Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo-and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used 1-4 , no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay 5,6 , (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye 7-9 , (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM 10-12 , (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice 13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM 14 .In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications.Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study. ProtocolAll equipment and reagents have to be purchased sterile or needs to be heat or steam sterilized or sterilized with 70% ETOH.The authors state that experiments on animals were performed in accordance with the European Communities Council Directive (86/609/EEC), following the Guidelines of the NIH regarding the care and use of animals for experimental procedures and ...

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The surface design of titanium implants influences not only the local biological reactions but also affects at least the clinical result in orthopaedic application. During the last decades, strong efforts have been made to improve osteointegration and prevent bacterial adhesion to these surfaces. Following the rule of “smaller, faster, cheaper”, nanotechnology has encountered clinical application. It is evident that the hierarchical implant surface micro- and nanotopography orchestrate the biological cascades of early peri-implant endosseous healing or implant loosening. This review of the literature gives a brief overview of nanostructured titanium-base biomaterials designed to improve osteointegration and prevent from bacterial infection.

Nanoparticle (NP)-based delivery has gained importance for improving the potency of therapeutic agents. The bovine serum albumin (BSA) NPs, obtained by a coacervation process, was modified by electrostatic adsorption of cationic polyethylenimine (PEI) to NP surfaces for delivery of bone-inducing growth factor, bone morphogenetic protein-2 (BMP-2). Different concentrations of PEI were utilized for coating BSA NPs to stabilize the colloidal system and to control the release of BMP-2. The NPs were characterized by size and zeta potential measurements, as well as by Scanning Electron Microscopy and Atomic Force Microscopy. The encapsulation efficiency was typically [90% in all NP preparations. In vitro release kinetics showed that the PEI concentration used for coating the NPs efficiently controlled the release of BMP-2, demonstrating a gradual slowing, sustained release pattern during a 10-day study period. The bioactivity of the encapsulated BMP-2 and the toxicity of the NPs were examined by the alkaline phosphatase (ALP) induction assay and the MTT assay, respectively, using C2C12 cells. The results indicated that PEI was the primary determinant of NP toxicities, and BSA NPs coated with 0.1 mg/mL PEI demonstrated tolerable toxicity, retained the bioactivity of BMP-2, and efficiently slowed the release rate of BMP-2. We conclude that BMP-2 encapsulated in BSA NPs might be an efficient way to deliver the protein for in vivo bone induction.

Contrary to widespread belief, the regulation and mechanism of degradation for the mass of intracellular proteins (i.e. differential, selective protein turnover) in vertebrate tissues is still a major biological enigma. There is no evidence for the conclusion that ubiquitin plays any role in these processes. The primary function of the ubiquitin-dependent protein degradation pathway appears to lie in the removal of abnormal, misfolded, denatured or foreign proteins in some eukaryotic cells. ATP/ubiquitin-dependent proteolysis probably also plays a role in the degradation of some so-called 'short-lived' proteins. Evidence obtained from the covalent modification of such natural substrates as calmodulin, histones (H2A, H2B) and some cell membrane receptors with ubiquitin indicates that the reversible interconversion of proteins with ubiquitin followed by concomitant functional changes may be of prime importance.

If the degree of substitution of Sepharose 4 B with alpha-alkylamines is varied gels of different hydrophobicity are produced. Proteins can be adsorbed when a critical hydrophobicity (ca. 10-12 alkyl residues/Sepharose sphere) is reached. The enzymes phosphorylase kinase, phosphorylase phosphatase, 3',5'-cAMP dependent protein kinase, glycogen synthetase, and phosphorylase b are successively adsorbed as the hydrophobicity of the Sepharose is increased. The capacity of the gels for these enzymes and protein in general increases exponentially reaches plateau values as a function of the degree of substitution. There is no indication of a restriction of the hydrophobic centers for a given protein. The critical hydrophobicity needed to adsorb proteins can either be otained in the above manner or by elongation of the employed alkylamine at a constant degree of substitution. Additonally, as the hydrophobicity of a gel is increased higher binding forces result and desorption of proteins requires an augmentation of the salt concentration in the elution buffer. Elution of proteins from a hydrophobic matrix can be described in terms of salting-in phenomena since desorption is dependent on the type of salt employed and not on the ionic strength alone. This also rules out ionic interactions as a major factor in adsorption per se. By rationally controlling the hydrophobicity of a Sepharose gel the adsorption and elution of a protein may be thus establised that its purification or elimination can be optimally performed.

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