Infrared analysis of the mineral and matrix in bones of osteonectin-null mice and their wildtype controls.

Osteonectin function in bone was investigated by infrared analysis of bones from osteonectin-null (KO) and wildtype mice (four each at 11, 17, and 36 weeks). An increase in mineral content and crystallinity in newly formed KO bone and collagen maturity at all sites was found using FTIR microspectroscopy and imaging; consistent with osteonectin's postulated role in regulating bone formation and remodeling. Mineral and matrix properties of tibias of osteonectin-null mice and their age- and background-matched wildtype controls were compared using Fourier-transform infrared microspectroscopy (FTIRM) and infrared imaging (FTIRI) at 10- and 7-mm spatial resolution, respectively. The bones came from animals that were 11, 17, and 36 weeks of age. Individual FTIRM spectra were acquired from 20 x 20 microm areas, whereas 4096 simultaneous FTIRI spectra were acquired from 400 x 400 microm areas. The FTIRM data for mineral-to-matrix, mineral crystallinity, and collagen maturity were highly correlated with the FTIRI data in similar regions. In general, the osteonectin-null mice bones had higher mineral contents and greater crystallinity (crystal size and perfection) than the age-matched wildtype controls. Specifically, the mineral content of the newly forming periosteal bone was increased in the osteonectin-null mice; the crystallinity of the cortical bone was decreased in all but the oldest animals, relative to the wildtype. The most significant finding, however, was increased collagen maturity in both the cortical and trabecular bone of the osteonectin-null mice. These spectroscopic data are consistent with a mechanism of decreased bone formation and remodeling.     

Matrix Vesicles Promote Mineralization in a Gelatin Gel

Extracellular matrix vesicles (MVs) are associated with initial calcification in a variety of tissues, but the mechanisms by which they promote mineralization are not certain. In this study, MVs isolated from fourth passage rat growth plate chondrocyte cultures were included within a gelatin gel into which calcium and phosphate ions diffused from opposite ends. In this gel, apatite formation occurs by 3.5 days in the absence of mineralization promoters, allowing measurement of the ability of different factors to ``nucleate' apatite before this time or to assess the effects of molecules which modulate the rate and extent of mineral deposition. Mineral ion accumulation and crystal type are assayed at 5 days. In this study, MV protein content in the central band of a 10% gelatin gel was varied by including 100 μl of a Tris-buffered solution containing 0–300 μg/ml MV protein. There was a concentration-dependent increase in mineral accretion. Whereas 10 μg MV protein in the gel did not significantly promote apatite formation as compared with vesicle-free gels, 20 and 30 μg MV protein in the gel did promote apatite deposition. Inclusion of 10 mM β-glycerophosphate in the gels, along with MVs, did not significantly increase apatite formation despite the demonstrable alkaline phosphatase activity of the MVs. In contrast, MVs at all concentrations significantly increased apatite accumulation when proteoglycan aggregates or ATP, inhibitors of apatite formation and proliferation, were included in the gel. Slight increases in calcium, but not phosphate accumulation, were also noted when an ionophore was included with the MVs to facilitate Ca ion transport into the vesicles. FT-IR analysis of the mineral formed in the vesicle-containing gels revealed the presence of a bone-like apatite. These data suggest that MVs facilitate mineralization by providing enzymes that modify inhibitory factors in the extracellular matrix, as well as by providing a protected environment in which mineral ions can accumulate. Received: 28 January 1996 / Accepted: 9 August 1996     

Gastro-oesophageal reflux disease: Medical or surgical treatment? Report of an interactive workshop

Gastroesophageal reflux disease (GERD) is the most common disease of the upper gastrointestinal tract. With the introduction of proton pump inhibitors medical treatment of GERD has been significantly improved. However, the development of laparoscopic antireflux surgery resulted in an increasing interest of surgeons in this disease. An interactive meeting was organized in order to develop an agreement between gastoenterologists and surgeons regarding therapeutic decisions and this is the main topic of this paper.     

Beta-defensin genomic copy number is not a modifier locus for cystic fibrosis

Human beta-defensin 2 (DEFB4, also known as DEFB2 or hBD-2) is a salt-sensitive antimicrobial protein that is expressed in lung epithelia. Previous work has shown that it is encoded in a cluster of beta-defensin genes at 8p23.1, which varies in copy number between 2 and 12 in different individuals. We determined the copy number of this locus in 355 patients with cystic fibrosis (CF), and tested for correlation between beta-defensin cluster genomic copy number and lung disease associated with CF. No significant association was found.     

Missense FGFR3 mutations create cysteine residues in thanatophoric dwarfism type I (TD1)

Thanatophoric dwarfism (TD) is a sporadic lethal skeletal dysplasia with micromelic shortening of the limbs, macrocephaly, platyspondyly and reduced thoracic cavity. In the most common subtype (TD1), femurs are curved, while in TD2, straight femurs are associated with cloverleaf skull. Mutations in the fibroblast growth factor receptor 3 (FGFR3) gene were identified in both subtypes. While TD2 was accounted for by a single recurrent mutation in the tyrosine kinase 2 domain, TD1 resulted from either stop codon mutations or missense mutations in the extracellular domain of the gene. Here, we report the identification of FGFR3 mutations in 25/26 TD cases. Two novel missense mutations (Y373C and G370C) were detected in 8/26 and 1/26 TD1 cases respectively. Both mutations created cysteine residues in the juxta extramembrane domain of the receptor. Sixteen cases carried the previously reported R248C (9/26 cases), S249C (2/26 cases) or stop codon FGFR3 mutations (5/26 cases). Our results suggest that TD1 is a genetically homogeneous condition and give additional support to the view that newly created cysteine residues in the extracellular domain of the protein play a key role in the severity of the disease.      

Morphological analysis of degeneration and regeneration of syncytiotrophoblast in first trimester placental villi during organ culture

We have recently shown using dansyl-L-lysine exclusion studies that the release of human chorionic gonadotrophin (HCG) in conjunction with L- lactate dehydrogenase (LDH) from first trimester villi during organ culture is symptomatic of syncytiotrophoblast degeneration. The purpose of this study was to examine chorionic villi at the ultrastructural level in order to determine events occurring during organ culture. The tissue was sampled after 0, 24, 48 and 120 h in culture and processed for electron microscopy. In addition to confirming the previously recorded syncytial degeneration, the electron micrographs showed clearly the generation of a new syncytiotrophoblast layer. The new layer, derived from differentiating cytotrophoblast cells, was largely formed by 48 h and was maintained for at least 120 h in culture. This study demonstrates a model which provides an opportunity to study the differentiation of cytotrophoblast cells whilst they retain their anatomical relationships within the villous structure.