Original ArticlesIn Vivo Tracer Transport Through the Lacunocanalicular System of Rat Bone in an Environment Devoid of Mechanical Loading
Introduction
A prerequisite for metabolism in any living tissue is an adequate supply of nutrients for anabolic activity as well as a mechanism for removal of waste products resulting from catabolic activities—in short, molecular transport must be insured. Within soft tissues and organs, molecular diffusion through interstitial spaces is considered the principal mechanism for movement of various physiologic fluids and solutes. In contrast to the soft tissues of internal organs, approximately 85% of bone volume consists of solid material such as mineral and collagen. Thus, within the particularly dense tissue of compact bone, osteocytes are dependent on the transport pathways comprising the lacunocanalicular system for maintenance of “metabolic traffic and interchange.”10, 11, 57
Although several studies have shown that diffusion is a major contributing mechanism for the transport of molecules and ions across the endothelial layer of bone capillaries, diffusive mechanisms may in fact not be sufficient to insure adequate transport to and from osteocytes that are not in close vicinity of vascular canals. Furthermore, due to the dynamic structure of bone, patent metabolic pathways may become blocked during remodeling processes, resulting in osteocyte starvation.[2] It has been proposed that, during daily activities, mechanical loading of the fluid-filled porous matrix of bone results in microdeformation of the fluid spaces. The ensuing pressure gradients induce interstitial fluid displacements within the solid matrix of the tissue. This so-called load-induced fluid flow has been hypothesized to promote bone “perfusion” by convective mixing of fluids or enhancement of molecular transport from the blood supply to outermost osteocytes within a given osteon, thus helping to support metabolic function.32, 33, 34, 52 However, to prove experimentally the hypothetical role of load-induced fluid flow in promoting molecular transport in bone tissue, it is first necessary to study the primary role of diffusion and to establish a baseline for fluid transport whereby mechanical loading effects are negligible.
To date, the degree to which diffusive mechanisms provide key molecules to osteocytes for maintenance of metabolic activity as well as for activation or suppression of modeling processes remains unclear. Thus, the purpose of this study was to elucidate the pathways and extent of molecular transport through the bone in vivo in an environment devoid of mechanical loading. To this end, we established methods for the investigation of short-term (i.e., on the order of minutes) and long-term (i.e., on the order of hours) diffusion of molecular tracers within the metacarpi and tibiae of circa 60-day-old (i.e., skeletally immature) and 180-day-old (i.e., mature) rats. In general, the rate of transport for a given tracer decreases with increasing molecular weight and, for metabolically inactive species, smaller substances are transported more rapidly than larger ones. Thus, procion red, a low-molecular-weight tracer (MW 300–400), was employed for the study of short-term transport through the extravascular space. Conversely, microperoxidase, with a molecular weight six times higher than that of procion red (MW 1800) was chosen to investigate longer-term transport through the lacunocanalicular system. Horseradish peroxidase (MW 40,000) was utilized in a limited number of rats for comparative purposes. The rats were anesthetized during the entire experimental period to insure that mechanical loading effects could be excluded. Furthermore, we chose metabolically inert tracers not endogenous to bone to characterize diffusive transport without having to account for effects of reaction, uptake, and active transport mechanisms. In this article, the feasibility of the tracer methods, proper choice of an appropriate animal model, and exemplary results are discussed.
Section snippets
Animal Preparation
The preterminal experiments were conducted using 6, approximately 60-day-old (145–150 g, skeletally immature) and 12, approximately 180-day-old (250–300 g, skeletally mature) male and female Sprague–Dawley rats. All rats were preanesthetized in a plexiglass chamber filled with a mixture of halothane, O2, and N2O. Anesthesia followed, using Hypnorm (Janssen Pharmaceutica, Belgium) at a dose of 0.1–0.2 mL per 100 g body weight. Using a table equipped with a heat pump, rats were maintained at a
Results
In short-term studies with procion red tracer, the fluorescent dye could be observed in tissue sections from the metacarpus and tibia from both 60- and 180-day-old rats. In both immature (i.e., from 60-day-old animals) and mature (i.e., from 180-day-old animals) bone, no difference in tracer distribution was noted when comparing analogous specimens explanted after 10 min and 30 min circulation times. Furthermore, the tracer appeared to demarcate pathways of diffusive transport from the vascular
Discussion
The purpose of this study was to elucidate in vivo the pathways and extent of tracer transport through rat bone in an environment devoid of mechanical loading. It could be shown that the low-molecular-weight tracer, procion red, lends itself to short-term studies (i.e., on the order of minutes) of transport through the blood vessels and extracellular space of the lacunocanalicular system within the mature cortical bone of 180-day-old rats; this tracer appears less well suited for demarcating
Acknowledgements
The authors thank Professor Teruo Tanaka and Professor Brian Schofield for their helpful advice in implementing the histochemical protocols. In addition, we thank Monique de Jager for assistance during the experimental procedures, Iris Keller for preparing the histological sections, and Sabine Klein for technical assistance with the confocal microscope.
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