Preparation and Characterization of Biodegradable β-TCP-Based Composite Microspheres as Bone Tissue Engineering Scaffolds

Since a small globular particle was first used as support for three-dimensional (3D) growth of anchorage-dependent cells in suspended cultures, a variety of microspheres as tissue engineering scaffolds have been developed. In this paper, β-TCP and chitosan were selected as the components of microspheres due to their biodegradability and osteogenic properties. The biodegradable β-TCP/chitosan composite microspheres were prepared by a solid-in-water-in-oil (s/w/o) emulsion cross-linking method in this paper. The size distribution, surface morphology, and microstructure of the microspheres were evaluated. Scanning electron microscopy revealed that the size of the microspheres with good spherical morphology was distributed in the range of 50~200μm. In vitro immersion experiments were carried out to evaluate the degradability of the microspheres, and the results demonstrated that the chitosan/β-TCP composite microspheres were potential materials as tissue engineering scaffolds for bone repair.

Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose.

We identified four polypeptides of 47, 44, 40, and 35 kD that bind to profilin-Sepharose and elute with high salt. When purified by conventional chromatography using an antibody to the 47-kD polypeptide, these four polypeptides copurified as a stoichiometric complex together with three additional polypeptides of 19, 18, and 13 kD that varied in their proportions to the other polypeptides. Partial protein sequences showed that the 47-kD polypeptide is a homologue of S. pombe act2 and the 44-kD polypeptide is a homologue of S. cerevisiae ACT2, both unconventional actins. The 40-kD polypeptide contains a sequence similar to the WD40 motif of the G beta subunit of a trimeric G-protein from Dictyostelium discoideum. From partial sequences, the 35-, 19-, and 18-kD polypeptides appear to be novel proteins. On gel filtration the complex of purified polypeptides cochromatograph with a Stokes' radius of 4.8 nm, a value consistent with a globular particle of 220 kD containing one copy of each polypeptide. Cell extracts also contain components of the complex that do not bind the profilin column. Affinity purified antibodies localize 47- and 18/19-kD polypeptides in the cortex and filopodia of Acanthamoeba. Antibodies to the 47-kD unconventional actin cross-react on immunoblots with polypeptides of similar size in Dictyostelium, rabbit muscle, and conventional preparations of rabbit muscle actin but do not react with actin.

Brain dynein crossbridges microtubules into bundles

Cytoplasmic dynein was purified from pig brain, using a modified version of published procedures, in order to study its interaction with microtubules. Since the preparation produces ATP-dependent sliding of taxol-stabilized purified microtubules over glass and runs on SDS-containing gels as a major band exceeding 300,000 Mr plus a medium chain band at about 75,000 Mr, it is assumed to be identical to the mammalian brain dynein (MAP 1C) purified by Vallee and colleagues. When viewed by electron microscopy in negative stain, individual particles show two distinct configurations. Some are clearly similar to the two-headed bouquet structure already shown for MAP 1C. A larger number of molecules in the present preparation appear to have two heads fused together, forming a dimeric globular particle with two separate tails. They are referred to as phiparticles, because of their resemblance to the greek letter phi. A model for the structural relationship between the two molecular forms is presented. The stems of two associated dynein subunits may separate beyond the base, to form a bouquet, or they may remain fused to form the larger tail of a phi-particle. The smaller tail probably represents a combined pair of features equivalent to the ‘stalks’ shown to emanate from axonemal dynein heads by Goodenough and colleagues. Both tails of a phi-particle can bind to microtubules, even in the presence of ATP, and cause microtubule bundling. These results suggest a complete structural homology between axonemal and cytoplasmic dynein.

A Morphometric Freeze-Fracture-Etch (FFE) Analysis on The Nature of Intramembrane Particles

