Fabrication of cell container arrays with overlaid surface topographies

This paper presents cell culture substrates in the form of microcontainer arrays with overlaid surface topographies, and a technology for their fabrication. The new fabrication technology is based on microscale thermoforming of thin polymer films whose surfaces are topographically prepatterned on a micro- or nanoscale. For microthermoforming, we apply a new process on the basis of temporary back moulding of polymer films and use the novel concept of a perforated-sheet-like mould. Thermal micro- or nanoimprinting is applied for prepatterning. The novel cell container arrays are fabricated from polylactic acid (PLA) films. The thin-walled microcontainer structures have the shape of a spherical calotte merging into a hexagonal shape at their upper circumferential edges. In the arrays, the cell containers are arranged densely packed in honeycomb fashion. The inner surfaces of the highly curved container walls are provided with various topographical micro- and nanopatterns. For a first validation of the microcontainer arrays as in vitro cell culture substrates, C2C12 mouse premyoblasts are cultured in containers with microgrooved surfaces and shown to align along the grooves in the three-dimensional film substrates. In future stem-cell-biological and tissue engineering applications, microcontainers fabricated using the proposed technology may act as geometrically defined artificial microenvironments or niches.     

A titer plate-based polymer microfluidic platform for high throughput nucleic acid purification

A 96-well solid-phase reversible immobilization (SPRI) reactor plate was designed to demonstrate functional titer plate-based microfluidic platforms. Nickel, large area mold inserts were fabricated using an SU-8 based, UV-LIGA technique on 150 mm diameter silicon substrates. Prior to UV exposure, the prebaked SU-8 resist was flycut to reduce the total thickness variation to less than 5 μm. Excellent UV lithography results, with highly vertical sidewalls, were obtained in the SU-8 by using an UV filter to remove high absorbance wavelengths below 350 nm. Overplating of nickel in the SU-8 patterns produced high quality, high precision, metal mold inserts, which were used to replicate titer plate-based SPRI reactors using hot embossing of polycarbonate (PC). Optimized molding conditions yielded good feature replication fidelity and feature location integrity over the entire surface area. Thermal fusion bonding of the molded PC chips at 150°C resulted in leak-free sealing, which was verified in leakage tests using a fluorescent dye. The assembled SPRI reactor was used for simple, fast purification of genomic DNA from whole cell lysates of several bacterial species, which was verified by PCR amplification of the purified genomic DNA.     

The fabrication of elastin-based hydrogels using high pressure CO(2)

The aim of this study was to investigate the effect of high pressure CO(2) on the crosslinking of elastin-based polymers and the characteristics of the fabricated hydrogels. A hydrogel was fabricated by chemically crosslinking alpha-elastin with glutaraldehyde at high pressure CO(2). The effects of pressure, reaction time, and crosslinker concentration on the characteristics of the fabricated hydrogels were determined. The reaction time had negligible effect on either the swelling ratio or the pore size of the fabricated hydrogels. Increasing the processing pressure from 30bar to 150bar resulted in a 60% increase in the hydrogel swelling ratio. The crosslinked hydrogels displayed stimuli-responsive characteristics towards temperature and salt concentration. The dense gas process facilitated coacervation, expedited the crosslinking reaction, and dramatically affected the micro- and macrostructures of pores within the sample. The results of micro-CT scan and SEM images demonstrated that pore interconnectivity was substantially enhanced for alpha-elastin hydrogels fabricated using high pressure CO(2). Dense gas CO(2) reduced the wall thickness and size of the pores and importantly induced channels within the structure of the alpha-elastin hydrogels. In vitro cell culture studies demonstrated that the channels facilitated fibroblast penetration and proliferation within alpha-elastin structures.     

An inverted microcontact printing method on topographically structured polystyrene chips for arrayed micro-3-D culturing of single cells

