Recent technological innovation has opened new avenues in migration research – for instance, by allowing individual migratory animals to be followed over great distances and long periods of time, as well as by recording physiological information. Here, we focus on how technology – specifically applied to bird migration – has advanced our knowledge of migratory connectivity, and the behavior, demography, ecology, and physiology of migrants. Anticipating the invention of new and smaller tracking devices, in addition to the ways that technologies may be combined to measure and record the behavior of migratory animals, we also summarize major conceptual questions that can only be addressed once innovative, cutting‐edge instrumentation becomes available.

The Classical Least Square (CLS) fitting method was used to analyze the Raman spectra of living cells with the aim of identification of new phenotype-specific spectral markers for osteoblasts. The following chemicals were used for the CLS model: DNA, RNA, serum albumin, chymotrypsin and phosphatidyl choline. In this study we analyzed primary mature osteoblasts as well as two other cell types used as potential sources of osteoblasts: embryonic stem cells and fetal bone cells. The results obtained suggest that the Raman spectra of the cell types can be well approximated with a linear combination of the Raman spectra of the biopolymers used in the CLS model. The relative concentrations of the CLS components varied significantly between cell types, indicating that this analytical method could be used for phenotypic identification of osteoblasts.

Understanding the biochemical and biophysical properties of live cells is fundamental for unravelling the secrets of many diseases and developing new therapies. Raman micro-spectroscopy is a powerful non-invasive technique that allows in vitro studies of individual living cells or groups of cells without the use of any labels or contrast enhancing chemicals. We describe the use of various multivariate statistical methods, such as Principal Component Analysis (PCA), Linear Discriminant Analysis (LDA) and Classical Least Square (CLS) fitting, to extract biochemical information related to various cellular events. Such methods are required because of the high complexity of the Raman spectra obtained from living cells.PCA and LDA are used to discriminate between healthy and tumor cells. A leave-one-out cross-validation method indicated high prediction accuracy (95%) in identification of tumorogenic bone cells. The CLS fitting method using commercially available biopolymers makes it possible to monitor biochemical changes during the differentiation of embryonic stem cells and foetal bone cells. The results suggest that in both cases differentiated cells are characterised by lower concentrations of RNA compared to undifferentiated cells. These studies suggest that Raman micro-spectroscopy could become an invaluable tool for in vitro cellular biochemistry studies.

Raman microspectroscopy was used to determine biochemical markers during the differentiation of embryonic murine stem cells (mES) in vitro. Such markers are useful to determine the differentiation status of ES cells cultured on biomaterials. Raman spectra of mES cells as undifferentiated, spontaneously differentiated (4 days), and differentiated cells via formation of embryoid bodies (16, 20 days) were analyzed. Unsupervised hierarchical cluster analysis and principal component analysis were used to determine biochemical differences between mES cells in various states of differentiation. The undifferentiated cells were characterized by high scores of the first principal component (PC1, 49% variance). Similarity between the PC1 loading and the Raman spectrum of RNA indicated a high concentration of RNA in mES cells compared to differentiated cells. The ratio between the peak areas of RNA and proteins was used as a measure of mRNA translation. Using the same peak area ratio, it was possible to differentiate even between mES as undifferentiated and in early stages of differentiation (4 days). These findings were correlated with biological studies reporting high levels of nontranslated mRNA during early embryonic development. Therefore, the RNA translation obtained from the Raman spectra can be used as marker of differentiation state of mES cells.

The in vitro differentiation of embryonic stem (ES) cells into specific phenotypes plays an important role in the development of stem cell therapy, tissue engineering and regenerative medicine. Currently, there are no biological assays able to characterise and monitor in situ and in real-time ES cells during the differentiation process. We applied Raman micro-spectroscopy to compare undifferentiated murine ES cells with murine ES cells in the differentiation process. The most significant differences between undifferentiated and differentiated ES cells (16 days differentiation via formation of embryoid body) were related to the nucleic acids. The decrease in the magnitude of RNA 813cm−1 Raman peaks (25%) in the differentiated ES cells in comparison to undifferentiated ES cells, suggests that part of the RNA in the ES cells is used for the synthesis of specific proteins in the early stages of differentiation. In the same time, the DNA 786cm−1 Raman peaks were lower by 50%, indicating that the differentiated cells are more in the G1 phase than S, G2 or M phases of the cell cycle. This result suggests that the proliferation rate of differentiated cells is reduced following development of a mature phenotype. This study shows the feasibility of using Raman micro-spectroscopy to monitor in situ and in real-time the differentiation of ES cells by using the intensity of Raman peak of nucleic acids as differentiation markers.

