Imaging and quantifying solute transport across periosteum: Implications for muscle–bone crosstalk

Abstract

Muscle and bone are known to act as a functional unit and communicate biochemically during tissue development and maintenance. Muscle-derived factors (myokines) have been found to affect bone functions in vitro. However, the transport times of myokines to penetrate into bone, a critical step required for local muscle–bone crosstalk, have not been quantified in situ or in vivo. In this study, we investigated the permeability of the periosteum, a major barrier to muscle–bone crosstalk by tracking and modeling fluorescent tracers that mimic myokines under confocal microscopy. Periosteal surface boundaries and tracer penetration within the boundaries were imaged in intact murine tibiae using reflected light and time-series xz confocal imaging, respectively. Four fluorescent tracers including sodium fluorescein (376Da) and dextrans (3kDa, 10kDa and 40kDa) were chosen because they represented a wide range of molecular weights (MW) of myokines. We found that i) murine periosteum was permeable to the three smaller tracers while the 40kDa could not penetrate beyond 40% of the outer periosteum within 8h, suggesting that periosteum is semi-permeable with a cut-off MW of approximately 40kDa, and ii) the characteristic penetration time through the periosteum (~60μm thick) increased with tracer MW and fit well with a relationship tcs=−4.43×104−0.57×MWDa−4×104−8.65×108MWDa−4×104, from which, the characteristic penetration times of various myokines were extrapolated. To achieve effective muscle–bone crosstalk, likely signaling candidates should have shorter penetration time than their bioactive time, which we assumed to be 5 times of the molecule's half-lifetime in the body. Myokines such as PGE2, IGF-1, IL-15 and FGF-2 were predicted to satisfy this requirement. In summary, a novel imaging approach was developed and used to investigate the transport of myokine mimicking-tracers through the periosteum, enabling further quantitative studies of muscle–bone communication in physiologically normal and pathological conditions.