The primary use of vascular corrosion casting has been and likely will continue to be for describing the 3-dimensional anatomy of the microvasculature of organs and tissues. In these studies the assumption is made that the corrosion cast faithfully replicates the anatomy, dimensions, and distribution of the vasculature of these tissues and organs. If this assumption is accurate, and it appears to be nearly so in tissues where measurements have been made, it follows that the casts should lend themselves to quantitative measurements. I summarize here some simple quantitative measurements we have made from corrosion casts. For these studies, cannulas were inserted into arteries leading to the organ of interest in anesthetized animals, and the blood was removed by flushing with Ringer solution or buffered saline, at physiological temperatures and pressures. Resin consisting of Mercox (or Mercox diluted with methyl methacrylate monomer) and catalyst was infused via the same cannula until the onset of polymerization.

The interaction of cells with biomaterials is influenced by a number of factors including the chemical composition, surface charge, hydrophobicity, porosity and texture of the biomaterial. Changes in texture alone have been shown to result in reduced platelet adhesion on ventricular assist devices, reduced scar tissue formation around implanted prostheses, and enhanced biocompatability of vascular stents. In vitro growth of certain cells requires extracellular matrix components to provide the right physical and chemical stimuli for adherence, growth and differentiation. What role the texture of these materials plays, and the mechanisms responsible for the effects of substrate texture on the behavior of cells are not well understood. Previously we have used microcorrosion casting technology to replicate lymph node and other vasculature. More recently, we have used corrosion casting to replicate of the subendothelial matrix exposed when cells are damaged due to the passage of air emboli through a vessel.

Several authors have been studying the microvascularization pattern of the ovary in different animals. To determine the microvascularization pattern using the corrosive-resin casting method, casts of the vascular network were prepared and examined by scanning electron microscopy. However, the same procedure has rarely been used to study the microvascularization pattern of the uterine tube itself. The microvascularization of the uterine tube has always been associated microvascularization studies of the ovary. This study analyses the microvascularization of the uterine tube, pointing out differences between the region of the tube itself and it's infundibulum. In this study we used uterine tubes from adult female rabbits. Anesthesia was carried out with Ketalar (5mg/kg body weight); after that saline solution and 2,5% glutaraldehyde were injected through the left ventricle. This procedure was followed by mercox 2B injection. After polymerization the samples were collected and immersed in warm water (60°C). Then samples underwent tissue digestion.

The purpose of this study has been to undertake a three-dimensional analysis of the vascular network of the lamina propria in the lingual mucosa of the rat. Using the corrosive-resin casting method, casts of the vascular network were prepared and examined by scanning electron microscopy. We used 21th day normal wistar rats. They were anesthetized and perfused with saline solution and after with mercox (25ml of resin, 5ml of methyl methacrylate monomer). Then, the tongues were resected and placed in warm water bath at 60° C for 4 h. The tongue epithelium were digested with 20% NaOH for 4 days. The vascular resin casts washed in distilled water, several times. The dried vascular casts were mounted on the stubs with conductive paste, coated with gold and examined with a JEOL JSM- 6100 scanning electron microscope. In the SEM images, the lingual mucosa revealed arteriolae which divide into smaller branches entering into connective tissue papillae.

The application of two or more imaging-based technologies to portions of a sample prepared in parallel or preferably to the exact same sample in series is the basis for correlative microscopy. Simultaneous visualization of structure in conjunction with other cellular parameters such as cell motility, intracellular ion concentration, receptor expression, and protein distribution/movement can be followed in living cells by various forms of photon, acoustic, and force based microscopy. The same cells can subsequently be prepared by various physical or chemical based procedures and reexamined by other microscopic methodologies to gain additional qualitative and quantitative information often at significantly increased levels of resolution. Recent advances key to correlative microscopy have been the development of high sensitivity, high resolution video cameras, and computer image processing, image enhancement, image analysis, and reconstruction capabilities. New instrumentation such as video enhanced, rectified interference-based light microscopy,LM; confocal LM, and two and three photon LM also have been critical.

