Microfabricated Quill-Type Surface Patterning Tools for the Creation of Biological Micro/Nano Arrays

Novel quill-type cantilever-based surface patterning tools (SPTs) were designed and constructed for use in controlled placement of femtoliter volumes of biological molecules on surfaces for biological applications. These tools were fabricated from silicon dioxide using microelectromechanical systems (MEMS) techniques. They featured a 1 microm split gap, fluidic transport microchannels and self-replenishing reservoirs. Experimental trials were performed using these tools on NanoArrayer molecular deposition instrumentation. Cy3-streptavidin was loaded as a biological sample and patterned on an amine-reactive dithiobis-succinimidyl undecanoate (DSU) monolayer on gold. Results showed these tools were capable of generating high quality biological arrays with routine spot sizes of 2-3 microm. The spot size could potentially achieve sub-micron dimensions if these SPT designs are reduced in size by more precise microfabrication techniques. The geometric designs of these tools facilitated sample replenishment from the local reservoir on the cantilever which allowed printing of large numbers of spots without sample reloading.     

Direct laser machining-induced topographic pattern promotes up-regulation of myogenic markers in human mesenchymal stem cells

The engineering of tissue is preferably done with stem cells, which can be differentiated into the tissue of interest using biochemical or physical cues. While much effort has been focused on using biological factors to regulate stem cell differentiation, recently interest in the contribution of physical factors has increased. In this work, three-dimensional (3-D) microchannels with topographic micropatterns were fabricated by femtosecond laser machining on a biodegradable polymer (poly(L-lactide-co-ε-caprolactone)) substrate. Two substrates with narrow and wide channels respectively were created. Human mesenchymal stem cells (hMSCs) were cultured on the scaffolds for cell proliferation and cellular organization. Gene expression and the immunostaining of myogenic and neurogenic markers were studied. Both scaffolds improved the cell alignment along the channels as compared to the control group. Microfilaments within hMSCs were more significantly aligned and elongated on the narrower microchannels. The gene expression study revealed significant up-regulation of several hallmark markers associated with myogenesis for hMSCs cultured on the scaffold with narrow microchannels, while osteogenic and neurogenic markers were down-regulated or remained similar to the control at day 14. Immunostaining of myogen- and neurogen-specific differentiation markers were used to further confirm the specific differentiation towards a myogenic lineage. This study demonstrates that femtosecond laser machining is a versatile tool for generating controllable 3-D microchannels with topographic features that can be used to induce specific myogenic differentiation of hMSCs in vitro, even in the absence of biological factors.     

The role of electrosprayed apatite nanocrystals in guiding osteoblast behaviour

Apatite nanocrystals, which mimic the dimensions of natural bone mineral, were electrosprayed on glass substrates, as a suitable synthetic biomedical material for osteoblast outgrowth was explored. A variety of topographic patterns were deposited and the influence of these designs on osteoblast alignment and cell differentiation was investigated. Patterned cell growth and enhanced cell differentiation were seen. Osteoblasts were also cultured on apatite nanocrystals chemically modified with either carbonate or silicon ions. Enhanced cell proliferation and early formation of mineral nodules were observed on apatite nanocrystals with silicon addition. This work highlights the importance of the combined effects of surface topography and surface chemistry in the guidance of cell behaviour.     

质谱技术研究进展

质谱技术具有高灵敏度、高精确度等特点,已广泛应用于生物学、生物医学、生物化学等学科的研究,特别是在蛋白质等生物大分子的研究中作用越来越重要。对质谱技术的研究现状及新技术的研究进展作一综述,并对未来予以展望。     

