Human Annulus Cells Research Articles

Prostaglandin E1 and misoprostol increase epidermal growth factor production in 3D-cultured human annulus cells

Background context Epidermal growth factor (EGF) is a peptide known to modulate a number of cellular responses including embryogenesis, cell proliferation, and cell survival. Little is known about EGF and its regulation in human annulus cells. Previous work has identified EGF and its receptor in control outer annulus disc tissue, but not in herniated tissue. Purpose To determine if human annulus cells express EGF in vitro, to determine if the epidermal growth factor-receptor (EGF-r) was expressed in vivo and in vitro in disc cells, to test the effect of EGF on annulus cell proliferation and proteoglycan production in vitro, and to test the effect of prostaglandin E1 (PGE1) and misoprostol on disc cell production of EGF in vitro. Study design/setting Studies were approved by the authors' Human Subjects Institutional review Board. Human disc tissue was used for immunocytochemistry, and human annulus cells were tested in vitro. Patient sample Thirty-four disc specimens were used for studies of proteoglycan production, cell proliferation, and EGF production in vitro. An additional nine discs were used for EGF-r immunolocalization. Methods Disc tissue was used for immunocytochemical studies for the localization of EGF-r and as a source for cultured annulus cells. Monolayer culture was used to test proliferation responses to 0, 25, 50, or 75 ng/mL EGF over a 2-day culture period. Three-dimensional (3D) culture in a collagen sponge was used to test 100,000 cells cultured in a paired experimental design over 14 days for production of EGF and proteoglycans. Cells were exposed to control conditions, or to either misoprostol at 8 ng/mL or PGE1 at 10 −7 M. Conditioned media was harvested and assayed using an enzyme-linked immunosorbent assay (ELISA) assay with the Human Protein Cytokine Antibody Array I kit. Replicate EGF relative intensity values were averaged and normalized to controls assayed on the same membrane. 3D-cultured cells were also used to confirm EGF gene expression using microarray analysis. Standard statistical methods were used to analyze results. Results Microarray analysis of mRNA from annulus cells in 3D culture confirmed expression of EGF, and immunocytochemistry verified the presence of EGF-r in vitro and in vivo. PGE1, at a dose of 10 −7 M, and misoprostol (a synthetic PGE1 analog) at a dose of 8 ng/mL, both significantly increased EGF levels in annulus cells cultured in 3D compared with control levels (p=.031 and .034, respectively). No significant difference, however, was seen in cell proliferation or in total sulfated proteoglycan production in EGF-exposed annulus cells. Conclusions Data showed that EGF is expressed and produced by annulus cells in vivo and in 3D culture, with significantly greater in vitro EGF produced in the presence of PGE1 or the PGE1 analog misoprostol. Misoprostol, developed for prevention/treatment of nonsteroidal anti-inflammatory-induced gastropathy, has now been reported to have some interesting anabolic effects stimulating osteoblasts during fracture healing and during ovariectomy in animal models. Exogenous EGF did not increase cell proliferation in monolayer, or total production of proteoglycans in 3D culture. Additional work is needed to further delineate the role of EGF in the human disc.

Exogenous thymosin β4 prevents apoptosis in human intervertebral annulus cells in vitro

Exogenous thymosin β4 prevents apoptosis in human intervertebral annulus cells in vitro

Adipose-derived mesenchymal stem cells from the sand rat: transforming growth factor beta and 3D co-culture with human disc cells stimulate proteoglycan and collagen type I rich extracellular matrix

IntroductionAdult mesenchymal stem cell therapy has a potential application in the biological treatment of disc degeneration. Our objectives were: to direct adipose-derived mesenchymal stem cells (AD-MSC) from the sand rat to produce a proteoglycan and collagen type I extracellular matrix (ECM) rich in known ECM components of the annulus fibrosis of disc; and to stimulate proteoglycan production by co-culture of human annulus cells with AD-MSC.MethodsAD-MSC were isolated and characterised by adherence to plastic, appropriate expression of cluster of differentiation (CD) markers, and differentiation to osteoblasts and chondrocytes in vitro. AD-MSC were grown in three-dimensional (3D) culture and treated with or without transforming growth factor beta (TGFβ) to direct them to produce annulus-like ECM as determined by proteoglycan content and collagen expression. AD-MSC were co-cultured with human annulus cells and grown in 3D culture.ResultsAD-MSC produced a proteoglycan and collagen type I rich ECM after treatment with TGFβ in 3D culture as confirmed by a 48% increase in proteoglycan content assayed by 1,9-dimethylmethylene blue (DMB), and by immunohistochemical identification of ECM components. Co-culture of human annulus and sand rat AD-MSC in 3D culture resulted in a 20% increase in proteoglycan production compared with the predicted value of the sum of the individual cultures.ConclusionResults support the hypothesis that AD-MSC have potential in cell-based therapy for disc degeneration.

