Bovine Coccygeal Discs Research Articles

Alterations of bovine nucleus pulposus cells with aging.

Aging is one of the major etiological factors driving intervertebral disc (IVD) degeneration, the main cause of low back pain. The nucleus pulposus (NP) includes a heterogeneous cell population, which is still poorly characterized. Here, we aimed to uncover main alterations in NP cells with aging. For that, bovine coccygeal discs from young (12 months) and old (10-16 years old) animals were dissected and primary NP cells were isolated. Gene expression and proteomics of fresh NP cells were performed. NP cells were labelled with propidium iodide and analysed by flow cytometry for the expression of CD29, CD44, CD45, CD146, GD2, Tie2, CD34 and Stro-1. Morphological cell features were also dissected by imaging flow cytometry. Elder NP cells (up-regulated bIL-6 and bMMP1 gene expression) presented lower percentages of CD29+, CD44+, CD45+ and Tie2+ cells compared with young NP cells (upregulated bIL-8, bCOL2A1 and bACAN gene expression), while GD2, CD146, Stro-1 and CD34 expression were maintained with age. NP cellulome showed an upregulation of proteins related to endoplasmic reticulum (ER) and melanosome independently of age, whereas proteins upregulated in elder NP cells were also associated with glycosylation and disulfide bonds. Flow cytometry analysis of NP cells disclosed the existence of 4 subpopulations with distinct auto-fluorescence and size with different dynamics along aging. Regarding cell morphology, aging increases NP cell area, diameter and vesicles. These results contribute to a better understanding of NP cells aging and highlighting potential anti-aging targets that can help to mitigate age-related disc disease.

Angiopoietin-1 receptor Tie2 distinguishes multipotent differentiation capability in bovine coccygeal nucleus pulposus cells.

BackgroundThe intervertebral disc (IVD) has limited self-healing potential and disc repair strategies require an appropriate cell source such as progenitor cells that could regenerate the damaged cells and tissues. The objective of this study was to identify nucleus pulposus-derived progenitor cells (NPPC) and examine their potential in regenerative medicine in vitro.MethodsNucleus pulposus cells (NPC) were obtained from 1-year-old bovine coccygeal discs by enzymatic digestion and were sorted for the angiopoietin-1 receptor Tie2. The obtained Tie2– and Tie2+ fractions of cells were differentiated into osteogenic, adipogenic, and chondrogenic lineages in vitro. Colony-forming units were prepared from both cell populations and the colonies formed were analyzed and quantified after 8 days of culture. In order to improve the preservation of the Tie2+ phenotype of NPPC in monolayer cultures, we tested a selection of growth factors known to have stimulating effects, cocultured NPPC with IVD tissue, and exposed them to hypoxic conditions (2 % O2).ResultsAfter 3 weeks of differentiation culture, only the NPC that were positive for Tie2 were able to differentiate into osteocytes, adipocytes, and chondrocytes as characterized by calcium deposition (p < 0.0001), fat droplet formation (p < 0.0001), and glycosaminoglycan content (p = 0.0095 vs. Tie2– NPC), respectively. Sorted Tie2– and Tie2+ subpopulations of cells both formed colonies; however, the colonies formed from Tie2+ cells were spheroid in shape, whereas those from Tie2– cells were spread and fibroblastic. In addition, Tie2+ cells formed more colonies in 3D culture (p = 0.011) than Tie2– cells. During expansion, a fast decline in the fraction of Tie2+ cells was observed (p < 0.0001), which was partially reversed by low oxygen concentration (p = 0.0068) and supplementation of the culture with fibroblast growth factor 2 (FGF2) (p < 0.0001).ConclusionsOur results showed that the bovine nucleus pulposus contains NPPC that are Tie2+. These cells fulfilled formally progenitor criteria that were maintained in subsequent monolayer culture for up to 7 days by addition of FGF2 or hypoxic conditions. We propose that the nucleus pulposus represents a niche of precursor cells for regeneration of the IVD.

Effect of Compression and Anisotropy on the Diffusion of Glucose in Annulus Fibrosus

