We examined the correlation between the activation of
nuclear factor kappaB (NFkappaB), stimulated by environmental factors involving
cytokines and
growth factors in ligament cells, and the onset of ossification of the spinal ligaments (OSL) or
diffuse idiopathic skeletal hyperostosis (DISH). Aseptic samples were taken carefully from non-ossified sites during surgery (75 patients). We carried out preliminary
hematoxylin and
eosin and
toluidine blue staining, using five portions of each specimen, and excluded samples containing chondrocytic, osteoblastic, or inflammatory cells (n = 25). We used specimens from the remaining 50 patients (35 men and 15 women, ranging in age from 45-81 years); average age, 59.5 years (18 nuchal ligament specimens, and 32 yellow ligament specimens). OSL or DISH had occurred in 25 patients, 20 patients were in the non-OSL group (8 with cervical spondylotic
myelopathy, and 12 with lumbar canal
stenosis), and the remaining 5 samples were collected from patients with injury. For culture study, we used portions of the 14 largest samples from the above 50 patients. We extracted
nuclear proteins and cytoplasmic
proteins from non-ossified spinal ligaments in 50 patients and detected p65RelA/NFkappaB by Western blotting.
Tumor necrosis factor-alpha (
TNF alpha),
interleukin 1beta (IL-1beta),
platelet-derived growth factor BB (
PDGF-BB) and
transforming growth factor-beta1 (TGF-beta1) in cytoplasm were quantified by
enzyme-linked
immunosorbent assays (ELISA). Cultured cells from the 14 samples were then stimulated with 10, 100, 250, or 500 ng/ml of recombinant human (rh)PDGF-B or TGFbeta1. A control experiment was performed without
rhPDGF-BB or TGFbeta1 stimulation.
Alkaline phosphatase (ALP) activity was standardized by the
DNA content of the cells. The number of NFkappaB-positive samples was significantly higher in patients with OSL or DISH than in non-OSL patients. This tendency was obvious in the case of OSL or DISH with
non-insulin-dependent diabetes mellitus (
NIDDM). In OSL and in DISH patients, significantly greater amounts of
PDGF-BB and TGFbeta1 were seen in ligament cells than in non-OSL patients (P < 0.05). There was a positive correlation between the detection of p65RelA/NFkappaB band and the content of
PDGF-BB and TGFbeta1 in ligament cells (P < 0.05). ALP activity tended to be higher in cells in the OSL group not receiving any other treatment. Our results indicate the possibility that NFkappaB, stimulated by environmental factors involving
PDGF-BB and TGFbeta1 in ligament cells, influences the osteoblastic differentiation of undifferentiated mesenchymal cells.