Atomic force microscopy (AFM) provides unique opportunities to study cell-surface and molecular scale interactions in three dimensions under aqueous conditions. Two plasma proteins, von Willebrand Factor (vWf) and fibrinogen, play central roles in the regulation of hemostasis and thrombosis by participating in coagulation and facilitating adhesion and aggregation of activated platelets. vWf and fibrinogen are believed to facilitate platelet adhesion under regions of relatively high and low vascular wall shear stress, respectively. Consequently, elucidating vWf and fibrinogen structure-function relations under shear is of considerable importance in developing a comprehensive understanding of the pathophysiology of thrombogenesis. Previously, we reported on molecular level AFM images of human vWf during shear-induced structural transition and human fibrinogen under aqueous conditions when both proteins were adsorbed on a hydrophobic surface. This presentation will report on the shear dependent interactions of dimeric and multimeric vWf with a hydrophobic surface and the substrate-dependent interactions of fibrinogen.

Although significant advances have been made in the development of biocompatible materials, currently available biomaterials still present a number of problems for in vivo applications. One of the attempts to improve the biocompatibility, especially blood-compatibility, of biomaterials has been surface modification. Typically, poly(ethylene glycol) (PEG), albumin, heparin, and phospholipid molecules are grafted to the surface to prevent protein adsorption and cell adhesion. We have been modifying biomaterial surfaces by covalent grafting of PEG and albumin. The control and modified surfaces were examined using an atomic force microscope (AFM). In this study, we examined the surface topography changes by surface modification using PEO grafting to glass as a model system.

Glass surfaces were modified with PEO using (N-triethoxysilylpropyl)-Omonomethoxy PEG urethane (PEG-Si), a PEG derivative containing a hydrophobic carbon chain and triethoxysilyl group at one end of the PEG chain. The presence of the hydrophobic carbon chain allowed self-assembly on the surface and triethoxysilyl resulted in covalent bonding to glass surfaces

The ability to adhere biomaterials to tooth tissue has drastically changed today's dental restorative practice. Modern adhesive techniques enable dentists to restrict operative procedures to the sole removal of diseased tooth tissue without further destruction of the remaining sound tissue. However, adhesive restorations have only a short lifetime of ca. 5 years. Extending the restoration lifetime is therefore one of the primary goals of present research in dental materials science. In practical relevance and direct benefit to that objective are studies that contribute to a better understanding of the interfacial phenomena that take place between restorative material and tooth tissue. Therefore, the resolution of instruments available to elucidate this interfacial interaction is essential. Besides the transmission electron microscope (TEM), a more recent powerful tool to study tooth-biomaterial interaction with high resolution is the atomic force microscope (AFM).

When studying connective tissues, it is often necessary to develop an understanding of their internal mechanics. In the aortic valve, studying of the internal strains of the leaflet material has provided insight into the means through which these largely avascular tissues survive up to 3 billion loading cycles during their functional lifespan. Even the inert, “dead” bioprosthetic valve replacements can withstand hundreds of millions of cycles before wearing out. The durability of the natural and prosthetic valves is therefore highly dependent on their microstructure and internal micromechanics.

Conventional microscopy has shown the aortic valve leaflet to consist of two structural layers, the fibrosa and ventricularis, separated by a loose central spongiosa rich in proteoglycans (figure 1). Both layers are highly organized, consisting of layers of elastin and collagen fibers. As the valve leaflets stretch and bend during opening and closure, these collagen fiber layers elongate, compress and shear in response to the applied external forces.

Septacin is a sustained-release antibiotic currently under development by the Hospital Products Division of Abbott Laboratories. The product is designed to be used as an anti-infective implant in orthopedic surgical procedures with a sustained drug release for up to six weeks in vivo. It consists of gentamicin sulfate formulated with a bioerodable polyanhydride copolymer. The polymer is biodegradable and has been approved by the FDA for human clinical trials. The final product is obtained by mixing 20% gentamicin sulfate with molten polymer and injection molding it to form cylindrical Septacin beads.

The microstructure of drug particles and polymer matrix is critical to the performance of sustained release products, thus scanning electron microscopy (SEM) and polarized light microscopy (PLM) were utilized in this study. SEM has proven useful for evaluating the microstructure of drug formulations3 and was used to examine the drug-polymer matrix structure. Average drug particle size and distribution were determined, and the drug-polymer boundary was evaluated.

