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PubMed2004

DNA sequencing and genotyping in miniaturized electrophoresis systems.

Advances in microchannel electrophoretic separation systems for DNA analyses have had important impacts on biological and biomedical sciences, as exemplified by the successes of the Human Genome Project (HGP). As we enter a new era in genomic science, further technological innovations promise to provide other far-reaching benefits, many of which will require continual increases in sequencing and genotyping efficiency and throughput, as well as major decreases in the cost per analysis. Since the high-resolution size- and/or conformation-based electrophoretic separation of DNA is the most critical step in many genetic analyses, continual advances in the development of materials and methods for microchannel electrophoretic separations will be needed to meet the massive demand for high-quality, low-cost genomic data. In particular, the development (and commercialization) of miniaturized genotyping platforms is needed to support and enable the future breakthroughs of biomedical science. In this review, we briefly discuss the major sequencing and genotyping techniques in which high-throughput and high-resolution electrophoretic separations of DNA play a significant role. We review recent advances in the development of technology for capillary electrophoresis (CE), including capillary array electrophoresis (CAE) systems. Most of these CE/CAE innovations are equally applicable to implementation on microfabricated electrophoresis chips. Major effort is devoted to discussing various key elements needed for the development of integrated and practical microfluidic sequencing and genotyping platforms, including chip substrate selection, microchannel design and fabrication, microchannel surface modification, sample preparation, analyte detection, DNA sieving matrices, and device integration. Finally, we identify some of the remaining challenges, and some of the possible routes to further advances in high-throughput DNA sequencing and genotyping technologies.

Animals↗

PubMed2005

Capillary electrophoresis of polycationic poly(amidoamine) dendrimers.

Generation 2 to generation 5 poly(amidoamine) (PAMAM) dendrimers having different terminal functionalities were analyzed by capillary electrophoresis (CE). Polyacrylamide gel electrophoresis was also used to assess the composition of the individual generations for comparison with the CE results. Separation of PAMAMs can be accomplished by either using uncoated silica or silanized silica capillaries, although reproducibility is poor using the uncoated silica capillary. To improve run-to-run reproducibility, silanized capillary was used and various internal standards were also tested. Relative and normalized migration times of primary amine terminated PAMAM dendrimers were then determined using 2,3-diaminopyridine (2,3-DAP) as an internal standard. Using silanized capillaries and internal standards, the relative and normalized migration times are fully reproducible and comparable between runs. Apparent dimensionless electrophoretic mobilities were determined and the results were compared to theoretical calculations. It is concluded that for PAMAMs a complex separation mechanism has to be considered in CE, where the movement of the ions is due to the electric field, but the separation is rather the consequence of the adsorption/desorption equilibria on the capillary wall ("electrokinetic capillary chromatography"). The described method may be used for quality control and may serve as an effective technique to analyze polycationic PAMAM dendrimers and their derivatives with different surface modifications.

Aminopyridines↗

PubMed2005

Static adsorptive coating of poly(methyl methacrylate) microfluidic chips for extended usage in DNA separations.

A simple and robust static adsorptive (dynamic) coating process using 2% hydroxyethylcellulose was developed for surface modification of poly(methyl methacrylate) (PMMA) microfluidic chips for DNA separations, suitable for usage over extended periods, involving hundreds of runs. The coating medium was also used as a sieving matrix for the DNA separations following the coating process. Four consecutive static treatments, by simply filling the PMMA chip channels with sieving matrix once every day, were required for obtaining a stable coating and optimum performance. The performance of the coated chips at different phases of the coating process was studied by consecutive gel electrophoretic separations with LIF detection using a PhiX-174/HaeIII DNA digest sample. The coated chip, with daily renewal of the sieving matrix, showed high stability in performance during a 25-day period of systematic study, involving more than 100 individual runs. The performance of the coated chip also remained almost the same after 3 months of continuous usage, during which over 200 separations were performed. The average precision of migration time for the 603-bp fragment was 1.31% RSD (n = 6) during the 25-day study, with a separation efficiency of 6.5 x 10(4) plates (effective separation length 5.4 cm).