Abstract Over a period of 19 years, we studied 237 breeding attempts of Spanish Imperial Eagles (Aquila adalberti) in Doñana National Park (southwestern Spain), including 29 pairs with at least one immature member, to investigate age-related effects on population fecundity. Without considering effect of territory quality, adult pairs were significantly more productive than immature pairs. Highly significant differences in breeding performance among territories were independent of age-classes of birds occupying them. Low-quality territories were more frequently occupied by immature pairs, whereas high-quality territories were occupied mostly or exclusively by adult birds. Therefore, age and territory quality appear to be interrelated. We found no effect on average population fecundity due to breeding by immature pairs.

Both Fourier Transform Infrared (FTIR) and Raman spectroscopy have been applied to the in vitro characterisation of biomaterials, mainly surface reactions leading to the formation of a biologically active hydroxycarbonate apatite (HCA) layer on the sample surface when immersed in simulated body fluids (SBF). The HCA layer indicates the degree of bioactivity of the sample, because it leads to a strong bond between the biomaterial and living tissue. Reflection measurements using FTIR allow quick, non-destructive detection of the HCA layer for solid and powder samples. Due to the low Raman scattering efficiency and low absorption of water in the visible-near infrared region, Raman micro-spectroscopy was successfully used for the in situ characterisation of 20 and 40 µ m diameter 45S5 Bioglass ® fibres. The in situ capabilities of the Raman micro-spectrometer have also been extended to the characterisation of living cells attached on bioinert silica and bioactive 45S5 Bioglass ® and 58S substrates. Using a high power 785 nm laser, living cells in physiological conditions can be real-time sampled over long periods of time without inducing cell damage and with good signal strength. Cell death can be monitored because it proved to induce strong changes in the Raman signature in the spectral regions 1000–1150 cm –1 and 1550–1650 cm –1 .

Objectives. Tissue engineering methods can be applied to regenerate diseased, or congenitally missing, urinary tract tissues. Urinary tract tissue cell cultures must be established in vitro and adequate matrices, acting as cell carriers, must be developed. Although degradable and nondegradable polymer matrices offer adequate mechanical stability, they are not optimal for cell adherence and growth. To overcome this problem, extracellular matrix proteins, permitting cell adhesion and regulation of cell proliferation and differentiation, can be adsorbed to the surface-modified polymer.Methods. In this study, nondegradable polymer films, poly(ethylene terephthalate), were used as an experimental model. Films were modified by graft polymerization of acrylic acid to subsequently allow collagen type I and III immobilization. The following adhesion, proliferation of human urothelial cells, and induction of their stratification were analyzed.Results. Collagen adsorption on 0.2 mug/cm(2) poly(acrylic acid)-grafted polymer films rendered the matrix apt for human urothelial cell adhesion and proliferation. Furthermore, stratification of urothelial cells was demonstrated on these surface-modified matrices.Conclusions. These results have shown that surface-modified polymer matrices can be used to act as cell carriers for cultured human urothelial cells. Such a cell-matrix construct could be applied in reparative surgery of the urinary tract. (C) 2002, Elsevier Science Inc.

Graft polymerization of acrylic acid onto plasma treated poly(ethylene terephthalate) (PET) films was carried out to develop surfaces for protein immobilization and smooth muscle cell seeding. Films with various graft densities were characterized by contact angle measurements, attenuated total reflectance infrared spectroscopy, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The contact angle was observed to decrease from 72.9 degrees for the virgin PET films to between 26 degrees and 33 degrees depending on the graft density. Storage of grafted films led to an increase in the contact angle, suggesting molecular rearrangement at the surface. However, films with the lowest graft levels showed maximum enhancement in the contact angle up on storage. XPS confirmed the presence of the polyacrylic acid grafts at the film surface and AFM showed a marked increase in the wavelength of the surface roughness as the graft density increased. The amount of collagen immobilized at the surface of the grafted films also increased as the graft density increased. The collagen immobilized films provided an excellent substrate for the growth of human smooth muscle cells.

