Objective. Prior reports document macrophage and lymphocyte infiltration with proinflammatory cytokine expression in pathologic intervertebral disc (IVD) tissues. Nevertheless, the role of the Th17 lymphocyte lineage in mediating disc disease remains uninvestigated. We undertook this study to evaluate the immunophenotype of pathologic IVD specimens, including interleukin-17 (IL-17) expression, from surgically obtained IVD tissue and from nondegenerated autopsy control tissue.Methods. Surgical IVD tissues were procured from patients with degenerative disc disease (n ‫؍‬ 25) or herniated IVDs (n ‫؍‬ 12); nondegenerated autopsy control tissue was also obtained (n ‫؍‬ 8) from the anulus fibrosus and nucleus pulposus regions. Immunohistochemistry was performed for cell surface antigens (CD68 for macrophages, CD4 for lymphocytes) and various cytokines, with differences in cellularity and target immunoreactivity scores analyzed between surgical tissue groups and between autopsy control tissue regions.Results. Immunoreactivity for IL-4, IL-6, IL-12, and interferon-␥ (IFN␥) was modest in surgical IVD tissue, although expression was higher in herniated IVD samples and virtually nonexistent in control samples. The Th17 lymphocyte product IL-17 was present in >70% of surgical tissue fields, and among control samples was detected rarely in anulus fibrosus regions and modestly in nucleus pulposus regions. Macrophages were prevalent in surgical tissues, particularly herniated IVD samples, and lymphocytes were expectedly scarce. Control tissue revealed lesser infiltration by macrophages and a near absence of lymphocytes.Conclusion. Greater IFN␥ positivity, macrophage presence, and cellularity in herniated IVDs suggests a pattern of Th1 lymphocyte activation in this pathology. Remarkable pathologic IVD tissue expression of IL-17 is a novel finding that contrasts markedly with low levels of IL-17 in autopsy control tissue. These findings suggest involvement of Th17 lymphocytes in the pathomechanism of disc degeneration.

The goal of this study was to evaluate the potential suitability of collagen Vitrigel (CV) membrane as a substrate for the separate reconstruction of the three main cellular layers of the cornea. Limbal explants, keratocytes, and endothelial cells were cultured on transparent membranes made of type I collagen. The resulting cell sheets were evaluated using RT-PCR, in addition to light and electron microscopy. Tensile testing was also performed to examine the mechanical properties of CV. Limbal explant cultures resulted in partially stratified epithelial sheets with upregulation of the putative stem cell marker p63. Keratocytes cultured in serum on CV exhibited stellate morphology along with a marked increase in expression of corneal crystallin ALDH and keratocan, (a keratan sulphate proteoglycan: KSPG), compared to identical cultures on tissue culture plastic. Endothelial cells formed dense monolayers with uniform cell size, tight intercellular junctions, and expression of voltage-dependent anion channels VDAC2 and VDAC3, chloride channel protein CLCN2, and sodium bicarbonate transporter NBC1. Epithelial and endothelial cells exhibited adhesive structures (desmosomes and hemidesmosomes) and evidence of apical specialization (microplicae), while endothelial cells also produced a Descemet's membrane-like basal lamina. CV was found to possess ultimate tensile strengths of 6.8 +/- 1.5 MPa when hydrated and 28.6 +/- 7.0 MPa when dry. Taken together, these results indicate that CV holds promise as a substrate for corneal reconstruction.

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Galectin-1 (Gal-1), an endogenous β-galactoside-binding protein, binds to laminins, which are highly expressed in the nucleus pulposus (NP) of the intervertebral disc (IVD). The objective of this study is to evaluate the expression of Gal-1 protein in IVD tissues during aging and the effect of Gal-1 on IVD cell adhesion to laminins. Tissues from rat, porcine and human (scoliosis or disc degeneration) IVDs were used to evaluate Gal-1 expression via immunostaining, RT-PCR and Western Blot analysis. Attachment of isolated IVD cells (porcine and human) on select laminin isoforms (LM-111 and LM-511) was compared with/without pre-incubation with exogenous Gal-1. A biotinylated Gal-1(B-Gal-1) was used to evaluate for binding to IVD cells and to select for IVD cells by magnetic activated cell sorting (MACS). NP cells expressed high levels of Gal-1 protein as compared to anulus fibrosus (AF) cells in immature tissues, while exogenous Gal-1 increased both NP and AF cell attachment to laminins and exhibited a similar binding to both cell types in vitro. With aging, Gal-1 levels in NP tissue appeared to decrease. In addition, incubation with B-Gal-1 was able to promote the retention of more than 50% of IVD cells via MACS. Our results provide new findings for the presence and functional role of Gal-1 within IVDs. Similar staining patterns for Gal-1 and LM-511 in IVD tissue suggest that Gal-1 may serve as an adhesion molecule to interact with both cells and laminins. This MACS protocol may be useful for selecting pure IVD cells from mixed cells of pathological tissue.

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