Electron microscopic observation of established chondrocytes derived from human intervertebral disc hernia (KTN-1) and role of macrophages in spontaneous regression of degenerated tissues.
BACKGROUND
Biological and pathological cell processes during the degeneration of intervertebral discs are as yet poorly understood.


OBJECTIVE
An electron microscope was used to observe disc hernia degeneration at the cellular level as expressed in extruded tissue from a human intervertebral disc and in cultured chondrocytes. The mechanism of spontaneous regression was analyzed in order to investigate the effects of homologous macrophages, and the results of this analysis may be developed into a clinical therapy.


METHODS
Extruded tissue specimens excised during surgery on human intervertebral disc hernia and cultured chondrocytes isolated from the excised tissue were observed by means of electron microscopy. Extracellular matrix metalloproteinase-3 (MMP-3) and its antagonist, tissue inhibitor of metalloproteinases-1 (TIMP-1), were observed by means of immune electron microscopy. Macrophages confirmed by CD68 immunostaining were added to the chondrocyte culture and observed by means of electron microscopy.


METHODS
All control subjects and patients gave written consent to the study.


METHODS
KTN-1 was directly observed without culture, and nuclei degeneration, the development of chromatin granules, changes in the osmotic pressure of the nuclear membrane and rough-surfaced endoplasmic reticulum, and the development of fat droplets were observed.


METHODS
Tissues excised during surgery were divided, a part of the tissues were fixed in various fixatives for electron microscopy and immune electron microscopy analysis, and the other part was treated with collagenase. In addition, chondrocytes were isolated and cultured. Human peripheral blood mononuclear cells were separated using the Ficoll method. After culturing the cells, macrophages were collected, added to the chondrocyte culture, and observed under an electron microscope. CD68 positivity of the macrophages was confirmed by CD68 immunostaining.


RESULTS
Freshly isolated chondrocytes in the hernia's extruded region differed markedly from cultured chondrocytes. By means of immunoelectron microscopy, MMP-3 and TIMP-1 were localized at the endoplasmic reticulum of the cultured chondrocytes. Infiltration of macrophages among the chondrocytes was observed in the mixed culture.


CONCLUSIONS
The tissue extruded from the intervertebral disc showed obvious signs of degeneration, such as changes in osmotic pressure. Macrophages were observed to be the mechanism of spontaneous regression.
Tsuru
M
M
Department of Orthopedic Surgery, Kurume University School of Medicine, 67 Asahi-machi, 830-0011 Kurume City, Japan.
Nagata
K
K
Ueno
T
T
Jimi
A
A
Irie
K
K
Yamada
A
A
Nishida
T
T
Sata
M
M
D002460:Cell Line; D019902:Chondrocytes / Q000737:chemistry / Q000648:ultrastructure*; D006801:Humans; D007403:Intervertebral Disc / Q000473:pathology*; D008264:Macrophages / Q000473:pathology*; D019278:Matrix Metalloproteinase 3 / Q000032:analysis; D016253:Microscopy, Immunoelectron
D019278:Matrix Metalloproteinase 3