With the goal to investigate the relation of shape and function of single cells or clusters of cells in a 3-dimensional (3-D) microenvironment, we present a novel platform technology to create arrays of microwells on polystyrene (PS) chips for hosting cells in a local microenvironment characterized by controlled shape and surface chemistry. The micro-3-D cell culturing combines 2-dimensional chemical patterning with topographical microstructuring presenting to the cells a local 3-D host structure. Microwells of controlled dimensions were produced by a two-step replication process, based on standard microfabrication of Si, replica molding into poly(dimethylsiloxane), and hot embossing of PS. This allowed the production of large numbers of microstructured surfaces with high reproducibility and fidelity of replication. Using inverted micro contact printing, the plateau surface between the microwells was successfully passivated to block adsorption of proteins and prevent cell attachment by transfer of a graft-copolymer, poly(l-lysine)-g-poly(ethylene glycol). The surface inside the microwells was subsequently modified by spontaneous adsorption of proteins or functionalized PLL-g-PEG/PEG-X (X=biotin or specific, cell-interactive peptide) to elicit specific responses inside the wells. Preliminary cell experiments demonstrated the functionality of such a device to host single epithelial cells (MDCK II) inside the functionalized microwells and thus to control their 3-D shape. This novel platform is useful for fundamental cell-biological studies and applications in the area of cell-based sensing.     

骨科钛板表面TiO2纳米管阵列的制备和结构表征

目的 在骨科钛板表面制备TiO2纳米管阵列,并对其表征,为骨科内植物表面修饰或涂层提供载体,也为防治骨科内植物感染寻求一新途径.方法 利用阳极氧化法,以甘油体系为电解液,骨科临床钛板为阳极,铂电极为阴极,通过调整一定的参数,在骨科钛板表面制备TiO2纳米管阵列,并对其表征.利用X射线光电子能谱仪(XPS)分析纳米管元素组成,并通过X射线衍射仪(XRD)观察其在300 ℃高温下形态是否变化.结果 以甘油体系为电解液,使用阳极氧化法24 h可在骨科临床钛板表面制备出管径为100～200 nm、管长在3μm以内的TiO2纳米管阵列.宏观直视下可见骨科钛板表面由银白色变为深蓝色.XPS分析骨科钛板表面的TiO2纳米管阵列主要由Ti元素(75.88％)、O元素(20.16％)和F元素(3.96％)组成.此外,经马弗炉加温至300℃的TiO2纳米管阵列形态稳定.结论 利用阳极氧化法可制备出表面均匀的TiO2纳米管阵列.该阵列形态稳定,内部中空状,底部封闭,具有较大的比表面积,且可耐受高温,可为骨科内植物表面修饰或涂层提供载体.     

The effects of combined micron-/submicron-scale surface roughness and nanoscale features on cell proliferation and differentiation

Titanium (Ti) osseointegration is critical for the success of dental and orthopedic implants. Previous studies have shown that surface roughness at the micro- and submicro-scales promotes osseointegration by enhancing osteoblast differentiation and local factor production. Only relatively recently have the effects of nanoscale roughness on cell response been considered. The aim of the present study was to develop a simple and scalable surface modification treatment that introduces nanoscale features to the surfaces of Ti substrates without greatly affecting other surface features, and to determine the effects of such superimposed nano-features on the differentiation and local factor production of osteoblasts. A simple oxidation treatment was developed for generating controlled nanoscale topographies on Ti surfaces, while retaining the starting micro-/submicro-scale roughness. Such nano-modified surfaces also possessed similar elemental compositions, and exhibited similar contact angles, as the original surfaces, but possessed a different surface crystal structure. MG63 cells were seeded on machined (PT), nano-modified PT (NMPT), sandblasted/acid-etched (SLA), and nano-modified SLA (NMSLA) Ti disks. The results suggested that the introduction of such nanoscale structures in combination with micro-/submicro-scale roughness improves osteoblast differentiation and local factor production, which, in turn, indicates the potential for improved implant osseointegration in vivo.     

Fabrication of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) microstructures using soft lithography for scaffold applications

This paper reports two soft lithographic methods, micromolding and hot embossing, to produce biodegradable poly (3-hydroxybutyrate-co-3-ftydroxyhexanoate) (PHBHHx) arrays of microstructures for hosting and culturing cells in a local microenvironment by controlled shape. Silicon masters with high-aspect-ratio microfeatures were fabricated using KOH and DRIE anisotropic etching. These silicon masters were used as molds to construct PHBHHx microstructures using micromolding and hot embossing. Using silicon rather than conventional PDMS as molds allowed microstructures with feature size of 20 microm and height of 100 microm to be realized. PHBHHx microstructures with different configurations including circles, rectangles, and octagons were fabricated to investigate the effects of topography on cell culture. Mouse fibroblast cell lines L929 were cultured on PHBHHx microstructures in vitro to investigate the biocompatibility. This study demonstrates the feasibility of microfabrication of PHBHHx structures with micro-scale feature size using soft lithography, and the results show that PHBHHx microstructures can be created to mimic cellular microenvironment for cell culture, providing a convenient means to investigate relationships of microstructures and cell functions.     

