Spatial and temporal localization of transforming growth factor-beta, fibroblast growth factor-2, and osteonectin, and identification of cells expressing alpha-smooth muscle actin in the injured anulus fibrosus: implications for extracellular matrix repair.

Abstract

The spatial and temporal localization of fibroblast growth factor-2, transforming growth factor-beta, osteonectin, and alpha-smooth muscle cell actin in the injured anulus fibrosus was investigated. To assess the involvement of fibroblast growth factor-2, transforming growth factor-beta, osteonectin, and alpha-smooth muscle cell actin in anulus fibrosus repair. Fibroblast growth factor-2 and transforming growth factor-beta have been localized to disc herniation tissue, and alpha-smooth muscle cell actin has been identified in a number of mesenchymal cell types, but their roles have not been evaluated in repair processes in the experimentally injured anulus fibrosus. For this study, 32 two adult merinos received a 4-mm deep standard annular incision in their L1L2 and L3L4 discs (lesion group). A similar number of sham-surgery animals served as control subjects. Osteonectin, fibroblast growth factor-2, transforming growth factor-beta, and alpha-smooth muscle cell actin were immunolocalized in sagittal disc sections 3, 6, 12, and 26 months after the operation. Selected specimens also were stained with hematoxylin and eosin, Masson-trichrome, toluidine blue, and picrosirius red. Early focal depletion of proteoglycan was evident in the anulus fibrosus and reorganization of outer annular lamellas 3 to 6 months after the operation. Blood vessel ingrowth and fibroblast infiltration from the outer anulus fibrosus along the plane of the annular defect were maximal 12 months after the operation. Focal upregulation in alpha-smooth muscle cell actin expression was evident with maximal staining in the 12-month lesion samples near infiltrating blood vessels at the lesion site, and also in cells well away from these vessels. Some of the anulus fibrosus cells of the sham sections also stained positively for alpha-smooth muscle cell actin, but this staining was significantly less than in the lesion samples. Staining for fibroblast growth factor-2, transforming growth factor-beta, and osteonectin was strongly localized to blood vessels and cells in the vicinity of the annular lesion. It was maximal 12 months after the operation and diminished by 26 months after the operation. Osteonectin expression also was significantly elevated in outer anulus fibrosus cells distant from the lesion site and its associated blood vessels. In the sham discs, immunoreactivity to fibroblast growth factor-2, transforming growth factor-beta, osteonectin, and alpha-smooth muscle cell actin was confined to sparsely distributed cells in the anulus fibrosus. No matrix staining was observed. Immunoreactivity for the noted agents was strongly associated with regions of the annular lesions undergoing matrix reorganization consistent with an active repair response. This response extended as far as the middle third of the anulus fibrosus, which also demarcated the extent of blood vessel ingrowth and cellular infiltration in this model. The alpha-smooth muscle cell actin expression suggested an active involvement of myofibroblasts in the anulus fibrosus repair processes.