Engineered articular cartilage: influence of the scaffold on cell phenotype and proliferation

Articular cartilage defects do not heal. Biodegradable scaffolds have been studied for cartilage engineering in order to implant autologous chondrocytes and help cartilage repair. We tested some new collagen matrices differing in collagen type, origin, structure and methods of extraction and purification, and compared the behavior of human chondrocytes cultured on them. Human chondrocytes were grown for three weeks on four different equine type I collagen matrices, one type I, III porcine collagen matrix and one porcine type II collagen matrix. After 21 days, samples were subjected to histochemical, immunohistochemical and histomorphometric analysis to study phenotype expression and cell adhesion. At 7, 14 and 21 days cell proliferation was studied by incorporation of [3H]-thymidine. Our data evidence that the collagen type influences cell morphology, adhesion and growth; indeed, cellularity and rate of proliferation were significantly higher and cells were rounder on the collagen II matrix than on either of the collagen I matrices. Among the collagen I matrices, we observed a great variability in terms of cell adhesion and proliferation. The present study allowed us to identify one type I collagen matrix and one type II collagen matrix that could be usefully employed as a scaffold for chondrocyte transplantation.     

Microtopography of the eye surface of the crab Carcinus maenas: an atomic force microscope study suggesting a possible antifouling potential

Marine biofouling causes problems for technologies based on the sea, including ships, power plants and marine sensors. Several antifouling techniques have been applied to marine sensors, but most of these methodologies are environmentally unfriendly or ineffective. Bioinspiration, seeking guidance from natural solutions, is a promising approach to antifouling. Here, the eye of the green crab Carcinus maenas was regarded as a marine sensor model and its surface characterized by means of atomic force microscopy. Engineered surface micro- and nanotopography is a new mechanism found to limit biofouling, promising an effective solution with much reduced environmental impact. Besides giving a new insight into the morphology of C. maenas eye and its characterization, our study indicates that the eye surface probably has antifouling/fouling-release potential. Furthermore, the topographical features of the surface may influence the wettability properties of the structure and its interaction with organic molecules. Results indicate that the eye surface micro- and nanotopography may lead to bioinspired solutions to antifouling protection.     

Optimum design criteria for elastic structures subject to random dynamic loads

A new stochastic optimization method, which makes use of a constraint on structural reliability, is proposed for structures subject to dynamic random loads. A minimum weight problem is posed, in which a constraint condition imposes that the failure probability must be smaller than a given admissible level. The failure is determined by the first crossing outside the safe domain of a suitable structural response vector. The method is used to find the optimal shape of an elastic vertical column supporting a fixed mass positioned on the top, subject to a Gaussian filtered stationary stochastic horizontal acceleration process. The column, with variable annular cross-section, is described by a deterministic elastic multi-degree-of-freedom system. It is assumed that failure is reached when its lateral displacement exceeds an acceptable threshold value. Under this constraint, the structural weight is minimized and the optimal shape is determined for different structural conditions.     

One-step substrate nanofabrication and patterning of nanoparticles by lithographically controlled etching

We propose an integrated top-down and bottom-up approach to single-step nanofabrication of complex nanostructures made of different materials. The process, termed lithographically controlled etching (LCE), starts with a drop of an etching solution cast on the surface to be patterned. By placing a polymeric mold on the substrate, the stamp protrusions come into contact with the surface, thus protecting it, whereas the surface beneath the mold recesses is exposed to a thin layer of etching solution, allowing the surface to be etched. By dispersing nanoparticles into the etching solution, these can be deposited and self-organize in the recesses on the substrate as these are excavated. We demonstrate here the fabrication of complex structures and nanowires 30 nm wide. Moreover, by exploiting capillary forces, it is possible to deposit nanoparticles at precise positions with respect to optically addressable microstructures, thus realizing a multiscale functional pattern.     

Chemically induced residual stresses in dental composites

In several European countries, dental composites are replacing mercury-containing amalgams as the most common restorative materials. One problem with dental composites is residual stresses which may lead to poor performance of the restoration. In the present study, a combined modeling and materials characterization approach is presented and predictions compare well with experimental data on residual stresses. The model takes stress relaxation into account through the complete relaxation time spectrum of the resin. The approach allows for detailed parametric studies where resin and composite composition as well as cure conditions may be tailored with respect to residual stress generation.     

Minimum Hellinger distance based inference for scalar skew-normal and skew-t distributions

The skew-normal is a parametric model that extends the normal family by the addition of a shape parameter to account for skewness. As well, the skew-t distribution is generated by a perturbation of symmetry of the basic Student’s t density. These families share some nice properties. In particular, they allow a continuous variation through different degrees of asymmetry and, in the case of the skew-t, tail thickness, but still retain relevant features of the perturbed symmetric densities. In both models, a problem occurs in the estimation of the skewness parameter: for small and moderate sample sizes, the maximum likelihood method gives rise to an infinite estimate with positive probability, even when the sample skewness is not too large. To get around this phenomenon, we consider the minimum Hellinger distance estimation technique as an alternative to maximum likelihood. The method always leads to a finite estimate of the shape parameter. Furthermore, the procedure is asymptotically efficient under the assumed model and allows for testing hypothesis and setting confidence regions in a standard fashion.     