Brain-derived neurotrophic factor and its receptor in the human and the sand rat intervertebral disc

IntroductionBrain-derived neurotrophic factor (BDNF) was first identified in the intervertebral disc (IVD) when its molecular upregulation was observed in sections of nucleus pulposus cultured under conditions of increased osmolarity. BDNF is now known to be involved in a number of biologic functions, including regulation of differentiation/survival of sensory neurons, regulation of nociceptive function and central pain modulation, and modulation of inflammatory pain hypersensitivity. In addition, more recent investigations show that BDNF can induce the recruitment of endothelial cells and the formation of vascular structures. The objectives of the present study were to use immunocytochemistry to determine the distribution of BDNF and its receptor (BDNF-tropomyosine receptor kinase B) in the human IVD, and to test for gene expression of BDNF and its receptor in cultured human annulus fibrosus cells.MethodsWe studied immunohistochemical localization of BDNF and its receptor in the human annulus, quantified the percentage of outer annulus and inner annulus cells and nucleus cells positive for BDNF immunolocalization, and studied the gene expression of BDNF and its receptor using microarray analysis.ResultsThe percentage (mean ± standard error of the mean) of cells positive for BDNF localization was significantly greater in the outer annulus (32.3 ± 2.7%, n = 22) compared with either the inner annulus (8.1 ± 1.5%, n = 6) or the nucleus (10.4 ± 2.8%, n = 3) (P < 0.0001). BDNF-receptor immunolocalization showed a pattern similar to that of BDNF, but was not quantitatively assessed. BDNF gene expression levels from cultured annulus cells showed a significant positive correlation with increasing levels of IVD degeneration (P = 0.011).ConclusionThese findings provide data on the presence of BDNF and its receptor in the human IVD at the translational level, and on the expression of BDNF and its receptor by cultured human annulus cells. Our findings point to the need for further studies to define the role of BDNF in the human IVD and to investigate regulatory events within the disc that control the expression of BDNF and its receptor.

IGF-1 rescues human intervertebral annulus cells from in vitro stress-induced premature senescence

The aging human intervertebral disc contains a sizeable population of senescent cells. Since senescent cells cannot divide, senescence reduces the disc's ability to generate new cells to replace existing ones lost to necrosis or apoptosis. The objectives of the present work were: (1) to develop a reliable in vitro model for stress-induced premature senescence in human annulus cells, and (2) to investigate the potential for insulin-like growth factor-1 (IGF-1) to prevent or ameliorate senescence in vitro. The developed experimental model employs a 2 h exposure to 50 μM hydrogen peroxide; immunocytochemical localization of senescence associated-beta-galactosidase at pH 6.0 was used as the marker for senescent cells, and the percentage of senescent cells quantified after 3 days of culture. Nine sets of annulus cells were obtained from eight human surgical disc specimens; cells were tested with 0, 50, 100 or 500 ng/ml IGF-1. Although 50 or 100 ng/ml IGF-1 did not significantly alter the percentage of senescent cells, a significant reduction was present following exposure to 500 ng/ml IGF-1 (control, 56.3% ± 8.5 (9); mean ± SEM, (n) vs. treated, 39.6% ± 6.6 (9), p = 0.0009). These novel findings point to the value of continued research towards development of future biologic therapies designed to reduce cell senescence in degenerating human discs.

