Nutrient transport in human annulus fibrosus is affected by compressive strain and anisotropy.

The avascular intervertebral disc (IVD) receives nutrition via transport from surrounding vasculature; poor nutrition is believed to be a main cause of disc degeneration. In this study, we investigated the effects of mechanical deformation and anisotropy on the transport of two important nutrients--oxygen and glucose--in human annulus fibrosus (AF). The diffusivities of oxygen and glucose were measured under three levels of uniaxial confined compression--0, 10, and 20%--and in three directions--axial, circumferential, and radial. The glucose partition coefficient was also measured at three compression levels. Results for glucose and oxygen diffusivity in AF ranged from 4.46 × 10(-7) to 9.77 × 10(-6) cm(2)/s and were comparable to previous studies; the glucose partition coefficient ranged from 0.71 to 0.82 and was also similar to previous results. Transport properties were found to decrease with increasing deformation, likely caused by fluid exudation during tissue compression and reduction in pore size. Furthermore, diffusivity in the radial direction was lower than in the axial or circumferential directions, indicating that nutrient transport in human AF is anisotropic. This behavior is likely a consequence of the layered structure and unique collagen architecture of AF tissue. These findings are important for better understanding nutritional supply in IVD and related disc degeneration.

Novel technique for online characterization of cartilaginous tissue properties.

The goal of tissue engineering is to use substitutes to repair and restore organ function. Bioreactors are an indispensable tool for monitoring and controlling the unique environment for engineered constructs to grow. However, in order to determine the biochemical properties of engineered constructs, samples need to be destroyed. In this study, we developed a novel technique to nondestructively online-characterize the water content and fixed charge density of cartilaginous tissues. A new technique was developed to determine the tissue mechano-electrochemical properties nondestructively. Bovine knee articular cartilage and lumbar annulus fibrosus were used in this study to demonstrate that this technique could be used on different types of tissue. The results show that our newly developed method is capable of precisely predicting the water volume fraction (less than 3% disparity) and fixed charge density (less than 16.7% disparity) within cartilaginous tissues. This novel technique will help to design a new generation of bioreactors which are able to actively determine the essential properties of the engineered constructs, as well as regulate the local environment to achieve the optimal conditions for cultivating constructs.

Difference in Energy Metabolism of Annulus Fibrosus and Nucleus Pulposus Cells of the Intervertebral Disc.

Low back pain is associated with intervertebral disc degeneration. One of the main signs of degeneration is the inability to maintain extracellular matrix integrity. Extracellular matrix synthesis is closely related to production of adenosine triphosphate (i.e. energy) of the cells. The intervertebral disc is composed of two major anatomical regions: annulus fibrosus and nucleus pulposus, which are structurally and compositionally different, indicating that their cellular metabolisms may also be distinct. The objective of this study was to investigate energy metabolism of annulus fibrosus and nucleus pulposus cells with and without dynamic compression, and examine differences between the two cell types. Porcine annulus and nucleus tissues were harvested and enzymatically digested. Cells were isolated and embedded into agarose constructs. Dynamically loaded samples were subjected to a sinusoidal displacement at 2 Hz and 15% strain for 4 h. Energy metabolism of cells was analyzed by measuring adenosine triphosphate content and release, glucose consumption, and lactate/nitric oxide production. A comparison of those measurements between annulus and nucleus cells was conducted. Annulus and nucleus cells exhibited different metabolic pathways. Nucleus cells had higher adenosine triphosphate content with and without dynamic loading, while annulus cells had higher lactate production and glucose consumption. Compression increased adenosine triphosphate release from both cell types and increased energy production of annulus cells. Dynamic loading affected energy metabolism of intervertebral disc cells, with the effect being greater in annulus cells.

Mechanical loading affects the energy metabolism of intervertebral disc cells.

Research has shown that mechanical loading affects matrix biosynthesis of intervertebral disc (IVD) cells; however, the pathway(s) to this effect is currently unknown. Cellular matrix biosynthesis is an energy demanding process. The objective of this study was to investigate the effects of static and dynamic compressive loading on energy metabolism of IVD cells. Porcine annulus fibrosus (AF) and nucleus pulposus (NP) cells seeded in 2% agarose were used in this experiment. Experimental groups included 15% static compression and 0.1 and 1 Hz dynamic compression at 15% strain magnitude for 4 h. ATP, lactate, glucose, and nitric oxide (NO) contents in culture media, and ATP content in cell-agarose construct were measured using biochemical assays. While the total ATP content of AF cells was promoted by static and dynamic loading, only 1 Hz dynamic loading increased total ATP content of NP cells. Increases in lactate production and glucose consumption of AF cells suggest that ATP production via glycolysis is promoted by dynamic compression. ATP release and NO production of AF and NP cells were significantly increased by dynamic loading. Thus, this study clearly illustrates that static and dynamic compressive loading affect IVD cell energy production while cellular responses to mechanical loading were both cell type and compression type dependent.

