Journal: Journal of Clinical Medicine
Article Title: Rotator Cuff Tenocytes Differentiate into Hypertrophic Chondrocyte-Like Cells to Produce Calcium Deposits in an Alkaline Phosphatase-Dependent Manner
Figure Lengend Snippet: Assessment of mineral deposition induced by tenocyte-like cells (TLC) after 21 days in the osteogenic medium and characterization of mineralizing cells. ( A ) Assessment of mineral deposition by alizarin red S staining after 21 days ( N = 5). Quantification of the bound stain after solubilization with formic acid for the two conditions (reading at 450 nm) and representative images of the wells. CT: control medium; TLC: tenocyte-like cells; OM: osteogenic medium. Scale bar = 1 mm. ( B ) Gene expression by TLC cultured 21 days in the osteogenic medium or in the control medium (N = 3 to 5). 1: Study of TNAP (tissue non-specific alkaline phosphatase) and ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) levels of expression. 2: Study of osteoblast markers: RUNX2, BGLAP (bone gamma-carboxyglutamate protein), BSP (Bone SialoProtein) and SPP1 (secreted phosphoProtein 1). 3: Study of chondrocyte markers: SOX9, COL2A1, ACAN (aggrecan), COMP (cartilage oligomeric matrix protein), COL10A1, and MMP13 (matrix metallopeptidase 13). 4: Study of tenocyte markers: SCX (scleraxis), MKX (Mohawk homeobox), TNMD (tenomodulin), COL1A1 and COL3A1. * = p < 0.05.
Article Snippet: Sections were incubated with primary antibodies targeting RUNX2 (Novus Biologicals NBP1-77461, 1/200), SOX9 (ab3697, 1/400, Abcam, UK), Type II collagen (COL2A1) (sc-52658, 1/1000, Santa Cruz Biotechnology, USA), Type X collagen (COL10) (ab49945, 1/1000, Abcam), tissue non-specific alkaline phosphatase (TNAP) (ab108337, 1/1000, Abcam), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) (ab40003, 1/2000, Abcam), Mohawk homeobox (MKX) (NBP2-45863, 1/400, Novus Biologicals, USA), CD31 (ab28364, 1/100, Abcam), CD68 (M0876, 1/400, Dako, USA), and cleaved caspase-3 (9664, 1/400, Cell Signaling, Netherlands).
Techniques: Staining, Expressing, Cell Culture