Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Architecture of the Interfrontal Suture Revealed by Transmitted Electron Microscopy
Jolanta M. Topczewska, PhD, Rupa Mirmira, BS; Chad A. Purnell, MD; Arun Gosain, MD
Northwestern Feinberg School of Medicine, Chicago, IL

PURPOSE:
The resolving power of TEM and the small size of zebrafish cranium prompted us to investigate architecture of the interfrontal suture using this type of microscopy. It is a complementary approach to functional and genetic studies conducted in our laboratory. Our aim is to identify characteristic cellular structures or organelles typical for osteoblasts residing in the sutural tissue of wild type and koliber mutant. In addition we aim to characterize ultrastructural organization of zebrafish sutural tissue including identity and morphology of specific structural features that might be associated with the pathological development.
METHODS:
Dissected calvaria from 3.5 months old fish were collected for TEM imaging (n=4). Tissue was process using standard EM protocol that include primary fixation in glutaraldehyde followed by brief decalcifying step in 0.5M EDTA and secondary fixation in osmium tetroxide. Samples were embedded in epoxy resin. 1 μm-thick section were collected and stained with Toluidine Blue for tissue evaluation; 70nm thick sections were collected and processed for TEM imaging. This work was conducted using the Cell Imaging Facility at Northwestern University Feinberg School of Medicine.
RESULTS:
The osteogenic cells identified under TEM can be classified into two general categories. The first group includes osteoblasts being in direct contact with the frontal bone. These micrographs revealed cells with extensive endoplasmic reticulum associated with ribosomes (RER) usually located on the bone site of the cell and connected to the prominent nucleus and the Golgi complex, which is surrounded by vesicular structures. Lysosomes with membranous inclusions and mitochondria are scattered among elements of the RER. Such subcellular organization is often described for cells intensively producing proteins. In addition we have observed the electron-dense mitochondrial granules in some of the bone attached cells, which presumably represent the inorganic material required for the bone mineralization. The second category of cells is mostly embedded in the ECM without direct contact to the bone. These cells are smaller, with the centrally located nucleus and reduced RER. The abundant presence of fibrillar collagen type I is the most striking difference between the wild type and koliber mutant sibling. Rather loosely organized within the ECM of the wild type, the collagen fibrils form large bundles in the mutant, varying in diameters; these bundles intertwine between cells as they connect two frontal bones.
CONCLUSION:
After a preliminary phase of descriptive work we will focus on relation between the ultrastructure of osteoblasts and expression of their genetic markers to better understand the structure-function relationship. As one of the central functions of the osteoblast is the mineralization process we will search for morphological changes in the osteoblast that relate to this process.


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