Alicia R. Jackson

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 × 10−6 cm2/s (n = 10) at 0%, 1.00 ± 0.070 × 10−6 cm2/s (n = 10) at 10%, and 7.65 ± 0.552 × 10−7 cm2/s (n = 10) at 20% compression. For radial specimens, the apparent glucose diffusivity was determined to be 9.17 ± 1.12 × 10−7 cm2/s (n = 10), 7.29 ± 0.863 × 10−7 cm2/s (n = 10), and 5.43 ± 1.16 × 10−7 cm2/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.

Effect of endplate calcification and mechanical deformation on the distribution of glucose in intervertebral disc: a 3D finite element study

The intervertebral disc (IVD) is avascular, receiving nutrition from surrounding vasculature. Theoretical modelling can supplement experimental results to understand nutrition to IVD more clearly. A new, 3D finite element model of the IVD was developed to investigate effects of endplate calcification and mechanical deformation on glucose distributions in IVD. The model included anatomical disc geometry, non-linear coupling of cellular metabolism with pH and oxygen concentration and strain-dependent properties of the extracellular matrix. Calcification was simulated by reducing endplate permeability (∼79%). Mechanical loading was applied based on in vivo disc deformation during the transition from supine to standing positions. Three static strain conditions were considered: supine, standing and weight-bearing standing. Minimum glucose concentrations decreased 45% with endplate calcification, whereas disc deformation led to a 4.8–63% decrease, depending on the endplate condition (i.e. normal vs. calcified). Furthermore, calcification more strongly affected glucose concentrations in the nucleus compared to the annulus fibrous region. This study provides important insight into nutrient distributions in IVD under mechanical deformation.

TRANSPORT PROPERTIES OF CARTILAGINOUS TISSUES

Cartilaginous tissues, such as articular cartilage and intervertebral disc, are avascular tissues which rely on transport for cellular nutrition. Comprehensive knowledge of transport properties in such tissues is therefore necessary in the understanding of nutritional supply to cells. Furthermore, poor cellular nutrition in cartilaginous tissues is believed to be a primary source of tissue degeneration, which may result in osteoarthritis (OA) or disc degeneration. In this mini-review, we present an overview of the current status of the study of transport properties and behavior in cartilaginous tissues. The mechanisms of transport in these tissues, as well as experimental approaches to measuring transport properties and results obtained are discussed. The current status of bioreactors used in cartilage tissue engineering is also presented.

Cell viability in intervertebral disc under various nutritional and dynamic loading conditions: 3d finite element analysis.

In this study, a new cell density model was developed and incorporated into the formulation of the mechano-electrochemical mixture theory to investigate the effects of deprivation of nutrition supply at boundary source, degeneration, and dynamic loading on the cell viability of intervertebral disc (IVD) using finite element methods. The deprivation of nutrition supply at boundary source was simulated by reduction in nutrition level at CEP and AF boundaries. Cases with 100%, 75%, 60%, 50% and 30% of normal nutrition level at both CEP and AF boundaries were modeled. Unconfined axial sinusoidal dynamic compressions with different combinations of amplitude (u=10%± 2.5%, ± 5%) and frequency (f=1, 10, 20 cycle/day) were applied. Degenerated IVD was modeled with altered material properties. Cell density decreased substantially with reduction of nutrition level at boundaries. Cell death was initiated primarily near the NP-AF interface on the mid-plane. Dynamic loading did not result in a change in the cell density in non-degenerated IVD, since glucose levels did not fall below the minimum value for cell survival; in degenerated IVDs, we found that increasing frequency and amplitude both resulted in higher cell density, because dynamic compression facilitates the diffusion of nutrients and thus increases the nutrition level around IVD cells. The novel computational model can be used to quantitatively predict both when and where cells start to die within the IVD under various kinds of nutritional and mechanical conditions.

3D Finite Element Analysis of Nutrient Distributions and Cell Viability in the Intervertebral Disc: Effects of Deformation and Degeneration

The intervertebral disc (IVD) receives important nutrients, such as glucose, from surrounding blood vessels. Poor nutritional supply is believed to play a key role in disc degeneration. Several investigators have presented finite element models of the IVD to investigate disc nutrition; however, none has predicted nutrient levels and cell viability in the disc with a realistic 3D geometry and tissue properties coupled to mechanical deformation. Understanding how degeneration and loading affect nutrition and cell viability is necessary for elucidating the mechanisms of disc degeneration and low back pain. The objective of this study was to analyze the effects of disc degeneration and static deformation on glucose distributions and cell viability in the IVD using finite element analysis. A realistic 3D finite element model of the IVD was developed based on mechano-electrochemical mixture theory. In the model, the cellular metabolic activities and viability were related to nutrient concentrations, and transport properties of nutrients were dependent on tissue deformation. The effects of disc degeneration and mechanical compression on glucose concentrations and cell density distributions in the IVD were investigated. To examine effects of disc degeneration, tissue properties were altered to reflect those of degenerated tissue, including reduced water content, fixed charge density, height, and endplate permeability. Two mechanical loading conditions were also investigated: a reference (undeformed) case and a 10% static deformation case. In general, nutrient levels decreased moving away from the nutritional supply at the disc periphery. Minimum glucose levels were at the interface between the nucleus and annulus regions of the disc. Deformation caused a 6.2% decrease in the minimum glucose concentration in the normal IVD, while degeneration resulted in an 80% decrease. Although cell density was not affected in the undeformed normal disc, there was a decrease in cell viability in the degenerated case, in which averaged cell density fell 11% compared with the normal case. This effect was further exacerbated by deformation of the degenerated IVD. Both deformation and disc degeneration altered the glucose distribution in the IVD. For the degenerated case, glucose levels fell below levels necessary for maintaining cell viability, and cell density decreased. This study provides important insight into nutrition-related mechanisms of disc degeneration. Moreover, our model may serve as a powerful tool in the development of new treatments for low back pain.

Relationship between solute transport properties and tissue morphology in human annulus fibrosus.

Poor nutritional supply to the intervertebral disc is believed to be an important factor leading to disc degeneration. However, little is known regarding nutritional transport in human annulus fibrosus (AF) and its relation to tissue morphology. We hypothesized that solute diffusivity in human AF is anisotropic and inhomogeneous, and that transport behaviors are associated with tissue composition and structure. To test these hypotheses, we measured the direction‐dependent diffusivity of a fluorescent molecule (fluorescein, 332 Da) in three regions of AF using a fluorescence recovery after photobleaching (FRAP) technique, and associated transport results to the regional variation in water content and collagen architecture in the tissue. Diffusivity in AF was anisotropic, with higher values in the axial direction than in the radial direction for all regions investigated. The values of the diffusion coefficient ranged from 0.38 ± 0.25 × 10−6 cm2/s (radial diffusivity in outer AF) to 2.68 ± 0.84 × 10−6 cm2/s (axial diffusivity in inner AF). In both directions, diffusivity decreased moving from inner to outer AF. Tissue structure was investigated using both scanning electron microscopy and environmental scanning electron microscopy. A unique arrangement of microtubes was found in human AF. Furthermore, we also found that the density of these microtubes varied moving from inner to outer AF. A similar trend of regional variation was found for water content, with the highest value also measured in inner AF. Therefore, we concluded that a relationship exists among the anisotropic and inhomogeneous diffusion in human AF and the structure and composition of the tissue.

Anisotropic ion diffusivity in intervertebral disc: an electrical conductivity approach.

Study Design. Investigation of the transport behavior of ions in intervertebral disc using an electrical conductivity method. Objectives. To determine the electrical conductivity and ion diffusivity of nucleus pulposus and anulus fibrosus in 3 major directions (axial, circumferential, and radial). Summary of Background Data. 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. Methods. 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. Results. 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. Conclusions. Electrical conductivity and ion diffusivity of anulus fibrosus are anisotropic.

