Apoptosis and gene expression of collagenases but not gelatinases in rabbit disc fragment cultures.
OBJECTIVE
The object of this study was to characterize the biological response of isolated intervertebral disc fragments to in vitro culture conditions with respect to cell death and inflammatory and catabolic changes. The acquired data could help to gain a better understanding of the biological reaction of disc tissue when exposed to environmental changes along with altered nutritional and osmotic conditions, as are encountered in different in vitro disc models or disc diseases in vivo.


METHODS
Intervertebral disc anulus fragments were isolated from Burgundy rabbits and cultured in standard media for 3 days. The disc fragments were analyzed for their swelling properties, proteoglycan loss on histological studies, lactate dehydrogenase activity, apoptosis, gene expression of collagenases and gelatinases, and for proinflammatory (MCP-1, IL-8, and IL-6) and apoptosis-associated (TNF-alpha, Fas-L, and caspase 3) genes.


RESULTS
The results demonstrate that disc specimens were swelling, and a loss of proteoglycans with disarrangement of anulus architecture was observed. The disc cells underwent rapid apoptosis with upregulation of various proinflammatory genes. Both collagenases, matrix metalloproteinase (MMP)-1 and MMP-13, were increasingly transcribed, whereas the gelatinases MMP-2 and MMP-9 did not respond or were downregulated.


CONCLUSIONS
Cultured disc fragments swell and undergo necrotic and apoptotic cell death combined with a catabolic gene response and gene expression of proinflammatory and chemoattractant proteins. Some of these findings have been demonstrated before in various spinal disorders. In addition, disc fragments are not suitable for long-term culture if a stable disc metabolism is desired, and the described changes have to be considered when using isolated disc material for in vitro cultures.
MEM Research Center for Orthopaedic Surgery, Institute for Surgical Technology and Biomechanics, University of Bern, Switzerland. Daniel.Haschtmann@MEMcenter.unibe.ch
Daniel
D
Haschtmann
Stephen J
SJ
Ferguson
Jivko V
JV
Stoyanov
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3 / Q000032:analysis; D018932:Chemokine CCL2 / Q000032:analysis; D017364:Collagenases / Q000032:analysis*; D004195:Disease Models, Animal; D053222:Fas Ligand Protein / Q000032:analysis; D018093:Gelatinases / Q000032:analysis*; D015971:Gene Expression Regulation, Enzymologic; D018836:Inflammation Mediators / Q000032:analysis; D015850:Interleukin-6 / Q000032:analysis; D016209:Interleukin-8 / Q000032:analysis; D007403:Intervertebral Disc / Q000201:enzymology / Q000473:pathology*; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D020781:Matrix Metalloproteinase 1 / Q000032:analysis; D053509:Matrix Metalloproteinase 13 / Q000032:analysis; D020778:Matrix Metalloproteinase 2 / Q000032:analysis; D020780:Matrix Metalloproteinase 9 / Q000032:analysis; D011509:Proteoglycans / Q000032:analysis; D011817:Rabbits; D046509:Tissue Culture Techniques; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis
D018932:Chemokine CCL2; D053222:Fas Ligand Protein; D018836:Inflammation Mediators; D015850:Interleukin-6; D016209:Interleukin-8; D011509:Proteoglycans; D014409:Tumor Necrosis Factor-alpha; D007770:L-Lactate Dehydrogenase; D053148:Caspase 3; D017364:Collagenases; D018093:Gelatinases; D053509:Matrix Metalloproteinase 13; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D020781:Matrix Metalloproteinase 1