Solute transport in intervertebral disc: experiments and finite element modeling.

Loss of nutrient supply to the human intervertebral disc (IVD) cells is thought to be a major cause of disc degeneration in humans. To address this issue, transport of molecules of different size have been analyzed by a combination of experimental and modeling studies. Solute transport has been compared for steady-state and transient diffusion of several different solutes with molecular masses in the range 3-70 kDa, injected into parts of the disc where degeneration is thought most likely to occur first and into the blood supply to the disc. Diffusion coefficients of fluorescently tagged dextran molecules of different molecular weights have been measured in vitro using the concentration gradient technique in thin specimens of disc outer annulus and nucleus pulposus. Diffusion coefficients were found to decrease with molecular weight following a nonlinear relationship. Diffusion coefficients changed more rapidly for solutes with molecular masses less than 10 kDa. Although unrealistic or painful, solutes injected directly into the disc achieve the largest disc coverage with concentrations that would be high enough to be of practical use. Although more practical, solutes injected into the blood supply do not penetrate to the central regions of the disc and their concentrations dissipate more rapidly. Injection into the disc would be the best method to get drugs or growth factors to regions of degeneration in IVDs quickly; else concentrations of solute must be kept at a high value for several hours in the blood supply to the discs.

Cytochrome c oxidase levels in chondrocytes during monolayer expansion and after return to three dimensional culture.

OBJECTIVE: Here we investigate whether monolayer culture or culture at 21% oxygen influences activity of cytochrome c oxidase, the terminal enzyme in the respiratory chain whose activity is essential for oxidative metabolism and whether return to three dimensional (3-D) culture restores cytochrome c oxidase activity to original levels. METHODS: Primary bovine articular chondrocytes were cultured in alginate beads (3-D) for 4 weeks or in monolayer under 1% and 21% oxygen for up to 9 days and then returned to 3-D culture for up to 4 weeks. Cells were stained to localise cytochrome c oxidase within the cells. Mitochondrial protein content and cytochrome c oxidase enzymatic activity were determined. Expression of cytochrome c oxidase subunits, COXI and COXIV, was assessed by qRT-PCR. RESULTS: Cytochrome c oxidase staining remained minimal in chondrocytes cultured in alginate for 4 weeks under 21% oxygen. Mitochondrial protein content and cytochrome c oxidase activity increased significantly during 9 days of chondrocyte expansion in monolayer, accompanied by up-regulation of the COXI mitochondrial gene but not the COXIV nuclear-encoded gene. Cytochrome c oxidase staining increased from day 5 of monolayer culture and remained high even after the cells were returned to 3-D culture for 4 weeks. CONCLUSIONS: Culture of chondrocytes in monolayer leads to a rapid increase in mitochondrial protein content and cytochrome c oxidase activity. The increase in cytochrome c oxidase activity is not reversed even after chondrocytes are returned to 3-D culture for 4 weeks; high oxygen tension alone does not appear to stimulate cytochrome c oxidase activity.

Microdialysis for monitoring the process of functional tissue culture.

Continuous monitoring is important during tissue culture. However, there are still technical difficulties in monitoring the internal status of cells or tissues. In this paper, microdialysis is adopted to monitor functional tissue growth in a bioreactor. Explanted bovine caudal intervertebral disc (IVD) was used as the test tissue. A microdialysis membrane probe of 100 kDa molecular weight cut-off was employed and in situ calibration methods with phenol red and fluorescent 40 kDa dextran were developed to measure the relative recovery of the solute of interest, and membrane fouling, respectively. Tissue metabolism was monitored successfully. At the same time soluble macromolecules were picked up by the probe and were detected and quantified by Fast Protein Liquid Chromatography (FPLC) and/or Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). These proteins were believed to be associated with biofunction of engineered tissue. Monitoring of phenol red content in the dialysate indicated that there was no significant fouling of the membrane probe during a 7-day culture period and the Relative Recovery of macromolecules of interests remained roughly 9%. We concluded that microdialysis could be used to sample a wide range of molecular species released during cell metabolism and extracellular matrix turnover, which were direct or indirect indications of cell and tissue functions. The application of the developed system could be extended to monitor tissue repair in vivo, and the development of the engineered tissue.

Differential effects of aging on transport properties of anterior and posterior human sclera.

The transport properties and composition of 44 pairs of human sclera, 37-91 years were compared. Solute transport, diffusion and partition coefficients of posterior sclera for solutes ranging in mass from 0.023-70kDa were higher than those of anterior sclera; the posterior region was also more hydrated. The differences in partition coefficient between anterior and posterior sclera became more pronounced as solute molecular weight increased. Partition coefficients and hydration of both regions decreased with increasing age. Chondroitinase ABC digestion, which removed the majority of glycosaminoglycans, increased partition coefficients of both regions significantly. These results suggest that for regions of equal scleral thickness, neglecting the influence of vascular factors, drug delivery will be more readily achieved across the posterior sclera than the anterior sclera in the age group studied and that, for both regions, ease of delivery will decrease with decreasing age.

The effect of hydration and matrix composition on solute diffusion in rabbit sclera.

There is increasing interest in the possibility for drug delivery into the vitreous humor across the conjunctiva and sclera as an alternative route to the conjunctiva-cornea pathway. As a preliminary to human studies we have investigated the influence of scleral composition and hydration on solute transport in the rabbit sclera. Intermuscular sclera was excised from adult New Zealand rabbits. Tissue samples were either examined directly (controls), digested using chondroitinase ABC or crosslinked using glutaraldehyde. The effect of these treatments on the ultrastructural appearance of the sclera was assessed. Diffusion and partition coefficients for solutes of different molecular weights [sodium chloride (23 MW),(14)C sucrose (342 MW) and dextran-fluoresceins (3, 10, 40 and 70 kDa)] were measured in relation to tissue treatment. The results were used to determine the effect of tissue structure and composition on solute movement. We have found that: (1) diffusion and partition coefficients are sensitive to solute MW, decreasing as MW increases; (2) diffusion and partition coefficients are sensitive to tissue hydration, increasing as hydration increases; (3) crosslinking of the sclera by glutaraldehyde reduced the partition coefficients significantly for solutes with MW over 3 kDa; and (4) removal of glycosaminoglycans has only a small effect on either diffusion or the partition coefficient.