We have recently completed (J. Cell Biol, in press) a morphometric analysis of FFE preparations of a membrane fraction from mammalian urinary bladder epithelial cells that gives a new insight into the nature of intramembrane particles (IMPs). The preparation contains vesicles of a membrane with paracrystalline arrays of external particles in placques 0.2-0.5μm in diameter. In transverse sections of placques (Fig. l) particles ~65Å thick by 100Å wide are seen spaced ~140Å center-to-center making the lattice membrane ~130Å thick. The interplacque membrane is an ordinary unit membrane ~75Å thick. We have prepared specimens for FFE study by centrifuging vesicles onto a glass cover slip cationized with alcian blue. The unattached membrane fragments were washed away with water and a small copper disc was placed on the cover slip before freezing by immersion in liquid propane by a special technique giving unusually high freezing rates. he specimens were fractured under liquig nitrogen and freeze-dried at -100° in a Denton FFE unit operated at 5 x 10-8 Torr. Platinum-carbon replicas were prepared by arc sublimation at -193°C. Unusual fracture planes sometimes occurred revealing EF, PF and PS surfaces as in Fig. 7. Two distinctly different patterns were observed in the EF faces: 1) placques consisting of globular particles each ~100-120Å in diameter spaced in a regular lattice corresponding to the external particle lattice (Fig. 4 - GP and Fig. 7). 2) placques entirely free of such particles in which a regular domain pattern was found with a lattice constant of 160Å (Fig. 1 - SP). The repeating unit in the latter was a domain consisting of a partial ring of metal ~20-25Å thick surrounding a shadow in the center of which there was a particle of metal about 20-25Å in diameter (filtered image inset to lower left in Fig. 4). Diffraction patterns from each type of lattice (insets Fig. 1) show the same lattice constants with evidence of shadowing directionality in the smooth lattice (absent diffraction spots) and prominent decoration in the globular lattice to the right (equal 1,0 spots). At the edges of placques the smooth lattice domains cast smooth shadows onto the glass (Fig. 5) whereas the globular particles cast spiked shadows (Fig. 6). Using the glass surface as a reference and employing stereoscopic techniques, measurements were made of the heights of the globular particle and the smooth domain lattices above the glass. The particle heights were analysed statistically and it was found that the mean globular particle height above the glass was 163(±24)Å. The total mean thickness of the unfractured lattice membrane was 146(±14)Å. The mean height of the smooth domain lattice above the glass was found to be 108(±17)Å. A model derived from thin sections corrected for 150Å shrinkage predicts the total lattice membrane thickness to be 1508 and the interplacque membrane to be 86Å. The model predicts the height of the EF face above the glass to be 107Å. The measurements indicate that the smooth domain lattice could represent a real structure and that the globular particles are artifacts. The PF faces in these membranes was studied in whole cells as well as membranes fractured on glass and found to be devoid of pits of sufficient size to accomodate the globular EF particles. The pattern in the PF faces (Fig. 2) consisted of domains in a hexagonal lattice with a lattice constant of 160Å. In this case each domain consisted of a complete ring of metal about 40Å thick surrounding a shadow around a central spot of metal about 608 in diameter. Fig. 3 is a filtered image of Fig. 2 showing this. The EF and PF faces are complementary in the sense that they contain similar structures; however, the ring shadows in each are mirror images. The globular EF lattice pattern was completely uncomplementary to the PF lattice in keeping with the conclusion that the globular particle is an artifact. It is probably produced by a combination of plastic deformation and decoration. Supported by NIH Grant #5 P01 GM 23911.

Lipid-protein globules of avian egg yolk. Isolation and properties of globules stable in concentrated sodium chloride solution

A new type of globular particle, the ‘insoluble yolk globule’, was isolated from the egg yolk of three avian species (hen, duck, and emu) by centrifugation or gel-filtration chromatography. These globules are stable in NaCl and urea solutions at concentrations that dissolve or disrupt other constituents of yolk, The isolated globules are about 1% of the dry yolk of hen's and duck's eggs but about 8% emu's-egg yolk. Most of these globules are less than 2 micrometer in diameter. Electron micrographs of sections show a preponderance of globules in the range 0.125-0.25 micrometer, each with a thick shell surrounding a feature-less anterior. Globules with the same appearance were seen in sections of unfractionated yolk. Two kinds of larger particles were also observed: (i) particles with a distinct outer membrane and a vesiculated interior; (ii) featureless spheres, possibly of lipid. The insoluble yolk globules comprise protein (8-11% by dry wt.), phospholipid (31-35% total lipid), triacylglycerols (49-53%), cholesterol (8%) and cholesteryl esters (2-3%); the variations being among species. The phospholipid is accessible to phospholipase C. The isolated protein is heterogeneous and resembles the apoprotein from the yolk low-density lipoprotein.

MICROTUBULE SURFACE LATTICE AND SUBUNIT STRUCTURE AND OBSERVATIONS ON REASSEMBLY

Neuronal microtubules have been reassembled from brain tissue homogenates and purified. In reassembly from purified preparations, one of the first structures formed was a flat sheet, consisting of up to 13 longitudinal filaments, which was identified as an incomplete microtubule wall. Electron micrographs of these flat sheets and intact microtubules were analyzed by optical diffraction, and the surface lattice on which the subunits are arranged was determined to be a 13 filament, 3-start helix. A similar, and probably identical, lattice was found for outer-doublet microtubules. Finally, a 2-D image of the structure and arrangement of the microtubule subunits was obtained by processing selected images with a computer filtering and averaging system. The 40 x 50 Å morphological subunit, which has previously been seen only as a globular particle and identified as the 55,000-dalton tubulin monomer, is seen in this higher resolution reconstructed image to be elongated, and split symmetrically by a longitudinal cleft into two lobes.