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.

Imbalanced Protein Expression Patterns of Anabolic, Catabolic, Anti-Catabolic and Inflammatory Cytokines in Degenerative Cervical Disc Cells: New Indications for Gene Therapeutic Treatments of Cervical Disc Diseases

“…This might occur instead because of undesirable phenotypic changes in NP cells. Previous studies of lumbar NP cells have also indicated similar cell densities in adult age of different disc degeneration grades [24], [28]. Conversely, some studies of lumbar NP cells reported a decline of NP cell densities with increasing degeneration grades [29]–[31].…”

Angle-ply biomaterial scaffold for annulus fibrosus repair replicates native tissue mechanical properties, restores spinal kinematics, and supports cell viability

“…Increasing grades of IVDD have been positively correlated with increasing concentrations of proteases. More specifically, mean concentrations of MMP-1 (collagenases type I) have been shown to increase with increasing grades of IVDD (grade III: 133 pg/ml, grade IV: 279 pg/ml, grade V: 319 pg/ml) [40]. Therefore, any ECM-based biomaterial placed in this caustic environment must resist accelerated degeneration so that it can maintain its mechanical integrity and function.…”

“…Moreover, even fewer have been assessed for their contribution to restoring functional spinal unit (FSU) kinematics following injury and repair; arguably one of the most important functional outcomes of any motion preserving/sparing spinal implant. Finally, biomaterials to be implanted into a damaged IVD must demonstrate resistance to accelerated degradation as investigations have illustrated increased concentrations of destructive proteases which could jeopardize their mechanical integrity [38–40]. This is of particular importance for biomaterials composed of extracellular matrix (ECM) components, which often have to be chemically crosslinked to impart resistance to accelerated enzymatic degradation, yet should demonstrate cytocompatibility.…”

Prolyl-4-hydroxylase Domain Protein 2 Controls NF-κB/p65 Transactivation and Enhances the Catabolic Effects of Inflammatory Cytokines on Cells of the Nucleus Pulposus

“…Intervertebral disc degeneration is characterized by expression of inflammatory molecules that elicit catabolic changes in proteins and proteoglycans of the NP extracellular matrix (32)(33)(34)(35)(36)(37)(38)(39)(40). That PHD2 mediates this response was evident because silencing significantly down-regulated TNF-␣-induced expression of MMP-3, MMP-13, SDC4, COX2, PHD3, and ADAMTS5; these catabolic factors promote degradation of the disc extracellular matrix, and their expression is closely tied to the pathogenesis of disc disease (29,35,41,42).…”