In tissue engineering, degradable or non-degradable polymer matrices can act as cell-carrier-scaffolds. Cell adhesion and growth on these scaffolds can be promoted by immobilizing extracellular matrix proteins. Therefore, in this study, polymer poly(ethylene terephthalate) (PET) films were surface modified by graft polymerization of acrylic acid, to subsequently allow collagen (types I and III) immobilization and human smooth muscle cell expansion. The surfaces of PET were activated by plasma, followed by acrylic acid graft polymerization, resulting in covalently bound brushes, containing an average of either 0.22+/-0.1 or 5.93+/-0.87 microg/cm2 of poly(acrylic acid) (PAA). Subsequent electrostatic adsorption of collagen gave a surface concentration of 4.96 and 17.2 microg/cm2, respectively, as determined using radiolabelled 125I collagen. Both PET films grafted with 0.22 microg/cm2 of PAA with or without adsorbed collagen were apt for smooth muscle cell adhesion and proliferation. However, films grafted with 5.93 microg/cm2 were not. PAA-grafted PET films, onto which serum proteins of the culture medium adsorbed spontaneously, proved to be better matrices than films on which collagen has been immobilized. It, therefore, can be speculated that other serum proteins are more important than collagen for the human smooth muscle cell adhesion and growth on surface-modified polymer matrices.

Graft polymerization of acrylic acid onto plasma-treated poly(ethylene terephthalate) (PET) films was used to prepare surfaces suitable for Collagen immobilization by dip-coating. Such surfaces could be used as matrices for smooth muscle cell cultures in tissue engineering. Contact angle measurements showed that plasma-treated and grafted PET films undergo considerable surface reorganization during storage under ambient conditions. However, after Collagen immobilization the contact angle remained relatively stable. The amount of Collagen initially attached to the film surface increased with increasing poly(acrylic acid) graft density, but subsequent washing in water led to significant Collagen loss. This loss could nevertheless be substantially reduced by thermal crosslinking of the Collagen in the range 110-130 degreesC. Atomic force microscopy (AFM) observations suggested that the washed crosslinked Collagen has a very similar structure to that of the un-crosslinked Collagen. (C) 2002 Wiley Periodicals, Inc.

The graft polymerization of acrylic acid was carried out onto poly(ethylene terephthalate) films that had been pretreated with argon plasma and subsequently exposed to oxygen to create peroxides. The influence of synthesis conditions, such as plasma treatment time, plasma power, monomer concentration, temperature, and the presence of Mohr's salt, on the degree of grafting was investigated. The observed initial increase in grafting with monomer concentration accelerated at about 20% monomer. The grafting reached a maximum at 40% monomer and subsequently decreased with further increases in monomer concentration. The reaction temperature had a pronounced effect on the degree of grafting. The initial rate of grafting increased with increasing temperature, but the degree of grafting showed a maximum at 50 degreesC. The activation energy of the grafting obtained from an Arrhenius plot was 29.1 kJ/mol. The addition of Mohr's salt to the reaction medium not only led to a homopolymer-free grafting reaction but also diminished the degree of grafting. The degree of grafting increased with increasing plasma power and plasma treatment time. (C) 2001 John Wiley & Sons, Inc.

