Second harmonic generation imaging microscopy studies of osteogenesis imperfecta.

We have used quantitative second harmonic generation (SHG) imaging microscopy to investigate the collagen matrix organization in the oim mouse model for human osteogenesis imperfecta (OI). OI is a heritable disease in which the type I collagen fibrils are either abnormally organized or small, resulting in a clinical presentation of recurrent bone fractures and other pathologies related to collagen-comprised tissues. Exploiting the exquisite sensitivity of SHG to supramolecular assembly, we investigated whether this approach can be utilized to differentiate normal and oim tissues. By comparing SHG intensity, fibrillar morphology, polarization anisotropy, and signal directionality, we show that statistically different results are obtained for the wild type (WT) and disease states in bone, tendon, and skin. All these optical signatures are consistent with the collagen matrix in the oim tissues being more disordered, and these results are further consistent with the known weaker mechanical properties of the oim mouse. While the current work shows the ability of SHG to differentiate normal and diseased states in a mouse model, we suggest that our results provide a framework for using SHG as a clinical diagnostic tool for human OI. We further suggest that the SHG metrics described could be applied to other connective tissue disorders that are characterized by abnormal collagen assembly.

Selective retention and degradation of molecules with a single mutant alpha1(I) chain in the Brtl IV mouse model of OI.

We investigated the secretion, matrix incorporation and interactions of molecules with one and two mutant alpha1(I) collagen chains in the Brtl IV murine model for Osteogenesis Imperfecta, carrying a Gly-349 to Cys substitution in one col1a1 allele. We detected a significant deviation from the expected 25 and 50% content of the molecules with no (37-46%) and one (26-40%) mutant chains in skin and bone as well as in fibroblast and osteoblast cell culture media. Steady-state labeling with (35)S-Cys demonstrated incomplete secretion of the mutant collagen in cell culture, particularly molecules containing one mutant chain. Pulse and pulse-chase experiments revealed slower secretion of the latter. An enlargement of endoplasmic reticulum in skin fibroblasts from Brtl IV mice, clearly visible by electron microscopy, supported the abnormal secretion identified by biochemical studies. We observed increased susceptibility of molecules with one mutant chain to proteolytic degradation in vitro, but we did not detect significant selective degradation in cell culture media. Mutant collagen molecules incorporated from the media into newly deposited fibers and into fully crosslinked and mature matrix in the same ratio as they were secreted. Specific labeling of reactive -SH demonstrated that about half of the Cys349-SH groups in the mutant molecules were exposed and potentially available for aberrant interactions with other molecules inside or outside the cells. Based on these and our previous findings, we argue that the outcome in Brtl IV may be significantly affected by cellular stress and malfunction caused by the retention and degradation of newly synthesized mutant collagen.

[Bone tissue in osteogenesis imperfecta: light and electron microscopic studies].

Osteogenesis imperfecta was studied by light and electron microscopic techniques in 36 cases, of them there were 10 postmortem and 26 bone biopsies. The persons' age was from 0 to 38 years. There were 25 males and 11 females. The microscopic studies indicated a decrease in the basic substance and an increase in the number of osteocytes. The electron microscopic studies revealed a reduction in the granular endoplasmic reticulum, as well as matrix swelling in the mitochondria, their crista degeneration, and the presence of mitochondrial hydroxyappatite; non-uniform mineralization of collagenous structures, their disintegration, a change in the diameter of collagenous fibril, and a decrease in their transversal lines.

Effect of ultrastructural changes on the toughness of bone.

The ultrastructure of bone can be considered as a conjunction between the biology and the biomechanics of the tissue. It is the result of cellular and molecular activities of bone formation, and its organization dominates the mechanical behavior of bone. Following this perspective, the objective of this review is to provide a current understanding of bone ultrastructure and its relationships with the toughness of the tissue. Therefore, we first provide a discussion on the organization of bone constituents, namely collagen, mineral, and water. Then, we present evidence on how the toughness of bone relates to its ultrastructure through the formation of micro damage. In addition, attention is given to how damage accumulation serves as a toughening mechanism. Finally, we describe how changes in the ultrastructure-caused by osteogenesis imperfecta, gamma irradiation, fluoride treatment, and aging affect the toughness and competence of bone.

The otodystrophies: diagnosis and differential diagnosis.

The imaging evaluation of hearing loss is usually focused on inflammatory entities, especially chronic otitis/ cholesteatoma and cerebellopontine angles lesions, particularly acoustic tumors. This discussion concerns several developmental and acquired entities. The developmental entities referred to as the otodystrophies include otosclerosis, Paget's disease, fibrous dysplasia, and osteogenisis imperfecta. Otosyphilis and postradiation temporal bone changes are also included because of the overlapping imaging appearances.

Ultrastructural localization of proteoglycans in bone in osteogenesis imperfecta as demonstrated by Cuprolinic Blue staining.

The role of proteoglycans in bone in osteogenesis imperfecta (OI) has been examined. Using Cuprolinic Blue staining of whole fetal bone tissue and examining the tissue in the transmission electron microscope, the presence of proteoglycans was observed. Quantitative comparative image-analysis of the proteoglycans from electron micrographs was performed, with measurement of sizes and number of proteoglycan particles. A significant increase in the total number of proteoglycan particles in OI bone osteoid was observed when compared with normal, matched controls. The area of the proteoglycan particles, as measured by pixel-area, using image analysis, was also increased in OI bone osteoid. These findings further suggest a role for proteoglycans in mineral formation by the possible inhibition of mineral growth and alteration of collagen nucleation sites. The increased number and size of proteoglycan particles may be a contributing factor to the previously reported poor mineral formation with subsequent loss of bone strength, making it more prone to fracture, in OI.