Adsorption↗

PubMed2005

A concept for miniaturized 3-D cell culture using an extracellular matrix gel.

This paper presents a novel method to embed, anchor, and cultivate cells in a controlled 3-D flow-through microenvironment. This is realized using an etched silicon pillar flow chamber filled with extracellular matrix (ECM) gel mixed with cells. At 4 degrees C, while in liquid form, ECM gel is mixed with cells and injected into the chamber. Raising the temperature to 37 degrees C results in a gel, with cells embedded. The silicon pillars both stabilize and increase the surface to volume ratio of the gel. During polymerization the gel shrinks, thus creating channels, which enables perfusion through the chip. The pillars increase the mechanical stability of the gel permitting high surface flow rates without surface modifications. Within the structure cells were still viable and proliferating after 6 days of cultivation. Our method thus makes it possible to perform medium- to long-term cultivation of cells in a controlled 3-D environment. This concept opens possibilities to perform studies of cells in a more physiological environment compared to traditional 2-D cultures on flat substrates.

Animals↗

PubMed2006

Organic and inorganic di-cations for capillary silica coating and EOF modulation in CE: Example of application in PEG analysis.

EOF measurements, by using 1,4-di-(4-aza-1-azonia-bicyclo[2.2.2]octane)butane diiodide, barium and strontium tetraborate as silica wall modifiers, are reported and, as an example of application, analysis of PEG (PEG 400-2000) polydisperse preparations in free solution CZE is shown. PEGs have been derivatized with phthalic anhydride so as to form singly or doubly charged derivatives with strong UV absorbance at 214 nm. Whereas separations in plain tetraborate buffer, pH 9.0, without any EOF control, did not lead to good resolution of all-size oligomers and suffered from long analysis times, excellent resolution of all oligomers up to 40 ethylene oxide (EO) units could be obtained under EOF control. Such EOF modulation was engendered by addition of 1 mM M7C4M7, a doubly charged organic cation able to stick tenaciously to the silica wall. Further modulation of EOF and silica surface modification could be achieved also by addition of inorganic cations, notably those of group II, whereas monovalent cations did not seem to affect much the EOF flux. Among the doubly charged cations investigated, Ca++, Mg++, Sr++ and Ba++, the latter did seem to offer best EOF control and reproducible runs. A judicious blend of M7C4M7 (0.33-1 mM range) with barium (10-20 mM range) allowed baseline resolution of all PEG oligomers investigated up to PEG 2000 and >40 EO units in length. In this last case, best results in terms of reproducibility and separation efficiency of the more heavy homologues were obtained using Li+ salt in small amounts.

Aza Compounds↗

PubMedDate not supplied

Two-dimensional gel electrophoretic analysis of rat sperm membrane interaction with cauda epididymal fluid.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to analyze the polypeptide composition of rat cauda epididymal fluid, blood serum and membrane-enriched fractions of caput, corpus, and cauda epididymal spermatozoa. Several polypeptides were found in both cauda fluid and blood serum, and in both cauda fluid and epididymal spermatozoa. Prominent cauda epididymal fluid polypeptides that were associated with caput, corpus, and cauda sperm membranes were 32 and 33 kDa. Passage of spermatozoa from the caput to the cauda epididymidis was characterized by the loss of three glycopolypeptides of 32, 30 and 29 kDa, and by the addition of a 37-kDa glycopolypeptide. Incubation of intact caput, corpus and cauda spermatozoa with cauda epididymal fluid revealed major changes in the polypeptide maps of the incubation fluid and the membrane-enriched fractions of caput and corpus, but not cauda spermatozoa. The incubation of cauda fluid with caput and corpus sperm cells was characterized by a loss of several polypeptides and the addition of a 24-kDa glycopolypeptide. The most striking change in spermatozoa incubated with cauda epididymal fluid was the addition of two glycopolypeptides of 32 and 33 kDa to the polypeptide maps of caput sperm cells. These data demonstrate that rat spermatozoa undergo surface modifications during epididymal maturation and that these modifications can be influenced by epididymal fluid.

