title: A combinatorial relative mass value evaluation of endogenous bioactive proteins in three-dimensional cultured nucleus pulposus cells of herniated intervertebral discs: identification of potential target proteins for gene therapeutic approaches.
abstract: Painful degenerative disc diseases have been targeted by different biological treatment approaches. Nucleus pulposus (NP) cells play a central role in intervertebral disc (IVD) maintenance by orchestrating catabolic, anabolic and inflammatory factors that affect the extracellular matrix. IVD degeneration is associated with imbalances of these factors, resulting in a catabolic inflammatory metabolism. Therefore, accurate knowledge about their quantity and quality with regard to matrix synthesis is vital for a rational gene therapeutic approach. NP cells were isolated from 63 patients operated due to lumbar disc herniation (mean age 56 / range 29 - 84 years). Then, three-dimensional culture with low-glucose was completed in a collagen type I scaffold for four weeks. Subsequently cell proliferation evaluation was performed using 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and intracellular concentration of 28 endogenously expressed anabolic, catabolic, inflammatory factors and relevant matrix proteins was determined by enzyme-linked immunosorbent assay. Specimen-related grades of degeneration were confirmed by preoperative magnetic resonance imaging. Independent from gender, age and grade of degeneration proliferation rates remained similar in all groups of NP cells. Progressive grades of degeneration, however, showed a significant influence on accumulation of selective groups of factors such as disintegrin and metalloproteinase with thrombospondin motifs 4 and 5, matrix metalloproteinase 3, metalloproteinase inhibitor 1 and 2, interleukin-1β and interleukin-1 receptor. Along with these changes, the key NP matrix proteins aggrecan and collagen II decreased significantly. The concentration of anabolic factors bone morphogenetic proteins 2, 4, 6 and 7, insulin-like growth factor 1, transforming growth factor beta 1 and 3, however, remained below the minimal detectable quantities. These findings indicate that progressive degenerative changes in NP may be problematic with regard to biologic treatment strategies. Hence, gene therapeutic interventions regulating relevant bioactive factors identified in this work might contribute to the development of regenerative treatment approaches for degenerative disc diseases.
authors.affiliation: Department of Neurosurgery, Innsbruck Medical University, Innsbruck, Austria ; Department of Neurosurgery, University Medical Center Mannheim, Heidelberg University, Mannheim, Germany.
authors.forename: Demissew S
authors.initials: DS
authors.lastname: Mern
authors.forename: Johann
authors.initials: J
authors.lastname: Fontana
authors.forename: Anja
authors.initials: A
authors.lastname: Beierfuß
authors.forename: Claudius
authors.initials: C
authors.lastname: Thomé
authors.forename: Aldemar A
authors.initials: AA
authors.lastname: Hegewald
mesh_terms: D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism; D000071121:ADAMTS4 Protein; D000071116:ADAMTS5 Protein; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D005109:Extracellular Matrix / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology*; D055959:Intervertebral Disc Degeneration / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism / Q000628:therapy; D008279:Magnetic Resonance Imaging; D008297:Male; D053505:Matrix Metalloproteinases, Secreted / Q000235:genetics / Q000378:metabolism; D008875:Middle Aged; D011348:Procollagen N-Endopeptidase / Q000235:genetics / Q000378:metabolism; D020543:Proteome; D019714:Tissue Inhibitor of Metalloproteinases / Q000235:genetics / Q000378:metabolism
chemical_list: D016326:Extracellular Matrix Proteins; D020543:Proteome; D019714:Tissue Inhibitor of Metalloproteinases; D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human; D053505:Matrix Metalloproteinases, Secreted; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein