Self-supporting nanoporous alumina membranes as substrates for hepatic cell cultures

Membranes made from nanoporous alumina exhibit interesting properties for their use in biomedical research. They show high porosity and the pore diameters can be easily adjusted in a reproducible manner. Nanoporous alumina membranes are thus ideal substrates for the cultivation of polar cells (e.g., hepatocytes) or the establishment of indirect co-cultures. The porous nature of the material allows supply of nutrients to both sides of adherent cells and the exchange of molecules across the membrane. However, it is well-known that surface features in the nanometer range affect cellular behavior. In this study, the response of HepG2 cells to nanoporous alumina membranes with three different pore diameters, ranging from 50 to 250 nm, has been evaluated. The cellular interactions with the nanoporous materials were assessed by investigating cell adhesion, morphology, and proliferation. Cell functionality was measured by means of albumin production. The membranes supported good cell adhesion and spreading. Compared to tissue culture plastic, the cells on the porous substrates developed distinct focal adhesion sites and actin stress fibers. Additionally, electron microscopical investigations revealed the penetration of cellular extensions into pores with diameters bigger than 200 nm. Furthermore, cell proliferation significantly increased with an increase in pore diameter, whereas the albumin production followed a reverse trend. Thus, it seems to be possible to direct cellular behavior of HepG2 cells growing on nanoporous alumina by changing the pore diameter of the material. Hence, nanoporous alumina membranes can be useful culture substrates to develop new approaches in the field of liver tissue engineering.     

The biocompatibility of porous silicon in tissues of the eye

In this report, we explore the biocompatibility of thermally-oxidised, aminosilanised porous silicon membranes and their potential to support human ocular cells in vitro and in vivo, in the rat eye. A colorimetric assay for silicic acid showed that membranes with pore sizes of 40–60 nm slowly dissolved, but the material could be maintained in tissue culture medium in vitro for at least two weeks without visible degradation. When implanted under the rat conjunctiva, the material did not erode the underlying or overlying tissue. The implant underwent slow dissolution, but remained visible at the operating microscope for over 8 weeks. End-stage histology indicated the presence of a thin fibrous capsule surrounding the implant, but little evidence of any local accumulation of acute inflammatory cells or vascularization. Human lens epithelial cells and primary human corneal explants adhered to the porous silicon membranes, where they remained viable and underwent division. Primary corneal epithelial cells supported on membranes were labelled with a cell tracker dye and implanted under the rat conjunctiva. Seven days later, labelled cells had moved from the membrane into the ocular tissue spaces. A porous silicon membrane may have value as a biomaterial that can support the delivery of cells to the ocular surface and improve existing therapeutic options in patients with corneal epithelial stem cell dysfunction and ocular surface disease.     

The scale of substratum topographic features modulates proliferation of corneal epithelial cells and corneal fibroblasts

The cornea is a complex tissue composed of different cell types, including corneal epithelial cells and keratocytes. Each of these cell types are directly exposed to rich nanoscale topography from the basement membrane or surrounding extracellular matrix. Nanoscale topography has been shown to influence cell behaviors, including orientation, alignment, differentiation, migration, and proliferation. We investigated whether proliferation of SV40-transformed human corneal epithelial cells (SV40-HCECs), primary human corneal epithelial cells (HCECs), and primary corneal fibroblasts is influenced by the scale of topographic features of the substratum. Using basement membrane feature sizes as our guide and the known dimensions of collagen fibrils of the corneal stroma (20-60 nm), we fabricated polyurethane molded substrates, which contain anisotropic feature sizes ranging from 200-2000 nm on pitches ranging from 400 to 4000 nm (pitch = ridge width + groove width). The planar regions separating each of the six patterned regions served as control surfaces. Primary corneal and SV40-HCEC proliferation decreased in direct response to decreasing nanoscale topographies down to 200 nm. In contrast to corneal epithelial cells, corneal fibroblasts did not exhibit significantly different response to any of the topographies when compared with planar controls at 5 days. However, decreased proliferation was observed on the smallest feature sizes after 14 days in culture. Results from these experiments are relevant in understanding the potential mechanisms involved in the control of proliferation and differentiation of cells within the cornea.     

Cell adhesion, spreading, and proliferation on surface functionalized with RGD nanopillar arrays

In this paper, a method was introduced for the fabrication of vertically and spatially-controlled peptide nanostructures that enhance cell adhesion, proliferation, spreading on artificial surfaces. The RGD nanostructures with different heights were fabricated on gold surfaces by self-assembly technique through a nanoporous alumina mask composed of nanoscale-controlled pores. Pore diameter and spatial distribution were controlled by manipulating the pore widening time at a constant voltage during the mask fabrication process. Two-dimensional RGD nanodot, three-dimensional RGD nanorod, and RGD nanopillar arrays were carried out using various concentrations of RGD peptide solution, self-assembly times, and pore sizes, which were 74 nm, 63 nm, and 43 nm in diameter, respectively. The fabricated RGD nanodot, nanorod, and nanopillar arrays were utilized as a cell adhesion layer to evaluate the cell adhesion force, adhesion speed, spreading assay, and phosphorylation of cofilin protein in PC12, HeLa, and HEK293T normal cells. Among the three different nanostructures, RGD nanopillar arrays were found to be suitable for cellular attachment, spreading, and proliferation due to the proper arrangement of the RGD motif, which mimics in vivo conditions. Hence, our newly fabricated RGD nanostructured array can be successfully applied as a bio-platform for improving cellular functions and in in vitro tissue engineering.     

Fabrication and evaluation of nanoporous alumina membranes for osteoblast culture

An understanding of osteoblast response to surface topography is essential for successful bone tissue engineering applications. Alumina has been extensively used as a substrate for bone tissue constructs. However, current techniques do not allow precise surface topography and orientation of the material. In this research, a two-step anodization process was optimized for the fabrication of nanoporous alumina membranes with uniform pore dimension and distribution. The anodization voltage can be varied to create nanoporous alumina membranes with pore sizes ranging from 30 to 80 nm in diameter. The impact of the nanoscale pores on osteoblast response was studied by evaluating cell adhesion, morphology, and matrix production. Scanning electron microscopy and atomic force microscopy were used to characterize the nanoporous alumina membranes. Osteoblast adhesion and morphology were investigated using scanning electron microscopy images and matrix production was characterized using energy dispersive spectroscopy. This research combined the advantages of using alumina, a material with proven biocompatibility and current orthopedic clinical applications, and incorporated porous features on the nanoscale which have been reported to improve osteoblast response.     

Morphological zeta-potential variation of nanoporous anodic alumina layers and cell adherence

Nanoscale surface modification of biomedical implant materials offers enhanced biological activity concerning protein adsorption and cell adherence. Nanoporous anodic alumina oxide (AAO) layers were prepared by electrochemical oxidation of thin Al-seed layers in 0.22 M C₂H₂O₄, applying anodization voltages of 20–60 V. The AAO layers are characterized by a mean pore diameter varying from 15 to 40 nm, a mean pore distance of 40–130 nm, a total porosity of ∼10% and a thickness of 560 ± 40 nm. Zeta potential and isoelectric point (iep) were derived from streaming potential measurements and correlated to the topology variation of the nanoporous AAO layers. With decreasing pore diameter a shift of iep from ∼7.9 (pore diameter 40 nm) to ∼6.7 (pore diameter 15 nm) was observed. Plain alumina layers, however, possess an iep of ∼9. Compared to the plain alumina surface an enhanced adherence and activity of hFOB cells was observed on the nanoporous AAO after 24 h culture with a maximum at a pore size of 40 nm. The topology-induced change of the electrochemical surface state may have a strong impact on protein adsorption as well as on cell adhesion, which offers a high potential for the development of bioactive AAO coatings on various biomaterial substrates.     

Enhancement of retinal pigment epithelial culture characteristics and subretinal space tolerance of scaffolds with 200 nm fiber topography

Tissue engineered retinal pigment epithelial (RPE) transplantation is a promising cell-based therapy for age-related macular degeneration. The aim of this work is to develop a supportive scaffold with a favorable topography to aid functional RPE monolayer maintenance while being tolerated underneath the retina. To this end, films and electrospun substrates with fiber diameters ranging from 200 to 1000 nm were made of polyethylene terephthalate or poly(l-lactide-co-ε-caprolactone), and then tested using human fetal RPE cells in vitro and transplanted subretinally in rabbits. The results indicated that RPE on both 200 nm fiber variants showed the highest cell densities, adherent monolayers achieved deeper pigmentation, and more uniform hexagonal tight junctions. Facile subretinal implantation of flat 200 nm fiber membranes was achieved by electrospinning them onto a porous rigid-elastic carrier. Spectral-domain optical coherence tomography showed a reattached, slightly thinned retina overlying the implants over 2 weeks observation. Histology demonstrated native RPE variably migrated onto the nanofibers, and a reactive gliosis with some photoreceptor degeneration. In conclusion, scaffolds with 200 nm fiber topography enhanced RPE culture, showed subretinal biocompatibility, and should thus be considered for future cell-based therapies in blinding retinal diseases.     

Modulation of corneal epithelial stratification by polymer surface topography.

The topography and porosity of a polymer may affect the epithelialization of a corneal implant. We used an in vitro model to examine the effect of polymer surface topography on corneal epithelial tissue stratification and the deposition of proteins associated with epithelial adhesion. A range of topographies was provided by polycarbonate membranes with nominal pore diameters of 0.1, 0.4, 0.8, 1.0, 2.0, or 3.0 microm and a nonporous surface. Stratification of epithelial tissue outgrowth on these surfaces was evaluated using light and electron microscopy. Deposition of proteins associated with basement membrane and adhesion complex formation at the tissue-polymer interface was assessed using immunohistochemistry. Surfaces with pores in the 0.1-0.8-microm-diameter range supported superior stratification and protein deposition compared with those containing pores of > or = 1.0 microm. Cytoplasmic processes penetrated single pores 2.0 and 3.0 microm in diameter and fused pores 1.0 microm in diameter. Tissue on the nonporous surface had a lower level of stratification compared with surfaces with pores 0.1-0.8 microm in diameter. These results point to the significance of surface topography in biomaterial applications that require persistent epithelialization.     

Capture and alignment of phi29 viral particles in sub-40 nanometer porous alumina membranes

Bacteriophage phi29 virus nanoparticles and its associated DNA packaging nanomotor can provide for novel possibilities towards the development of hybrid bio-nano structures. Towards the goal of interfacing the phi29 viruses and nanomotors with artificial micro and nanostructures, we fabricated nanoporous Anodic Aluminum Oxide (AAO) membranes with pore size of 70 nm and shrunk the pores to sub 40 nm diameter using atomic layer deposition (ALD) of Aluminum Oxide. We were able to capture and align particles in the anodized nanopores using two methods. Firstly, a functionalization and polishing process to chemically attach the particles in the inner surface of the pores was developed. Secondly, centrifugation of the particles was utilized to align them in the pores of the nanoporous membranes. In addition, when a mixture of empty capsids and packaged particles was centrifuged at specific speeds, it was found that the empty capsids deform and pass through 40 nm diameter pores whereas the particles packaged with DNA were mainly retained at the top surface of the nanoporous membranes. Fluorescence microscopy was used to verify the selective filtration of empty capsids through the nanoporous membranes.     

Influence of polymer membrane porosity on C3A hepatoblastoma cell adhesive interaction and function

The effect of the porosity of acrylonitrile-N-vinylpyrrolidone copolymer membranes on human C3A hepatoblastoma cell adhesive interaction and functioning is investigated on four membranes with an average pore size ranging between 6 and 12 nm. Adhesion of C3A cells was quantified and characterized by studying overall cell morphology and focal adhesion formation. Cell-cell interactions were characterized by E-cadherin expression and organization. Cell growth, fibronectin synthesis and cytochrome P450 activity were estimated as criteria of functional cell activity. The results suggest that membrane porosity influences the initial cell-surface interactions since an increasing pore size augmented cell adhesion and aggregate formation. Cell growth after 7 d was diminished on membranes with an average pore size of 12 nm. The activity of P450 measured by 7-ethoxycoumarin conversion at day 7 was influenced by membrane topography representing a clear optimum in the range of 7-10 nm pore size. These results indicate that membrane porosity is a determinant for the function of hepatocytes in extracorporal liver assist devices.     

Neuronal cell biocompatibility and adhesion to modified CMOS electrodes

The use of CMOS (Complementary Metal Oxide Semiconductor) integrated circuits to create electrodes for biosensors, implants and drug-discovery has several potential advantages over passive multi-electrode arrays (MEAs). However, unmodified aluminium CMOS electrodes may corrode in a physiological environment. We have investigated a low-cost electrode design based on the modification of CMOS metallisation to produce a nanoporous alumina electrode as an interface to mammalian neuronal cells and corrosion inhibitor. Using NG108-15 mouse neuroblastoma x rat glioma hybrid cells, results show that porous alumina is biocompatible and that the inter-pore distance (pore pitch) of the alumina has no effect on cell vitality. To establish whether porous alumina and a cell membrane can produce a tight junction required for good electrical coupling between electrode and cell, we devised a novel cell detachment centrifugation assay to assess the long-term adhesion of cells. Results show that porous alumina substrates produced with a large pore pitch of 206 nm present a significantly improved surface compared to the unmodified aluminium control and that small pore-pitches of 17 nm and 69 nm present a less favourable surface for cell adhesion.     

Multiscale three-dimensional scaffolds for soft tissue engineering via multimodal electrospinning

A novel (scalable) electrospinning process was developed to fabricate bio-inspired multiscale three-dimensional scaffolds endowed with a controlled multimodal distribution of fiber diameters and geared towards soft tissue engineering. The resulting materials finely mingle nano- and microscale fibers together, rather than simply juxtaposing them, as is commonly found in the literature. A detailed proof of concept study was conducted on a simpler bimodal poly(ε-caprolactone) (PCL) scaffold with modes of fiber distribution at 600 nm and 3.3 μm. Three conventional unimodal scaffolds with mean diameters of 300 nm and 2.6 and 5.2 μm, respectively, were used as controls to evaluate the new materials. Characterization of the microstructure (i.e. porosity, fiber distribution and pore structure) and mechanical properties (i.e. stiffness, strength and failure mode) indicated that the multimodal scaffold had superior mechanical properties (Young’s modulus ～40 MPa and strength ～1 MPa) in comparison with the controls, despite the large porosity (～90% on average). A biological assessment was conducted with bone marrow stromal cell type (mesenchymal stem cells, mTERT-MSCs). While the new material compared favorably with the controls with respect to cell viability (on the outer surface), it outperformed them in terms of cell colonization within the scaffold. The latter result, which could neither be practically achieved in the controls nor expected based on current models of pore size distribution, demonstrated the greater openness of the pore structure of the bimodal material, which remarkably did not come at the expense of its mechanical properties. Furthermore, nanofibers were seen to form a nanoweb bridging across neighboring microfibers, which boosted cell motility and survival. Lastly, standard adipogenic and osteogenic differentiation tests served to demonstrate that the new scaffold did not hinder the multilineage potential of stem cells.     

A novel Bruch's membrane-mimetic electrospun substrate scaffold for human retinal pigment epithelium cells

Various artificial membranes have been used as scaffolds for retinal pigment epithelium cells (RPE) for monolayer reconstruction, however, long-term cell viability and functionality are still largely unknown. This study aimed to construct an ultrathin porous nanofibrous film to mimic Bruch's membrane, and in particular to investigate human RPE cell responses to the resultant substrates. An ultrathin porous nanofibrous membrane was fabricated by using regenerated wild Antheraea pernyi silk fibroin (RWSF), polycaprolactone (PCL) and gelatin (Gt) and displayed a thickness of 3–5 μm, with a high porosity and an average fiber diameter of 166 ± 85 nm. Human RPE cells seeded on the RWSF/PCL/Gt membranes showed a higher cell growth rate (p < 0.05), and a typical expression pattern of RPE signature genes, with reduced expression of inflammatory mediators. With long-term cultivation on the substrates, RPE cells exhibited characteristic polygonal morphology and development of apical microvilli. Immunocytochemisty demonstrated RPE-specific expression profiles in cells after 12-weeks of co-culture on RWSF/PCL/Gt membranes. Interestingly, the cells on the RWSF/PCL/Gt membranes functionally secreted polarized PEDF and phagocytosed labeled porcine POS. Furthermore, RWSF/PCL/Gt membranes transplanted subsclerally exhibited excellent biocompatibility without any evidence of inflammation or rejection. In conclusion, we established a novel RWSF-based substrate for growth of RPE cells with excellent cytocompatibility in vitro and biocompatibility in vivo for potential use as a prosthetic Bruch's membrane for RPE transplantation.     

Low-aspect ratio nanopatterns on bioinert alumina influence the response and morphology of osteoblast-like cells

Topographical features on the nanometer scale are known to influence cellular behavior. The response of specific cell types to various types of surface structures is currently still being investigated. Alumina ceramics play an important role as biomaterials, e.g., in medical and dental applications. In this study, we investigated the influence of nanoscale surface features with low aspect ratio (< 0.1) on the response of osteoblast-like MG-63 cells. To this end, low-energy ion irradiation was employed to produce shallow nanoscale ripple patterns on Al₂O₃(0001) surfaces with lateral periodicities of 24 nm and 179 nm and heights of only 0.7 and 11.5 nm, respectively. The nanopatterning was found to increase the proliferation of MG-63 cells and may lead to pseudopodia alignment along the ripples. Furthermore, focal adhesion behavior and cell morphology were analyzed. We found that MG-63 cells are able to recognize surface nanopatterns with extremely low vertical variations of less than 1 nm. In conclusion, it is shown that surface topography in the sub-nm range significantly influences the response of osteoblast-like cells.     

The effect of substrate surface nanotopography on the behavior of multipotnent mesenchymal stromal cells and osteoblasts

Hexagonally arranged Gold nanoparticles with controllable diameters and inter-particle distances were deposited on thick SiO₂ layers on top of Si wafers and used as masks during subsequent reactive ion etching. In this way, arrays of nanopillars are obtained with well-defined diameters (10/30 nm), inter-pillar distances (50–120 nm) and heights (20–35 nm), all on the nanoscale. Such nanotopographies served as substrate for multipotent mesenchymal stromal cells (MSC) and human osteoblasts (OB) allowing to study cellular responses to purely topographically patterned interfaces. Focus was put on adhesion, proliferation and differentiation of the cells. It turned out experimentally that adhesion is comparable for both cell types practically independent of topographical details at the substrate surface. Topography induced proliferation enhancement, however, is again independent of geometrical details in case of MSC, but significantly sensitive to pillar height in case of OB with a clear preference towards short nanopillars (20 nm). A high sensitivity to topographic details is also observed for osteogenic differentiation of MSC, in that case with a preference towards higher nanopillars (50 nm). The present experimental data also allow the important conclusion that cell proliferation and differentiation can be optimized simultaneously by fine-tuning nanoscaled topographical parameters.     

Persistent adhesion of epithelial tissue is sensitive to polymer topography.

The persistent adhesion of corneal epithelial tissue to the surface of a porous polymer is of interest in the development of a corneal onlay. Using an in vitro model system, this study examined the effect of polymer surface topography on the assembly of basement membrane and hemidesmosomes. Corneal epithelial tissue was grown on polycarbonate surfaces with a range of pore sizes (0.1-3.0 micron, pore diameter) and an equivalent nonporous surface. The ultrastructure of the tissue-polymer interface was evaluated using electron microscopy. On the porous surfaces, the tissue responded to a balance between the size of the discontinuity (pores) and the amount of polymer surface between the pores. Continuous basement membrane and a regular pattern of hemidesmosomal plaque occurred only on the 0.1 micron surface, where both the pores and the total surface area covered by pores were relatively small. The assembly of adhesive structures on surfaces with pore diameters between 0.4-2.0 microns was restricted to regions of polymer between pores. No adhesive structures assembled on the nonporous or on the 3.0-micron surface. These results demonstrate that, in addition to porosity, surface topography is a significant factor in the formation of structures involved in the persistent adhesion of stratified epithelial tissue on a polymer.     

