Differential Response of Bovine Mature Nucleus Pulposus and Notochordal Cells to Hydrostatic Pressure and Glucose Restriction.

OBJECTIVE: The nucleus pulposus of the human intervertebral disc contains 2 cell types: notochordal (NC) and mature nucleus pulposus (MNP) cells. NC cell loss is associated with disc degeneration and this process may be initiated by mechanical stress and/or nutrient deprivation. This study aimed to investigate the functional responses of NC and MNP cells to hydrostatic pressures and glucose restriction. DESIGN: Bovine MNP and NC cells were cultured in 3-dimensional alginate beads under low (0.4-0.8 MPa) and high (1.6-2.4 MPa) dynamic pressure for 24 hours. Cells were cultured in either physiological (5.5 mM) glucose media or glucose-restriction (0.55 mM) media. Finally, the combined effect of glucose restriction and high pressure was examined. RESULTS: Cell viability and notochordal phenotypic markers were not significantly altered in response to pressure or glucose restriction. MNP cells responded to low pressure with an increase in glycosaminoglycan (GAG) production while high pressure significantly decreased ACAN gene expression compared with atmospheric controls. NC cells showed no response in matrix gene expression or GAG production with either loading regime. Glucose restriction decreased NC cell TIMP-1 expression but had no effect on MNP cells. The combination of glucose restriction and high pressure only affected MNP cell gene expression, with decreased ACAN, Col2α1, and ADAMTS-5 expression. CONCLUSION: This study shows that NC cells are more resistant to acute mechanical stresses than MNP cells and provides a strong rationale for future studies to further our understanding the role of NC cells within the disc, and the effects of long-term exposure to physical stresses.

Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state.

BACKGROUND: Bone erosion is a frequent complication of gout and is strongly associated with tophi, which are lesions comprising inflammatory cells surrounding collections of monosodium urate (MSU) crystals. Osteocytes are important cellular mediators of bone remodeling. The aim of this study was to investigate the direct effects of MSU crystals and indirect effects of MSU crystal-induced inflammation on osteocytes. METHODS: For direct assays, MSU crystals were added to MLO-Y4 osteocyte cell line cultures or primary mouse osteocyte cultures. For indirect assays, the RAW264.7 macrophage cell line was cultured with or without MSU crystals, and conditioned medium from these cultures was added to MLO-Y4 cells. MLO-Y4 cell viability was assessed using alamarBlue® and LIVE/DEAD® assays, and MLO-Y4 cell gene expression and protein expression were assessed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Histological analysis was used to examine the relationship between MSU crystals, inflammatory cells, and osteocytes in human joints affected by tophaceous gout. RESULTS: In direct assays, MSU crystals reduced MLO-Y4 cell and primary mouse osteocyte viability but did not alter MLO-Y4 cell gene expression. In contrast, conditioned medium from MSU crystal-stimulated RAW264.7 macrophages did not affect MLO-Y4 cell viability but significantly increased MLO-Y4 cell expression of osteocyte-related factors including E11, connexin 43, and RANKL, and inflammatory mediators such as interleukin (IL)-6, IL-11, tumor necrosis factor (TNF)-α and cyclooxygenase-2 (COX-2). Inhibition of COX-2 in MLO-Y4 cells significantly reduced the indirect effects of MSU crystals. In histological analysis, CD68+ macrophages and MSU crystals were identified in close proximity to osteocytes within bone. COX-2 expression was also observed in tophaceous joint samples. CONCLUSIONS: MSU crystals directly inhibit osteocyte viability and, through interactions with macrophages, indirectly promote a shift in osteocyte function that favors bone resorption and inflammation. These interactions may contribute to disordered bone remodeling in gout.

Inhibition of glutamate regulated calcium entry into leukemic megakaryoblasts reduces cell proliferation and supports differentiation.

Human megakaryocytes release glutamate and express glutamate-gated Ca(2+)-permeable N-methyl-D-aspartate receptors (NMDARs) that support megakaryocytic maturation. While deregulated glutamate pathways impact oncogenicity in some cancers, the role of glutamate and NMDARs in megakaryocytic malignancies remains unknown. The aim of this study was to determine if NMDARs participate in Ca(2+) responses in leukemic megakaryoblasts and if so, whether modulating NMDAR activity could influence cell growth. Three human cell lines, Meg-01, Set-2 and K-562 were used as models of leukemic megakaryoblasts. NMDAR components were examined in leukemic cells and human bone marrow, including in megakaryocytic disease. Well-established NMDAR modulators (agonists and antagonists) were employed to determine NMDAR effects on Ca(2+) flux, cell viability, proliferation and differentiation. Leukemic megakaryoblasts contained combinations of NMDAR subunits that differed from normal bone marrow and the brain. NMDAR agonists facilitated Ca(2+) entry into Meg-01 cells, amplified Ca(2+) responses to adenosine diphosphate (ADP) and promoted growth of Meg-01, Set-2 and K-562 cells. Low concentrations of NMDAR inhibitors (riluzole, memantine, MK-801 and AP5; 5-100µM) were weakly cytotoxic but mainly reduced cell numbers by suppressing proliferation. The use-dependent NMDAR inhibitor, memantine (100µM), reduced numbers and proliferation of Meg-01 cells to less than 20% of controls (IC50 20µM and 36µM, respectively). In the presence of NMDAR inhibitors cells acquired morphologic and immunophenotypic features of megakaryocytic differentiation. In conclusion, NMDARs provide a novel pathway for Ca(2+) entry into leukemic megakaryoblasts that supports cell proliferation but not differentiation. NMDAR inhibitors counteract these effects, suggesting a novel opportunity to modulate growth of leukemic megakaryoblasts.

