Fluid flow and convective transport of solutes within the intervertebral disc

Previous experimental and analytical studies of solute transport in the intervertebral disc have demonstrated that for small molecules diffusive transport alone fulfils the nutritional needs of disc cells. It has been often suggested that fluid flow into and within the disc may enhance the transport of larger molecules. The goal of the study was to predict the influence of load-induced interstitial fluid flow on mass transport in the intervertebral disc.An iterative procedure was used to predict the convective transport of physiologically relevant molecules within the disc. An axisymmetric, poroelastic finite-element structural model of the disc was developed. The diurnal loading was divided into discrete time steps. At each time step, the fluid flow within the disc due to compression or swelling was calculated. A sequentially coupled diffusion/convection model was then employed to calculate solute transport, with a constant concentration of solute being provided at the vascularised endplates and outer annulus. Loading was simulated for a complete diurnal cycle, and the relative convective and diffusive transport was compared for solutes with molecular weights ranging from 400 Da to 40 kDa.Consistent with previous studies, fluid flow did not enhance the transport of low-weight solutes. During swelling, interstitial fluid flow increased the unidirectional penetration of large solutes by approximately 100%. Due to the bi-directional temporal nature of disc loading, however, the net effect of convective transport over a full diurnal cycle was more limited (30% increase). Further study is required to determine the significance of large solutes and the timing of their delivery for disc physiology.     

Dynamic compression augments interstitial transport of a glucose-like solute in articular cartilage

Solute transport through the extracellular matrix is essential for cellular activities in articular cartilage. Increased solute transport via fluid convection may be a mechanism by which dynamic compression stimulates chondrocyte metabolism. However, loading conditions that optimally augment transport likely vary for different solutes. To investigate effects of dynamic loading on transport of a bioactive solute, triangular mechanical loading waveforms were applied to cartilage explants disks while interstitial transport of a fluorescent glucose analog was monitored. Peak-to-peak compression amplitudes varied from 5-50% and frequencies varied from 0.0006-0.1 Hz to alter the spatial distribution and magnitude of oscillatory fluid flow. Solute transport was quantified by monitoring accumulation of fluorescence in a saline bath circulated around the explant. Individual explants were subjected to a series of compression protocols, so that effects of loading on solute desorption could be observed directly. Maximum increases in solute transport were obtained with 10-20% compression amplitudes at 0.1 Hz; similar loading protocols were previously found to stimulate chondrocyte metabolism in vitro. Results therefore support hypotheses relating to increased solute transport as a mediator of the cartilage biological response to dynamic compression, and may have application in mechanical conditioning of cartilage constructs for tissue engineering.     

Effects of dynamic loading on solute transport through the human cartilage endplate

Nutrient and metabolite transport through the cartilage endplate (CEP) is important for maintaining proper disc nutrition, but the mechanisms of solute transport remain unclear. One unresolved issue is the role of dynamic loading. In comparison to static loading, dynamic loading is thought to enhance transport by increasing convection. However, the CEP has a high resistance to fluid flow, which could limit solute convection. Here we measure solute transport through site-matched cadaveric human lumbar CEP tissues under static vs. dynamic loading, and we determine how the degree of transport enhancement from dynamic loading depends on CEP porosity and solute size. We found that dynamic loading significantly increased small and large solute transport through the CEP: on average, dynamic loading increased the transport of sodium fluorescein (376 Da) by a factor of 1.85 ± 0.64 and the transport of a large dextran (4000 Da) by a factor of 4.97 ± 3.05. Importantly, CEP porosity (0.65 ± 0.07; range: 0.47–0.76) strongly influenced the degree of transport enhancement. Specifically, for both solutes, transport enhancement was greater for CEPs with low porosity than for CEPs with high porosity. This is because the CEPs with low porosity were susceptible to larger improvements in fluid flow under dynamic loading. The CEP becomes less porous and less hydrated with aging and as disc degeneration progresses. Together, these findings suggest that as those changes occur, dynamic loading has a greater effect on solute transport through the CEP compared to static loading, and thus may play a larger role in disc nutrition.     

Validation of theoretical framework explaining active solute uptake in dynamically loaded porous media

Solute transport in biological tissues is a fundamental process necessary for cell metabolism. In connective soft tissues, such as articular cartilage, cells are embedded within a dense extracellular matrix that hinders the transport of solutes. However, according to a recent theoretical study (Mauck et al., 2003, J. Biomech. Eng. 125, 602–614), the convective motion of a dynamically loaded porous solid matrix can also impart momentum to solutes, pumping them into the tissue and giving rise to concentrations which exceed those achived under passive diffusion alone. In this study, the theoretical predictions of this model are verified against experimental measurements. The mechanical and transport properties of an agarose–dextran model system were characterized from independent measurements and substituted into the theory to predict solute uptake or desorption under dynamic mechanical loading for various agarose concentrations and dextran molecular weights, as well as different boundary and initial conditions. In every tested case, agreement was observed between experiments and theoretical predictions as assessed by coefficients of determination ranging from R²=0.61 to 0.95. These results provide strong support for the hypothesis that dynamic loading of a deformable porous tissue can produce active transport of solutes via a pumping mechanisms mediated by momentum exchange between the solute and solid matrix.     

Preservation and analysis of nonequilibrium solute concentration distributions within mechanically compressed cartilage explants

Solute transport within articular cartilage is of central importance to tissue physiology, and may mediate effects of mechanical compression on cell metabolism. We therefore developed and applied a freeze-substitution method for fixation of cartilage explant disks which had been compressed axially during radial solute desorption. Dextrans were used as model solutes. Explant morphology was well preserved and nonequilibrium solute concentration distributions were stable for several hours at room temperature. For desorption from explants compressed statically to 0-46% strain, analysis of laser confocal images and comparison to a theoretical model permitted measurement of effective diffusivities. Results were consistent with previous studies suggesting a role for transport limitations in mediating the decreases of chondrocyte metabolic rates associated with static compression. In explants compressed dynamically (23+/-5% strain at 0.001 Hz), evidence was obtained for the augmentation of effective transport rate of 3 kDa dextrans by oscillatory interstitial fluid flows. This suggests that augmented solute transport may play a role in mediating the increases of chondrocyte metabolic rates associated with dynamic compression. Methods appear suitable for quantitative studies of transport within mechanically compressed cartilage-like tissues, and may be valuable for identification of loading environments which optimize solute transport in tissue engineering applications.     

Effects of tension-compression nonlinearity on solute transport in charged hydrated fibrous tissues under dynamic unconfined compression

Cartilage is a charged hydrated fibrous tissue exhibiting a high degree of tension-compression nonlinearity (i.e., tissue anisotropy). The effect of tension-compression nonlinearity on solute transport has not been investigated in cartilaginous tissue under dynamic loading conditions. In this study, a new model was developed based on the mechano-electrochemical mixture model [Yao and Gu, 2007, J. Biomech. Model Mechanobiol., 6, pp. 63-72, Lai et al., 1991, J. Biomech. Eng., 113, pp. 245-258], and conewise linear elasticity model [Soltz and Ateshian, 2000, J. Biomech. Eng., 122, pp. 576-586; Curnier et al., 1995, J. Elasticity, 37, pp. 1-38]. The solute desorption in cartilage under unconfined dynamic compression was investigated numerically using this new model. Analyses and results demonstrated that a high degree of tissue tension-compression nonlinearity could enhance the transport of large solutes considerably in the cartilage sample under dynamic unconfined compression, whereas it had little effect on the transport of small solutes (at 5% dynamic strain level). The loading-induced convection is an important mechanism for enhancing the transport of large solutes in the cartilage sample with tension-compression nonlinearity. The dynamic compression also promoted diffusion of large solutes in both tissues with and without tension-compression nonlinearity. These findings provide a new insight into the mechanisms of solute transport in hydrated, fibrous soft tissues.     

