Sol-Gel-Deposited Ti-Doped ZnO: Toward Cell Fouling Transparent Conductive Oxides.

Ti-doped ZnO thin films were obtained with the aim of tailoring ZnO film bioadhesiveness and making the optoelectronic properties of ZnO materials transferable to biological environments. The films were prepared on silicon substrates by sol-gel spin-coating and subsequent annealing. A Ti-O segregation limits the ZnO crystallite growth and creates a buffer out-layer. Consequently, the Ti-doped ZnO presents slightly increased resistivity, which remains in the order of 10-3 Ω·cm. The strong biochemical interference of Zn2+ ions released from pure ZnO surfaces was evidenced by culturing Staphylococcus epidermidis with and without the Zn2+ coupling agent clioquinol. The Ti-doped ZnO surfaces showed a considerable increase of bacterial viability with respect to pure ZnO. Cell adhesion was assayed with human mesenchymal stem cells (hMSCs). Although hMSCs find difficulties to adhere to the pure ZnO surface, they progressively expand on the surface of ZnO when the Ti doping is increased. A preliminary microdevice has been built on the Si substrate with a ZnO film doped with 5% Ti. A one-dimensional micropattern with a zigzag structure shows the preference of hMSCs for adhesion on Ti-doped ZnO with respect to Si. The induced contrast of surface tension further induces a cell polarization effect on hMSCs. It is suggested that the presence of Ti-O covalent bonding on the doped surfaces provides a much more stable ground for bioadhesion. Such fouling behavior suggests an influence of Ti doping on film bioadhesiveness and sets the starting point for the selection of optimal materials for implantable optoelectronic devices.

Nanotopography enhanced mobility determines mesenchymal stem cell distribution on micropatterned semiconductors bearing nanorough areas.

Surface micropatterns are relevant instruments for the in vitro analysis of cell cultures in non-conventional planar conditions. In this work, two semiconductors (Si and TiO2) have been micropatterned by combined ion-beam/chemical-etching processes leading to selective areas bearing nanorough features. A preferential affinity of human mesenchymal stem cells (hMSCs) for planar areas versus nanotopographic ones is observed. Fluorescence microscopy after ß-catenin staining suggests that hMSCs adhesion is inhibited on nanostructured porous silicon areas. This has a direct impact in the development of actin fibers and suggests different cell migration mechanisms on the materials of a micropattern. hMSCs organization on nanotopographic micropatterns has been modeled by using a simplified random walk approach. The model attributes preferential cell mobilities on the nanotopographic areas with respect to the planar and considers purely stochastic movement with no inertial term. Simulations of the cell distribution have been run on 1D and 2D micropatterns and compared with the real hMSC cultures. The simulations allow defining two regimes for cell organization as a function of cell density. hMSCs ordering on planar areas is diffusion-induced in most micropatterns but constriction forced disorder appears for high cell densities. The relative mobility on the planar versus nanotopographic areas can be used as a quality indicator of the nanotopography contrasts in the diffusion induced ordering regime. It is shown that the relative mobility is favorable for the TiO2 versus the Si based system, and allows envisaging its use for the calibrated design of nanotopography based micropatterned materials.

Physicochemical modifications accompanying UV laser induced surface structures on poly(ethylene terephthalate) and their effect on adhesion of mesenchymal cells.

This work reports on the formation of different types of structures on the surface of polymer films upon UV laser irradiation. Poly(ethylene terephthalate) was irradiated with nanosecond UV pulses at 193 and 266 nm. The polarization of the laser beam and the irradiation angle of incidence were varied, giving rise to laser induced surface structures with different shapes and periodicities. The irradiated surfaces were topographically characterized by atomic force microscopy and the chemical modifications induced by laser irradiation were inspected via micro-Raman and fluorescence spectroscopies. Contact angle measurements were performed with different liquids, and the results evaluated in terms of surface free energy components. Finally, in order to test the influence of surface properties for a potential application, the modified surfaces were used for mesenchymal stem cell culture assays and the effect of nanostructure and surface chemistry on cell adhesion was evaluated.