Attachment and growth of fibroblasts on poly(L-lactide/epsilon-caprolactone) scaffolds prepared in supercritical CO2 and modified by polyethylene imine grafting with ethylene diamine-plasma in a glow-discharge apparatus

In this study, a copolymer of L-lactide and epsilon-caprolactone (Mn: 73,523, Mw: 127,990 and PI: 1.74) was synthesized by ring-opening polymerization by using stannous octoate as the catalyst. FTIR, 1H-NMR and DSC confirmed the copolymer formation. The copolymer films were prepared and a novel method was developed to produce highly porous sponges for potential use in tissue engineering. Films were subjected to supercritical CO2 at 3300 psi and 70 degrees C to create porous structures for production of possible tissue engineering scaffolds. The pore sizes were in the range of 40-80 microm. The copolymer films were pre-wetted with polyethylene imine (PEI) and then treated with ethylene diamine (EDA)-plasma in glow-discharge apparatus. Gas plasma surface modification of three-dimensional scaffolds fabricated by supercritical carbon dioxide technique was demonstrated to enhance cell adhesion, proliferation, and differentiation over 6 days in culture using L929 fibroblast cell line. Alkaline phosphatase (ALP) activity and glucose uptake in cell culture medium were followed in the cell culture experiments. Fibroblastic cell attachment and growth on the EDA-plasma treated scaffolds were rather low. However, both cell attachment and growth were significantly increased by PEI pre-treatment before EDA-plasma. The changes in ALP activity and glucose uptake also supported the cell growth behavior on these PEI and EDA-plasma treated scaffolds.     

Interaction of fibroblasts on polycarbonate membrane surfaces with different micropore sizes and hydrophilicity

Surface topography appears to be an important but often neglected factor in implant performance. In this study, fibroblasts were cultured on a range of porous polycarbonate (PC) membranes with well defined surface topography (track-etched micropores, 0.2-8.0 microm in diameter) and wettability gradients. The wettability gradient on the PC membrane surfaces was produced by treating the surfaces with corona from a knife-type electrode whose power increased gradually along the sample length. The PC membrane surfaces were characterized by scanning electron microscopy (SEM) and the water contact angle measurement. Fibroblasts were cultured on the corona-treated PC membrane surfaces with different micropore sizes for 1 and 2 days. The cells attached on the membrane surfaces were examined by SEM and the cell density on the surfaces was estimated by counting the number of attached cells along the wettability gradient. It was observed that the cells were adhered and grew more on the hydrophilic positions of the membrane surfaces than the more hydrophobic ones, regardless of micropore size. It was also observed that cell adhesion and growth decreased gradually with increasing micropore size of the membrane surfaces. It seems that the cell adhesion and growth were progressively inhibited as the membrane surfaces had micropores with increasing size, probably due to surface discontinuities produced by tract-etched pores. On the membrane surfaces with smaller micropore sizes, the cells seemed to override these surface discontinuities.     

Hot embossing for micropatterned cell substrates

This paper reports the development of a technique for preparing microtextured polymer substrates for cell growth and studies the response of osteoblast cells grown on these surfaces. The surfaces were manufactured with hot embossing, where a silicon micromachined printing master was pressed into a thermoplastic polymer substrate at elevated temperature, forming a regular microgroove pattern in the polymer. The grooves were approximately 5 microm deep, 4 microm wide, and had a periodicity of 34 microm. The polymer substrate was polyimide, which can be spincast and printed in its uncured form, and is mechanically rigid and chemically nonreactive after full cure. Osteoblast cells were grown on the textured polymer substrate and their responses to grooved and smooth surfaces were observed with fluorescence microscopy. Alignment and aspect ratio were analyzed for the cell body, cell nucleus, and focal adhesions. Cell membrane body, cell nucleus, and focal adhesions all strongly aligned with the microgrooves, while only the cell body shape changed on the microgrooved surface. This novel substrate preparation technique offers the opportunity for low-cost and rapid manufacture of microtextured surfaces that can be used to control cell shape and alignment.     