Solute transport in cartilage undergoing cyclic deformation

There are no blood vessels in cartilage to transport nutrients and growth factors to chondrocytes dispersed throughout the cartilage matrix. Insulin-like growth factor-I (IGF-I) is a large molecule with an important role in cartilage growth and metabolism, however, it first must reach the chondrocytes to exert its effect. While diffusion of IGF-I through cartilage is possible, it has been speculated that cyclic loading can enhance the rate of solute transport within cartilage. To better understand this process, here a one-dimensional axisymmetric mathematical model is developed to examine the transport of solutes through a cylindrical plug of cartilage undergoing cyclic axial deformation in the range of 10(-3) -1 Hz. This study has revealed the role of timescales in interpreting transport results in cartilage. It is shown that dynamic strains can either enhance or inhibit IGF-I transport at small timescales (< 20 min after onset of loading), depending on loading frequency. However, on longer timescales it is found that dynamic loading has negligible effect on IGF-I transport. Most importantly, in all cases examined the steady state IGF-I concentration did not exceed the fixed boundary value, in contrast to the predictions of Mauk et al. (2003).     

Solute convection in dynamically compressed cartilage

Chondrocytes depend upon solute transport within the avascular extracellular matrix of articular cartilage for many of their biological activities. Alterations to solute transport parameters may therefore mediate the cell response to tissue compression. While interstitial solute transport may be supplemented by convection during dynamic tissue compression, matrix compression is also associated with decreased diffusivities. Such trade-offs between increased convection and decreased diffusivities of solutes in dynamically compressed cartilage remain largely unexplored. We measured diffusion and convection coefficients of a wide range of solutes in mature bovine cartilage explant disks subjected to radially unconfined axial ramp compression and release. Solutes included approximately 500 Da fluorophores bearing positive and negative charges, and 10 kDa dextrans bearing positive, neutral, and negative charges. Significantly positive values of convection coefficients were measured for several different solutes. Findings therefore support a role for solute convection in mediating the cartilage biological response to dynamic compression.     

A review of experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms

Experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms are reviewed. Effective diffusive permeabilities, the parameter appropriate to the analysis of reaction-diffusion interactions, depend on solute type and biofilm density. Three categories of solute physical chemistry with distinct diffusive properties were distinguished by the present analysis. In order of descending mean relative effective diffusive permeability (De/Daq) these were inorganic anions or cations (0.56), nonpolar solutes with molecular weights of 44 or less (0.43), and organic solutes of molecular weight greater than 44 (0.29). Effective diffusive permeabilities decrease sharply with increasing biomass volume fraction suggesting a serial resistance model of diffusion in biofilms as proposed by Hinson and Kocher (1996). A conceptual model of biofilm structure is proposed in which each cell is surrounded by a restricted permeability envelope. Effective diffusion coefficients, which are appropriate to the analysis of transient penetration of nonreactive solutes, are generally similar to effective diffusive permeabilities in biofilms of similar composition. In three studies that examine diffusion of very large molecular weight solutes (>5000) in biofilms, the average ratio of the relative effective diffusion coefficient of the large solute to the relative effective diffusion coefficient of either sucrose or fluorescein was 0.64, 0.61, and 0.36. It is proposed that large solutes are effectively excluded from microbial cells, that small solutes partition into and diffuse within cells, and that ionic solutes are excluded from cells but exhibit increased diffusive permeability (but decreased effective diffusion coefficients) due to sorption to the biofilm matrix.     

Dynamic loading of immature epiphyseal cartilage pumps nutrients out of vascular canals

The potential influence of mechanical loading on transvascular transport in vascularized soft tissues has not been explored extensively. This experimental investigation introduced and explored the hypothesis that dynamic mechanical loading can pump solutes out of blood vessels and into the surrounding tissue, leading to faster uptake and higher solute concentrations than could otherwise be achieved under unloaded conditions. Immature epiphyseal cartilage was used as a model tissue system, with fluorescein (332 Da), dextran (3, 10, and 70 kDa) and transferrin (80 kDa) as model solutes. Cartilage disks were either dynamically loaded (± 10% compression over a 10% static offset strain, at 0.2 Hz) or maintained unloaded in solution for up to 20 h. Results demonstrated statistically significant solute uptake in dynamically loaded (DL) explants relative to passive diffusion (PD) controls for all solutes except unbound fluorescein, as evidenced by the DL:PD concentration ratios after 20 h (1.0 ± 0.2, 2.4 ± 1.1, 6.1 ± 3.3, 9.0 ± 4.0, and 5.5 ± 1.6 for fluorescein, 3, 10, and 70 kDa dextran, and transferrin). Significant uptake enhancements were also observed within the first 30s of loading. Termination of dynamic loading produced dissipation of enhanced solute uptake back to PD control values. Confocal images confirmed that solute uptake occurred from cartilage canals into their surrounding extracellular matrix. The incidence of this loading-induced transvascular solute pumping mechanism may significantly alter our understanding of the interaction of mechanical loading and tissue metabolism.     