Optical Manipulation of Objects and Biological Cells in Microfluidic Devices

In this paper, we review optical techniques used for micro-manipulation of small particles and cells in microfluidic devices. These techniques are based on the object's interaction with focused laser light (consequential forces of scattering and gradient). Inorganic objects including polystyrene spheres and organic objects including biological cells were manipulated and switched in and between fluidic channels using these forces that can typically be generated by vertical cavity surface emitting laser (VCSEL) arrays, with only a few mW optical powers. T-, Y-, and multi-layered X fluidic channel devices were fabricated by polydimethylsiloxane (PDMS) elastomer molding of channel structures over photolithographically defined patterns using a thick negative photoresist. We have also shown that this optical manipulation technique can be extended to smaller multiple objects by using an optically trapped particle as a handle, or an optical handle. Ultimately, optical manipulation of small particles and biological cells could have applications in biomedical devices for drug discovery, cytometry and cell biology research.     

Micropatterning of mammalian cells on inorganic-based nanosponges

Developing artificial scaffolding structures in vitro in order to mimic physiological-relevant situations in vivo is critical in many biological and medical arenas including bone and cartilage generation, biomaterials, small-scale biomedical devices, tissue engineering, as well as the development of nanofabrication methods. We focus on using simple physical principles (photolithography) and chemical techniques (liquid vapor deposition) to build non-cytotoxic scaffolds with a nanometer resolution through using silicon substrates as the backbone. This method merges an optics-based approach with chemical restructuring to modify the surface properties of an IC-compatible material, switching from hydrophilicity to hydrophobicity. Through this nanofabrication-based approach that we developed, hydrophobic oxidized silicon nanosponges were obtained. We then probed cellular responses-examining cytoskeletal and morphological changes in living cells through a combination of fluorescence microscopy and scanning electron microscopy-via culturing Chinese hamster ovary cells, HIG-82 fibroblasts and Madin-Darby canine kidney cells on these silicon nanosponges. This study has demonstrated the potential applications of using these silicon-based nanopatterns such as influencing cellular behaviors at desired locations with a micro-/nanometer level.     

Thin films of glass and their application to biomedical sensors

Thin films of glass are attractive as a means for protecting integrated circuits for use in biological systems and are especially suitable for use with biomedical sensors. They offer advantages over polymer films since they can be deposited in thin, uniform layers and can be photoengraved using conventional techniques. This paper first reviews the requirements for film quality in using thin glassy films in these applications and then reviews the techniques available for film deposition. One technique which has been found especially suitable for biomedical work is described in detail, and the characterization of the resulting films is discussed. Films of silicon dioxide and silicon nitride 0·2–0·3 μm thick have exhibited adequate adhesion and continuity for use with sensors fabricated using integrated circuit technology. Furthermore, they have provided excellent corrosion protection for unencapsulated transistors implanted in the brain for periods of 4 months. Such glassy films also provide good electrical insulation for applied voltages less than 1 V; however, when higher voltages are present, thicker films are required to avoid excessive leakage currents. Applications to specific sensors are considered.     

Rapid Biocompatible Micro Device Fabrication by Micro Electro-Discharge Machining

Fabrication of a biocompatible micro device is predominantly done by silicon micromachining techniques. The lithographic and etching techniques require preparation and the use of masks which are time consuming and costly. Since bio research involves highly complex mechanisms, the modeling and simulation is difficult and experimental study is inevitable. To incorporate frequent design changes and to realize the hardware quickly, fabrication processes, complementary to the silicon micromachining techniques are required. In the present work the feasibility of using micro electro-discharge machining (EDM) for the fabrication of biocompatible microdevice has been studied. Micro channels with feature size as small as 25 microm are realized. The process is further improved by the introduction of ultrasonic vibration of the workpiece and the total time taken for the hardware realization is about 4 hours. The effects of ultrasonic vibration on the roughness of the spark eroded surface has been studied and reported. The potential of using micro EDM for making biocompatible devices for bio experiments is demonstrated and the surface finish achieved is well within the recommended Rz and Ra values of 3.4 and 0.4 microm respectively for biological studies like implant abutment.     

