Aims

Inflammatory response plays a pivotal role in the pathophysiological process of intervertebral disc degeneration (IDD). A20 (also known as tumour necrosis factor alpha-induced protein 3 (TNFAIP3)) is a ubiquitin-editing enzyme that restricts nuclear factor-kappa B (NF-κB) signalling. A20 prevents the occurrence of multiple inflammatory diseases. However, the role of A20 in the initiation of IDD has not been elucidated. The aim of the study was to investigate the effect of A20 in senescence of TNF alpha (TNF-α)-induced nucleus pulposus cells (NPCs).

Introduction: Tissue engineered scaffolds require vascularization to 1) enhance nutrient exchange and 2) provide cells needed to build new tissue. Cell-seeded scaffolds; bioreactors-- require rapid penetration of vessels or enhanced fluid percolation to keep their contents alive until normal nutrient exchange can be established. Bone fluid flow depends on a pumping system which drives percolation through its own matrix. Recent interest in the pumping mechanism has resulted in bone fluid flow models, which link the pumps to bending of bone by muscle contraction and compression-tension cycles from weight-bearing during locomotion. The present authors have proposed that capillary filtration, the source of the percolating fluid, is sufficiently enhanced by soliton pressure waves in blood driven by the muscle pump during exercise to provide a significant hydraulic pressure component to bone fluid percolating through bone and any bone-implanted scaffold. A proposal and some preliminary results from a pilot project suggesting enhancement of capillary filtration by the muscle pump is presented.

Materials and Methods: Optical bone chambers were implanted in adult New Zealand White female rabbits. Chamber construction and implantation were as usual1. At the third week post-op, chamber ends were exposed and weekly intravital microscopy commenced. Transcutaneous electrical stimulation was administered with a ToneATronic® TENS at 85V, 80mA and 2Hz. The stimulator was applied externally over the gastrocnemius muscle. A fluorescence digital image was obtained before 30 minutes of application of transcutaneous electrical nerve stimulation (TENS) after injection of FITC-D70. Blood samples were obtained from an aural vein in the ear opposite that being injected with the fluorescent dye after each injection. Blood concentration of dye was determined with a SPEX Fluoromax-3 spectrofluorometer for both serum (absolute concentration) and whole blood (to detect differences which would make fluorescence in vessels an inaccurate indicator of red blood cell color contamination). For analysis, four vessels were chosen and the average dye concentration profiles before and after 30 minutes of stimulation were obtained.

Results: Results are shown in Figure 1. Extravasated dye levels in TENS rabbits were markedly higher than those in controls. Analysis of profiles using an erfc-based diffusion-convection discrimination model2 showed that extravasation was convective.

Discussion: These data are consistent with significant contribution to convective percolation of bone fluid through implanted scaffolds by muscle pump-driven extravasating fluid. They do not, however, answer two critical questions: 1) Is the magnitude of this convection a major component of flow through the scaffold? 2) What are the relative contributions of skeletal muscle-generated intravascular pressure solitons and incompressible fluid transmission of bone bending pressure to the convective flow observed? Additional studies with released gastrocnemius muscles are in progress.