Investigation of the effect of static compression and anisotropy on the apparent diffusivity of glucose in bovine annulus fibrosus (AF). OBJECTIVE. To determine the apparent glucose diffusivity in 2 directions (axial and radial) of bovine AF under 3 levels of compressive strain (0%, 10%, and 20%). Knowledge of diffusivity of small molecules is important for understanding nutritional supply in intervertebral discs and the mechanisms of disc degeneration. However, little is known regarding the strain-dependent and anisotropic behavior of glucose diffusivity in intervertebral discs. Apparent glucose diffusivity measurements were performed on 10 axial and 10 radial AF specimens from bovine coccygeal discs. The dependence of diffusivity on compression was determined using 3 levels of strain (0%, 10%, and 20%). The apparent glucose diffusivity (mean +/- standard deviation) of the bovine AF in the axial direction was 1.38 +/- 0.015 x 10 cm/s (n = 10) at 0%, 1.00 +/- 0.070 x 10 cm/s (n = 10) at 10%, and 7.65 +/- 0.552 x 10 cm/s (n = 10) at 20% compression. For radial specimens, the apparent glucose diffusivity was determined to be 9.17 +/- 1.12 x 10 cm/s (n = 10), 7.29 +/- 0.863 x 10 cm/s (n = 10), and 5.43 +/- 1.16 x 10 cm/s (n = 10) for 0%, 10%, and 20% compressions, respectively. A significant decrease in diffusivity with increasing strain was found for both axial and radial specimens [analysis of variance (ANOVA), P < 0.05]. Diffusion in the radial direction was determined to be significantly less than that in the axial direction (ANOVA, P < 0.05). A significant interaction was found between the level of strain and the direction of diffusion (ANOVA, P < 0.05). Diffusion of glucose in bovine AF is dependent on strain and the direction of diffusion.

Anisotropic Ion Diffusivity in Intervertebral Disc: An Electrical Conductivity Approach

Investigation of the transport behavior of ions in intervertebral disc using an electrical conductivity method. To determine the electrical conductivity and ion diffusivity of nucleus pulposus and anulus fibrosus in 3 major directions (axial, circumferential, and radial). Knowledge of diffusivity of small molecules is important for understanding nutrition supply in intervertebral disc and disc degeneration. However, little is known on the anisotropic behaviors of ion diffusivity and of electrical conductivity in intervertebral disc. Electrical conductivity measurement was performed on 24 axial, circumferential, and radial anulus fibrosus specimens and 24 axial nucleus pulposus specimens from bovine coccygeal discs. The diffusivity of Na and Cl were estimated by the analysis of conductivity data. The electrical conductivity (mean +/- standard deviation; n = 24) of the bovine anulus fibrosus was 4.70 +/- 1.08 mS/cm in the axial, 2.86 +/- 0.83 mS/cm in the radial, and 4.38 +/- 1.25 mS/cm in the circumferential direction. For nucleus pulposus, the electrical conductivity (mean +/- standard deviation; n = 24) was 8.95 +/- 0.89 mS/cm. The mean value for nucleus pulposus was significantly higher than that of anulus fibrosus (t test, P < 0.05). For anulus fibrosus, the conductivity in the radial direction was significantly lower than in axial or circumferential directions. Similar trends were found for both Na and Cl diffusivities. Both electrical conductivity and ion diffusivity were highly sensitive to water content. Electrical conductivity and ion diffusivity of anulus fibrosus are anisotropic.

Cell Deformation and Micromechanical Environment in the Intervertebral Disc

The purpose of this research was to explore the in situ anatomic and mechanical environment of disc cells. Laser scanning confocal microscopy was used to characterize three-dimensional morphology of intervertebral disc cells, micromechanical deformation and interaction with extracellular matrix, and functional intercellular communication. Bovine coccygeal discs were used for both the anatomic and micromechanical investigations. Anulus fibrosus cells had a complex morphology with sinuous processes woven into the extracellular matrix, particularly in the outer aspect of the anulus where they were also interconnected via functional gap junctions. They were also found in an extensive pericellular matrix of type-VI collagen, joining as many as ten cells into linear cell arrays that could be extracted from the matrix as functional units. Mechanically, collagen fibril sliding was demonstrated to govern cell mechanics and strain transfer in the anulus fibrosus during loading activities. Lamellar cells were largely protected from direct tensile strains in the matrix, with minimal intercellular strains. However, intercellular strains between lamellar cells in adjacent arrays were large, illustrating shearing between linear cell arrays. Appreciable shear was observed across the lamellar cell bodies as well as to the cellular processes woven into the matrix. These findings demonstrated the morphologic and micromechanical complexity of anulus fibrosus cells. The knowledge of the in situ environment of disc cells will provide a base to investigate the mechanical implications of disc degeneration on the cellular environment and to better understand how mechanical and genetic risk factors can impact the cells that are essential to maintaining the intervertebral disc.