Bioprinted gelatin hydrogel platform promotes smooth muscle cell contractile phenotype maintenance

Three dimensional (3D) bioprinting has been proposed as a method for fabricating tissue engineered small diameter vascular prostheses. This technique not only involves constructing the structural features to obtain a desired pattern but the morphology of the pattern may also be used to influence the behavior of seeded cells. Herein, we 3D bioprinted a gelatin hydrogel microchannel construct to promote and preserve the contractile phenotype of vascular smooth muscle cells (vSMCs), which is crucial for vasoresponsiveness. The microchanneled surface of a gelatin hydrogel facilitated vSMC attachment and an elongated alignment along the microchannel direction. The cells displayed distinct F-actin anisotropy in the direction of the channel. The vSMC contractile phenotype was confirmed by the positive detection of contractile marker gene proteins (α-smooth muscle actin (α-SMA) and smooth muscle-myosin heavy chain (SM-MHC)). Having demonstrated the effectiveness of the hydrogel channels bioprinted on a film, the bioprinting was applied radially to the surface of a 3D tubular construct by integrating a rotating mandrel into the 3D bioprinter. The hydrogel microchannels printed on the 3D tubular vascular construct also orientated the vSMCs and strongly promoted the contractile phenotype. Together, our study demonstrated that microchannels bioprinted using a transglutaminase crosslinked gelatin hydrogel, could successfully promote and preserve vSMC contractile phenotype. Furthermore, the hydrogel bioink could be retained on the surface of a rotating polymer tube to print radial cell guiding channels onto a vascular graft construct.     

Biological significance of piezoelectricity in relation to acupuncture, Hatha Yoga, osteopathic medicine and action of air ions

Piezoelectric properties of biological macromolecules such as proteins, nucleic acids and mucopolysaccharides are reviewed in this paper. It is indicated that the structural elements of the human body composed of these piezoelectric substances are capable of transducing a mechanical energy into an electric current. Such a transduction may be brought about by movements of an acupuncture needle, osteopathic manipulations; Hatha Yoga postures or action of negatively charged air irons. It is postulated that electric current induced by stimulation of the specific sites on the surface of human body flows towards the internal organs along the semiconductive channels of biologic macromolecules. Electric current induced either by the piezoelectric transduction or directly applied from an external source may in turn stimulate individual cells in the target organ. Involvement of electrical phenomena in regulatory mechanisms on cellular and molecular levels is discussed.     

Transport pathways for macromolecules in the aortic endothelium: I. Transendothelial channels revealed by three-dimensional reconstruction using serial sections

Three-dimensional organization of structures labeled by horseradish peroxidase as a tracer molecule in rat aortic endothelium was examined to elucidate the transport pathways for macromolecules. Aortic endothelium was divisible morphologically into four distinct parts, i.e., the parajunctional region (JR), peripheral region (PR), organelle region (OR), and nuclear region (NR). Almost all vesicles, intercellular clefts, and phagosomes were labeled by horseradish peroxidase. Peroxidase-positive vesicles tended to gather in the PR, occasional JR, and the upper part of the NR. Ultrathin serial micrographs revealed the transcellular channels composed of vesicles in the PR and JR, and the paracellular channels composed of the intercellular cleft without constrictions of tight and gap junctions. Transcellular channels were subdivided into three morphologically different types, where not only vesicles but invagination of abluminal membrane and intercellular cleft occasionally participated in their formation. Three-dimensional reconstructions from three series of consecutive electron micrographs revealed that almost all peroxidase-positive vesicles were connected directly or indirectly with the cell surface. These results indicate that the transcellular and paracellular channels are the transport pathways for macromolecules in the aortic endothelium and may suggest that the “shuttle” hypothesis is unsuitable to explain transport system for macromolecules because it postulates the existence of many free vesicles in the cytoplasm. © 1993 Wiley-Liss, Inc.     