Leptin expression by annulus cells in the human intervertebral disc

Background context It is now known that leptin acts not only as a metabolic signal related to energy homeostasis but also as an endocrine hormone regulating traditional endocrine systems and neuroendocrine function in various cells. It participates in bone remodeling and acts as a growth factor stimulating proliferation. Expression of leptin and the presence of leptin receptors have not been explored in disc tissue. Purpose To determine (1) whether leptin is produced by cells in the human annulus in vivo, (2) whether annulus cells have leptin receptors in vivo and in vitro, and (3) whether measurable amounts of leptin are produced during three-dimensional culture of human annulus cells. Study design/setting Studies were approved by the human subjects Institutional Review Board. Surgical and donor disc tissue was obtained and assessed by using immunocytochemistry of paraffin-embedded disc tissue. Annulus cells were also cultured from disc specimens and conditioned media assessed for the production of leptin during three-dimensional culture. Patient sample Disc tissue was examined from 7 young subjects and 29 adult subjects. Outcome measures Immunodetection of leptin and leptin receptors in cells of the human annulus; conditioned media was analyzed for production of leptin in vitro by human disc cells. Methods Human annulus tissue and cultured cells were examined by using immunohistochemical methods to identify the presence of leptin and leptin receptors. Human disc cells were assayed for leptin production in three-dimensional culture. Results Immunocytochemistry showed the presence of intracellular leptin and the presence of leptin receptors in some (but not all) annulus cells in the human disc. Production of leptin by annulus cells was further confirmed by assays of conditioned media from three-dimensional annulus cell culture. Conclusions These novel studies identify the presence of a heretofore unrecognized cytokine/hormone and its receptor in human annulus cells. Because of the mitogenic role of leptin in other tissues, the present work points to the importance of future studies to explore whether leptin has a mitogenic function in maintaining disc cell numbers.

4:24 Human annulus cell response to vibratory loading and modulation of the effect by interleukin-1B

Purpose of study: Chronic exposure to whole body vibration correlates with increased risk of lumbar degenerative disorders. Our laboratory has demonstrated rabbit annulus cells respond to vibratory stimulation with an influx of extracellular calcium through stretch-activated Ca2+ channels. The objective of this study is to determine if human annular cells react similarly to vibratory loading, and what affect the presence of an inflammatory mediator interleukin (IL)-1B, has on the response.Methods used: Annulus tissue was obtained from subjects having surgery for herniated nucleus pulposus. Cells were isolated by means of collagenase digestion and cultured. Cultures were loaded with a Ca2+ sensitive fluorescent dye and fixed to a vibration jig. A computer-operated microscope was used to determine intracellular calcium concentrations ([Ca2+]ic). Vibration trials were for 15, 30 45 or 60 seconds. Ten mM ATP was used as a positive control. Separate cultures were vibrated in Ca2+ free EBSS to determine the need for extracellular calcium. Additional cultures were pretreated with 10 mM ATP, a purinoceptor agonist, or either 10 mM verapamil or 10 mM gadolinium, Ca2+ channel antagonists. All of the vibration trials were then repeated after incubation in 1 nM IL-1B. Data were analyzed with one-way analysis of variance and paired Student t test.of findings: Baseline [Ca2+]ic was 185 nM. Increases in [Ca2+]ic were seen after the 30- and 45-second vibration trials (p<.01). No change in [Ca2+]ic was seen after 15- or 60-second trials. Trials in Ca2+ free EBSS demonstrated no change in [Ca2+]ic with vibration but maintained response to ATP. Pretreatment with 10 uM ATP inhibited the response to vibration. Incubation of the culture in 10 mM verapamil, a slow Ca2+ channel antagonist, did not inhibit the response to vibration or ATP. Incubation in 10 mm gadolinium, a stretch-activated Ca2+ channel antagonist, inhibited the response to vibration but not the response to ATP. Vibration trials after IL-1B incubation demonstrated increased [Ca2+]ic at all vibration intervals (p<.01). In addition, the response in each trial was significantly greater than the corresponding trials not pretreated with IL-1B (p<.05). Again, gadolinium, but not verapamil, blocked the response to vibration in the IL-1B groups.Relationship between findings and existing knowledge: This study demonstrates human annulus fibrosus cells respond to vibratory stimuli with increases in [Ca2+]ic by means of an influx of extracellular calcium through stretch-activated Ca2+ channels. Exposure to ATP results in an increase in [Ca2+]ic by means of the release of intracellular Ca2+ stores. This suggests that vibration exerts a direct cellular effect on annulus cells with the activation of membrane Ca2+ channels. Activation of annulus cell surface purinoceptors by ATP inhibits the mechanisms triggered by vibration. However, the trials performed after pretreatment with the inflammatory mediator IL-1B clearly demonstrate that inflammation exaggerates the response of annulus cells to vibratory load.Overall significance of findings: These results suggest mechanisms involving intracellular calcium are involved in the response of intervertebral disc tissues to vibration, and extracellular purines and inflammatory mediators modulate the signaling cascade. As the mechanism of increasing intracellular calcium is different for ATP and vibration, this does not simply represent a depletion of calcium stores. We are currently working to delineate the downstream effects of this signaling mechanism and if purinoceptor and inflammatory modulation is protective or deleterious.Disclosures: No disclosures.Conflict of interest: Anthony Russo, grant research support. CREF 99-016NIH-AR38121.