Cellular reactions of osteoblasts to micron- and submicron-scale porous structures of titanium surfaces

Osteoblast reactions to topographic structures of titanium play a key role in host tissue responses and the final osseointegration. Since it is difficult to fabricate micro- and nano-scale structures on titanium surfaces, little is known about the mechanism whereby the topography of titanium surfaces exerts its effects on cell behavior at the cellular level. In the present study, the titanium surface was structured in micron- and submicron-scale ranges by anodic oxidation in either 0.2 M H3PO4 or 0.03 M calcium glycerophosphate with 0.15 calcium acetate. The average dimensions of pores in the structured surface were about 0.5 and 2 microm in diameter, with roughness averaging at 0.2 and 0.4 microm, respectively. Enhanced attachment of cells (SaOS-2) was shown on micron- and submicron-scale structures. Initial cell reactions to different titanium surfaces, e.g. the development of the actin-containing structures, are determined by the different morphology of the surfaces. It is demonstrated that on either micron- or submicron-structured surfaces, many well-developed filopodia were observed to be primary adhesion structures in cell-substrate interactions, and some of them entered pores using their distinct tips or points along their length for initial attachment. Therefore, porous structures at either micro- or submicrometre scale supply positive guidance cues for anchorage-dependent cells to attach, leading to enhanced cell attachment. In contrast, the cells attached to a smooth titanium surface by focal contacts around their periphery as predominant adhesion structures, since repulsive signals from the environment led to retraction of the filopodia back to the cell bodies. These cells showed well-organized stress fibres, which exert tension across the cell body, resulting in flattened cells.     

Periodontal ligament and gingival fibroblast adhesion to dentin-like textured surfaces

It is known that the (micro-) structure of a substrate surface is of major influence on the growth behaviour of adherent cells. In the current study, we aimed to produce a surface that exactly mimics the structure of natural dentin, and to describe the effect of this surface on the growth behaviour of primary periodontal ligament fibroblasts (PDLF) or gingival fibroblasts (GF). First, we used scanning electron microscopy (SEM) and morphometric techniques to analyse the porous dentin structure. Then, using a template made by photolithographic techniques, cell culture dishes with similar surface structure were made. On these dishes, and on smooth controls, primary PDLF and GF were seeded and assayed up to 14 days for proliferation, alkaline phosphatase (ALP) activity, and collagen content. Also, cell morphology was observed with SEM and transmission electron microscopy (TEM). Results showed that GF showed significantly less ALP activity than PDLF. Abundant collagen fibres were only formed by GF grown on the textured surfaces. SEM assessment showed equal spreading of both cell types on smooth and textured surfaces. TEM showed a preferential deposition of ECM material in the texture porosity. From our study we can conclude that dentin-like surfaces have no negative effect on either cell type, and could be used to enhance extracellular matrix deposition in GF formation. However, considerable differences were observed between primary cells from different animals. Therefore, final efficacy of the surfaces remains to be proven in implantation experiments.     

Laser Sintered Porous Ti–6Al–4V Implants Stimulate Vertical Bone Growth

The objective of this study was to examine the ability of 3D implants with trabecular-bone-inspired porosity and micro-/nano-rough surfaces to enhance vertical bone ingrowth. Porous Ti–6Al–4V constructs were fabricated via laser-sintering and processed to obtain micro-/nano-rough surfaces. Male and female human osteoblasts were seeded on constructs to analyze cell morphology and response. Implants were then placed on rat calvaria for 10 weeks to assess vertical bone ingrowth, mechanical stability and osseointegration. All osteoblasts showed higher levels of osteocalcin, osteoprotegerin, vascular endothelial growth factor and bone morphogenetic protein 2 on porous constructs compared to solid laser-sintered controls. Porous implants placed in vivo resulted in an average of 3.1 ± 0.6 mm³ vertical bone growth and osseointegration within implant pores and had significantly higher pull-out strength values than solid implants. New bone formation and pull-out strength was not improved with the addition of demineralized bone matrix putty. Scanning electron images and histological results corroborated vertical bone growth. This study indicates that Ti–6Al–4V implants fabricated by additive manufacturing to have porosity based on trabecular bone and post-build processing to have micro-/nano-surface roughness can support vertical bone growth in vivo, and suggests that these implants may be used clinically to increase osseointegration in challenging patient cases.     