Animals↗

PubMed1994

Fatigue behavior of titanium femoral hip prosthesis with proximal sleeve-stem modularity.

Modular hip prostheses are increasing in variety and utilization. Component stability, high endurance limit, and minimal particulate debris generation are critical for long-term clinical success. The purpose of this study was to characterize the fatigue response and evaluate the in vitro potential for component motion and wear of the S-ROM¿, a Ti-6Al-4V hip prothesis with a modular design based on a Morse taper connection. A fatigue jib was designed to simulate fixation of the device at the sleeve-bone interface only with distal support mainly against the lateral endosteal cortex. Two series of tests were performed in air at room temperature: one with direct vertical loading (to produce high bending moments in the coronal plane) and one with a compounding loading angle directed at 15 degrees out-of-plane (to include torsional physiological loads). Applied loads using a servohydraulic test machine ranged from 5 x BW (body weight) to 9 x BW (1 x BW = 73 kg, approximately 160 lb) at 10 Hz on an Instron apparatus. No mechanical failures were observed on the 11-mm size stems below 6 x BW for in-plane vertical loading, and at or below 7 x BW for out-of-plane loading. Using displacement monitoring with a sensitivity of 35 mum, no measurable slippage or relative motion was detected between the stem and sleeve when they were properly assembled. Examination of the contact areas with scanning electron microscopy releaved random surface modification (an indication of fretting or burnishing) with occasional evidence of transfer of material between stem and sleeve.(ABSTRACT TRUNCATED AT 250 WORDS)

Alloys↗

PubMed1991

Asymmetric membrane filters for the removal of leukocytes from blood.

As part of a study on the mechanisms of leukocyte filtration, the influence of pore size distribution on filter efficiency was investigated. Conventional leukocyte filters are not suitable for model studies, as these filters are composed of tightly packed synthetic fibers, with a poorly defined porous structure. Therefore, open cellular polyurethane membranes with pore size distributions varying from approximately 15 to 65 microns were prepared. Filtration experiments with stacked packages of these membranes showed that leukocytes are best removed (greater than 99%) by filters with a pore size distribution of 11-19 microns. These pore sizes approach the size of leukocytes (6-12 microns). However, due to fast clogging, blood flow through these filters is rapidly reduced, which results in a low filter capacity. With an asymmetric membrane filter, in which the pore size decreases from about 65 to 15 microns in the direction of blood flow, both moderate removal of leukocytes (greater than 80%) and maintenance of flow (approximately 0.2 mL/s) are obtained. This results in efficient leukocyte removal. From cell analysis of both filtrate and filter, it is concluded that adhesion rather than sieving is the major filtration mechanism. Thus, further optimization of the filter may be achieved by surface modification.

Cell Separation↗

PubMed1992

Albumin-binding surfaces for implantable devices.

Surfaces of implantable and blood contact-devices accumulate adsorbed and denatured proteins. This anomalous layer of proteins may help trigger unwanted events such as activation of coagulation systems and, perhaps, chronic inflammation. Because, in many experimental systems, the purposeful coating of surfaces with albumin will biologically "passivate" materials, we have attempted to develop polymers which, when exposed to blood or body fluids, will spontaneously, selectively, and reversibly adsorb host albumin. We report here a novel derivatization technique for increasing the albumin affinity of implantable polyetherurethane (PU). The technique is based on the incorporation of high-molecular-weight dextran to which the albumin-binding dye Cibacron Blue is covalently attached. Somewhat surprisingly, the amounts of human albumin adsorbed by Blue Dextran-modified and unmodified PU are quite similar. There are, however, important differences. First, the binding of albumin to derivatized PU is specific and not readily blocked by proteins in albumin-depleted human serum. Second, the majority of albumin associated with derivatized PU appears to be reversibly bound. Third, the binding of albumin to derivatized PU evidently is mediated primarily through ligand-specific binding of the protein to the albumin-binding dextran-dye conjugate. We conclude that it is possible to produce implantable polymers having surfaces which display albumin-binding dyes that selectively and reversibly bind albumin. Materials with this property, when implanted or exposed to blood, should form an infinitely renewable coating of albumin derived from physiologic fluids. This surface modification strategy may spawn a new generation of implantable materials with improved biologic compatibility.

