퇴행 비후된 척추 후관절이 황색인대의 섬유화 및 골화에 미치는 영향

Abstract

Study Design In vitro experimental study

Objectives To examine the effect of a synovial supernatant on the cell viability, osteogenic phenotype, mRNA expression of the types collagen and various transcriptional factors on osteogenesis in ligamentum flavum (LF) cells stimulated with synovial fluid from a degenerated facet joint.

Literature Review In degenerative lumbar spinal stenosis, hypertrophied LF or osteoarthritic hypertrophy of a facet joint often causes neurogenic claudication. The facet joint is a synovial joint with hyaline cartilage on each side. Therefore, osteoarthritis of a facet joint eventually occurs with aging and other degenerative conditions of the spine. In lumbar spinal degeneration, inflammatory mediators or cytokines are released from the facet joint tissue, which consequently affects the adjacent LF because the LF covers posterolateral aspect of the spinal canal near facet joints. However, there are no reports on the relationship between a degenerated facet joint fluid and the LF in the lumbar spine.

Materials and Methods LF surgical specimens were obtained from patients with a lumbar spine stenosis, and the cells were isolated by enzymatic digestion. Each of the synovium tissues were weighed and recorded. Each tissue was cut into small pieces with a pair of scissors and then washed 3 times with PBS. The washed tissue pieces were then cultured for 96 hr at 37℃, 5% CO2 in DMEM/F-12-0.1% FBS with a density of 200 mg/ml medium. The supernatant was collected after 96 hr. In order to measure quantitatively the proliferation of cells, the AlamarBlue assay was used. The total cellular RNA was extracted from the cells and amplification reactions specific to the following types of cDNA were performed: the osteogenic master transcription factors, Dlx5, Runx2, osterix, and types collagen and osteocalcin. Alkaline phosphatase staining for the biochemical assay and western blotting for osteocalcin protein expression were performed.

Results Human LF cells cultured with the supernatant from the facet synovium showed a slightly stronger AlamarBlue staining than the intensity of the control culture. RT-PCR revealed the upregulation of the osteogenic master transcription factors, Dlx5, Runx2, and osterix in the synovium supernatant group from one hour to 72 hours, and an increase in osteocalcin, types collagen I, III, V, XI levels from one hour to one week. LF cells cultured with the supernatant from the facet synovium showed positive staining for alkaline phosphatase. The level of the osteocalcin protein in the LF cells cultured with the supernatant from the facet synovium was higher than the control group.

Conclusions The supernatant of the facet joint from patients with degenerative spinal stenosis affects LF cells by increasing the level of cellular proliferation, upregulating the mRNA expression of osteocalcin, types of collagen, osteogenic transcription factors, positive alkaline phosphatase staining, and osteocalcin protein expression. Therefore, degenerated synovial fluid from the facet joint is an important mechanism of LF hypertrophy and ossification.