Quantification of Extracellular Matrix Proteins in the Nucleus Pulposus and Anulus Fibrosus of the Human Intervertebral Disc: Variation with Aging

*Singh, K; *Masuda, K; *Thonar, E.; *An, H; +*Cs-Szabo, G +* Rush University Medical Center, Chicago, IL [email protected] INTRODUCTION: The functions of the intervertebral disc (IVD) as a unit, and of the annulus fibrosus (AF) and nucleus pulposus (NP) individually, are determined by the molecular composition of their extracellular matrices (ECM). The outerand inner-AF consists of large collagen fibers in oblique layers, whereas the central NP contains a loose matrix rich in proteoglycans (PGs). Small-PGs (biglycan, decorin, lumican, and fibromodulin) bind to collagens, growth factors and other matrix components thereby regulating the assembly of the ECM and repair after injury. Loss by proteolytic fragmentation of large and small PGs may induce or inhibit essential inflammatory response pathways altering the ability of the ECM to maintain its structural integrity. Aging is considered a primary factor in symptomatic IVD degeneration. Since age-related changes in ECM molecules may influence matrix quality potentially contributing to IVD degeneration, the purpose of this study was to quantify the age-related changes of a select group of ECM molecules, including collagen and PGs, in the AF and NP of human IVDs. METHODS: IVD specimens obtained (through the Gift of Hope Organ and Tissue Donor Network) from both male and female thoracolumbar spines (T11-L5) represent a range of ages from 32-80. Samples used for this study met the approval of the Institutional Review Board. All specimens were analyzed by decade of life. Only specimens that were classified as Thompson Grade II according to MR evaluation were analyzed in this study. IVDs were dissected and the AF and NP were separated. DNA (Hoechst assay), proteoglycan (DMMB assay) and collagen (OH-proline HPLC assay) contents were determined for all samples. Select sets of samples (5-8 samples per decade of life) were processed for protein extraction [1,2]. Levels of biglycan, decorin, fibromodulin and lumican were semi-quantified by comparative Western blotting [1,2] by measuring the density of the immuno-stained bands on the transfer membranes with comparison to an internal control. Standard deviations within each decade and the significance of change during aging were calculated using ANOVA analysis. RESULTS: Changes in Collagen and total PG content Collagen content (normalized to DNA) decreased with aging in both AF and NP. The lowest concentration was observed in the 70-80 year range (p <0.05) in both AF and NP. Proteoglycan content (normalized to DNA) showed a similar decrease. The lowest content of PG occurred in the 70-80 year range (p < 0.05). (Figure 1)