Introduction: Insufficiency of poroelastic bone bending as the sole mechanism driving bone interstitial fluid flow (BIFF) to account for the shear stress required to activate mechanoreceptors on osteocytes and osteoblasts, has stimulated a search for alternative or complementary mechanisms in the quest for a comprehensive bone remodeling model. Some investigators, noting that a substantial amount of interstitial fluid is exchanged with blood and lymphatic capillaries, have suggested that this exchange may play a substantial role in both microtransport through the collagen matrix and lacunar-canalicular transport. In order to accept the vascular system as a significant source of transport driving BIFF, it must first be demonstrated that capillary filtration, the proc ess by which fluid is transported from blood vasculature, is sufficiently convective to drive interstitial percolation. We have proposed that while, as shown by Otter et al., resting transmural vascular pressures are sufficient to generate streaming potentials across cortical bone, it is likely that these forces must be complemented by muscle pump contractions during exercise to generate convective percolation flows which will develop the required mechanotransducer shear stress activation threshold. To determine a minimal baseline for a muscle pump driven BIFF (MPD-BIFF) model, we have investigated the role of repetitive skeletal muscle contractions, uncoupled from gravitational loading, on blood flow and capillary filtration in cortical bone of the rabbit tibia. We tested the hypothesis that these effects increased when the muscle pump was activated.

Materials & Methods: The experimental model combined non-invasive, low magnitude transcutaneous neuromuscular stimulation (TENS), with real-time measurements from intravital microscopy (IVM) of optical bone chamber implants. Sling suspension of rabbits was utilized to eliminate gravitational reaction forces throughout TENS and data collection. TENS-induced muscle contraction forces were measured in situ, resultant bone strains were calculated, and systemic circulatory parameters were monitored, in order to eliminate these factors as contributors to blood flow changes. Blood flow rates and capillary filtration were measured by video-image analysis of 1 μm fluorescent microspheres and dextran-conjugated fluorescein isothiocyanate (FITC) and rhodamine (ITC) injected intravascularly during IVM. Bone formation, angiogenesis, and mineral apposition rates (tetracycline labelling) were analyzed from weekly microscopy pictures. Changes in bone mineral content and density were determined with CT scans obtained at implantation and termination.

Results: Mechanical loading and baseline systemic circulation did not significantly contribute to the findings. Rhythmic muscle contractions were shown to increase cortical blood flow, rate of capillary filtration, rate of bone apposition, and angiogenesis.

Discussion: The hypothesis was supported by the data. However, since no measurements were made on single capillaries, we could not confirm previous reports by this laboratory of convective extravasation.

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.