Dynamic loading of deformable porous media can induce active solute transport

Active solute transport mediated by molecular motors across porous membranes is a well-recognized mechanism for transport across the cell membrane. In contrast, active transport mediated by mechanical loading of porous media is a non-intuitive mechanism that has only been predicted recently from theory, but not yet observed experimentally. This study uses agarose hydrogel and dextran molecules as a model experimental system to explore this mechanism. Results show that dynamic loading can enhance the uptake of dextran by a factor greater than 15 over passive diffusion, for certain combinations of gel concentration and dextran molecular weight. Upon cessation of loading, the concentration reverts back to that achieved under passive diffusion. Thus, active solute transport in porous media can indeed be mediated by cyclical mechanical loading.     

Purification, cloning, and functional expression of phenylalanine aminomutase: the first committed step in Taxol side-chain biosynthesis

The biomechanical functions of articular cartilage are governed largely by the composition and density of its specialized extracellular matrix. Relationships between matrix density and functional indices such as mechanical properties or interstitial solute diffusivities have been previously explored. However, direct correlations between mechanical properties and solute transport parameters have received less attention, despite potential application of this information for cartilage functional assessment both in vivo and in vitro. The objective of this study was therefore to examine relationships among solute diffusivities, mechanical properties, and matrix density of compressed articular cartilage. Matrix density varied due to natural variation among explants and due to applied static compression. Matrix density of statically compressed cartilage explants was characterized by glycoaminoglycan (GAG) weight fraction and fluid volume fraction, while diffusion coefficients of a wide range of solutes were measured to characterize the transport environment. Explant mechanical properties were characterized by a non-linear Young's modulus (axial stress-strain ratio) and a non-linear Poisson's ratio (radial-to-axial strain ratio). Solute diffusivities were consistently correlated with Young's modulus, as well as with explant GAG weight and fluid volume fractions. Therefore, in vitro mechanical tests may provide a means of assessing transport environments in cartilage-like materials, while in vivo measurements of solute transport (for example with magnetic resonance imaging) may be a useful complement in identifying localized differences in matrix density and mechanical properties.     

Effect of Dynamic Loading on the Transport of Solutes into Agarose Hydrogels

In functional tissue engineering, the application of dynamic loading has been shown to improve the mechanical properties of chondrocyte-seeded agarose hydrogels relative to unloaded free swelling controls. The goal of this study is to determine the effect of dynamic loading on the transport of nutrients in tissue-engineered constructs. To eliminate confounding effects, such as nutrient consumption in cell-laden disks, this study examines the response of solute transport due to loading using a model system of acellular agarose disks and dextran in phosphate-buffered saline (3 and 70 kDa). An examination of the passive diffusion response of dextran in agarose confirms the applicability of Fick's law of diffusion in describing the behavior of dextran. Under static loading, the application of compressive strain decreased the total interstitial volume available for the 70 kDa dextran, compared to free swelling. Dynamic loading significantly enhanced the rate of solute uptake into agarose disks, relative to static loading. Moreover, the steady-state concentration under dynamic loading was found to be significantly greater than under static loading, for larger-molecular-mass dextran (70 kDa). This experimental finding confirms recent theoretical predictions that mechanical pumping of a porous tissue may actively transport solutes into the disk against their concentration gradient. The results of this study support the hypothesis that the application of dynamic loading in the presence of growth factors of large molecular weight may result in both a mechanically and chemically stimulating environment for tissue growth.     