Adsorption↗

PubMed2004

Human alveolar bone cell adhesion and growth on ion-implanted titanium.

Surface characteristics play a vital role in determining the biocompatibility of materials used as bone implants. Calcium ion implantation of titanium was previously reported to enhance osseointegration and bone formation in vivo, although the lack of consistent and reproducible effects highlight the need to understand the basic mechanisms involved in the response of target cells to such surfaces. The aim of this study was therefore to measure the precise effects of ion implantation of titanium on bone cells in vitro. Alveolar bone cells were seeded on the surface of polished titanium disks implanted with calcium, potassium, and argon ions. Using radioisotopically tagged bone cells, the results showed that although the calcium ion implanted surface reduced cell adhesion, it nevertheless significantly enhanced cell spreading and subsequent cell growth. In contrast, few differences in bone cell behavior were observed between the potassium- and argon-implanted titanium and the control nonimplanted titanium disks. These findings suggest the possibility that the calcium-implanted surface may significantly affect the biocompatibility of titanium implants by enhancing bone cell growth. Surface modification by ion implantation could thus prove to be a valuable tool for improving the clinical efficacy of titanium for bone repair and regeneration in vivo.

Argon↗

PubMed2005

Gene expression in tissue associated with extracellular matrix modified ePTFE.

Previous studies have established that surface modification of ePTFE with extracellular matrix molecules promotes vascularization within and around the implanted material. To understand the molecular basis of this tissue response to modified ePTFE, we analyzed large-scale gene expression in nonmodified and extracellular matrix-modified ePTFE-associated healing. Using a microarray containing 15,000 unique mouse cDNAs and an ANOVA-based analysis, we identified 789 genes related to cell signaling, inflammation, matrix remodeling, and proliferation that were differentially expressed across time, between modifications, or both. Genes were clustered based upon similarity in gene expression, producing 7 unique temporal super-patterns of expression. The clustered data revealed 3 general expression patterns unique to tissue surrounding the nonmodified ePTFE, while 6 unique expression patterns were associated with extracellular matrix-modified ePTFE. The more diverse expression patterns associated with extracellular matrix-modified ePTFE suggests that the tissue surrounding the extracellular matrix-modified ePTFE is more dynamic in terms of transcriptional activity. Taken together, these clusters serve as a "genetic fingerprint" for tissue healing in response to a specific material or material modification. Use of these genetic profiles will aid in the pursuit of improved device biocompatibility and enhanced material function.

Animals↗

PubMed2006

Morphological features of ovine embryonic lung fibroblasts cultured on different bioactive scaffolds.

Tissue regeneration with autologous cell transplantation is one of the most important goals in clinical research. In this field, the development of bioactive materials that provide microenvironments for cell-matrix interactions mimicking biological conditions is required. In recent years, many synthetic materials have been developed as scaffolds and many procedures for the surface modification of these materials have been applied using biological molecules. In this study, we analyzed the morphology and the molecule production by ovine embryonic lung fibroblasts cultured on three different sponge-like matrices based on poly(L-lactic acid) (PLLA): agarose/PLLA, crosslinked and uncrosslinked gelatin/PLLA. The matrices were produced by using an emulsion freeze-drying method leading to the formation of sponge-like materials with high porosity and with interconnection between the pores. In vitro MTT test demonstrated that transplanted cells were viable and metabolically active. Morphological analysis revealed that fibroblasts adhered to and penetrated the polymeric structures. Moreover, all the different matrices supported fibroblast production of proteoglycans, glycoproteins, and matrix molecules such as elastin, collagen I, and fibronectin. These data suggest that the tested bioactive scaffolds may support the growth and extracellular matrix molecule production of fibroblasts allowing in vitro connective tissue regeneration.

