Study design: In vitro molecular research on the posterior longitudinal ligament fibroblasts.
Objective: To investigate different expression of old astrocyte specifically induced substance (OASIS) between spinal ligament fibroblasts from the patients with ossification of the posterior longitudinal ligament (OPLL) and from non-OPLL patients and demonstrate knockdown of OASIS protein expression by RNA interference inhibiting expression of type I collagen (COL I) in OPLL cells.
Summary of background data: OPLL is characterized by ectopic bone formation in spinal ligaments. Some evidence indicates that ligament fibroblasts from OPLL patients have osteogenic characteristics. However, the relevant intracellular signaling pathways remain unclear.
Methods: Spinal ligament cells were cultured using tissue fragment cell culture and identified by immunocytochemistry and immunofluorescence. The mRNA expression of osteoblast-specific genes of osteocalcin, alkaline phosphatase, and COL I were detected in OPLL and non-OPLL cells by semiquantitative reverse transcription-polymerase chain reaction. The protein expression of OASIS was detected by Western blotting. And then, after 72 hours, when RNA interference against OASIS was performed in OPLL cells, expression of the osteoblast-specific genes was compared again between the transfection group and the nontransfection group.
Results: Spinal ligament fibroblasts were observed 7 to 10 days after cell culture. Immunocytochemistry and immunofluorescence exhibited positive results of vimentin staining. The mRNA expressions of osteocalcin, alkaline phosphatase, and COL I and protein expressions of OASIS from OPLL cells were significantly greater than those from non-OPLL cells. In addition, knockdown of OASIS protein expression inhibited the mRNA expressions of COL I remarkably in the transfection group compared with the nontransfection group, at 72 hours after RNA interference targeting OASIS was performed in OPLL cells.
Conclusions: The cultured fibroblasts from OPLL patients exhibited osteogenic characteristics, and OASIS expression plays an important role in the development of OPLL through the expression of COL I.