Fluid transport and mechanical properties of articular cartilage: a review

This review is aimed at unifying our understanding of cartilage viscoelastic properties in compression, in particular the role of compression-dependent permeability in controlling interstitial fluid flow and its contribution to the observed viscoelastic effects. During the previous decade, it was shown that compression causes the permeability of cartilage to drop in a functional manner described by k = ko exp (epsilon M) where ko and M were defined as intrinsic permeability parameters and epsilon is the dilatation of the solid matrix (epsilon = tr delta u). Since permeability is inversely related to the diffusive drag coefficient of relative fluid motion with respect to the porous solid matrix, the measured load-deformation response of the tissue must therefore also depend on the non-linearly permeable nature of the tissue. We have summarized in this review our understanding of this non-linear phenomenon. This understanding of these flow-dependent viscoelastic effects are put into the historical perspective of a comprehensive literature review of earlier attempts to model the compressive viscoelastic properties of articular cartilage.     

Static compression of articular cartilage can reduce solute diffusivity and partitioning: implications for the chondrocyte biological response.

Chondrocytes depend upon solute transport within the avascular extracellular matrix of adult articular cartilage for many of their biological activities. Alterations to bioactive solute transport may, therefore, represent a mechanism by which cartilage compression is transduced into cellular metabolic responses. We investigated the effects of cartilage static compression on diffusivity and partitioning of a range of model solutes including dextrans of molecular weights 3 and 40 kDa, and tetramethylrhodamine (a 430 Da fluorophore). New fluorescence methods were developed for real-time visualization and measurement of transport within compressed cartilage explants. Experimental design allowed for multiple measurements on individual explants at different compression levels in order to minimize confounding influences of compositional variations. Results demonstrate that physiological levels of static compression may significantly decrease solute diffusivity and partitioning in cartilage. Effects of compression were most dramatic for the relatively high molecular weight solutes. For 40 kDa dextran, diffusivity decreased significantly (p<0.01) between 8% and 23% compression, while partitioning of 3 and 40 kDa dextran decreased significantly (p<0.01) between free-swelling conditions and 8% compression. Since diffusivity and partitioning can influence pericellular concentrations of bioactive solutes, these observations support a role for perturbations to solute transport in mediating the cartilage biological response to compression.     

Reversible permeabilization using high-intensity femtosecond laser pulses: applications to biopreservation

Non-invasive manipulation of live cells is important for cell-based therapeutics. Herein we report on the uniqueness of using high-intensity femtosecond laser pulses for reversibly permeabilizing mammalian cells for biopreservation applications. When mammalian cells were suspended in a impermeable hyperosmotic cryoprotectant sucrose solution, femtosecond laser pulses were used to transiently permeabilize cells for cytoplasmic solute uptake. The kinetics of cells exposed to 0.2, 0.3, 0.4, and 0.5 M sucrose, following permeabilization, were measured using video microscopy, and post-permeabilization survival was determined by a dual fluorescence membrane integrity assay. Using appropriate laser parameters, we observed the highest cell survival for 0.2 M sucrose solution (>90%), with a progressive decline in cell survival towards higher concentrations. Using diffusion equations describing the transport of solutes, the intracellular osmolarity at the inner surface of the membrane (x = 10 nm) and to a diffusive length of x = 10 microm was estimated, and a high loading efficiency (>98% for x = 10 nm and >70% for x = 10 microm) was calculated for cells suspended in 0.2 M sucrose. This is the first report of using femtosecond laser pulses for permeabilizing cells in the presence of cryoprotectants for biopreservation applications.     