Same-species phenotypic comparison of notochordal and mature nucleus pulposus cells.

PURPOSE: The ratio of notochordal (NC) cells to mature nucleus pulposus (MNP) cells in the nucleus pulposus varies with species, age and health. Studies suggest that loss of NC cells is a key component of intervertebral disc degeneration. However, few studies have examined the phenotypes of these two cell populations. Therefore, this study aimed to isolate NC and MNP cells from the same intervertebral disc and study phenotypic differences in extracellular matrix production and cell morphology in 3D culture over 7 days. METHODS: Sequential mechanical dissociation and enzymatic digestion were used to isolate NC cell clusters and single MNP cells from bovine caudal discs. Cells were cultured in alginate beads and subsequently analysed for viability, cytokeratin-8 expression, GAG production and extracellular matrix gene expression. RESULTS: Mechanical dissociation allowed NC cells to be extracted as intact cell clusters. NC cells represented 8% of the NP cell population and expressed both cytokeratin-8 and vimentin. MNP cells expressed vimentin, only. Both cells types were viable for 7 days. In addition to morphological differences, NC cells produced up to 30 times more total proteoglycan than MNP cells. NC cells had significantly higher aggrecan and brachyury expression. CONCLUSIONS: NC and MNP cells can be isolated from the same bovine disc and maintain their distinct phenotypes in 3D culture.

Primary cilia mediate mechanotransduction through control of ATP-induced Ca2+ signaling in compressed chondrocytes.

We investigated the role of the chondrocyte primary cilium in mechanotransduction events related to cartilage extracellular matrix synthesis. We generated conditionally immortalized wild-type (WT) and IFT88(orpk) (ORPK) mutant chondrocytes that lack primary cilia and assessed intracellular Ca(2+) signaling, extracellular matrix synthesis, and ATP release in response to physiologically relevant compressive strains in a 3-dimensional chondrocyte culture system. All conditions were compared to unloaded controls. We found that cilia were required for compression-induced Ca(2+) signaling mediated by ATP release, and an associated up-regulation of aggrecan mRNA and sulfated glycosaminosglycan secretion. However, chondrocyte cilia were not the initial mechanoreceptors, since both WT and ORPK cells showed mechanically induced ATP release. Rather, we found that primary cilia were required for downstream ATP reception, since ORPK cells did not elicit a Ca(2+) response to exogenous ATP even though WT and ORPK cells express similar levels of purine receptors. We suggest that purinergic Ca(2+) signaling may be regulated by polycystin-1, since ORPK cells only expressed the C-terminal tail. This is the first study to demonstrate that primary cilia are essential organelles for cartilage mechanotransduction, as well as identifying a novel role for primary cilia not previously reported in any other cell type, namely cilia-mediated control of ATP reception.

Development of a method for the measurement of primary cilia length in 3D.

BACKGROUND: Primary cilia length is an important measure of cell and tissue function. While accurate length measurements can be calculated from cells in 2D culture, measurements in tissue or 3D culture are inherently difficult due to optical distortions. This study uses a novel combination of image processing techniques to rectify optical distortions and accurately measure cilia length from 3D images. METHODS: Point spread functions and experimental resolutions were calculated from subresolution microspheres embedded in 3D agarose gels for both wide-field fluorescence and confocal laser scanning microscopes. The degree of axial smearing and spherical aberration was calculated from xy:xz diameter ratios of 3D image data sets of 4 µm microspheres that had undergone deconvolution and/or Gaussian blurring. Custom-made 18 and 50 µm fluorescent microfibers were also used as calibration objects to test the suitability of processed image sets for 3D skeletonization. Microfiber length in 2D was first measured to establish an original population mean. Fibers were then embedded in 3D agarose gels to act as ciliary models. 3D image sets of microfibers underwent deconvolution and Gaussian blurring. Length measurements within 1 standard deviation of the original 2D population mean were deemed accurate. Finally, the combined method of deconvolution, Gaussian blurring and skeletonization was compared to previously published methods using images of immunofluorescently labeled renal and chondrocyte primary cilia. RESULTS: Deconvolution significantly improved contrast and resolution but did not restore the xy:xz diameter ratio (0.80). Only the additional step of Gaussian blurring equalized xy and xz resolutions and yielded a diameter ratio of 1.02. Following image processing, skeletonization successfully estimated microfiber boundaries and allowed reliable and repeatable measurement of fiber lengths in 3D. We also found that the previously published method of calculating length from 2D maximum projection images significantly underestimated ciliary length. CONCLUSIONS: This study used commercial and public domain image processing software to rectify a long-standing problem of 3D microscopy. We have shown that a combination of deconvolution and Gaussian blurring rectifies optical distortions inherent in 3D images and allows accurate skeletonization and length measurement of microfibers and primary cilia that are bent or curved in 3D space.