IL‐1β sensitizes intervertebral disc annulus cells to fluid‐induced shear stress

Chronic inflammation and altered mechanical loading are implicated as contributors to intervertebral disc degeneration. Biomechanical and biochemical factors play a role in disc degeneration but have received limited study. Mechanically, intervertebral discs are sheared during bending or twisting of the trunk. Biochemically, IL-1beta, detected in degenerative discs, promotes metalloproteinase expression. We hypothesized that disc cells might respond to shear stress and IL-1beta in a calcium signaling response. We measured the effect of single and combined stimuli on intracellular calcium concentration ([Ca2+]ic) and signaling. Cells were isolated from annulus tissue, cultured to quiescence, plated on collagen-bonded Culture Slips and incubated with Fura-2AM. Cells then were incubated in IL-1beta. Cell response to the effects of fluid flow was tested using FlexFlo, a laminar flow device. Human annulus (hAN) cells responded to laminar fluid flow with a one to three-fold increase in [Ca2+]ic. IL-1beta alone produced a small, transient stimulation. hAN cells pretreated with IL-1beta responded to shear with a more dramatic and sustained increase in [Ca2+]ic, six to ten-fold over basal level, when compared to shear then IL-1beta or shear and IL-1beta alone (P<0.001 for all comparisons). This is the first study documenting synergism of a signaling response to biomechanical and biochemical stimuli in human disc cells. IL-1beta treatment appeared to "sensitize" annulus cells to mechanical load. This increased responsiveness to mechanical load in the face of inflammatory cytokines may imply that the sensitivity of annulus cells to shear increases during inflammation and may affect initiation and progression of disc degeneration.

Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation.

BackgroundThe relationship between cell shape, proliferation, and extracellular matrix (ECM) production, important aspects of cell behavior, is examined in a little-studied cell type, the human annulus cell from the intervertebral disc, during monolayer vs three-dimensional (3D) culture.ResultsThree experimental studies showed that cells respond specifically to culture microenvironments by changes in cell shape, mitosis and ECM production: 1) Cell passages showed extensive immunohistochemical evidence of Type I and II collagens only in 3D culture. Chondroitin sulfate and keratan sulfate were abundant in both monolayer and 3D cultures. 2) Cells showed significantly greater proliferation in monolayer in the presence of platelet-derived growth factor compared to cells in 3D. 3) Cells on Matrigel™-coated monolayer substrates became rounded and formed nodular colonies, a finding absent during monolayer growth.ConclusionsThe cell's in vivo interactions with the ECM can regulate shape, gene expression and other cell functions. The shape of the annulus cell changes markedly during life: the young, healthy disc contains spindle shaped cells and abundant collagen. With aging and degeneration, many cells assume a strikingly different appearance, become rounded and are surrounded by unusual accumulations of ECM products. In vitro manipulation of disc cells provides an experimental window for testing how disc cells from given individuals respond when they are grown in environments which direct cells to have either spindle- or rounded-shapes. In vitro assessment of the response of such cells to platelet-derived growth factor and to Matrigel™ showed a continued influence of cell shape even in the presence of a growth factor stimulus. These findings contribute new information to the important issue of the influence of cell shape on cell behavior.