Superparamagnetic bead interactions with functionalized surfaces characterized by an immunomicroarray

Magneto-resistive sensors capable of detecting superparamagnetic micro-/nano-sized beads are promising alternatives to standard diagnostic assays based on absorbance or fluorescence and streptavidin-functionalized beads are widely used as an integral part of these sensors. Here we have developed an immunomicroarray for systematic studies of the binding properties of 10 different micro-/nano-sized streptavidin-functionalized beads to a biotin substrate immobilized on SiO₂ with or without surface modification. SiO₂ surface cleaning, immobilized substrate concentration and surface blocking conditions were optimized. Polyethylene glycol-based surfaces with different end groups on the anchor molecule, 2,4,6-trichloro-1,3,5-triazine (TsT), were synthesized and compared with the standard (3-aminopropyl)triethoxysilane (APTS)/glutaraldehyde chemistry. APTS/glutaraldehyde, directly linked TsT and bare H₂O₂-activated SiO₂ performed better than polyethylene glycol-modified surfaces. Two beads, Masterbeads and M-280 beads, were found to give superior results compared with other bead types. Antibody/antigen interactions, illustrated by C-reactive protein, were best performed with Masterbeads. The results provide important information concerning the surface binding properties of streptavidin-functionalized beads and the immunomicroarray can be used when optimizing the performance of bead-based biosensors.     

Bioinspired superhydrophobic poly(L-lactic acid) surfaces control bone marrow derived cells adhesion and proliferation

The aptitude of a cell to adhere, migrate, and differentiate on a compact substrate or scaffold is important in the field of tissue engineering and biomaterials. It is well known that cell behavior can be controlled and guided through the change in micro- and nano-scale topographic features. In this work, we intend to demonstrate that special topographic features that control wettability may also have an important role in the biological performance of biodegradable substrates. Poly(L-lactic acid) surfaces with superhydrophobic characteristics were produced, based on the so-called Lotus effect, exhibiting dual micro- and nano-scale roughness. The water contact angle could be higher than 150 degrees and a value of that order could be kept even upon immersion in a simulated body fluid solution for more than 20 days. Such water repellent surfaces were found to prevent adhesion and proliferation of bone marrow derived cells previously isolated from the femurs of 6-week-old male Wistar rats, when compared with smoother surfaces prepared by simple solvent casting. Such results demonstrate that these superhydrophobic surfaces may be used to control cell behavior onto biodegradable substrates.     

Collagen-coupled poly(2-hydroxyethyl methacrylate)-Si(111) hybrid surfaces for cell immobilization

To improve the biocompatibility of silicon-based implantable micro- and nanodevices, and to tailor silicon surfaces for controlled cell immobilization, well-defined functional polymer-Si(111) hybrids, consisting of nearly monodispersed poly(2-hydroxyethyl methacrylate [P(HEMA)] with covalently coupled collagen and tethered (Si-C bonded) on the silicon surfaces, were prepared. HEMA was graft polymerized on the hydrogen-terminated Si(111) surface (Si-H surface) via surface-initiated atom transfer radical polymerization (ATRP) to give rise to the Si-g-P(HEMA) hybrid. The active chloride end groups preserved throughout the ATRP process and the chloride groups converted from some (approximately 20%) of the OH groups of the P(HEMA) brushes were used as the leaving groups for nucleophilic reaction with the -NH2 groups of collagen to give rise to the Si-g-P(HEMA)-collagen surface conjugates. These hybrid surfaces were evaluated by culturing 3T3 fibroblasts. The biocompatible Si-g-P(HEMA) hybrid surface resisted attachment and growth of this cell line. The Si-g-P(HEMA)-collagen hybrid surfaces, on the other hand, exhibited good cell adhesion and growth characteristics, and the extent of cell immobilization could be controlled by adjusting the amount of immobilized collagen. Thus, incorporating the collagen-coupled P(HEMA) onto silicon surfaces via robust Si-C bonds may endow the silicon substrates with new and interesting properties for potential applications in silicon-based implantable devices, such as molecular sensors and biochips.     

Syntheses and properties of elastic copoly(ester-urethane)s containing a phospholipid moiety and the fabrication of nanosheets

In these years, we have investigated the syntheses of novel diamine and diol monomers containing phosphorylcholine (PC) group to obtain biocompatible polymers, the backbone components of which were thermally stable and mechanically strong. In this study, the preparations of elastic copoly(ester-urethane)s containing PC group and polycarbonate segment were carried out by polycondensation and polyaddition using a diol monomer containing PC group and polycarbonate diol. It was found that the obtained polymers exhibited the high-thermal stability up to 200?°C and the elasticity derived from the soft segment. The introduction of PC group was effective to improve the resistance to the adhesions of proteins and platelets on the polymer films, which was the result of surface properties derived from the PC moiety. In addition, we tried to prepare ultra-thin polymer films composed of copoly(ester-urethane)s, so-called nanosheets. As a result, the desired nanosheets were successfully fabricated and the obtained nanosheets exhibited the high adhesive strength, indicating that the nanosheets could conform closely to the desired surfaces due to their exquisite flexibility and low roughness.     