PURPOSE:Gastric or intestinal patches, commonly used for reconstructive cystoplasty, may induce severe metabolic complications. The use of bladder tissues reconstructed in vitro could avoid these complications. We compared cellular differentiation and permeability characteristics of human native with in vitro cultured stratified urothelium. MATERIALS AND METHODS:Human stratified urothelium was induced in vitro. Morphology was studied with light and electron microscopy and expression of key cellular proteins was assessed using immunohistochemistry. Permeability coefficients were determined by measuring water, urea, ammonia and proton fluxes across the urothelium. RESULTS:As in native urothelium the stratified urothelial construct consisted of basal membrane and basal, intermediate and superficial cell layers. The apical membrane of superficial cells formed villi and glycocalices, and tight junctions and desmosomes were developed. Immunohistochemistry showed similarities and differences in the expression of cytokeratins, integrin and cellular adhesion proteins. In the cultured urothelium cytokeratin 20 and integrin subunits alpha6 and beta4 were absent, and symplekin was expressed diffusely in all layers. Uroplakins were clearly expressed in the superficial umbrella cells of the urothelial constructs, however, they were also present in intermediate and basal cells. Symplekin and uroplakins were expressed only in the superficial cells of native bladder tissue. The urothelial constructs showed excellent viability, and functionally their permeabilities for water, urea and ammonia were no different from those measured in native human urothelium. Proton permeability was even lower in the constructs compared to that of native urothelium. CONCLUSIONS:Although the in vitro cultured human stratified urothelium did not show complete terminal differentiation of its superficial cells, it retained the same barrier characteristics against the principal urine components. These results indicate that such in vitro cultured urothelium, after being grown on a compliant degradable support or in coculture with smooth muscle cells, is suitable for reconstructive cystoplasty.

Poly(acrylic acid) (PAA) was grafted onto RF plasma treated PET films and onto SiO2 covered glass surfaces. PET films with various amounts of grafted PAA (0.4, 5, 11 and 19 μm/cm2) were immersed into a solution of collagen to allow for polyionic complex formation as a method for protein immobilization. It was found that the amount of complexed collagen was close to proportional to the amount of PAA. A closer investigation of the optical density of the PAA brushes close to the glass surface wa was performed using Reflectometric Inteference Spectroscopy. The supression of possibilities of extension and collaps of the brushes upon variation in pH is suggested to be caused by polyionic crosslinking between protonated collagen and deprotonated PAA. Such PAA-collagen surfaces with PAA concentrations lower that 10 μm/cm2 were shown to be suitable substrates for growth of human bladder smooth muscle cells.

Vesico-ureteral reflux, a common pathology in children, can be treated cystoscopically by injection of a bulking material underneath the most distal, intramural ureter, which forces the latter to do a detour, increasing its submucosal path. This increase of the length of the submucosal path of the ureter within the bladder is directly responsible for the anti-reflux effect. So far Teflon and collagen paste have been commonly used as bulking materials. We suggest replacing these materials by living tissue consisting of bladder smooth muscle, normally present at this location. The aim of this work is to provide a long-term effective treatment by producing bioresorbable microspheres which can act as a support matrix and an entrapment substance for bladder smooth muscle cells, with the goal of an in vivo transfer of the in vitro cultured cells with a minimal surgical procedure. By the use of Spinning Disk Atomization, which has specifically been developed for this purpose, we have shown two methods for the preparation of porous poly(lactic acid) microspheres with tunable sizes from 160 to 320 microm. The controlled solvent burst method has shown the advantage over the crystal leaching method in the direct creation of microspheres with large closed pores, by atomizing the polymer solution in controlled temperature conditions. Microspheres with various closed pore structures have thus been prepared. The innovation of this work is in the direct and rapid formation of porous microspheres with a pore morphology which is designed to create cavities suitable for adherence and growth of cells by adapting the temperature conditions of atomization. Injection tests have shown promising results in using these cell-loaded microspheres for future non-invasive tissue engineering.

Congenital bladder disorders, such as bladder and cloacal exstrophy as well as acquired pathologies such as traumatic neurogenic bladder, chronic interstitial cystistis, and possibly bladder cancer, require the increase in size or the replacement of the bladder. This to improve or to create adequate storage capacity, and/or to definitively treat the bladder disease.

A new method for surface functionalization of inherently reactive polymers such as polyesters has been developed. It is based on the direct nucleophilic attack on the ester group in the polyethyleneterephtalate backbone by the acid moiety of polyacrylic acid (PAA) catalyzed by titaniumisopropoxide. The PAA grafted surfaces were characterized by X-ray photoelectron spectroscopy to demonstrated the presence of a thin grafted layer rendering the substrates hydrophilic. Subsequent acid group activation using N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide hydrochloride allowed for in-situ collagen immobilization rendering the surfaces cell adhesive. Human smooth muscle and urothelial cells successfully seeded these surfaces, reaching confluence after 7 days.