Measurement of reactive oxygen species in the culture media using Acridan Lumigen PS-3 assay.

Reactive oxygen species (ROS) are generated continuously during aerobic metabolism. ROS are highly reactive molecules and in excessive amounts, can lead to protein and DNA oxidation, protein cross-linking, and cell death. Cell-culture models provide a valuable tool in understanding the mechanisms that lead to cell death. Accumulation of ROS within cells and/or their release into the culture media are highly cell type-specific. The ability to estimate ROS levels in the culture media is an important step in understanding the mechanisms contributing to disease processes. In this paper, we describe the optimization of a simple method to estimate ROS levels in the culture media using the Acridan Lumigen PS-3 reagent provided in the Amersham ECL Plus kit (GE Healthcare, UK). We have shown that the Acridan Lumigen PS-3 assay generates ROS-specific chemiluminescence in fresh as well as media stored at -20°C, in as little as 10-20 µl of samples. The method was able to detect the dose (of stimulants)- and time (acute and chronic)-dependent changes in ROS levels in media collected from various cell types. Our results suggest that the kit reagents, PBS buffer, and various media did not contribute significantly to the overall chemiluminescence generated in the assay; however, we suggest that the unused medium specific for each cell type should be used as blanks and final readings of test samples normalized against these readings. As this method uses commonly available laboratory equipment and commercially available reagents, we believe this assay is convenient, economical, and specific in estimating ROS released extracellularly into the culture media.

Mechanical loading modulates chondrocyte primary cilia incidence and length.

The pathways by which chondrocytes of articular cartilage sense their mechanical environment are unclear. Compelling structural evidence suggests that chondrocyte primary cilia are mechanosensory organelles. This study used a 3D agarose culture model to examine the effect of compressive strain on chondrocyte cilia. Chondrocyte/agarose constructs were subjected to cyclic compression (0-15%; 1 Hz) for 0.5-48 h. Additional constructs were compressed for 48 h and allowed to recover for 72 h in uncompressed free-swelling conditions. Incidence and length of cilia labelled with anti-acetylated alpha-tubulin were examined using confocal microscopy. In free-swelling chondrocytes, these parameters increased progressively, but showed a significant decrease following 24 or 48 h compression. A 72 h recovery partially reversed this effect. The reduced cilia incidence and length were not due to increased cell division. We therefore propose that control of primary cilia length is an adaptive signalling mechanism in response to varying levels and duration of mechanical loads during joint activity.

Does milk increase mucus production?

Excessive milk consumption has a long association with increased respiratory tract mucus production and asthma. Such an association cannot be explained using a conventional allergic paradigm and there is limited medical evidence showing causality. In the human colon, beta-casomorphin-7 (beta-CM-7), an exorphin derived from the breakdown of A1 milk, stimulates mucus production from gut MUC5AC glands. In the presence of inflammation similar mucus overproduction from respiratory tract MUC5AC glands characterises many respiratory tract diseases. beta-CM-7 from the blood stream could stimulate the production and secretion of mucus production from these respiratory glands. Such a hypothesis could be tested in vitro using quantitative RT-PCR to show that the addition of beta-CM-7 into an incubation medium of respiratory goblet cells elicits an increase in MUC5AC mRNA and by identifying beta-CM-7 in the blood of asthmatic patients. This association may not necessarily be simply cause and effect as the person has to be consuming A1 milk, beta-CM-7 must pass into the systemic circulation and the tissues have to be actively inflamed. These prerequisites could explain why only a subgroup of the population, who have increased respiratory tract mucus production, find that many of their symptoms, including asthma, improve on a dairy elimination diet.

The cellular composition and morphological organization of the rostral migratory stream in the adult human brain.

