region and subject as random variables. The linear mixed model predicted that the collagen content of the meniscus cell microenvironment decreases progressively from Pauli grade 2 to grade 4 degeneration states (Fig. 1C, Pauli 1: 20.9 [95%CI: 17.4–24.3] AU; Pauli 2: 24.2 [22.5–25.9] AU; Pauli 3: 21.6 [20.0–23.2] AU; Pauli 4: 17.4 [15.3–19.5] AU). We observed a gradual decrease in collagen content in the cell microenvironment as meniscal degeneration progresses, consistent with previous tissue-level studies. This collagen loss may impair cell response to mechanical stimuli and disrupt optimal mechanotransduction. However, with only one healthy meniscus analyzed, we cannot determine whether collagen loss in the cell microenvironment occurs early and precedes extracellular matrix degeneration. For any figures or tables, please contact the authors directly.