RESUMO
Characterizing the iron distribution in tissue sections is important for several pathologies. Iron content in excised tissue is typically analyzed via histochemical stains, which are dependent on sample preparation and staining protocols. In our recent studies, we examined how magnetic properties of iron can also be exploited to characterize iron distribution in tissue sections in a label free manner. To enable a histomagnetic characterization of iron in a wide variety of available tissues, it is important to extend it to samples routinely prepared for histochemical staining, which often involve use of chemical fixatives. In this study, we took a systematic approach to determine differences between unfixed and formalin-fixed murine spleen tissues in histomagnetic characterization of iron. Superconducting quantum interference device (SQUID) magnetometry and magnetic force microscopy (MFM) were used for macro- and micro-scale histomagnetic characterization. Perl's stain was used for histochemical characterization of ferric (Fe3+) iron on adjacent sections as that used for MFM analysis. While histochemical analysis revealed a substantial difference in the dispersion of the stain between fixed versus unfixed samples, histomagnetic characterization was not dependent on chemical fixation of tissue. The results from this study reveal that histomagnetic characterization of iron is free from staining artifacts which can be present in histochemical analysis.
RESUMO
Collagen fibrils serve as the major template for mineral deposits in both biologically derived and engineered tissues. In recent years certain non-collagenous proteins have been elucidated as important players in differentially modulating intra vs. extra-fibrillar mineralization of collagen. We and others have previously shown that the expression of the collagen receptor, discoidin domain receptor 2 (DDR2) positively correlates with matrix mineralization. The objective of this study was to examine if the ectodomain (ECD) of DDR2 modulates intra versus extra-fibrillar mineralization of collagen independent of cell-signaling. For this purpose, a decellularized collagenous substrate, namely glutaraldehyde fixed porcine pericardium (GFPP) was subjected to biomimetic mineralization protocols. GFPP was incubated in modified simulated body fluid (mSBF) or polymer-induced liquid precursor (PILP) solutions in the presence of recombinant DDR2 ECD (DDR2-Fc) to mediate extra or intra-fibrillar mineralization of collagen. Thermogravimetric analysis revealed that DDR2-Fc increased mineral content in GFPP calcified in mSBF while no significant differences were observed in PILP mediated mineralization. Electron microscopy approaches were used to evaluate the quality and quantity mineral deposits. An increase in the matrix to mineral ratio, frequency of particles and size of mineral deposits was observed in the presence of DDR2-Fc in mSBF. Von Kossa staining and immunohistochemistry analysis of adjacent sections indicated that DDR2-Fc bound to both the matrix and mineral phase of GFPP. Further, DDR2-Fc was found to bind to hydroxyapatite (HAP) particles and enhance the nucleation of mineral deposits in mSBF solutions independent of collagen. Taken together, our results elucidate DDR2 ECD as a novel player in the modulation of extra-fibrillar mineralization of collagen.