Microfabrication of cylindrical microfluidic channel networks for microvascular research

Current methods for formation of microvascular channel scaffolds are limited with non-circular channel cross-sections, complicated fabrication, and less flexibility in microchannel network design. To address current limitations in the creation of engineered microvascular channels with complex three-dimensional (3-D) geometries in the shape of microvessels, we have developed a reproducible, cost-effective, and flexible micromanufacturing process combined with photolithographic reflowable photoresist and soft lithography techniques to fabricate cylindrical microchannel and networks. A positive reflowable photoresist AZ P4620 was used to fabricate a master microchannel mold with semi-circular cross-sections. By the alignment and bonding of two polydimethylsiloxane (PDMS) microchannels replicated from the master mold together, a cylindrical microchannel or microchannel network was created. Further examination of the channel dimensions and surface profiles at different branching levels showed that the shape of the microfluidic channel was well approximated by a semi-circular surface, and a multi-level, multi-depth channel network was created. In addition, a computational fluidic dynamics (CFD) model was used to simulate shear flows and corresponding pressure distributions inside of the microchannel and channel network based on the dimensions of the fabricated channels. The fabricated multi-depth cylindrical microchannel network can provide platforms for the investigation of microvascular cells growing inside of cylindrical channels under shear flows and lumen pressures, and work as scaffolds for the investigation of morphogenesis and tubulogenesis.     

Influence of channel width on alignment of smooth muscle cells by high-aspect-ratio microfabricated elastomeric cell culture scaffolds

Engineered smooth muscle tissue requires ordered configurations of cells to reproduce native function, and microtechnology offers possibilities for physically and chemically controlling cell organization with high spatial resolution. In this work, poly(dimethylsiloxane) microchannel scaffolds, modified by layer-by-layer self-assembly of polyelectrolytes to promote cell adhesion, were evaluated for use as substrates for the culture of aligned smooth muscle cells. The hypothesis that narrower channels would result in better alignment was tested using channel width dimensions of 20, 30, 40, 50, and 60 microm, in addition to flat (control) surfaces. Alignment of cells was assessed by two different methods, each sensitive to a different aspect of cell alignment from fluorescence micrographs. Two-dimensional fast Fourier transform analysis was performed to analyze the orientation distribution of actin filaments in cells. This was complemented by connectivity analysis of stained nuclei to obtain nuclear orientation distributions. Both methods produced consistent data that support the hypothesis that narrow microchannels promote a highly aligned culture of smooth muscle cells, and the degree of alignment is dependent on the microchannel width. Precise replication of in vivo cell alignment in engineered tissue, with the ability to tailor specific surface chemistries of the scaffold to the desired application, will potentially allow the production of artificial tissue that more closely duplicates the structure and function of native tissue.     

Engineering of adult human neural stem cells differentiation through surface micropatterning

Interaction between differentiating neural stem cells and the extracellular environment guides the establishment of cell polarity during nervous system development. Developing neurons read the physical properties of the local substrate in a contact-dependent manner and retrieve essential guidance cues. To restore damage brain area by tissue engineering, the biomaterial scaffold has to mimic this microenvironment to allow organized tissue regeneration. To establish the validity of using microgrooved surfaces in order to simultaneously provide to primary adult human neural stem cells a permissive growth environment and a guide for neurite outgrowth in a pre-established direction, we have studied the long-term culture of adult human neural stem cells from patient biopsies on microgrooved polymers. By exploiting polymer moulding techniques, we engineered non-cytotoxic deep microstructured surfaces of polydimethylsiloxane (PDMS) exhibiting microchannels of various widths. Our results demonstrate that precoated micropatterned PDMS surfaces can serve as effective neurite guidance surfaces for human neural stem cells. Immunocytochemistry analysis show that channel width can impact strongly development and differentiation. In particular we found an optimal microchannel width, that conciliates a high differentiation rate with a pronounced alignment of neurites along the edges of the microchannels. The impact of the microstructures on neurite orientation turned out to be strongly influenced by cell density, attesting that cell/surface interactions at the origin of the alignment effect, are in competition with cell/cell interactions tending to promote interconnected networks of cells. Considering all these effects, we have been able to design appropriate structures allowing to obtain neuron development and differentiation rate comparable to a plane unpatterned surface, with an efficient neurite guidance and a long-term cell viability.     

