An Improved Method for Assessing Macroscale Diffusion Through Osteonal Bone

The characterization of diffusion through biological tissues has played an important role in fundamental medical research and product development. Understanding the diffusion phenomena allows for the identification of new concepts in fundamental science, evolving medical knowledge and improving future standards and protocols. To illustrate, the structure of cortical bone changes upon the onset of osteoporosis, altering the limited porous compartment through which nutrients and essential signaling molecules travel to bone cells. Estrogen hormone replacement therapy (HRT) is one of the gold standard treatments to attempt to mitigate the effects that this structural change exerts in menopausal osteoporosis patients; however, HRT effectiveness is often variable in these patients, likely due to variability in bone structure and physiology, and thus transport rates. Scientists have studied diffusion in cortical bone tissue for decades. Current methodological standards include fluorescence recovery after photobleaching and computed tomography finite element analysis. Both techniques limit areas of tissue to microscale (1-100 μm2) analysis - only examining a few osteocytes within the structure at a time - and adopt assumptions that oversimplify in vivo tissue structure and transport phenomena. Also, the range of diffusion tracers is limited by the sensitivities of the analytical equipment, typically requiring tracer concentrations in the micromolar range. Herein is described a novel device for directly assessing the diffusion coefficient of 3H-estradiol at 37°C in macroscale osteonal bone specimens (1.4 cm2) - assessing a much larger portion of the total tissue than previously reported - while using radioisotope tracers for much higher sensitivity, thus achieving physiologically relevant estradiol concentrations. The current diffusion chamber device represents a cost-effective and validated method to mitigate these shortcomings. The device provides long-term diffusion data through macroscale (greater than 1 mm2) tissue areas, presenting a more physiologically accurate way to assess cortical bone diffusion. The device can assess solute diffusion through other tissues or materials and may easily be scaled up to run multiple diffusion experiments simultaneously. The diffusion chamber device represents a cost-effective and validated method to assess solute diffusion through solid materials. Specifically, it demonstrates that this novel device provides long-term diffusion data through macroscale tissue samples at nanomolar concentrations, presenting a precise way to address the effects of tissue structures on diffusion. This device can be applied to other tissues or engineered materials, offering a methodology that is easily scaled up to allow simultaneous assessment of multiple material samples.