Mutations in ANTXR1 cause GAPO syndrome.

The genetic cause of GAPO syndrome, a condition characterized by growth retardation, alopecia, pseudoanodontia, and progressive visual impairment, has not previously been identified. We studied four ethnically unrelated affected individuals and identified homozygous nonsense mutations (c.262C>T [p.Arg88*] and c.505C>T [p.Arg169*]) or splicing mutations (c.1435-12A>G [p.Gly479Phefs*119]) in ANTXR1, which encodes anthrax toxin receptor 1. The nonsense mutations predictably trigger nonsense-mediated mRNA decay, resulting in the loss of ANTXR1. The transcript with the splicing mutation theoretically encodes a truncated ANTXR1 containing a neopeptide composed of 118 unique amino acids in its C terminus. GAPO syndrome's major phenotypic features, which include dental abnormalities and the accumulation of extracellular matrix, recapitulate those found in Antxr1-mutant mice and point toward an underlying defect in extracellular-matrix regulation. Thus, we propose that mutations affecting ANTXR1 function are responsible for this disease's characteristic generalized defect in extracellular-matrix homeostasis.

Nanocomposite Ti/hydrocarbon plasma polymer films from reactive magnetron sputtering as growth support for osteoblast-like and endothelial cells.

Nanocomposite Ti/hydrocarbon plasma polymer (Ti/ppCH) films were deposited by DC magnetron sputtering of titanium target in n-hexane, argon, or a mixture of these two gases. The resultant films were heterogeneous, with inorganic regions of nanometer scale distributed within a plasma polymer matrix. The titanium content was controlled by adjusting the argon/n-hexane ratio in the working gas. In the pure n-hexane atmosphere, the Ti concentration was found to be below 1 at %, whereas in pure argon it reached 20 at %, as measured by Rutherford backscattering spectroscopy and elastic recoil detection analysis (RBS/ERDA). A high level of titanium oxidation is detected with TiO(2), substoichiometric titania, and titanium carbide, composing an inorganic phase of the composite films. In addition, high hydrogen content is detected in films rich with titanium. Ti-deficient and Ti-rich films proved equally good substrates for adhesion and growth of cultured human osteoblast-like MG 63 cells. In these cells, the population densities on days 1, 3, and 7 after seeding, spreading area on day 1, formation of talin-containing focal adhesion plaques as well as concentrations of talin and osteocalcin (per mg of protein) were comparable to the values obtained in cells on the reference cell culture materials, represented by microscopic glass coverslips or a polystyrene dish. An interesting finding was made when the Ti/ppCH films were seeded with calf pulmonary artery endothelial cells of the line CPAE. The cell population densities, the spreading area and also the concentration of von Willebrand factor, a marker of endothelial cell maturation, were significantly higher on Ti-rich than on Ti-deficient films. On Ti-rich films, these parameters were also higher or similar in comparison with the reference cell culture materials. Thus, both types of films could be used for coating bone implants, of which the Ti-rich film remains effective in enhancing the endothelialization of blood contacting artificial materials.

The influence of pore size on colonization of poly(L-lactide-glycolide) scaffolds with human osteoblast-like MG 63 cells in vitro.

A degradable copolymer of L-lactide and glycolide (PLG) was synthesized by ring opening polymerization using zirconium acetylacetonate [Zr(acac)(4)] as a biocompatible initiator. The structure of the copolymer was studied by nuclear magnetic resonance spectroscopy (NMR) and gel permeation chromatography (GPC). Porous scaffolds of defined microstructure were prepared by solvent casting/salt particulate leaching, which resulted in the creation of three types of scaffolds with the same porosity (87%+/-1%) but with different diameters of the pores (600, 200 and 40 microm) and degree of interconnectivity. The potential of the scaffolds for cell colonization was tested in a conventional static cell culture system using human osteoblast-like MG 63 cells. As revealed by conventional fluorescence and confocal microscopy on days 5 and 7 after seeding, the cells on the scaffolds of large or medium pore size infiltrated the inside part of the material, whereas on the scaffolds of small pore size, the cells were retained on the material surface. On day 7 after seeding, the highest number of cells was found on the scaffolds of the largest pore size (more than 120,000 cells per sample of the diameter 15 mm and thickness 2 mm), whereas on the scaffolds with medium and smallest pore diameter, the number of cells was almost three times lower and similar for both pore sizes. These results corresponded well with the incorporation of bromodeoxyuridine into newly synthesized DNA, which was significantly higher in cells on scaffolds of the largest pore size than on the material with medium and smallest pore diameter. As indicated by the MTT test, the mitochondrial activity in cells on scaffolds with medium pore size was similar to that on the material with the highest pore size, and significantly higher than on scaffolds of the smallest pore diameter. These results suggest that PLG scaffolds with the largest pore diameter (600 microm) and better pore interconnectivity are the most suitable for colonization with osteogenic cells.