IVF within microfluidic channels requires lower total numbers and lower concentrations of sperm

BACKGROUND: Microfluidic technology has been utilized in numerous biological applications specifically for miniaturization and simplification of laboratory techniques. We sought to apply microfluidic technology to murine IVF. METHODS: Microfluidic devices measuring 500 microm wide, 180 microm deep, and 2.25 cm in length were designed and fabricated using poly(dimethylsiloxane) (PDMS). Controls were standard centre-well culture dishes with 500 microl of media, half of which also contained PDMS as a material control. Denuded mouse oocytes were placed into microchannels or centre-well dish controls in groups of 10, then co-incubated overnight with epididymal mouse sperm at various concentrations. Fertilization was assessed and Fisher's exact test was used for statistical analysis (P < 0.05 significant). RESULTS: Fertilization rates between the two control groups (42%, no PDMS; 41%, with PDMS; not significant) were similar. Fertilization rates for denuded oocytes at standard mouse insemination sperm concentration (1 degrees 10(6) sperm/ml) was poorer in microchannels (12%) than controls (43%; P < 0.001). As insemination concentrations decreased, fertilization rates improved in microchannels with a plateau between 8 degrees 10(4) and 2 degrees 10(4) sperm/ml (4000-1000 total sperm). At these concentrations, combined fertilization rate for denuded oocytes was significantly higher in microchannels than centre-well dishes (27 versus 10%, respectively; P < 0.001), and was not significantly different from corresponding controls with a sperm concentration of 1 degrees 10(6) (37%; P = 0.06). CONCLUSIONS: Murine IVF can be conducted successfully within microfluidic channels. Lower total numbers and concentrations of sperm are required. Microfluidic devices may ultimately be useful in clinical IVF.     

Immobilization of RGD to < 1 1 1 > silicon surfaces for enhanced cell adhesion and proliferation

The ability of biomaterial surfaces to regulate cell behavior requires control over surface chemistry and microstructure. One of the greatest challenges with silicon-based biomedical microdevices such as those recently developed for neural stimulation, implantable encapsulation, biosensors, and drug delivery, is to improve biocompatibility and tissue integration. This may be achieved by modifying the exposed silicon surface with bioactive peptides. In this study, Arg-Gly-Asp (RGD) peptide conjugated surfaces were prepared and characterized. The effect of these surfaces on fibroblast adhesion and proliferation was examined over 4 days. Silicon surfaces coupled with a synthetic RGD peptide, as characterized with X-ray photoelectron spectroscopy and atomic force microscopy, display enhanced cell proliferation and bioactivity. Results demonstrate an almost three-fold greater cell attachment! proliferation on RGD immobilized surfaces compared to unmodified (control) silicon surfaces. Modulating the biological response of inorganic materials such as silicon will allow us to design more appropriate interfaces for implantable diagnostic and therapeutic silicon-based microdevices.     

