title: Effects of mechanical strain on ANK, ENPP1 and TGF-β1 expression in rat endplate chondrocytes in vitro.
abstract: We investigated the effects of mechanical strain on the progressive ankylosis (ANK) gene and extracellular nucleotide phosphatase/phosphodiesterase (ENPP)1 mRNA expression and TGF-β1 protein expression in rat endplate chondrocytes in vitro. Endplate chondrocytes were isolated and cultured in vitro. Following identification with toluidine blue and immunocytochemical staining, chondrocytes were subjected to 10% elongation with various frequencies (0.5, 1, 1.5 and 2 Hz) using a Flexercell Tension Plus system at various intervals (3, 6, 12, 24, 36 and 48 h). As a control, cells that had been cultured statically on the same type of plate but were not subjected to stretch were also observed. Real-time reverse transcription-polymerase chain reaction and the enzyme-linked immunosorbent assay were used to study the effects of mechanical strain on ANK and ENPP1 mRNA expression and TGF-β1 concentration in the supernatant, respectively. Following treatment, the shape of the chondrocytes displayed a significant change from the original polygon to a typical spindle cell morphology; and the arrangement of the cells exhibited a change from a haphazard arrangement to an alignment with a certain direction. In the 0.5 Hz, 24-h group, the ANK gene expression was significantly increased compared to the control group (P<0.05); whereas in the other groups, the ANK and ENPP1 expression levels were reduced. With the increased frequencies in the 24-h group, the ANK gene expression gradually reduced. Changes in the expression of ANK and ENPP1 followed similar trends. TGF-β1 in the supernatant increased gradually in each frequency group, with a clear increase in the 0.5 Hz group. We conclude that various frequencies of mechanical strain can affect the expression of ANK, ENPP1 and endogenous TGF-β1 in endplate chondrocytes. Our results indicate that 0.5 Hz, 24 h may be the optimal stimulation condition to prevent calcification occurrence and to maintain the function of endplate chondrocytes.
authors.affiliation: Department of Orthopedic Surgery, Yijishan Hospital, Wannan Medical College, Wuhu, Anhui 241001, P.R. China. xuhg@medmail.com.cn
authors.forename: Hong-Guang
authors.initials: HG
authors.lastname: Xu
authors.forename: Chun-Jiang
authors.initials: CJ
authors.lastname: Hu
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Ping
authors.initials: P
authors.lastname: Liu
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Yang
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.forename: Ling-Ting
authors.initials: LT
authors.lastname: Wang
mesh_terms: D000818:Animals; D048430:Cell Shape; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000201:enzymology*; D005786:Gene Expression Regulation; D008297:Male; D009045:Motor Endplate / Q000166:cytology*; D010641:Phenotype; D028061:Phosphate Transport Proteins / Q000235:genetics / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013314:Stress, Mechanical; D013347:Subcellular Fractions / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
chemical_list: C498769:Ankh protein, mouse; D028061:Phosphate Transport Proteins; D012333:RNA, Messenger; C508962:Tgfb1 protein, rat; D053773:Transforming Growth Factor beta1; D010727:Phosphoric Diester Hydrolases; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; D011755:Pyrophosphatases