A conductivity approach to measuring fixed charge density in intervertebral disc tissue.

A new method for measuring the fixed charge density (FCD) in intervertebral disc (IVD) tissues employing a two-point electrical conductivity approach was developed. In this technique, the tissue is first confined and equilibrated in a potassium chloride (KCl) solution, and the tissue conductivity is then measured. This is then repeated with a second concentration of KCl solution. The FCD can be determined from the conductivity measurements. Using this method, the FCD values of bovine annulus fibrosus (AF) and nucleus pulposus (NP) tissues were determined to be 0.060 +/- 0.027 mEq/g wet tissue and 0.19 +/- 0.039 mEq/g wet tissue, respectively. The FCD of AF was significantly lower than that of NP tissue, similar to results in the literature for human IVD tissues. In order to verify the accuracy of the new method, the glycosaminoglycan (GAG) contents of the tissues were measured and used to estimate the tissue FCD. A strong correlation (R (2) = 0.84-0.87) was found to exist between FCD values measured and those estimated from GAG contents, indicating that the conductivity approach is a reliable technique for measuring the FCD of IVD tissues.

Intracellular Flow Cytometric Measurement of Extracellular Matrix Components in Porcine Intervertebral Disc Cells.

The objective of this study was to develop and demonstrate the utility of a novel method of evaluating intracellular levels of extracellular matrix (ECM) components in intervertebral disc (IVD) cells using flow cytometry. By using this method, this study discriminated between cell populations in porcine IVD and examined the response of IVD cells to monolayer cultures, a traditional method of cell expansion, by measuring phenotypic attributes of ECM component production. It was found that monolayer cultures affected collagen production of IVD cells while there were differences in collagen type II production between the cells isolated from the annulus fibrosus (AF) and nucleus pulposus (NP) regions of IVD. Size distributions of fresh and cultured cells were also presented while the relationships between cell size and intracellular collagen level revealed heterogeneous cell populations in AF and NP regions. Furthermore, this study showed that the intracellular collagen signals of IVD cells were significantly enhanced by the treatments of Brefeldin-A and ascorbic acid. This suggests that Brefeldin-A and ascorbic acid could be used to increase the sensitivity of flow cytometric analysis on intracellular collagen levels by maximizing collagen accumulation inside cells. Since a unique feature of the flow cytometric screening tool is the ability to discriminate between various cell populations in a single sample, the flow cytometric method developed in this study may have the potential to identify specific collagen-producing cell populations from tissues or cell cultures.

Effect of compression and anisotropy on the diffusion of glucose in annulus fibrosus.

STUDY DESIGN: 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%). SUMMARY OF BACKGROUND DATA: 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. METHODS: 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%). RESULTS: 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). CONCLUSION: Diffusion of glucose in bovine AF is dependent on strain and the direction of diffusion.

Effects of low glucose concentrations on oxygen consumption rates of intervertebral disc cells.

STUDY DESIGN: Investigation of the effects of low glucose concentrations on the oxygen consumption rates of intervertebral disc cells. OBJECTIVES: To determine the oxygen consumption rate of porcine anulus fibrosus (AF) cells at different glucose concentrations and to examine the differences in the oxygen consumption rate between AF and nucleus pulposus (NP) cells at different glucose levels. SUMMARY OF BACKGROUND DATA: Poor nutrient supply has been suggested as a potential mechanism for degeneration of the intervertebral disc (IVD). Distribution of nutrients in the IVD is strongly dependent on transport properties of the tissue and cellular metabolic rates. Previous studies have shown dependence of oxygen consumption rate of IVD cells on oxygen tension, pH levels, and glucose levels outside the physiologic range. However, the oxygen consumption rate of AF cells at in vivo glucose levels has not been investigated. METHODS: IVD cells were isolated from the outer AF and NP of 4- to 5-month-old porcine lumbar discs. The changes in oxygen tension were recorded when cells were cultured in sealed metabolism chamber. The oxygen consumption rate of cells was determined by theoretical curve fitting using the Michaelis-Menten equation. RESULTS: The outer AF cells cultured in high glucose medium (25 mmol/L) exhibited the lowest oxygen consumption rate, whereas no significant differences in oxygen consumption rates were found among outer AF cells cultured at physiologic glucose levels (i.e., 1 mmol/L, 2.5 mmol/L, 5 mmol/L). The oxygen consumption rate of NP cells was significantly greater than that of outer AF cells. CONCLUSION: Since the oxygen consumption rates determined in this study are comparable to the findings in the literature, this study has developed a new alternative method for determining oxygen consumption rate. The oxygen consumption rates of IVD cells reported in this study will be valuable for theoretically predicting local oxygen concentrations in IVD, which can provide a better understanding of transport of oxygen in the discs.