Animals↗

PubMed2006

Characterization of nano hydroxyapatite/collagen surfaces and cellular behaviors.

Osseointegration at the bone-implant interface is a prerequisite for endosseous implants to succeed in achieving and maintaining their long-term stability in bone tissue. The achievement of osseointegration is significantly affected by surface nature of implants. To optimize osseointegration, this study presents the characterization of synthesized nanocrystalline hydroxyapatite (nano HA) and in vitro studies on nano HA, nano-HA/collagen, and titanium surfaces. Voids were found within the grain of nano HA, which consisted of the shell and the core. The finding assists the clarification of microstructures of nano HA. By low-temperature mixing nano-HA sol with collagen gel (nano-HA/collagen 80:20), nano HA, and nano-HA/collagen coated on pure titanium or porous anodic titanium oxides resulted in higher wettability and lower roughness. The in vitro studies showed that porous structures produced by anodic oxides on titanium served as positive anchorage sites for cell filopodia to connect, and nano HA decreased cell attachment of osteoblasts and induced well-developed long filopodia and broad lamellipodia, thereby enhancing cellular motility. Collagen involvement enhanced cell adhesion to nano HA. Cell reactions to nano HA, nano-HA/collagen, native, and porous titanium surfaces provide some guidance for an optimal osseointegration by their application in surface modifications for implants.

Cell Line↗

PubMed2006

Construction of antibacterial multilayer films containing nanosilver via layer-by-layer assembly of heparin and chitosan-silver ions complex.

Antibacterial multilayer films containing nanosilver were prepared via layer-by-layer fashion. PET film was aminolyzed with 1,6-hexanediamine to introduce amino groups on PET film surface; chitosan-silver nitrate complex and heparin were alternately deposited onto an aminolyzed PET film surface, and subsequently, the silver ions within the multilayer films were reduced with ascorbic acid to form silver nanoparticles. UV-visible spectroscopy and transmission electron microscopy confirmed the formation of well-dispersed nanosilver particles with sizes (10-40 nm) that depended on the initial concentration of silver ions in chitosan solution and the pH of ascorbic acid solution. The chitosan/heparin multilayer films were possessed of bactericidal effect on Escherichia coli (E. coli), and this antibacterial effect could be significantly enhanced by the incorporation of silver nanoparticles into the multilayer films. The multilayer films containing nanosilver were not only effective as antibacterial but also as anticoagulant coating. And cell toxicity evaluation suggested that the multilayer films containing nanosilver did not show any cytotoxicity. The multilayer films containing nanosilver may have good potentials for surface modification of medical devices, especially for cardiovascular implants.

Animals↗

PubMed2006

Heparin-immobilized biodegradable scaffolds for local and sustained release of angiogenic growth factor.

Heparin-immobilized porous biodegradable scaffolds were fabricated to release basic fibroblast growth factor (bFGF) in a sustained manner. Heparin was covalently conjugated onto the surface of macroporous PLGA scaffolds fabricated by a gas-foaming/salt-leaching method. Sustained release of bFGF was successfully achieved for over 20 days due to high affinity of bFGF onto the immobilized heparin. It appears that bFGF release rate was regulated by the specific interaction between bFGF and heparin. The bFGF fraction released from the scaffolds maintained its bioactivity, as judged from determining the proliferation extent of human umbilical vein endothelial cells (HUVECs) in vitro. When heparin-immobilized scaffolds loaded with bFGF were implanted subcutaneously in vivo, they effectively induced the formation of blood vessels in the vicinity of the implant site. This study demonstrated that local and sustained delivery of angiogenic growth factor for tissue regeneration could be achieved by surface modification of porous scaffolds with heparin.

Absorbable Implants↗

PubMed2007

Three-dimensional collagen fiber remodeling by mesenchymal stem cells requires the integrin-matrix interaction.

