Increase of TNFα-stimulated osteoarthritic chondrocytes apoptosis and decrease of matrix metalloproteinases 9 by NF-κB inhibition.

OBJECTIVE: To investigate the in vitro effect of caffeic acid phenethyl ester (CAPE), a NF-κB inhibitor, on the apoptosis of osteoarthritic (OA) chondrocytes and on the regulation of the gelatinases matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9). METHODS: Annexin V-FITC/propidium iodide (PI) labeling and western blotting were used to observe and determine the apoptosis in TNFα-stimulated primary cultured osteoarthritic chondrocytes. Also, gelatin zymography was applied to examine MMP-2 and MMP-9 activities in supernatants. RESULTS: It was confirmed by both flow cytometry and western blotting that chondrocytes from OA patients have an apoptotic background. Use of CAPE in combination with 10 ng/mL of TNFα for 24 h facilitated the apoptosis. MMP-9 in the supernatant could be autoactivated (from proMMP-9 to active MMP-9), and the physiologic calcium concentration (2.5 mmol/L) could delay the autoactivation of MMP-9. The activities of MMP-2 and MMP-9 in the fresh supernatant increased significantly in response to stimulation by 10 ng/mL of TNFα for 24 h. The stimulatory effect of TNFα just on proMMP-9 was counteracted significantly by CAPE. CONCLUSION: NF-κB could prevent chondrocytes apoptosis though its activation was attributed to the increase of proMMP-9 activity induced by TNFα (a pro-apoptotic factor). Therefore, therapeutic NF-κB inhibitor was a 'double-edged swords' to the apoptosis of chondrocytes and the secretion of MMP-9.

[Primary culture and adipogenic process of pre-adipocytes from infrapatellar fat pad of osteoarthritic patients].

OBJECTIVE: To investigate the primary culture and adipogenic process of pre-adipocytes from infrapatellar fat pad of osteoarthritic patients. METHODS: The pre-adipocytes were isolated by enzymatic digestion. The morphological changes of cultured cells were observed and the growth curve was drawn by CCK-8 method. During the adipogenic process, the intracytoplasmic lipid of differentiated cells was determined by oil red O staining. And the adiponectin levels in the culture supernatants were measured by ELISA (enzyme-linked immunosorbent assay). RESULTS: The primary cultured fibroblast-like cells were spindle-shaped. In the process of adipogenesis, the intracytoplasmic lipid droplets were observed at Day 3 and over 80% of the cells differentiated into adipocytes at Day 21. With the increasing number of adipocytes, the adiponectin levels in the culture supernatant elevated and peaked at Week 3. The differentiated cells were proven to be adipocytes functioning actively. CONCLUSION: The primary culture and adipogenic process of pre-adipocytes in infrapatellar fat pad of osteoarthritic patients has been successfully established. Thus it may provide an ideal model for the study of endocrine function of infrapatellar fat pad and understanding its role in the pathogenesis of osteoarthritis.

[The Biomechanics Influence of unilateral laminectomy and discectomy surgery on the adjacent different grades of Disc Degeneration].

OBJECTIVES: To study the stress distribution of the adjacent different grades of disc degeneration underwenting unilateral laminectomy and discectomy surgery using non-linear finite element analysis. METHODS: Based on the lumbar CT scans, the finite element model (FEM) of lumbar spinal segment (L3-L5) was established. According to L3-L4 intervertebral disc degeneration, different grades of disc degeneration (healthy, mild, moderate and severe) models were established and unilateral laminectomy and discectomy surgery were also established. Physiological action such as flexion, extension, lateral bending and lateral rotation was simulated and the von Mises stress in the nucleus pulposus and annulus fibrosus matrix of L3-L4 disc was investigated. RESULTS: After unilateral laminectomy and discectomy surgery, the extremum value of von Mises stress of nucleus pulposus and annulus fibrosus matrix was maximum during extension and minimus left bending in the healthy intervertebral disc. Compared with healthy disc, the increment of extremum value was found during left bending in the mildly degenerated disc. When the value decreased in the moderately degenerated disc, but still higher than that in the healthy disc. When the adjacent disc is severely degenerated, the extremum value of nucleus pulposus decreased, in addition to axial rotation, and even lower than that of healthy disc. The value of annulus matrix decreased and still higher than that of healthy disc, especially during left bending. CONCLUSIONS: After unilateral laminectomy and discectomy surgery, avoiding lateral bending will reduce the abnormal stress in the degenerated disc and decreased the risk of accelerating disc degeneration.