The objective of the present study was to examine the relation between gene expression and the shape of human intervertebral disc cells cultured in vitro in three-dimensional (3D) scaffolds. Disc cells from 19 subjects were seeded into either a collagen sponge or collagen gel and cultured for 10 days. In situ hybridization was performed on serial sections of paraffin embedded specimens and assessed for expression of selected genes important for extracellular matrix formation: Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase. Rounded cells grown in collagen gel showed expression of Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase; expression of these genes was absent in spindle shaped cells. Cells in the collagen sponge that lay on the sponge margin were frequently spindle shaped; these cells expressed type I collagen, but not type II collagen, aggrecan or chondroitin-6 sulfotransferase. Results presented here provide novel data concerning disc cell gene expression with collagen 3D constructs. This information is useful for future tissue engineering studies that have the challenging goal of selectively modulating gene expression.

IL-17 is expressed in a number of tissues including the intervertebral disc, where it exerts strong inflammatory properties. We evaluated IL-17 using immunolocalization in herniated and non-herniated human discs, IL-17 gene expression, and the production of IL-17 by annulus cells cultured in three dimensions in the presence of IL-1ß or TNF-α. There was no difference in the percentage of IL-17 positive cells in annulus or nucleus in herniated vs. non-herniated disc specimens. Molecular studies confirmed expression of IL-17 in disc tissue, with significantly increased expression in more degenerated discs; there was no difference in expression between herniated vs. non-herniated discs. Exposure to IL-1ß or TNF-α resulted in significantly greater production of IL-17. Our findings expand understanding of IL-17 production by disc cells and reveal the importance of non-canonical IL-17 production in the disc. Significantly greater expression of IL-17 in more degenerated discs adds to our understanding of the changes in disc cell function with advancing stages of disc degeneration.

Matrix metalloproteinases (MMPs) degrade components of the extracellular matrix of the disc, but the presence of MMP-19 has not been explored. In other tissues, MMP-19 is known to act in proteolysis of the insulin-like growth factor (IGF) binding protein-3, thereby exposing this protein to make it available to influence cell behavior. MMP-19 also has been shown to inhibit capillary-like formation and thus play a role in the avascular nature of the disc. Using immunohistochemistry, normal discs from six subjects aged newborn through 10 years and 20 disc specimens from control donors or surgical patients aged 15-76 (mean age 40.2 years) were examined for immunolocalization of MMP-19; six Thompson grade I discs, five Thompson grade II, eight Thompson grade III, five Thompson grade IV, and one Thompson grade V discs were analyzed. The results indicate that in discs from young subjects, MMP-19 was uniformly localized in the outer annulus. In discs from adult donors and surgical patients, outer and inner annulus cells only occasionally showed MMP-19 localization. The greatest expression of MMP-19 was observed in young discs, and little expression was seen in older or degenerating discs. Because MMP-19 has been shown to regulate IGF-mediated proliferation in other tissues, its decline in the aging/degenerating disc may contribute to the age-related decrease in disc cell numbers.

Periostin, a matricellular protein in the fasciclin family, is expressed in tissues subjected to constant mechanical stress. Periostin modulates cell-to-extracellular matrix interactions and can bind to collagen, fibronectin, tenascin-C and several integrins. Our objective was to evaluate whether periostin is expressed in the human intervertebral disc. Immunohistochemical localization of periostin was carried out in tissue of human lumbar discs and lumbar discs of the sand rat (Psammomys obesus). Human discs also were examined for periostin gene expression. Immunohistochemical localization demonstrated periostin in the cytoplasm of annulus and nucleus cells, and occasionally in the surrounding pericellular and interterritorial extracellular matrix. Periostin distribution in the human disc was distinctive. Outer annulus contained the highest proportion of periostin-positive cells (88.8%), whereas inner annulus contained only 61.4%. The nucleus pulposus contained the fewest periostin-positive cells (18.5%). There was a significant negative correlation between the percentage of cells positive for periostin in the inner annulus and subject age. Periostin gene expression in the human disc also was confirmed using molecular microarray analysis. Because work by others has shown that periostin plays an important role in the biomechanical properties of other connective tissues (skin, tendon, heart valves), future research is needed to elucidate the role of periostin in disc, loading, aging and degeneration.

The pericellular region of the extracellular matrix (ECM) contains collagens, proteoglycans and other noncollagenous matrix proteins. Although such specialized pericellular ECM has been well studied in articular cartilage, little is known about the pericellular matrix in the disc. In the study reported here, pericellular matrix was studied in annulus tissue from 52 subjects ranging in age from 17-74 years. In aging/degenerating intervertebral discs, cells were identified that formed a distinctive cocoon of encircling pericellular ECM. Immunohistochemical studies identified types I, II, III and VI collagen in these pericellular sites with diverse morphological features. Similar types of changes in the pericellular matrix were observed in both surgical specimens and control donor discs. Results indicate the need for future studies to address why such specialized matrix regions form around certain disc cells and to determine the consequences of these unusual matrix regions on annular lamellar organization and function.