Model porous surfaces for systematic studies of material-cell interactions

A model system for studying cell-surface interactions, based on microfabricated cell culture substrates, has been developed and is described here. Porous surfaces consisting of interconnecting channels with openings of subcellular dimensions are generated on flat, single crystal, silicon substrates. Channel size (width, depth), distribution, and surface coating can be varied independently and used for systematic investigation of how topographical, chemical, and elastic surface properties influence cell or tissue biological responses. Model porous surfaces have been produced by using two different microfabrication methods. Submicron-sized channels with very high depth-to-width aspect ratios (up to 30) have been made by using electron beam lithography and anisotropic reactive ion etching into single-crystal silicon. Another method uses thick-resist photolithography, which can be used to produce channels wider than 1 microm and with depth-to-width aspect ratios below 20 in an epoxy polymer. Preliminary cell culture tests show that fibroblasts bridge 0.8- to 1.8-microm-wide channels with very few exceptions (i.e., a continuous space below the cell-surface interface is created). It has also been shown that variation of channel periodicity significantly affects fibroblast morphology and attachment density. With this model system, it is possible to load the channels with bioactive substances intended to interact with cells at or near the surface in a time-dependent manner.     

Guided corona generates wettability patterns that selectively direct cell attachment inside closed microchannels

We present a method to create plasma mediated linear protein patterns along the lengths of simple one-inlet-one-outlet straight polydimethylsiloxane microchannels by biasing the delivery of corona discharge at the capillary openings. Pattern widths ranging from 500–1,000 μm were generated in 2 mm wide microchannels with lengths of 0.5, 1.0, or 1.5 cm. Corona-treated surfaces enabled the spatial alignment of C2C12 myoblasts to the adhesive protein-coated regions, facilitating myoblast differentiation into myotubes. Although limited in precision, this protein patterning technique offers the advantages of simplicity and low cost, making it attractive for educational and research environments that lack access to extensive microfabrication facilities. The results also provide a cautionary note to those using corona discharge to increase wettability of microchannels; the surface modification may not be uniform, even within single microchannels being treated depending on settings and positioning of the corona device tips.     

The lubricative function of artificial joint material surfaces by confocal laser scanning microscopy. Comparison with natural synovial joint surface

The purpose of this study was to observe and compare the effect of the behavior of different lubricating surfaces, including articular cartilage and several artificial joint materials, under the physiological loading by confocal laser scanning microscopy (CLSM) to clarify the mechanism of lubrication in natural joints and subsequently improve the quality of artificial joints. In our experiment, even with considerable loading, natural articular cartilage exhibited a synovial fluid area and an area of direct and solid contact. In the region between these two areas, a liquid crystal layer was observed. On the other hand, the materials used for artificial joints (metal and polyethylene, which are now in use, and polyvinyl alcohol-hydrogel polymer which is being developed), did not exhibit neither a clear fluid pool area nor the intermediary area with liquid crystal formation. These results suggest that natural articular cartilage surface has a particular characteristic which builds up a synovial pooling area and liquid crystal formation in the third area by interaction with macromolecules in synovial fluid under the loading condition. These characteristics give natural articular cartilage its excellent lubricative function. To improve the quality of artificial joints, the characteristics of the implant material surface and the synovial macromolecules must be considered.     

Organoapatites: materials for artificial bone. I. Synthesis and microstructure.

We have synthesized a new family of materials we termed organoapatites which may be useful in the formulation of artificial bone. These materials are synthesized by nucleation and growth of apatite crystals in media containing poly(amino acids) or synthetic organic polyelectrolytes using strict atmospheric, temperature, and pH control. The macromolecules used to synthesize the organoapatites include poly(L-lysine), poly(L-glutamic acid), and poly(sodium acrylate). The products were characterized by x-ray diffraction, scanning electron microscopy, surface area measurements, elemental analysis, and spectroscopic techniques. Organoapatites were found to contain large surface area morphologies with small crystallites which mature slowly based on analysis of Ca/P ratios. The organic macromolecules are thought to induce nucleation of crystals but also to quench their growth, thus becoming intimately dispersed in a mineral network. The organomineral particles harvested from the reaction medium contain polymer-netted microcrystals, and for this reason the synthetic approach can be used to modulate crystal maturation and biological response. It is likely that the preparative approach mimics some aspects of natural bone matrix synthesis and could be specially useful in the preparation of mineral implants containing intimate dispersions of small amounts of biomolecules such as growth factors, special drugs, or bioadhesives.