The effect of a slow mode of BMP-2 delivery on the inflammatory response provoked by bone-defect-filling polymeric scaffolds

We investigated the inflammatory response to, and the osteoinductive efficacies of, four polymers (collagen, Ethisorb, PLGA and Polyactive) that bore either an adsorbed (fast-release kinetics) or a calcium-phosphate-coating-incorporated (slow-release kinetics) depot of BMP-2. Titanium-plate-supported discs of each polymer (n = 6 per group) were implanted at an ectopic (subcutaneous) ossification site in rats (n = 48). Five weeks later, they were retrieved for a histomorphometric analysis of the volumes of ectopic bone and foreign-body giant cells (a gauge of inflammatory reactivity), and the degree of polymer degradation. For each polymer, the osteoinductive efficacy of BMP-2 was higher when it was incorporated into a coating than when it was directly adsorbed onto the material. This mode of BMP-2 carriage was consistently associated with an attenuation of the inflammatory response. For coated materials, the volume density of foreign-body giant cells was inversely correlated with the volume density of bone (r(2) = 0.96), and the volume density of bone was directly proportional to the surface-area density of the polymer (r(2) = 0.97). Following coating degradation, other competitive factors, such as the biocompatibility and the biodegradability of the polymer itself, came into play.

Rapidly excised and cryofixed rat tissue

All preparation efforts of biological samples in electron microscopy are focused to preserve structures as close as possible to the native state. To achieve this goal with tissues, it is of advantage to have a very short time between excision and fixation. The most common approach is chemical fixation: cross-linking of the tissue samples with aldehydes followed by postfixation with osmium tetroxide. Here, the fastest approach for tissue samples is perfusion. However, the diffusion of the fixation solution from blood vessels into the depth of the tissue is still slow and does not allow an overall instant fixation of a single cell. As a result, osmotic effects become evident (swelling or shrinkage of cell organelles). Another possibility is to take a tissue sample from the experimental animal. Excision of tissue can last quite some time, which results in even more pronounced autolytic induced osmotic effects. Furthermore, the animal does not survive the procedure in most cases. Alternatively, microbiopsies are an elegant technique to rapidly excise small quantities of tissue. Some tissues, such as liver and muscle, may be obtained using a non-lethal approach. To avoid the artifacts introduced by chemical fixation, high-pressure freezing of microbiopsies (brain, liver, kidney, and muscle) is a powerful alternative to chemical fixation. Here, we describe the microbiopsy method, and high-pressure freezing/freeze-substitution (HPF/FS) as a follow-up procedure. Cryosectioning of high-pressure frozen samples is optimally preserving the ultrastructure; however, it is not considered to be a routine approach yet.

Lamellar body ultrastructure revisited: high-pressure freezing and cryo-electron microscopy of vitreous sections

Lamellar bodies are the storage sites for lung surfactant within type II alveolar epithelial cells. The structure-function models of lamellar bodies are based on microscopic analyses of chemically fixed tissue. Despite available alternative fixation methods that are less prone to artifacts, such as cryofixation by high-pressure freezing, the nature of the lung, being mostly air filled, makes it difficult to take advantage of these improved methods. In this paper, we propose a new approach and show for the first time the ultrastructure of intracellular lamellar bodies based on cryo-electron microscopy of vitreous sections in the range of nanometer resolution. Thus, unspoiled by chemical fixation, dehydration and contrasting agents, a close to native structure is revealed. Our approach uses perfluorocarbon to substitute the air in the alveoli. Lung tissue was subsequently high-pressure frozen, cryosectioned and observed in a cryo-electron microscope. The lamellar bodies clearly show a tight lamellar morphology. The periodicity of these lamellae was 7.3 nm. Lamellar bifurcations were observed in our cryosections. The technical approach described in this paper allows the examination of the native cellular ultrastructure of the surfactant system under near in vivo conditions, and therefore opens up prospectives for scrutinizing various theories of lamellar body biogenesis, exocytosis and recycling.

Muscle unloading potentiates the effects of acetyl-L-carnitine on the slow oxidative muscle phenotype

The effect of acetyl-L-carnitine (ALCAR) supplementation to 3-month-old rats in normal-loading and unloading conditions has been here investigated by a combined morphological, biochemical and transcriptional approach to test whether ALCAR might cause a remodeling of the metabolic/contractile phenotype of soleus muscle. Morphological assessment demonstrated an increase of type I oxidative fiber content and cross-sectional area in ALCAR-treated animals both in normal-loading and in unloading conditions. ALCAR prevented loss of mitochondrial mass in unloaded animals whereas no ALCAR-dependent increase of mitochondrial mass occurred in normal-loaded muscle. Validated microarray analysis delineated an ALCAR-induced maintenance of a slow-oxidative expression program only in unloaded soleus muscle. Indeed, the muscle adjustment of the expression profile of factors underlying mitochondrial oxidative metabolism, protein turnover, fiber type differentiation and an adaptation of voltage-gated ion channel expression was distinguishable with respect to the loading status. This selectivity may suggest a key role of muscle loading status in the manifestation of ALCAR effects. The results extend to a broader level of biological informations the previous notion on ALCAR positive effect in rat soleus muscle during unloading and point to a role of ALCAR for the maintenance of its slow-oxidative fiber character.

