Intermittent Cyclic Mechanical Tension-Induced Calcification and downregulation of ankh gene expression of end plate chondrocytes.
METHODS
Intermittent Cyclic Mechanical Tension (ICMT) was applied to end plate chondrocytes by using an FX-4000T Flexercell Tension Plus unit (Flexcell International Corporation, Hillsborough, NC). Changes of end plate chondrocytes were observed after ICMT stimulation.


OBJECTIVE
To investigate the relationship between mechanical stimulation and calcification of end plate chondrocytes.


BACKGROUND
Previous study showed that end plate calcification was related to mechanical stress, but there was no clear evidence to indicate whether or not mechanical stimulation could induce calcification of end plate chondrocytes in vitro.


METHODS
Rat end plate chondrocytes were cultured and ICMT (strain at 0.5 Hz sinusoidal curve at 10% elongation) was applied for 25 days, 4 hours a day and continued to culture for 5 days. End plate chondrocytes were incubated for 12 hours in the presence or absence of 10 ng/mL of transforming growth factor-β1 (TGF-β1) (prepared from a stock solution at 10 μg/mL in 2 mM citric acid containing 2 mg/mL bovine serum albumin) in MEM/F-12 containing a final concentration of 1% FCS. End plate chondrocytes calcification was stained by alizarin red S (AR-S). End plate chondrocytes viability was examined by LIVE/DEAD viability/cytotoxicity kit (Invitrogen, Carlsbad, CA). Related gene expression was examined by reverse transcription-polymerase chain reaction and Western blot.


RESULTS
LIVE/DEAD assay verified that the nonloading (NC) group and the ICMT group end plate chondrocytes remained adherent, with no change in viability after the application of ICMT. Alizarin red staining showed that ICMT induced the calcification of end plate chondrocytes. Real-time reverse transcription-polymerase chain reaction showed that mRNA expression of endogenous TGF-β1 decreased and mRNA expression of type I, type X, osteocalcin and osteopontin increased after ICMT. The ankh gene expression of both mRNA and protein levels decreased in the ICMT stimulation. The ankh gene expression of both mRNA and protein levels increased in TGF-β1 stimulation. Compared with NC group, the alkaline phosphatase activities significantly increased in ICMT group.


CONCLUSIONS
Our results directly showed that ICMT induced the calcification and downregulation of ankh gene expression of end plate chondrocytes, which may be caused by the endogenous TGF-β1.
Department of Orthopedic Surgery, Yijishan Hospital, Wannan Medical College, Wuhu, Anhui, China. xuhg@medmail
Hong-guang
HG
Xu
Xiao-hai
XH
Zhang
Hong
H
Wang
Ping
P
Liu
Ling-ting
LT
Wang
Chi-jian
CJ
Zuo
Wen-xue
WX
Tong
Xiao-ling
XL
Zhang
D000818:Animals; D015153:Blotting, Western; D002113:Calcification, Physiologic; D002470:Cell Survival; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D024121:Collagen Type X / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D005786:Gene Expression Regulation; D006132:Growth Plate / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism; D028061:Phosphate Transport Proteins / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013314:Stress, Mechanical; D013997:Time Factors; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
D024042:Collagen Type I; D024121:Collagen Type X; D028061:Phosphate Transport Proteins; D053773:Transforming Growth Factor beta1; D015675:Osteocalcin; D053495:Osteopontin