Surgical treatment of atherosclerotic lesions of subclavian artery: carotid-subclavian bypass versus subclavian-carotid transposition

BACKGROUND: The aim of this retrospective study is to analyze the short and long term results of two different surgical treatments in patients with subclavian lesions: common carotid-subclavian artery bypass (CSB) versus transposition of subclavian artery on the common carotid artery (SCT). METHODS: From 1981 until 1995, 40 non randomized patients with symptomatic subclavian steal underwent 20 CSBs and 20 SCTs. Risk factor rates were equally balanced in the two groups. Surgery was carried out routinely under general anesthesia, with electroencephalic continuous monitoring. Patency of revascularization was assessed by physical examination, brachial blood pressure determinations, ultrasound sonography and angiography whenever recurrence of symptoms developed or when the function of repair was in doubt. Patients were examined every year. In Spring 1996 (range 9-189 mos, average 7 years) a general clinical-instrumental follow-up was performed. RESULTS: In the short term (<30 days) mortality was 5%: one death (5%) for pulmonary embolism in a patient with CSB and one for myocardial infarction in a patient with SCT. The early thrombosis rate was 5% (1 CSB and 1 common carotid artery distal to a patent SCT). During follow-up 10 patients (25%) died and 6 were lost. The six-year actuarial patency rate was 100% for SCT and 66% for CSB. Moreover there were 3 thromboses of the vertebral artery homolateral to patent CSBs. CONCLUSIONS: In conclusions SCT should be considered the surgical technical choice for the treatment of proximal subclavian artery lesions.     

Modeling of the time-dependency of in vitro drug cytotoxicity and resistance

For potential clinical extrapolation of in vitro findings, it is of interest to relate the measured effect of an anticancer agent to concentration and exposure time. The Hill model (A. V. Hill, J. Physiol., 40: iv-vii, 1910) is commonly used to describe pharmacodynamic (PD) effects, including drug-induced growth inhibition of cancer cells in vitro. The IC(X)n x T = k relationship, in which IC(X) is the concentration of agent required to reduce cell growth by X%, T is the exposure time, and n and k are estimable parameters, was first applied to bacterial disinfectant action and then was successfully used to model anticancer drug potency as a function of exposure time (D. J. Adams, Cancer Res., 49: 6615-6620, 1989). Our goal was to create a new global PD modeling paradigm to facilitate the quantitative assessment of the growth-inhibitory effect of anticancer agents as a function of concentration and exposure time. Wild-type human ovarian A2780 and ileocecal HCT-8 carcinoma cells and sublines that were resistant to cisplatin (A2780/CP3), doxorubicin (A2780/DX5B), and raltitrexed (RTX) (HCT-8/DW2) were exposed to various anticancer agents, cisplatin, doxorubicin, paclitaxel, trimetrexate, RTX, methotrexate, and AG2034, for periods ranging from 1 to 96 h. Cell growth inhibition was measured with the sulforhodamine B protein dye assay. Patterns of time-dependency of drug potency, slope of the concentration-effect curves, and relative degree of resistance were characterized. Empirical mathematical expressions were built into a global concentration-time-effect model. The global PD model was then fit to the concentration-time-effect data with iteratively reweighted nonlinear regression. Under specific treatment conditions, the examination of the slope and the shape of the concentration-effect curves revealed a large heterogeneity in drug response, e.g., shallow concentration-effect curve or double or triple Hill "roller coaster" concentration-effect curve. These patterns, which were observed at intermediate exposure times in parental and resistant cells for paclitaxel and trimetrexate or only in resistant HCT-8/DW2 cells for RTX, methotrexate, and AG2034, revealed mechanistic insights for the former cases but possible methodological artifacts for the latter cases. The comprehensive PD modeling of the cytotoxic effect of anticancer agents showed that it was possible to modulate drug effect, response heterogeneity, and drug resistance by altering the time of exposure to the agents. This approach will be useful for: (a) describing complex concentration-time-effect surfaces; (b) refining biological interpretations of data; (c) providing insights on mechanisms of drug action and resistance; and (d) generating leads for clinical use of anticancer drugs.     

UGUS, a reporter for use with destabilizing N-termini

Due to constraints in vector construction, reporter polypeptides often carry N-terminal sequences of extraneous origin. Since protein half-life can be influenced by small determinants in the N-terminus, such foreign sequences can destabilize proteins and compromise results of reporter-based studies. We provide a real-life example of this problem (destabilizing sequences derived from a ribosomal protein) and show that it can be solved with the ubiquitin fusion technique, in which ubiquitin sequences are placed upstream of the reporter, in our case beta-glucuronidase. Post-translational processing by characterized pathways removes the ubiquitin together with destabilizing sequences, generating a stable reporter whose N-terminus is constant.