Kinetics of a cloned special ginsenosidase hydrolyzing 3-O-glucoside of multi-protopanaxadiol-type ginsenosides, named ginsenosidase type III

In this paper, the kinetics of a cloned special glucosidase, named ginsenosidase type III hydrolyzing 3-O-glucoside of multi-protopanaxadiol (PPD)-type ginsenosides, were investigated. The gene (bgpA) encoding this enzyme was cloned from a Terrabacter ginsenosidimutans strain and then expressed in E. coli cells. Ginsenosidase type III was able to hydrolyze 3-O-glucoside of multi-PPD-type ginsenosides. For instance, it was able to hydrolyze the 3-O-β-D-(1-->2)-glucopyranosyl of Rb1 to gypenoside XVII, and then to further hydrolyze the 3-O-β-D-glucopyranosyl of gypenoside XVII to gypenoside LXXV. Similarly, the enzyme could hydrolyze the glucopyranosyls linked to the 3-O- position of Rb2, Rc, Rd, Rb3, and Rg3. With a larger enzyme reaction Km value, there was a slower enzyme reaction speed; and the larger the enzyme reaction Vmax value, the faster the enzyme reaction speed was. The Km values from small to large were 3.85 mM for Rc, 4.08 mM for Rb1, 8.85 mM for Rb3, 9.09 mM for Rb2, 9.70 mM for Rg3(S), 11.4 mM for Rd and 12.9 mM for F2; and Vmax value from large to small was 23.2 mM/h for Rc, 16.6 mM/h for Rb1, 14.6 mM/h for Rb3, 14.3 mM/h for Rb2, 1.81mM/h for Rg3(S), 1.40 mM/h for Rd, and 0.41 mM/h for F2. According to the Vmax and Km values of the ginsenosidase type III, the hydrolysis speed of these substrates by the enzyme was Rc>Rb1>Rb3>Rb2>Rg3(S)>Rd>F2 in order.     

Convection and Diffusion in Charged Hydrated Soft Tissues: A Mixture Theory Approach

The extracellular matrix of cartilage is a charged porous fibrous material. Transport phenomena in such a medium are very complex. In this study, solute diffusive flux and convective flux in porous fibrous media were investigated using a continuum mixture theory approach. The intrinsic diffusion coefficient of solute in the mixture was defined and its relation to drag coefficients was presented. The effect of mechanical loading on solute diffusion in cartilage under unconfined compression with a frictionless boundary condition was analyzed numerically using the model developed. Both strain-dependent hydraulic permeability and diffusivity were considered. Analyses and results show that (1) In porous media, the convective velocity for each solute phase is different. (2) The solute convection in tissue is governed by the relative convective velocity (i.e., relative to solid velocity). (3) Under the assumption that all the frictional interactions among solutes are negligible, the relative convective velocity for α-solute phase is equal to the relative solvent velocity multiplied by its convective coefficient (H ^α) which is also known as the hindrance factor in the literature. The relationship between the convective coefficient and the relative diffusivity of solute is presented. (4) Solute concentration profile within the cartilage sample depends on the phase of dynamic compression.     

Can ginsenosides protect human erythrocytes against free-radical-induced hemolysis?

Many studies have focused on the free-radical-initiated peroxidation of membrane lipid, which is associated with a variety of pathological events. Panax ginseng is used in traditional Chinese medicine to enhance stamina and capacity to deal with fatigue and physical stress. Many reports have been devoted to the effects of ginsenosides, the major active components in P. ginseng, on the lipid metabolism, immune function and cardiovascular system. The results, however, are usually contradictory since the usage of mixture of ginsenosides cannot identify the function of every individual ginsenosides on the experimental system. On the other hand, every individual ginsenosides is not compared under the same experimental condition. These facts motivate us to evaluate the antioxidant effect of various individual ginsenosides on the experimental system of free-radical-initiated peroxidation: the hemolysis of human erythrocyte induced thermally by water-soluble initiator, 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). The inhibitory concentration of 50% inhibition (IC(50)) of AAPH-induced hemolysis of the erythrocyte has been studied firstly and found that the order of IC(50) is Rb3 - Rb1Rc>Re>Rh1>R1>Rg2>Rb3. Rg3, Rd and Rh2, however, act as synergistic prooxidants in the above experimental system. Rg1 does not show any synergistic antioxidative property. Although the antioxidative and prooxidative mechanism of various ginsenosides with or without TOH in AAPH-induced hemolysis of human erythrocytes will be further studied in detail, this information may be useful in the clinical usage of ginsenosides.