Laser fabrication of porous silicon-based platforms for cell culturing.

In this study, we explore the selective culturing of human mesenchymal stem cells (hMSCs) on Si-based diffractive platforms. We demonstrate a single-step and flexible method for producing platforms on nanostructured porous silicon (nanoPS) based on the use of single pulses of an excimer laser to expose phase masks. The resulting patterns are typically 1D patterns formed by fringes or 2D patterns formed by circles. They are formed by alternate regions of almost unmodified nanoPS and regions where the nanoPS surface has melted and transformed into Si nanoparticles. The patterns are produced in relatively large areas (a few square millimeters) and can have a wide range of periodicities and aspect ratios. Direct binding, that is, with no previous functionalization of the pattern, alignment, and active polarization of hMSCs are explored. The results show the preferential direct binding of the hMSCs along the transformed regions whenever their width compares with the dimensions of the cells and they escape from patterns for smaller widths suggesting that the selectivity can be tailored through the pattern period. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2013.

Laser fabrication of porous silicon-based platforms for cell culturing.

In this study, we explore the selective culturing of human mesenchymal stem cells (hMSCs) on Si-based diffractive platforms. We demonstrate a single-step and flexible method for producing platforms on nanostructured porous silicon (nanoPS) based on the use of single pulses of an excimer laser to expose phase masks. The resulting patterns are typically 1D patterns formed by fringes or 2D patterns formed by circles. They are formed by alternate regions of almost unmodified nanoPS and regions where the nanoPS surface has melted and transformed into Si nanoparticles. The patterns are produced in relatively large areas (a few square millimeters) and can have a wide range of periodicities and aspect ratios. Direct binding, that is, with no previous functionalization of the pattern, alignment, and active polarization of hMSCs are explored. The results show the preferential direct binding of the hMSCs along the transformed regions whenever their width compares with the dimensions of the cells and they escape from patterns for smaller widths suggesting that the selectivity can be tailored through the pattern period.

Engineering of silicon surfaces at the micro- and nanoscales for cell adhesion and migration control.

The engineering of surface patterns is a powerful tool for analyzing cellular communication factors involved in the processes of adhesion, migration, and expansion, which can have a notable impact on therapeutic applications including tissue engineering. In this regard, the main objective of this research was to fabricate patterned and textured surfaces at micron- and nanoscale levels, respectively, with very different chemical and topographic characteristics to control cell-substrate interactions. For this task, one-dimensional (1-D) and two-dimensional (2-D) patterns combining silicon and nanostructured porous silicon were engineered by ion beam irradiation and subsequent electrochemical etch. The experimental results show that under the influence of chemical and morphological stimuli, human mesenchymal stem cells polarize and move directionally toward or away from the particular stimulus. Furthermore, a computational model was developed aiming at understanding cell behavior by reproducing the surface distribution and migration of human mesenchymal stem cells observed experimentally.

Prolactin's role in the early stages of liver regeneration in rats.

Liver regeneration after partial hepatectomy (PHx) is a complex process that is regulated by hemodynamic changes, the modulation of cytokines and growth factors, and the activation of immediate early transcription factors that lead to a round of hepatocyte mitosis. Among the factors involved, the pituitary hormone prolactin (PRL) has been shown to induce a hepatotrophic response after partial hepatectomy similar to that caused by phorbol esters; and in isolated hepatocytes PRL triggers a mitogenic response. However, it is becoming clear that PRL exerts a dual role acting in proliferation and differentiation processes. In this work, we have assessed the role of PRL in the early stages of liver regeneration in rats. To this end, three groups of rats were compared: Sham operated, regenerant and regenerant with PRL i.p. administration. Results show that PRL administration prior to partial hepatectomy caused an increase in the binding activity of several transcription factors involved in cell proliferation: AP-1, c-Jun and STAT-3, and in liver-specific differentiation and maintenance of energetic metabolism: CEBPalpha, HNF-1, HNF-4 at early time points and at later time points HNF-3. Hepatic sections show that PRL administration increases the number of proliferating cells within 5 h post-partial hepatectomy. The mRNA of the angiogenic and survival factors VEGF and HIF-1alpha, was also induced by PRL treatment. Data indicate that PRL triggers, either directly or indirectly, an acceleration of liver regeneration, preserving liver function and fulfilling a hepatoprotective role. J. Cell. Physiol. 219: 626-633, 2009. (c) 2009 Wiley-Liss, Inc.

