P111 Tenocytes extracted from rotator cuff tendons induce TNAP-dependent mineral deposition and express genes related to a hypertrophic chondrocyte differentiation
Authors: C Darrieutort-Laffite, P Arnolfo, A Najm, T Garraud, B Le Goff, F Blanchard
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Career situation of first and presenting author</h3> Resident. <h3>Introduction</h3> 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. Our previous results showed that calcific deposits are surrounded by chondrocyte-like cells expressing TNAP (Tissue Nonspecific Alkaline Phosphatase) and ENPP1 (Ectonucleotidepyrophosphatase/phosphodiesterase 1), two key enzymes involved in the mineralization process. <h3>Objectives</h3> To study the ability of cells extracted from rotator cuff tendons to produce apatite crystals and to analyze the phenotype of these mineralizing cells. <h3>Methods</h3> Tenocytes were extracted from rotator cuff tendons removed during shoulder total replacement. To evaluate their ability to mineralize, they were cultured in an osteogenic medium (OM) for 21 days. Mineral deposition then was assessed by staining with Alizarin red. Tenocytes total RNA was extracted and analyzed by RT-qPCRs. TNAP enzymatic activity was also assessed in the cells. A TNAP inhibitor was used to delineate its implication in the mineralization process. <h3>Results</h3> Tendon samples were obtained from 5 patients (age 69.6±5.13 years). Cells extracted from these tendons expressed collagen I, collagen III, Scleraxis and Mkx (Mohawk homeobox), as expected for tenocytes. However, Tenomodulin 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. qPCR analyses showed a significant increase of TNAP and ENPP1 expression by cells cultured in OM (p&lt;0.05). Osteoblast markers (Runx2, osteocalcin, osteopontin, BSP) were not increased by the OM. COMP (Cartilage Oligomeric Matrix Protein), a chondrocyte marker was significantly increased, as well as MMP13 (Matrix Metallopeptidase 13) and Collagen X suggesting a hypertrophic differentiation. In parallel, in the OM, TNAP enzymatic activity was significantly higher at 14 and 21 days compared to the control medium. An inhibitor of TNAP completely prevented mineral deposition in OM and reduced expression of the hypertrophic chondrocyte markers MMP13 and Collagen X. <h3>Conclusions</h3> Tenocyte-like cells extracted from tendons of the rotator cuff are able to induce mineralization in an osteogenic medium. The cells express genes associated with a hypertrophic chondrocyte phenotype (<i>TNAP, COL10</i> and <i>MMP13</i>) and TNAP seems to have a crucial role in the induced mineralization. These results suggest that tenocytes could differentiate into hypertrophic chondrocyte which induce TNAP-dependent apatite deposition in calcific tendonitis. <h3>Acknowledgements</h3> This study was supported by Inserm and the French Society of Rheumatology. <h3>Disclosure of Interest</h3> None declared.