Earliest mineral and matrix changes in force-induced musculoskeletal disease as revealed by Raman microspectroscopic imaging.

UNLABELLED: Craniosynostosis, premature fusion of the skull bones at the sutures, is the second most common human birth defect in the skull. Raman microspectroscopy was used to examine the composition, relative amounts, and locations of the mineral and matrix produced in mouse skulls undergoing force-induced craniosynostosis. Raman imaging revealed decreased relative mineral content in skulls undergoing craniosynostosis compared with unloaded specimens. INTRODUCTION: Raman microspectroscopy, a nondestructive vibrational spectroscopic technique, was used to examine the composition, relative amounts, and locations of the mineral and matrix produced in mouse skulls undergoing force-induced craniosynostosis. Craniosynostosis, premature fusion of the skull bones at the sutures, is the second most common birth defect in the face and skull. The calvaria, or flat bones that comprise the top of the skull, are most often affected, and craniosynostosis is a feature of over 100 human syndromes and conditions. MATERIALS AND METHODS: Raman images of the suture, the tips immediately adjacent to the suture (osteogenic fronts), and mature parietal bones of loaded and unloaded calvaria were acquired. Images were acquired at 2.6 x 2.6 microm spatial resolution and ranged in a field of view from 180 x 210 microm to 180 x 325 microm. RESULTS AND CONCLUSIONS: This study found that osteogenic fronts subjected to uniaxial compression had decreased relative mineral content compared with unloaded osteogenic fronts, presumably because of new and incomplete mineral deposition. Increased matrix production in osteogenic fronts undergoing craniosynostosis was observed. Understanding how force affects the composition, relative amounts, and location of the mineral and matrix provides insight into musculoskeletal disease in general and craniosynostosis in particular. This is the first report in which Raman microspectroscopy was used to study musculoskeletal disease. These data show how Raman microspectroscopy can be used to study subtle changes that occur in disease.

Effects of treatment protocols and subcutaneous implantation on bovine pericardium: a Raman spectroscopy study.

Using Raman microspectroscopy, we have studied mineral deposition on bovine pericardia, fixed according to three different protocols and either implanted subcutaneously or not implanted (controls). A lightly carbonated apatitic phosphate mineral, similar to that found in bone tissue, was deposited on the surface of a glutaraldehyde-fixed, implanted pericardium. Implanted pericardia fixed in glutaraldehyde followed by treatment in either an 80% ethanol or a 5% octanol/40% ethanol solution did not mineralize on implantation. Collagen secondary structure changes were observed on glutaraldehyde fixation by monitoring the center of gravity of the amide I envelope. It is proposed that the decrease in the amide I center of gravity frequency for the glutaraldehyde-fixed tissue compared to the nonfixed tissue is due to an increase in nonreducible collagen cross-links (1660 cm(-1)) and a decrease in reducible cross-links (1690 cm(-1)). The amide I center of gravity in the glutaraldehyde/ethanol-fixed pericardium was higher than the glutaraldehyde-fixed tissue center of gravity. This increase in center of gravity could possibly be due to a decrease in hydrogen bonding within the collagen fibrils following the ethanol pretreatment. In addition, we found a secondary structure change to the pericardial collagen after implantation: an increase in the frequency of the center of gravity of amide I is indicative of an increase in cross-links.

Mineralization of developing mouse calvaria as revealed by Raman microspectroscopy.

Raman microspectroscopy is a nondestructive vibrational spectroscopic technique that permits the study of organic and mineral species at micron resolution, offers the ability to work with hydrated and dehydrated specimens in vivo or in vitro, and requires minimal specimen preparation. We used Raman microspectroscopy to determine the composition of the mineral environments present in mouse calvaria, the flat bones that comprise the top of the skull. We have acquired Raman transects (lines of point spectra) from mouse calvaria during a developmental time course ranging from embryonic day 13.5 (E13.5; 6 days before birth) to 6 months of age. Exploratory factor analysis (FA) reveals the presence of a variety of apatitic mineral environments throughout the tissue series. The earliest mineral is observed in the fetal day 15.5 (F15.5) mice and is identified as a carbonated apatite. The presence of a heterogeneous mineralized tissue in the postnatal specimens suggests that ionic incorporation and crystal perfection in the lattice yary as the mouse develops. This variation is indicative of the presence of both recently deposited mineral and more matured remodeled mineral. Band area ratios reveal that the mineral/matrix ratio initially increases, reaches a plateau, and then increases again. The carbonate/phosphate band area ratio remains constant from F18.5 to postnatal day 3 (PN3) and then increases with age. Insights into the chemical species, the degree of mineralization, and the multiple mineral environments that are present in normal calvarial tissue will enable us to better understand both normal and abnormal mineralization processes.