ATP7B copper-regulated traffic and association with the tight junctions: copper excretion into the bile.

BACKGROUND & AIMS: The copper transporter ATP7B plays a central role in the elimination of excess copper by the liver into the bile, yet the site of its action remains controversial. The studies reported here examine the correspondence between the site of ATP7B action and distribution and the pathways of copper disposal by the liver. METHODS: Microscopy and cell fractionation studies of polarized Can 10 cells forming long-branched bile canaliculi have been used to study the cellular distribution of ATP7B. Copper excretion into the bile was studied in perfused rat liver. RESULTS: Copper excess provokes a massive download of the ATP7B retained in the trans-Golgi network into the bile canalicular membrane. Furthermore, a stable ATP7B pool is localized to the tight junctions that seal the bile canaliculi. The profile of Cu(64) excretion into the bile by isolated rat livers perfused under one-pass conditions provides evidence of copper excretion by 2 separate mechanisms, transcytosis across the hepatocyte and paracellular transport throughout the tight junctions. CONCLUSIONS: Whereas the ATP7B retained in the trans-Golgi-network is massively translocated to the bile canalicular membrane in response to increased copper levels, a pool of ATP7B associated with the tight junctions remains stable. In situ studies indicate that copper is excreted into the bile by 2 separate pathways. The results are discussed in the frame of the normal and impeded excretion of copper into the bile.

Properties of average score distributions of SEQUEST: the probability ratio method.

High throughput identification of peptides in databases from tandem mass spectrometry data is a key technique in modern proteomics. Common approaches to interpret large scale peptide identification results are based on the statistical analysis of average score distributions, which are constructed from the set of best scores produced by large collections of MS/MS spectra by using searching engines such as SEQUEST. Other approaches calculate individual peptide identification probabilities on the basis of theoretical models or from single-spectrum score distributions constructed by the set of scores produced by each MS/MS spectrum. In this work, we study the mathematical properties of average SEQUEST score distributions by introducing the concept of spectrum quality and expressing these average distributions as compositions of single-spectrum distributions. We predict and demonstrate in the practice that average score distributions are dominated by the quality distribution in the spectra collection, except in the low probability region, where it is possible to predict the dependence of average probability on database size. Our analysis leads to a novel indicator, the probability ratio, which takes optimally into account the statistical information provided by the first and second best scores. The probability ratio is a non-parametric and robust indicator that makes spectra classification according to parameters such as charge state unnecessary and allows a peptide identification performance, on the basis of false discovery rates, that is better than that obtained by other empirical statistical approaches. The probability ratio also compares favorably with statistical probability indicators obtained by the construction of single-spectrum SEQUEST score distributions. These results make the robustness, conceptual simplicity, and ease of automation of the probability ratio algorithm a very attractive alternative to determine peptide identification confidences and error rates in high throughput experiments.

Functional characterization of human mesenchymal stem cells that maintain osteochondral fates.