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.

STRAIN-DEPENDENT OXYGEN DIFFUSIVITY IN BOVINE ANNULUS FIBROSUS

The intervertebral disk (IVD) is the largest avascular structure in the human body. Transport of small molecules in IVD is mainly through diffusion from the endplates and the peripheral blood vessels surrounding IVD. Studies have investigated the structure, chemical components, and water content in IVD, but to our knowledge no study has investigated the effect of mechanical loading on oxygen transport in IVD. The objective of this study was to determine the strain-dependent behavior of oxygen diffusivity in IVD tissue. A one-dimensional steady-state diffusion experiment was designed and performed to determine the oxygen diffusivity in bovine annulus fibrosus (AF). The oxygen diffusivity was calculated using equation derived from Ficks law. A total of 20 AF specimens (d=6 mm, h approximately 0.5 mm) from bovine coccygeal IVD were used to determine oxygen diffusivity at three levels of compressive strain. The average oxygen diffusivity (mean+/-SD) of bovine AF in the axial direction was 1.43+/-0.242 x 10(-5) cm(2)/s (n=20) at 4.68+/-1.67% compressive strain level, 1.05+/-0.282 x 10(-5) cm(2)/s (n=20) at 14.2+/-1.50% strain level, and 7.71+/-1.63 x 10(-6) cm(2)/s (n=20) at 23.7+/-1.34% strain level. There was a significant decrease in oxygen diffusivity with increasing level of compressive strain (ANOVA, p<0.05). Oxygen diffusivity of bovine AF in the axial direction has been determined. The mechanical loading has a significant effect on oxygen transport in IVD tissues. This study is important in understanding nutritional transport in IVD tissues and related disk degeneration.

Effect of Mechanical Loading on Electrical Conductivity in Human Intervertebral Disk

The intervertebral disk (IVD), characterized as a charged, hydrated soft tissue, is the largest avascular structure in the body. Mechanical loading to the disk results in electromechanical transduction phenomenon as well as altered transport properties. Electrical conductivity is a material property of tissue depending on ion concentrations and diffusivities, which are in turn functions of tissue composition and structure. The aim of this study was to investigate the effect of mechanical loading on electrical behavior in human IVD tissues. We hypothesized that electrical conductivity in human IVD is strain-dependent, due to change in tissue composition caused by compression, and inhomogeneous, due to tissue structure and composition. We also hypothesized that conductivity in human annulus fibrosus (AF) is anisotropic, due to the layered structure of the tissue. Three lumbar IVDs were harvested from three human spines. From each disk, four AF specimens were prepared in each of the three principal directions (axial, circumferential, and radial), and four axial nucleus pulposus (NP) specimens were prepared. Conductivity was determined using a four-wire sense-current method and a custom-designed apparatus by measuring the resistance across the sample. Resistance measurements were taken at three levels of compression (0%, 10%, and 20%). Scanning electron microscopy (SEM) images of the human AF tissue were obtained in order to correlate tissue structure with conductivity results. Increasing compressive strain significantly decreased conductivity for all groups (p<0.05, analysis of variance (ANOVA)). Additionally, specimen orientation significantly affected electrical conductivity in the AF tissue, with conductivity in the radial direction being significantly lower than that in the axial or circumferential directions at all levels of compressive strain (p<0.05, ANOVA). Finally, conductivity in the NP tissue was significantly higher than that in the AF tissue (p<0.05, ANOVA). SEM images of the AF tissues showed evidence of microtubes orientated in the axial and circumferential directions, but not in the radial direction. This may suggest a relationship between tissue morphology and the anisotropic behavior of conductivity in the AF. The results of this investigation demonstrate that electrical conductivity in human IVD is strain-dependent and inhomogeneous, and that conductivity in the human AF tissue is anisotropic (i.e., direction-dependent). This anisotropic behavior is correlated with tissue structure shown in SEM images. This study provides important information regarding the effects of mechanical loading on solute transport and electrical behavior in IVD tissues.