Background/Purpose: Calcific tendonitis is a frequent cause of shoulder pain. The calcium deposits are composed of carbonated apatite and their origin stays still largely unknown. Our preliminary results showed that calcific deposits are surrounded by chondrocyte-like cells expressing TNAP ((Tissue Nonspecific Alkaline Phosphatase) and ENPP1 (Ectonucleotide pyrophosphatase/phosphodiesterase 1), two key enzymes involved in the mineralization process [1].

The purpose was to study the ability of cells extracted from rotator cuff tendons to produce apatite crystals and to analyze the phenotype of these mineralizing cells.

Methods: Tenocytes were extracted from rotator cuff tendons removed during shoulder total replacement. These cells were first characterized by their gene expression. To evaluate their ability to mineralize, they were cultured in an “osteogenic medium” (OM) containing dexamethasone 10^-7M, ascorbic acid 2P (250 µM) and β-glycerophosphate (10 mM) or in a control medium (DMEM 3%) for 21 days. At 21 days, mineral deposition was assessed by staining with Alizarin red solution. At 21 days, tenocytes total RNA was extracted and quantitative PCRs (qPCRs) were performed after first strand cDNA synthesis. Primers for the following genes were used: Runx2, Sox9, Collagen I, Collagen II, Collagen III, Collagen X, Osteopontin, Osteocalcin, Bone Sialo Protein (BSP), TNAP, ENPP1, Mohawk homeobox (Mkx), Scleraxis (SCX), Tenomodulin (TNMD), Cartilage Oligomeric Matrix Protein (COMP), Aggrecan, Matrix Metallopeptidase 13 (MMP13) and GAPDH. TNAP enzymatic activity was also assessed in the cells at 3, 7, 14 and 21 days.

Results:

Tendon samples were obtained from 5 patients (age 69.6 ± 5.13 years). Four were females. Cells extracted from these tendons expressed collagen I, collagen III, Scleraxis and Mkx, as expected for this type of cells. However, TNMD was very weakly expressed and lost after passage 1. These cells were able to mineralize in the OM although no mineralization was observed in the control medium. The mineralized surface was about 15% of surface of the well. qPCR analyses showed a significant increase of TNAP and ENPP1 expression by cells cultured in OM compared to control cells (p<0.05, Mann-Whitney test). Osteoblast markers (Runx2, osteocalcin, osteopontin, BSP) were not increased by the OM. COMP, a chondrocyte marker was significantly increased and there was a trend to an increase of MMP13 and Collagen X suggesting a hypertrophic orientation. In parallel, in the OM, TNAP enzymatic activity started to increase at 7 days and was significantly higher at 14 and 21 days compared to the control medium.

Conclusion: Cells extracted from tendons of the rotator cuff are able to mineralize in an osteogenic medium. The cells expressed genes associated with a chondrocyte phenotype and these results are interesting considering the histological data previously obtained. Further in vitro experiments are necessary to look at the key role of TNAP in the process.

[1] Darrieutort-Laffite C, et al. Rotator Cuff Calcific Tendinopathy: Chondrocyte-like Cells Surrounding Calcific Deposits Express TNAP and ENPP1, Two Key Enzymes of the Mineralization Process. [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10).

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/tenocytes-extracted-from-rotator-cuff-tendons-are-able-to-induce-mineral-deposition-in-vitro-and-express-genes-related-to-a-chondrocyte-differentiation/