The rostral migratory stream (RMS) is the major pathway by which progenitor cells migrate from the subventricular zone (SVZ) to the olfactory bulb (OB) in rodents, rabbits and primates. However, the existence of an RMS within the adult human brain has been elusive. Immunohistochemical studies utilising cell-type specific markers for early progenitor cells (CD133), proliferating cells (PCNA), astrocytes and type B cells (GFAP) and migrating neuroblasts (PSA-NCAM), reveal that the adult human RMS is organized into layers containing glial cells, proliferating cells and neuroblasts. In addition, the RMS is arranged around a remnant of the ventricular cavity that extends from the SVZ to the OB as seen by immunohistological staining analysis and electron microscopy, showing the presence of basal bodies and a typical 9+2 arrangement of tubulin in tufts of cilia from all levels of the RMS. Overall, these findings suggest that a pathway of migratory progenitor cells similar to that seen in other mammals is present within the adult human brain and that this pathway could provide for neurogenesis in the human forebrain. These findings contribute to the scientific understanding of adult neurogenesis and establish the detailed cytoarchitecture of this novel neurogenic niche in the human brain.

A novel histological technique for distinguishing between epithelial cells in forensic casework.

There are a number of forensic cases in which the identification of the epithelial cell type from which DNA originated would provide important probative evidence. This study aimed to develop a technique using histological staining of fixed cells to distinguish between skin, buccal and vaginal epithelium. First, 11 different stains were screened on formalin-fixed, wax-embedded cells from five women. Samples were analysed qualitatively by examining staining patterns (colour) and morphology (absence or presence of nuclei). Three of the staining methods--Dane's, Csaba's and Ayoub-Shklar--were successful in distinguishing skin epithelial cells from buccal and vaginal. Second, cells were smeared directly onto slides, fixed with one of five fixatives and stained with one of the three stains mentioned above. Methanol fixation, coupled with the Dane's staining method, specific to keratin, was the only technique that distinguished between all three cell types. Skin cells stained magenta, red and orange and lacked nuclei; buccal cells stained predominantly orange-pink with red nuclei; while vaginal cells stained bright orange with orange nuclei and a blue extracellular hue. This staining pattern in vaginal cells was consistent in samples collected from 50 women aged between 18 and 67. Identification of cell type from unlabelled micrographs by 10 trained observers showed a mean success rate of 95%. The results of this study demonstrate that histological staining may provide forensic scientists with a technique for distinguishing between skin, buccal and vaginal epithelial cells and thus would enable more conclusive analyses when investigating sexual assault cases.

Immunohistochemical staining as a potential method for the identification of vaginal epithelial cells in forensic casework.

There is currently no accurate method to identify vaginal epithelial cells uniquely. This study aimed to use a cell extraction procedure compatible with routine forensic sampling methods, and to investigate the expression of cytokeratin (CK), estrogen receptor-alpha (ERalpha), and phosphodiesterase 5 (PDE5) in order to distinguish between skin, buccal, vaginal, and external penile epithelial cells. Seminal fluid samples were also examined. Epithelial cell samples were fixed in formalin, embedded in agarose, and processed using histological methods. Antigen-antibody reactions were detected using the DAKO Envision+ detection system. CK was present in all cells from all five sources confirming the origin of cells as epithelial. Both ERalpha and PDE5 positively labeled vaginal, buccal, and skin epithelial cells. Although an antigen unique to vaginal epithelial cells was not identified, we have described a cell extraction procedure for use in the immunohistochemical detection of a wide range of antigens, an approach compatible with forensic diagnostics.

Localization of extracellular matrix receptors on the chondrocyte primary cilium.

A single primary cilium is found in chondrocytes and other connective tissue cells. We have previously shown that extracellular matrix (ECM) macromolecules such as collagen fibers closely associate with chondrocyte primary cilia, and their points of contact are characterized by electron-opaque plaques suggesting a direct link between the ECM and the cilium. This study examines the expression of receptors for ECM molecules on chondrocyte primary cilia. Embryonic chick sterna were fluorescently labeled with antibodies against alpha and beta integrins, NG2, CD44, and annexin V. Primary cilia were labeled using acetylated alpha-tubulin antibody. Expression of ECM receptors was examined on chondrocyte plasma membranes and their primary cilia using immunofluorescence and confocal microscopy. All receptors examined showed a punctate distribution on the plasma membrane. alpha2, alpha3, and beta1 integrins and NG2 were also present on primary cilia, whereas annexin V and CD44 were excluded. The number of receptor-positive cilia varied from 8/50 for NG2 to 43/50 for beta1 integrin. This is the first study to demonstrate the expression of integrins and NG2 on chondrocyte primary cilia. The data strongly suggest that chondrocyte primary cilia have the necessary machinery to act as mechanosensors, linking the ECM to cytoplasmic organelles responsible for matrix production and secretion.