Prosthetic mechanical heart valves (MHVs) made from pyrolytic carbon (PYC) are a highly successful biomaterial application due to their excellent durability and clinically adequate blood compatibility. Their thromboresistance has been attributed to a combination of hemodynamics, material inertness, and surface properties which minimize platelet adhesion and activation. These surface properties include that polished PYC is smooth and that it strongly adsorbs plasma albumin, which is well known to reduce platelet adhesion. However, recent evidence suggests that this explanation is incomplete. Low voltage SEM shows that PYC valve leaflets are actually quite rough at submicron scales and that PYC induces extensive platelet spreading in vitro in the presence of albumin. Surprisingly few studies have examined MHVs explanted from humans or research animals for platelet adhesion and thrombosis using SEM. Moreover, in most studies it appears that even routine sample preparation methods for the preservation of biological samples for SEM,

Introduction

We have previously described a quantitative in vitro method for the assessment of bone matrix deposition, employing the fluorescent antibiotic tetracycline1. In a present study we demonstrate the relationship of bone matrix formation by osteoblasts as a function of cell seeding density, and time, on calcium phosphate ceramic thin film substrates.

Materials and Methods

Substrata. Commercially available 3x1 inch quartz Osteologic™ chamber slides (Millenium Biologix Inc., Kingston, Ont.) previously shown to support bone growth, were used as substrates for osteoblastic cell growth2. These chamber slides are coated with a sintered sub-micron calcium phosphate ceramic thin film, and are fitted with a removable 16 well superstructure.

Rat Bone Marrow Cell Culture. Bone marrow cells from young adult male Wistar rat (115-120g) femora were cultured according to a previously described protocol. Each well of each Osteologic™ chamber slide (14/16 wells; 2 wells contained media, but without cells, to provide background for tetracycline fluorescence) was inoculated with a 250 μl of cell suspension.

We have developed an in vitro system to study the process by which osteoblasts attach and secrete extracellular matrix (ECM) proteins that subsequently mineralize in response to implant materials. In vivo mineralization is characterized by the sequential expression by osteoblasts of specific ECM proteins that are capable of calcifying in an orderly manner. A similar sequence of events is shown to occur in this in vitro system. The extent and pattern of mineralization was visualized by fluorescence light microscopy and transmission electron microscopy (TEM), and was dependent on the substrate to which osteoblasts adhered.

Materials and Methods

Disks (22 mm diameter) composed of Tivanium (Tiv) (Ti6AI4V, ASTM F-136) and Zimaloy (Zim)(CoCrMo, ASTM F-75), prepared identically to clinical orthopaedic implants, were supplied by Zimmer, Inc. Glass disks were used as controls. Osteoblast-like cells (Saos-2), derived from a human osteosarcoma, or primary human osteoblasts from a 41 year-old male patient were added to the disks in a-MEM medium with 10% fetal bovine serum,

Particulate wear debris found around joint arthroplasties has been linked to the premature failure of artificial joints. Cytokines and eicosanoids released by macrophages are known mediators of inflammation and osteolysis. Previous in vitro studies of wear debris-mediated inflammation have relied upon poorly characterized particulates and peripheral blood cell fractions of mixed cellular composition. The objective of this study was to utilize titanium particulates that closely match wear debris found around joint arthroplasties and to study their effects on the biosynthesis of inflammatory mediators by isolated monocytes.

To mimic wear debris that surrounds artificial joints, titanium alloy particulates (1-3 μm in diameter) were obtained. A submicron fraction of these particulates was isolated by differential sedimentation. The morphology, size and concentration of the resulting population was characterized utilizing scanning electron microscopy. Titanium particulate size was estimated from photomicrographs of calibrated latex microspheres mixed with titanium particles (Figure 1). A known volume of titanium particulates in suspension was applied to a carbon planchette and allowed to dry.

The human tooth is a functionally-gradient structural composite material composed of collagen and hydroxyapatite (HAP). The outer most region is a hard enamel layer consisting primarily of HAP. Underneath is dentin region, which is composed of HAP with a significant fraction of collagen fibers. It has been demonstrated that it is possible to perform nano-indentation on sub-micron regions of tooth using either a standard nanoindentor or modified cantilever type atomic force microscope tip (AFM). In this study, we report nano-mechanical properties of dentin and enamel regions of a human tooth, measured with a nanoindentor attached to an AFM using a vertical force, in correlation with the microstructures observed with TEM.

TEM and nano-indentation samples were prepared from a juvenile human incisor tooth. The tooth was first sectioned into 1 mm thick slices along its length, each containing both dentin and enamel regions. A slice was taken to prepare a TEM sample and an adjacent slice was used for nano-indentation measurements.

