Background/Purpose:

Calcific tendinopathy represents 10 to 42% of chronic painful shoulders. These calcium deposits are composed of carbonated apatite. Although the disease is frequent, its origin stays still largely unknown. Molecular and cellular mechanisms involved in this pathological mineralization process are not currently identified. The objective of the study was to analyze calcified tendon samples to understand the organization of the deposits and to characterize the cells potentially involved in their formation.

Methods:

Samples were collected from cadaveric subjects. Ultrasound was first used to detect calcified tendons. Then, tendons were collected and fixed in formalin 4% during 48h. Samples were then decalcified in EDTA, dehydrated and embedded in paraffin. Some samples were not decalcified to allow a better characterization of the calcific deposits. Hematoxylin and eosin (HE), Safranin O/Fast Green (SO/FG) and Von Kossa (no decalcified samples) staining were performed. Immunohistochemistry using anti-Runx2, anti-Sox9, anti-Collagen II and X, anti-CD31 and CD68 has been performed to characterize the cells and tissue around the calcifications. We used also used anti-TNAP (Tissue Nonspecific Alkaline Phosphatase) and ENPP1 (Ectonucleotide Pyrophosphatase/Phosphodiesterase 1) antibodies. Indeed, these two enzymes are essential in the physiological mineralization: extracellular inorganic pyrophosphates are provided by ENPP1 then hydrolyzed by TNAP to promote mineralization.

Results:

Six samples were collected (1 normal and 5 calcified). On HE staining, voluminous calcium deposits were encapsulated by a fibrocartilaginous tissue. In one sample, we observed an intra-tendinous osseous metaplasia. This fibrocartilaginous area presented a red coloration (proteoglycan specific) on SO/FG staining but was collagen II negative whereas the fibrocartilage at the tendon attachment was strongly positive. Within this area, cells with round nuclei and pericellular lacunae and different from tenocytes were observed as previously described (Uhthoff, 1975). These cells expressed Runx2 and Sox9 suggesting a chondrocyte differentiation but only a small number of them expressed type X collagen, hypertrophic chondrocytes-specific marker. These cells also expressed ENPP1 and TNAP. Interestingly, extracellular TNAP deposits were also present at the periphery of the deposits. We identified vessels surrounding the deposits on 4 of the 5 calcified samples. Finally, no CD68 positive cells were detected around the deposits.

Conclusion:

Histological analyses of whole calcified tendon tissues showed a fibrocartilaginous area surrounding the calcium deposits. Chondrocyte-like cells present within this area expressed ENPP1 and TNAP suggesting their crucial role in the deposition of apatite crystals. An osseous metaplasia was seen in one sample but probably does not represent the main mineralization process involved in the disease. Further analyses are necessary to understand the origin of these cells and the regulatory factors involved in their differentiation.

References: Uhthoff HK. Calcifying tendinitis, an active cell-mediated calcification. Virchows Arch A Pathol Anat Histol. 1975;366(1):51-8.

« Back to 2017 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/rotator-cuff-calcific-tendinopathy-chondrocyte-like-cells-surrounding-calcific-deposits-express-tnap-and-enpp1-two-key-enzymes-of-the-mineralization-process/