Tissue engineering aiming to repair or regenerate damaged tissues necessitates fabricating three-dimensional biomaterial scaffolds with controlled porosity for delivering cells. To facilitate cell distribution, a strategy using stem cell-based fabrication of biomaterials was tested in type II collagen fibers. Human mesenchymal stem cells when delivered in type II collagen assembled and reorganized these matrices and differentiated into spherical chondrocytes with the synthesis of cartilage proteins. The cell-mediated assembly and reorganization of collagen fibers was not limitless and only restricted to an appropriate ratio of cell number and collagen amount. The blocking of alpha2 or beta1-integrin function with specific antibodies significantly impeded the collagen-assembly effects. In vitro chondrogenesis or in vivo cartilage formation of human mesenchymal stem cells was also dependent on the interactions between cells and surrounding matrices. This method for three-dimensional fabricating collagen fibers may generally be applied to other biomaterials, when combined with surface modification or ligand addition for cell adhesion.

Biocompatible Materials↗

PubMed2004

Protein electrostatic self-assembly on poly(DL-lactide) scaffold to promote osteoblast growth.

The development of protein coating on 3D biodegradable scaffold based on electrostatic self-assembly (ESA) to promote osteoblast growth is reported. Poly (ethylenimine) (PEI) was employed to obtain a stable positively charged surface on poly(DL-lactide) (PDL-LA) substrate. An extracellular-matrix (ECM)-like biomacromolecule, gelatin, was chosen as the polyelectrolyte to deposit on the activated PDL-LA substrate via ESA technique. Osteoblast (MC3T3) was then cultured on unmodified and gelatin-modified PDL-LA scaffolds. Osteoblast testing regarding total intracellular protein content, total DNA content, cell activity, and cell morphology on the ECM-like multilayer-modified PDL-LA scaffold showed that osteoblast growth was promoted. It will be easy to replace the gelatin with osteoinductive proteins or other polyelectrolytes to promote specific osteoblast functions. In comparison with conventional coating methods, polyelectrolyte multilayers are easy and stable to prepare. They may be a good choice for the surface modification of complex biomedical devices, especially for the 3D tissue-engineering scaffold. These very flexible systems allow broad medical applications for drug delivery and tissue engineering.

3T3 Cells↗

PubMed2006

The effect of self-designed bifunctional RGD-containing fusion protein on the behavior of human keratinocytes and dermal fibroblasts.

In this study, self-designed bifunctional RGD-containing fusion protein (BFP) was grafted on the petri dish to evaluate its cytotoxicity and attachment efficiency on primary cultured keratinocytes and dermal fibroblasts. Two lengths of the GRGDS sequences were separately fused to the N-terminus and C-terminus of the Trichoderma koningii cellobiohydrolase I gene cellulose-binding domain, to serve as linking molecule between the cell and the substrate. The grafting procedure was no more labor-intensive and could be done just in aqueous condition itself. The epidermal keratinocytes and dermal fibroblasts, harvested and separated from human foreskin, were cultured in serum-free keratinocyte culture medium and DMEM, respectively. The BFP was dissolved in double-deionized water, and was prepared at different concentrations. The BFP solution was subsequently added into the petri dish for grafting. MTT assay, total DNA measurement, and lactate dehydrogenase analysis were used to evaluate the cell viability, cell proliferation, and cytotoxicity. The immunochemical stain and SEM examination were chosen to make sure that the cultured cells still kept in phenotype. The results showed that the self-designed BFP was successfully coated on the petri dish to improve the cells' adhesion. The whole coating procedure was just done in aqueous solution without any organic solvent being involved. This method was much simpler than the traditional one, and there was no possibility to damage the immobilized biomolecules. From the results of the study, BFP could enhance attachment of keratinocytes and dermal fibroblasts without losing normal cell morphology and keep keratinocytes on the desired differentiation pathway. We believe that coating BFP on petri dish not only enhanced the keratinocyte attachment but also promoted keratinocytes proliferation. We suggest that the self-designed BFP has a great potential to apply on surface modification for the tissue-engineering scaffolds in the future.

Amino Acid Sequence↗