Calcific tendonitis is a frequent cause of chronic shoulder pain. Its cause is currently poorly known. The objectives of this study were to better characterize the cells and mechanisms involved in depositing apatite crystals in human tendons. Histologic sections of cadaveric calcified tendons were analyzed, and human calcific deposits from patients undergoing lavage of their calcification were obtained to perform infrared spectroscopy and mass spectrometry-based proteomic characterizations. In vitro, the mineralization ability of human rotator cuff cells from osteoarthritis donors was assessed by alizarin red or Von Kossa staining. Calcifications were amorphous areas surrounded by a fibrocartilaginous metaplasia containing hypertrophic chondrocyte-like cells that expressed tissue non-specific alkaline phosphatase (TNAP) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which are two key enzymes of the mineralization process. Calcific deposits were composed of apatite crystals associated with proteins involved in bone and cartilage development and endochondral bone growth. In vitro, tenocyte-like cells extracted from the rotator cuff were able to mineralize in osteogenic cultures, and expressed TNAP, type X COLLAGEN, and MMP13, which are hypertrophic chondrocytes markers. The use of a TNAP inhibitor significantly prevented mineral deposits. We provide evidence that tenocytes have a propensity to differentiate into hypertrophic chondrocyte-like cells to produce TNAP-dependent calcium deposits. We believe that these results may pave the way to identifying regulating factors that might represent valuable targets in calcific tendonitis.
Background Excessive bone formation in the entheses is one of the features of peripheral spondyloarthritis (SpA). Complex pathological mechanisms connecting inflammation, mechanical stress, and ossification are probably involved. We focused on bone morphogenetic protein (BMP)-2, -4, and -7 as possible mediators of this process. Methods BMP-2, -4, and -7 concentration was measured by ELISA in synovial fluids (SFs) of SpA (n = 56) and osteoarthritic (n = 21) patients. Mouse organotypic ankle cultures were challenged by a pro-inflammatory cocktail. Mouse primary chondrocytes, osteoblasts, or tenocytes were treated with TNF-α, interleukin (IL)-17, or IL-22 and/or subjected to cyclic stretch, or with recombinant BMP-2 or -4. Results In SpA SFs, if BMP-7 was barely detectable, BMP-2 concentration was higher and BMP-4 was lower than in osteoarthritic samples, so that BMP-2/BMP-4 ratio augmented 6.5 folds (p < 0.001). In SpA patients, TNF-α, IL-6, and IL-17 levels correlated this ratio (n = 21). Bmp-2/Bmp-4 ratio was similarly enhanced by cytokine treatment in explant and cell cultures, at mRNA level. In particular, simultaneous application of TNF-α and cyclical stretch induced a 30-fold increase of the Bmp-2/Bmp-4 ratio in chondrocytes (p = 0.027). Blockade of prostaglandin E2 and IL-6 production had almost no effect on the stretch-induced regulation of Bmp-2 or -4. Osteoinductive effects of BMP-4, and to a lesser extend BMP-2, were identified on cultured chondrocytes and tenocytes. Conclusions Our results first settle that BMP factors are locally deregulated in the SpA joint. An unexpected decrease in BMP-4 could be associated to an increase in BMP-2, possibly in response to mechanical and/or cytokine stimulations.
Career situation of first and presenting authorResident.IntroductionCalcific 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.ObjectivesTo study the ability of cells extracted from rotator cuff tendons to produce apatite crystals and to analyze the phenotype of these mineralizing cells.MethodsTenocytes 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.ResultsTendon 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<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.ConclusionsTenocyte-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 (TNAP, COL10 and MMP13) 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.AcknowledgementsThis study was supported by Inserm and the French Society of Rheumatology.Disclosure of InterestNone declared.
Background:Calcific tendonitis of the rotator cuff is a frequent cause of chronic shoulder pain. It is due to apatite deposits within the tendons. Little data are currently available about proteins associated to crystals within deposits.Objectives:The aim of the study was to quantify 6 proteins in calcific powders obtained from patients who have undergone an ultrasound-guided percutaneous lavage (UGPL) of their calcification and to look for correlations between their concentration and patient characteristics.Methods:Calcific powders were obtained from patients included in the CALCECHO trial whose main objective was to compare post-procedure pain between two groups: methylprednisolone or placebo injected at the end of the lavage [1]. Based on preliminary proteomic data and literature data, the following proteins have been selected and quantified by ELISA: Pigment-epithelium Derived Factor (PEDF), Osteopontin (OPN), Periostin (POSTN), Activin A (ACT A), Osteoprotegerin (OPG) and Bone Morphogenic Protein-2 (BMP-2). The level of each protein was expressed in µg per pg of the total proteins present in the sample. These proteins have been selected for their link to the mineralization. Correlations between the level of each protein and radiographic and ultrasound appearance of the calcific deposits were sought. We also looked for correlations between level of each protein and duration of pain or response to UGPL (Mann-Whitney test).Results:Sixty-six samples were studied: mean age was 48.9 (+/- 9.7) and 68% were female. Mean duration of shoulder pain was 30 months with a mean VAS pain of 68/100 (+/-14). Mean calcification size was 1.8 cm. Results of ELISA were as follows: mean level of PEDF at 1097 pg/µg, mean level of OPG at 135 pg/µg, mean level of POSTN at 6.9 pg/µg, mean level of ACT A at 19.6 pg/µg and mean level of OPN at 49.6 pg/µg although BMP-2 was undetectable. There was no correlation between level of proteins and the size of the calcification or the duration of pain. There was no difference in protein levels between type A and type B calcifications on radiography (classification of the French Society for Arthroscopy). In contrast, levels of POSTN and OPN were significantly higher in nodular calcifications compared to the homogenous (p=0.003 and p=0.01 respectively) or fragmented types (p=0.03 and p=0.04 respectively). Furthermore, calcifications without acoustic shadowing were enriched in POSTN compared to those with (p=0.04). Finally, the periostin level was significantly higher in calcifications that have responded well to UGPL (p=0.02).Conclusion:In this cohort of patients treated by UGPL, we observed higher levels of POSTN and OPN in the less dense calcifications and POSTN enrichment appeared to be associated with a better response to UGPL. Considering these data, further studies will be necessary to better understand the role of this protein in calcific tendonitis.References:[1]Darrieutort-Laffite C, Varin S, Coiffier G, Albert JD, Planche L, Maugars Y, Cormier G, Le Goff B. Are Corticosteroid Injections Needed After Needling and Lavage of Calcific Tendinitis: A Randomized, Double-Blind, Non-Inferiority Trial. Ann Rheum Dis. 2019 Jun;78(6):837-843.Disclosure of Interests:None declared
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