Genetic characterization of slow bee paralysis virus of the honeybee (Apis mellifera L.)

Complete genome sequences were determined for two distinct strains of slow bee paralysis virus (SBPV) of honeybees (Apis mellifera). The SBPV genome is approximately 9 5 kb long and contains a single ORF flanked by 5'- and 3'-UTRs and a naturally polyadenylated 3' tail, with a genome organization typical of members of the family Iflaviridae The two strains, labelled `Rothamsted' and 'Harpenden', are 83% identical at the nucleotide level (94% identical at the amino acid level), although this variation is distributed unevenly over the genome. The two strains were found to co-exist at different proportions in two independently propagated SBPV preparations The natural prevalence of SBPV for 847 colonies in 162 apiaries across five European countries was <2%, with positive samples found only in England and Switzerland, in colonies with variable degrees of Varroa infestation

Dorsal rather than ventral visual pathways discriminate freezing status in Parkinson's disease

BACKGROUND: Although visuospatial deficits have been linked with freezing of gait (FOG) in Parkinson's disease (PD), the specific effects of dorsal and ventral visual pathway dysfunction on FOG is not well understood. METHOD: We assessed visuospatial function in FOG using an angle discrimination test (dorsal visual pathway bias) and overlapping figure test (ventral visual pathway bias), and recorded overall response time, mean fixation duration and dwell time. Covariate analysis was conducted controlling for disease duration, motor severity, contrast sensitivity and attention with Bonferroni adjustments for multiple comparisons. RESULTS: Twenty seven people with FOG, 27 people without FOG and 24 controls were assessed. Average fixation duration during angle discrimination distinguished freezing status: [F (1, 43) = 4.77 p < 0.05] (1-way ANCOVA). CONCLUSION: Results indicate a preferential dysfunction of dorsal occipito-parietal pathways in FOG, independent of disease severity, attentional deficit, and contrast sensitivity.

Au@Hg nanoalloy formation through direct amalgamation: Structural, spectroscopic, and computational evidence for slow nanoscale diffusion

Dynamic core-shell nanoparticles have received increasing attention in recent years. This paper presents a detailed study of Au-Hg nanoalloys, whose composing elements show a large difference in cohesive energy. A simple method to prepare Au@Hg particles with precise control over the composition up to 15 atom% mercury is introduced, based on reacting a citrate stabilized gold sol with elemental mercury. Transmission electron microscopy shows an increase of particle size with increasing mercury content and, together with X-ray powder diffraction, points towards the presence of a core-shell structure with a gold core surrounded by an Au-Hg solid solution layer. The amalgamation process is described by pseudo-zero-order reaction kinetics, which indicates slow dissolution of mercury in water as the rate determining step, followed by fast scavenging by nanoparticles in solution. Once adsorbed at the surface, slow diffusion of Hg into the particle lattice occurs, to a depth of ca. 3 nm, independent of Hg concentration. Discrete dipole approximation calculations relate the UV-vis spectra to the microscopic details of the nanoalloy structure. Segregation energies and metal distribution in the nanoalloys were modeled by density functional theory calculations. The results indicate slow metal interdiffusion at the nanoscale, which has important implications for synthetic methods aimed at core-shell particles.

Osseointegration: the slow delivery of BMP-2 enhances osteoinductivity

Although the placement of dental and orthopedic implants is now generally a safe, reliable and successful undertaking, the functional outcome is less assured in patients whose bone-healing capacity is compromised. To enhance peri-implant osteogenesis in these individuals, BMP-2 could be locally administered. However, neither a free suspension nor an implant-adsorbed depot of the agent is capable of triggering sustained bone formation. We hypothesize that this end could be achieved by incorporating BMP-2 into the three-dimensional crystalline latticework of a bone-mineral like, calcium-phosphate implant coating, where from it would be liberated gradually - as the inorganic layer undergoes osteoclast-mediated degradation - not rapidly, as from an implant-adsorbed (two-dimensional) depot. To test this postulate, we compared the osteoinductive efficacies of implant coatings bearing either an incorporated, an adorbed, or an incorporated and an adsorbed depot of BMP-2 at a maxillary site in miniature pigs. The implants were retrieved 1, 2 and 3 weeks after surgery for the histomorphometric analysis of bone formation within a defined 'osteoinductive' space. At each juncture, the volume of newly-formed bone within the osteoinductive space was greatest around implants that bore a coating-incorporated depot of BMP-2, peak osteogenic activity being attained during the first week and sustained thereafter. In the other groups, the temporal course of bone formation was variable, and the peak levels were not sustained. The findings of this study confirm our hypothesis: they demonstrate that we now have at our disposal a means of efficaciously augmenting and expediting peri-implant bone formation. Clinically, this possibility would render the process of implant placement a safer and a more reliable undertaking in patients whose bone-healing capacity is compromised, and would also permit a curtailment of the postoperative recovery period by a forestallment of the mechanical-loading phase.

Safe high-pressure freezing of infectious micro-organisms

We describe how high-pressure freezing of infectious biological material can safely be accomplished with the help of membrane carriers. The method described is easy to perform; however, careful manipulations are required. Existing safety regulations must still be followed. However, the procedure reduces the risk of dissemination of infectious material.