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 8 hours, 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 (((tc[sec]=−(4.43×104)−0.57×(MW[Da]−4×104)−8.65×108MW[Da]−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-life time 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.

Seeing through Musculoskeletal Tissues: Improving In Situ Imaging of Bone and the Lacunar Canalicular System through Optical Clearing

“…Cells of the musculoskeletal system reside within complex 3-D environments where their function and homeostasis depends critically on their interactions with neighboring cells and the surrounding ECM [ 1 – 4 ]. Studies directly interrogating the complex 3-D morphology, architecture, interactions among, and functions of musculoskeletal cells in situ have risen to the fore in the study of musculoskeletal physiology, homeostasis, and disease [ 12 – 16 , 63 – 70 ]. Numerous imaging techniques, including electron microscopy, serial sectioning and reconstruction, and en bloc imaging have been utilized to visualize and quantify 3-D musculoskeletal cell and tissue properties [ 3 , 17 , 19 , 71 , 72 ] in situ.…”

Skeletal Muscle, but not Cardiovascular Function, Is Altered in a Mouse Model of Autosomal Recessive Hypophosphatemic Rickets

“…Specifically, bone can secrete factors in a paracrine manner which has important implications for local biochemical crosstalk between bone and skeletal muscle. Using fluorescent tracers, the periosteum has been shown to be permeable to molecules under 40 kDa such as PGE2, IGF-1, IL-15, and FGF-2 (Lai et al, 2014 ). Our research group has shown that PGE2 is an important factor in MLO-Y4 osteocyte-like conditioned media which accounts for changes in myoblast growth, differentiation, and proliferation (Mo et al, 2012 , 2015 ).…”

Role of Muscle Stem Cells During Skeletal Regeneration

“…When treated with rhBMP2, the delayed bone regeneration in Pax7 CreERT2/1 ;DTA f/f mice was improved. Muscle stem cells may play a role, in addition to other cell types, by regulating the BMP-dependent activation of skeletal stem cells within periosteum and callus formation [31,39,50], providing a functional explanation for the critical interactions between muscle and bone that are required for periosteal activation. The role of muscle in periosteal activation via a BMP-dependent mechanism may be particularly relevant in the context of endochondral ossification, as we previously showed that cell fate within the periosteum was regulated by BMP2 [39].…”