In Vitro Organ Culture of the Bovine Intervertebral Disc

Whole bovine coccygeal discs were cultured under static load, with or without vertebral endplates (VEPs), and assessed for cell viability, biochemical stability, biosynthetic activity, and biosynthetic responsiveness to changes in mechanical load. To assess the effects of VEPs on biochemical and cellular stability of disc cells during in vitro culture of large disc explants. To determine whether cultured discs could respond to mechanical perturbation. Previous methods for culturing the intervertebral disc have focused on rabbit and rat discs, but the small size of these discs limits the relevance of these culture systems to the human condition. Bovine coccygeal discs have similar dimensions to the human lumbar disc (i.e., similar size and nominal stresses), but long-term culture of these discs has not been reported. Bovine coccygeal discs were harvested with or without VEPs, cultured under static load (5 kg, approximately 0.25 MPa, in situ swelling pressure) for up to 1 week, and evaluated for changes in hydration, glycosaminoglycan content, cell viability, and biosynthetic activity. Additionally, the biochemical and biosynthetic response of discs cultured without VEP to increasing the load to a 20-kg (approximately 1 MPa, the estimated stress in human lumbar disc during heavy lifting) static load for 6 hours was assessed. During the first 24 hours, culturing discs with endplates was moderately better with regards to maintaining in situ anulus hydration and nucleus glycosaminoglycan levels. The endplates, however, obstructed media flow to the disc, resulting in a marked decrease in cell viability after 1 week of culture. Nucleus pulposus cell viability was maintained in discs cultured without endplates, but there was a significant drop in biosynthetic activity within 2 days of culture. Despite this drop, the disc cells in the discs without VEP remained biosynthetically responsive to changes in mechanical loading. It is possible to maintain cell viability and the biosynthetic responsiveness of large discs for up to 1 week in vitro when the discs are cultured under static load and without VEP.

Value and Limitations of Using the Bovine Tail as a Model for the Human Lumbar Spine

The contents of DNA, proteoglycan, type II collagen, and denatured type II collagen in the bovine coccygeal intervertebral discs were examined in situ in relation to disc level, age, and tissue region. To determine whether bovine coccygeal discs are a suitable model to study human lumbar discs. Bovine coccygeal discs have been suggested as a suitable alternative model because they are readily available, in contrast to human discs, and represent a common source of tissue in the disc field. However, it is not known whether the changes in matrix contents in bovine coccygeal discs are similar to those found in the human lumbar spine. Intervertebral discs from bovine tails were dissected into the nucleus pulposus (NP) and anulus fibrosus (AF). Tissues were weighed and analyzed for matrix contents using specific assays. Similar to water content, the proteoglycan content was higher in the NP than in the AF. Water content of the bovine NP did not change with age, unlike the proteoglycan content, which decreased. type II collagen content was higher in the NP than in the AF, and both did not change overall significantly with age. The percent of denatured type II collagen decreased with age only in the NP. The DNA content did not vary with age in the AF and in the NP. Differences in matrix contents exist between the bovine coccygeal discs and the human lumbar spine. Thus, caution must be exercised when using the bovine tail as a model for the human lumbar spine in biochemical studies.

Effect of static load on matrix synthesis rates in the intervertebral disc measured in vitro by a new perfusion technique.

The effect of static loading on matrix synthesis was measured in intact bovine coccygeal discs. The discs were maintained at 37 degrees C in a humid atmosphere and were perfused across their upper and lower surfaces for as long as 8 hours with media containing radioisotopes (3H-proline and 35S-sulphate) via porous filter disks embedded in Perspex (Plexiglas) holders. Static loads were applied to the disc with use of weights. The activity of free, nonincorporated isotope in the centre of the disc was monitored continuously with a microdialysis probe. Incorporated tracer in different regions of the disc was determined at the end of each experiment from the activity of the nondialysable tracer. Free tracer activity rose steeply over the first 3-4 hours of perfusion, as tracer diffused into the disc, and reached a steady value after 5-6 hours, as expected from diffusion theory. The rate of tracer incorporation estimated from the integrated value of free tracer concentration was constant for as long as 8 hours. Incorporation rates varied across the disc: the highest rates were in the inner layer of the annulus fibrosus, and the lowest rates were in the outer layer of the annulus. The rates for both 35S-sulphate and 3H-proline varied with load. The rates were lowest in the nucleus pulposus and the inner layer of the annulus fibrosus with a 0.5 kg load, and they almost doubled as the load was increased to 5-10 kg. Heavier loads (15 kg) led to a decrease in incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of lactate and pH on proteoglycan and protein synthesis rates in the intervertebral disc.

The intervertebral disc is the largest avascular tissue in the body. Its metabolism is mainly anaerobic, and thus lactate is produced at a significant rate. As a result the lactate concentrations in the center of the disc may be 8-10 times as high as in the plasma. The pH in the disc center is thus acidic. Because low values of pH are known to affect proteoglycan synthesis in other cartilages, the authors measured the effect of lactate levels and pH on 35S-sulphate and 3H-proline incorporation rates in the nucleus of bovine coccygeal discs and in human disc obtained during percutaneous nucleotomy. The maximum incorporation rate occurred at pH 7.2-pH 6.9. Here the rate was 40-50% greater than at pH 7.4. Below pH 6.8 the rate fell steeply, more so for sulphate than for proline. At pH 6.3 the sulphate incorporation rate was around 20 percent that at pH 7.4. The results indicate that proteoglycan synthesis rates in particular are sensitive to extracellular pH, and that the peak rate occurs around the level of pH seen in vivo. Factors that cause lactate levels to rise, such as a fall in O2 levels as the result of smoking or vibration (Holm and Nachemson, 1988) could lead to a fall in proteoglycan synthesis rates, ultimately leading to a fall in proteoglycan content and to disc degeneration.