Background : Calcific tendinopathy is a frequent cause of chronic painful shoulders. It is caused by deposits of carbonated apatite in rotator cuff tendons. Although the disease is frequent, 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. They were fixed in formalin 4%, 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), Von Kossa (no decalcified samples) and Tartrate-Resistant Acid Phosphatase (TRAP) staining were performed. Immunohistochemistry using anti-Runx2, anti-Sox9, anti-Collagen II and X, anti-CD31 and CD68 antibodies has been performed. 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 : Five calcified samples were collected. 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 were observed as previously described. 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 or TRAP positive cells were detected around the deposits.
Conclusion : Histological analyses of whole calcified tendon tissues showed a fibrocartilaginous area surrounding the calcium deposits with chondrocyte-like cells expressing ENPP1 and TNAP suggesting their crucial role in the deposition of apatite crystals. Further analyses are necessary to understand the origin of these cells and the regulatory factors involved in their differentiation.