Adult stem cells are essential for tissue renewal, regeneration and repair, and their expansion in defined culture medium is on focus for regenerative medicine and genetic pathologies. The bone marrow has been shown to be very rich is pluripotent mesenchymal stem cells (MSCs) capable of forming bone, cartilage and also may give rise, to neurons and astrocytes in vivo and in vitro. MSCs can be isolated and expanded in culture, but human cells cannot be verified for a cartilage or a bone fate by transfer experiments. Accordingly, here we used different approaches to characterize hMSCs osteoblastic differentiation in vitro. hMSCs grown in culture in the presence of fetal bovine serum (FBS) expressed the bone-specific transcription factor Runx2/AML3. When cells were incubated in osteoblastic differentiation medium, cells expressed transcripts belonging to the signaling of Indian HH-PTHrP axis, GLI transcription factors, and bone target genes including osteopontin. The HH pathway proved to be functional since it induced cells to grow. Cells growing or differentiating to osteoblasts presented the Runx2/AML3 transcription factor, its partner CBFB, and Smad2/3 at the nuclei associated with the nuclear matrix. Furthermore, Runx2/AML3 was observed to co-localize with SC35 to the nuclear intermediary filaments. These data support the notion that hMSCs isolated from human bone are or become bone progenitor cells upon culture. In the absence of FBS and in the presence of insulin or prolactin, cells show cytoskeletal organization and an AP-1 transcription site activity resembling proliferative osteochondrocytes while cells in the presence of dexamethasone and added prolactin or TGF-beta resembled differentiated osteoblasts. These specific cellular conditions match those observed during endochondral bone formation.

Chondrocyte cloning in aging and osteoarthritis of the hip cartilage: morphometric analysis in transgenic mice expressing bovine growth hormone.

An arthritic disorder resembling human osteoarthritis occurs in transgenic mice expressing the fusion gene coding for bovine growth hormone (bGH). In these animals, we performed a morphometric evaluation of cellular density, clonal density and the relationship between both parameters in articular cartilage. These parameters were determined in the femoral head of bGH (+) mice at 1, 6 and 12 months of age and compared to values in the control mice. The transgenic mouse showed a reduction in cellular density of the superficial and middle zones of the articular cartilage with age. In the uncalcified cartilage at 6 and 12 months of age, cellular density was significantly lower in age-matched transgenic mice than in the control group. In the former, the changes in cellular density were accompanied by a gradual reduction in the clonal density in the superficial and middle zones. The clonal density/cellular density ratio was similar in transgenic mice and the control group at 12 months of age. These findings indicate that these transgenic mice develop an osteoarthritic process characterized by loss of cellularity and a gradual decline in chondrocyte cloning in the superficial and middle zones.

Prolactin is a component of the human synovial liquid and modulates the growth and chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.

The hormone prolactin (PRL) is the product of a single gene synthesized by pituitary and many extrapituitary tissues. In this study, we have purified and sequenced by mass spectrometry a 29 kDa protein from human synovial liquid, bound to the proteoglycan component of synovial liquid that showed an identical sequence in 20 amino acids to hPRL. We have also found PRL receptor (PRLR) in human knee tissues. The cartilage from osteoarthritic patients shows transcripts of the long PRLR isoform while synovial tissue expresses the intermediate PRLR isoform. Pluripotent mesenchymal stem cells (MSCs) can be isolated from adult bone marrow providing an excellent tool to study MSC-derived differentiation processes. We analyzed the expression of the PRL-PRLR system in hMSCs and during the acquisition of chondrocyte phenotype. We show by RT-PCR that intermediate PRLR isoform is expressed in hMSCs and that PRL exerts a significant increase in cell proliferation. In MSC aggregates cultured in chemically defined medium, we found that extrapituitary PRL transcripts are expressed and the receptor switches isoform expression from the intermediate to long isoform. Furthermore, in cell aggregates, PRL induces type II collagen and extrapituitary PRL expression. Histomorphologic analysis of cell aggregates showed that PRL induces the synthesis of proteoglycans and, in combination with glucocorticoids, a tissue structure with cells organized in longitudinal columns. Under the above conditions, electron microscopic observations show that PRL both downregulates the formation of fibrils of type II collagen and induces cell-cell interactions. All the results presented are consistent with a role of the PRL-PRLR system in bone/cartilage formation/repair processes.