Current dental restorative materials include tooth-colored materials called composite resins, which are made of a resin matrix and an inorganic filler coated with a silane coupling agent. Composite resins can shrink considerably upon setting. This shrinkage stress can generate gaps along the composite/dentin interface. Low-viscosity fluid resins and primers have been used to bond composite resins to dentin. The adhesive resin layer may be able to act as an elastic medium and compensate for the polymerization stresses that develop within the composite resin. The hypothesis tested in this study was that the use of a “stress-absorbing” intermediary dentin adhesive would result in lower incidence of gaps at the restoration/cavity interface and in a thicker resin/dentin interdifusion layer or hybrid layer.

Dentin disks with a thickness of 900 um±100 μm were obtained from extracted human molars and assigned to two adhesive systems (One-Step [OS] and Single Bond [SB]), three degrees of intermediary “stress-absorbing” treatments (none, Scotchbond Multi-Purpose [SBMP], and OptiBond FL [OB]) and two composite resins (Ælite-Flo [ÆF] and Z100

Alphaviruses are a group of enveloped viruses in the Togaviridae family. Studies of several alphaviruses, including Ross River, Sindbis and Semliki Forest viruses, by cryo-electron microscopy (cryo-EM), three-dimensional (3D) image resconstruction and other techniques have illustrated that these spherical viruses have a T=4, multi-layered structure.

Aura virus, which is closely related to Sindbis, was first isolated in South America. Unlike the other alphaviruses, both genomic RNA (12kb, 49S) and subgenomic RNA(4.2kb, 26S) are encapsidated efficiently and form mature virions. Studies on negatively-stained virus particles demonstrated that there are two major size classes. The first contains particles of ∼72nm diameter, which are most similar to wild type virus, whereas the second class includes particles of ∼62nm in diameter. The 72nm particles are believed to have one copy of genomic RNA or one to three copies of subgenomic RNA, and a T=4 structure. The 62nm particles probably only have a single copy of subgenomic RNA and are presumed to be T=3 structures.

Paramecium bursaria Chlorella virus l(PBCV-l), the type species of the Pycodnaviridae family, is a large, plaque-forming virus that replicates in certain unicellular, exsymbiotic, Chlorella-like green algae. The virion (∼lxl09 daltons) has a 330,740 base pair dsDNA genome encapsulated within an icosahedral shell of ∼190nm diameter. Purified virions contain more than 50 different proteins, which account for 65% of the total virion mass and range in size from 10 to more than 200kDa. Vp54, the major capsid protein (40% of total virion protein) is a myristilated glycoprotein but it is not phosphorylated. Three other proteins are located on the virus surface. Virions contain 5-10% lipid, located inside the glycoprotein shell, which is required for virus infectivity. Little is known about the detailed ultrastructure of PBCV-1 virions, though some information has been obtained with sectioned, negatively-stained, and metal-shadowed specimens. We have used cryo-electron microscopy and three-dimensional (3D) image reconstruction methods to examine the native morphology of PBCV-1.

Several structures of the Ryanodine receptor in different functional states with various bound ligands have been determined using electron cryo-microscopy in recent years. These structures have varied in resolution from 25-40Å, however, to date, none have been corrected for the CTF (contrast transfer function) of the microscope. The CTF can have a significant effect on the detailed structure of the molecule, causing features in the 40-70Å range to be unnaturally emphasized. This causes small holes to become larger and small protrusions to become elongated. Unfortunately due to zero points in the CTF, correction cannot be properly accomplished with individual images. Several approaches to correcting the CTF have been proposed and/or used by combining information from images taken at different defocus settings. We have taken the approach of performing CTF correction as a preprocessing step. There are both advantages and disadvantages to this method. There are 2 primary advantages: First, by combining data from identical regions of focal pairs,

The eukaryotic 20S proteasome (700 kDa) serves as the core complex for the breakdown of most cytosolic proteins by the ubiquitin-dependent pathway. This cylinder-shaped proteolytic assembly (17 nm long, 11 nm diameter) consists of four stacked heptameric rings that help to form a central cavity where degradation occurs. The catalytic sites are located on the cavity surface of the two middle β rings while the outer α rings serve as docking sites for various regulatory complexes.

PA700 (700 kDa, 20 protein subunits) is the major regulatory complex of the proteasome, forming a “cap” that attaches to either or both proteasome ends. The resulting superassembly structurally resembles the larger, more active 26S proteasome and exhibits both a higher intrinsic peptidase activity as well as a wider range of substrate specificity, including ubiquitin-tagged proteins. A second regulatory complex, the modulator (300 kDa), further stimulates the effects of PA700 at subsaturating concentrations of the activator with no detectable effect on the proteasome in the absence of PA700.