Simulation of in-vivo-equivalent epithelial barriers using a micro fluidic device

In biomedical approaches cell culture models do often not fully represent their biological counterparts. Often the methods used do not completely mimic the in-vivo situation, either by using only single-cell-type culture approaches, or by using inadequate culture conditions. We therefore developed a variable system based on individual modules to simulate in vitro equivalent cell-barriers (e.g. for mucous layers). This system allows the growth of different communicating cell types in micro channels. Hot embossing is used to fabricate the micro structured polymer sheets. The stamp for hot embossing is fabricated by UV-lithography/electroforming or by micro milling. The system consists of a container with micro fluidic modules and a pump-system for a continuous medium-supply. An individual module is made of two micro-structured polycarbonate-sheets separated by a transmissible polycarbonate membrane. The two sheets are arranged orthogonally to induce a cross flow. The system is highly variable by channel-geometry (height and width), capacity (number of micro fluidic modules), and pore sizes of the transmissible membranes. In a first approach we simulated the intestinal mucosa. Epithelial cells and primary neurons of the enteric nervous system were cultured on both sides of the transmissible membrane within the two different compartments. So the cells could be supplied with two different media. We kept a mono-culture of primary neurons or epithelial cells for 5 days and a co-culture between these two cell-types was established for 4 days. The proposed system delivers a sophisticated model for the simulation of various epithelial layers which takes the specific biological properties into account.     

Effects of surface molecular chirality on adhesion and differentiation of stem cells

Chirality is one of the most fascinating and ubiquitous cues in nature, especially in life. The effects of chiral surfaces on stem cells have, however, not yet been revealed. Herein we examined the molecular chirality effect on stem cell behaviors. Self assembly monolayers of l- or d-cysteine (Cys) were formed on a glass surface coated with gold. Mesenchymal stem cells (MSCs) derived from bone marrow of rats exhibited more adhering preference and thus less cell spreading on the l surface than on the d one at the confluent condition. More protein adsorption was observed on the l surface after immersed in cell culture medium with fetal bovine serum. After osteogenic and adipogenic co-induction at the confluent condition, a larger proportion of cells became osteoblasts on the d surface, while the adipogenic fraction on the l surface was found to be higher than on the d surface. In order to interpret how this chirality effect worked, we fabricated Cys microislands of two sizes on the non-fouling poly(ethylene glycol) hydrogel to pre-define the spreading areas of single cells. Then the differentiation extents did not exhibit a significant difference between l and d surfaces under a given area of microislands, yet very significant differences of osteogenesis and adipogenesis were found between different areas. So, the molecular chirality influenced stem cells, probably via favored adsorption of natural proteins on the l surface, which led to more cell adhesion; and the larger cell spreading area with higher cell tension in turn favored osteogenesis rather than adipogenesis. As a result, this study reveals the molecular chirality on material surfaces as an indirect regulator of stem cells.     

Endothelial vacuolization induced by highly permeable silicon membranes

Assays for initiating, controlling and studying endothelial cell behavior and blood vessel formation have applications in developmental biology, cancer and tissue engineering. In vitro vasculogenesis models typically combine complex three-dimensional gels of extracellular matrix proteins with other stimuli like growth factor supplements. Biomaterials with unique micro- and nanoscale features may provide simpler substrates to study endothelial cell morphogenesis. In this work, patterns of nanoporous, nanothin silicon membranes (porous nanocrystalline silicon, or pnc-Si) are fabricated to control the permeability of an endothelial cell culture substrate. Permeability on the basal surface of primary and immortalized endothelial cells causes vacuole formation and endothelial organization into capillary-like structures. This phenomenon is repeatable, robust and controlled entirely by patterns of free-standing, highly permeable pnc-Si membranes. Pnc-Si is a new biomaterial with precisely defined micro- and nanoscale features that can be used as a unique in vitro platform to study endothelial cell behavior and vasculogenesis.