78 - Embryonic Mouse Tissue Ultrastructure and Materials Properties

Poster Board Number: 78
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Abstract Body : Introduction and Objective. Bone and cartilage material properties are well characterized through investigations including atomic force microscopy (AFM), though most investigations focus on fully developed or mature bone being tested in air. In this research, we used AFM to characterize the cell-level environment of embryonic craniopharyngeal cartilage and bone with topographical and force vs. distance data transformed into elastic modulus in the laboratory mouse. The goal was to accumulate these data to aid in understanding the relative roles of craniopharyngeal bone and cartilage during embryogenesis.
Materials and Methods. We developed a liquid environment testing protocol for cryosections of embryonic mouse crania. A testing protocol was developed through an iterative design process resulting in a scanning environment that produces high-fidelity AFM scans and emulates the in vivo cellular environment. The first step in this process was to select an appropriate solvent for testing in liquid. Phosphate-buffered saline provided an electrolytic solvent with appropriate physiologic pH. Passing solvent through a 0.2-micron filter ensured no debris would cause significant surface charge interactions with samples or interfere with AFM probe calibration. A cylinder-bore silicone gasket provided a liquid well for solvent while also maintaining a large area surrounding the section with low boundaries to ensure unencumbered movement of the AFM probe. Sectioning with cryomatrix provided an embedding medium that was soluble and able to be removed in aqueous solutions. Cryosectioning onto positively charged slides ensured sections would remain fixed on glass slides without significant movement even in aqueous environments. Bone on cryosections was visualized by intraperitoneally injected calcein, a fluorescent dye that binds to calcium ions. Additionally, the elastic moduIus of demineralized bone was used as the threshold, and locations showing elastic moduli lower than the threshold were excluded from the analysis.
Results. Craniofacial bone and cartilage changes morphologically and mechanically between embryonic days 15.5 (E15.5) and 17.5 (E17.5) differentially according to location on the head (cranial vault versus lower jaw). In both the vault and the jaw, differences in bone and cartilage modulus increase and significantly diverge over embryonic time. We demonstrate the importance of packing and orientation of collagen fibers by comparing demineralized bone elastic modulus to intact bone elastic modulus.
Significance/Implication. The potential importance of embryonic craniopharyngeal cartilage in dermal bone formation has only recently been recognized by developmental biologists. These are the first data that measure the cell-level environment of embryonic cranipharyngeal cartilage and bone in the laboratory mouse. These data will be invaluable for understanding how the relationship between craniopharyngeal cartilage and bone contributes to the structural integrity of the head during embryonic development.
Funding Sources. This research was supported in part by NIDCR/NIH R01 DE027677, R01DE 029832, and R01DE031439.