Characterization of menisci, ligaments and synovial membranes in patients with osteoarthritis and calcium pyrophosphate deposition (CPPD) by histology and Raman spectroscopy
Silvia Sirotti (1), Paola Maroni (2), Giovanni Lombardi (2,3), Tom Niessink (4), Matthijs Janssen (4), Cees Otto (5), Tim L Jansen (4), Giuseppe Peretti (6), Georgios Filippou (1,7)
Affiliation(s):
1. Rheumatology Department, IRCCS Galeazzi – Sant’Ambrogio Hospital, Milan, Italy.
Laboratory of Experimental Biochemistry and Molecular Biology, IRCCS Galeazzi – Sant’Ambrogio Hospital, Milan, Italy.
2. Department of Athletics, Strength and Conditioning, Poznań University of Physical Education, Poznań, Poland
3. Rheumatology Department, VieCuri Medical Centre, Venlo, the Netherlands
4. Personalized Therapeutics and Diagnostics, Technical Medical Centre, University of Twente, Enschede, the Netherlands
5. Orthopedic Department, IRCCS Galeazzi – Sant’Ambrogio Hospital, Milan, Italy
6. Department of Biomedical and Clinical Sciences, University of Milan, Milan, Italy
Background: Calcium pyrophosphate (CPP) crystals are frequently observed in patients with osteoarthritis (OA). However, the processes underlying their formation and their role in OA pathogenesis remain unclear, and basic studies have predominantly focused on hyaline cartilage (HC). On the other hand, imaging studies have demonstrated that fibrocartilage is more frequently involved in CPPD, but much less is known on the changes of this tissue during CPPD course, as well as in tendons and ligaments, other commonly involved structures. This study aims to evaluate the histological characteristics and Raman spectroscopic findings in menisci, ligaments and synovial membranes in patients with OA and CPPD.
Methods: Menisci, cruciate ligaments and synovial membranes were obtained from consecutive OA patients undergoing total knee replacement surgery. For conventional light microscopy studies, all specimens were fixed 24h in 10% neutral buffered formalin, dehydrated in a graded ethanol series cleared with xylene, embedded in paraffin, and cut into 4µm sections. Samples were stained with haematoxylin and eosin (H&E) and analysed. Compensated polarized light microscopy was used for identifying the presence of CPP crystals. For immunohistochemistry, slices were treated, after antigen retrieval, for 10 min with 0.1% H2O2 and blocked with normal serum. Immunostaining was performed overnight at 4°C with anti-Collagen X (10µg/mL) and detected with a streptavidin-biotin system and diaminobenzidine.
An iRPolM integrated Raman polarized light microscope was used to locate and characterize birefringent material in unstained paraffin slides. This device operates a 532 nm Raman excitation laser with a 10.3 mW under-the-objective laser power. Crystals were measured with an integration time of 100 ms.
Results: 3 patients were enrolled in the study (2/3 F, mean age of 76.3 yrs). Polarized microscopy and H&E staining showed circumscribed structures filled with rhomboid-shaped crystals, characteristic of CPP, in the menisci of all 3 patients (Figure1), and in the cruciate ligaments of 2/3 patients (Figure2). No deposits were detected in the synovial membranes. H&E staining further identified the same foci of CPP crystals, surrounded by hypertrophic chondrocyte clusters, which are strongly positive for collagen X, a marker of hypertrophic chondrocytes (Figures).
Raman spectroscopic analysis of slides from 2 out of 3 patients confirmed the presence of CPP crystals in the menisci of both patients and in the cruciate ligaments of 1 patient, consistent with findings from H&E staining. In the synovial membranes of the 2 patients, a minimal amount of CPP crystals was identified, and in 1 patient, the coexistence of CPP and basic calcium phosphate within the same foci was observed. All identified CPP crystals were of the triclinic variant.
Conclusion: All consecutively enrolled OA patients in this study also exhibited CPPD, highlighting its high prevalence in final-stage OA. CPP deposition in fibrocartilage and ligaments showed histological characteristics similar to HC, and the presence of collagen X in these structures suggests a role of hypertrophic chondrocytes in the formation/deposition of CPP crystals also in fibrocartilage and ligaments. The identification of shared characteristics across various tissues can serve as a valuable starting point for understanding the mechanisms underlying CPP crystal production, which have not been fully elucidated to date.