Cultivation of hepatoma cell line HepG2 on nanoporous aluminum oxide membranes

Nanoporous aluminum oxide membranes were prepared by anodic oxidation of aluminum for application as novel cell culture substrates. Self-supporting as well as mechanically stabilized nanoporous membranes were produced from aluminum plates and micro-imprinted aluminum foils, respectively. Membranes of two different pore sizes (70 and 260 nm) were selected to investigate cellular interactions with such nanoporous substrates using cells of hepatoma cell line HepG2. The membranes express excellent cell-growth conditions. As shown by scanning electron microscopy investigations, the cells could easily adhere to the membranes and proliferate during a 4 day cell culture period. The cells exhibit normal morphology and were able to penetrate into pores with a diameter of 260 nm by small extensions (filopodia). On mechanically stabilized aluminum oxide membranes it was observed that the cells even adhere to the walls of the small cavities. It was demonstrated experimentally that the nanoporous aluminum oxide membranes are well suited as substrates in cell culture model systems for metabolic, pharmacological/toxicological research, tissue engineering and studies on pathogens as well as bioartificial liver systems.     

Silk film biomaterials for cornea tissue engineering

Biomaterials for corneal tissue engineering must demonstrate several critical features for potential utility in vivo, including transparency, mechanical integrity, biocompatibility and slow biodegradation. Silk film biomaterials were designed and characterized to meet these functional requirements. Silk protein films were used in a biomimetic approach to replicate corneal stromal tissue architecture. The films were 2 μm thick to emulate corneal collagen lamellae dimensions, and were surface patterned to guide cell alignment. To enhance trans-lamellar diffusion of nutrients and to promote cell–cell interaction, pores with 0.5–5.0 μm diameters were introduced into the silk films. Human and rabbit corneal fibroblast proliferation, alignment and corneal extracellular matrix expression on these films in both 2D and 3D cultures were demonstrated. The mechanical properties, optical clarity and surface patterned features of these films, combined with their ability to support corneal cell functions suggest that this new biomaterial system offers important potential benefits for corneal tissue regeneration.     

Systematic in vitro and in vivo study on porous silicon to improve the oral bioavailability of celecoxib

Mesoporous materials are promising candidates for improving dissolution rate of poorly water-soluble drugs in vitro and their bioavailability in vivo. In the present study, sixteen batches of celecoxib-loaded PSi particles with pore sizes ranging from 17 to 58 nm and celecoxib content from 5 to 36 w-% were prepared and a detailed physicochemical characterization of the drug was performed by several methods. Interaction between co-culture of Caco-2/HT29-MTX cells and unloaded PSi particles was tested in toxicity assays, and increased toxicity for particles with large pore size was observed. Dissolution rate of celecoxib was improved in vitro by lowering the drug loading degree which hindered the recrystallization of celecoxib on the external surface of the particles. The fastest permeation of loaded celecoxib through the co-culture monolayer as well as the highest bioavailability in rats was observed with the particles with small pore size and low loading degree. New insights were obtained on how various parameters of the mesoporous delivery system affect the state of the drug inside the pores and its release in vitro and in vivo.     

Investigation of cell-substrate interactions by focused ion beam preparation and scanning electron microscopy

Cell-substrate interactions, which are an important issue in tissue engineering, have been studied using focused ion beam (FIB) milling and scanning electron microscopy (SEM). Sample cross-sections were generated at predefined positions (target preparation) to investigate the interdependency of growing cells and the substrate material. The experiments focus on two cell culturing systems, hepatocytes (HepG2) on nanoporous aluminum oxide (alumina) membranes and mouse fibroblasts (L929) and primary nerve cells on silicon chips comprised of microneedles. Cross-sections of these soft/hard hybrid systems cannot be prepared by conventional techniques like microtomy. Morphological investigations of hepatocytes growing on nanoporous alumina membranes demonstrate that there is in-growth of microvilli from the cell surface into porous membranes having pore diameters larger than 200 nm. Furthermore, for various cell cultures on microneedle arrays contact between the cells and the microneedles can be observed at high resolution. Based on FIB milled cross-sections and SEM micrographs cells which are only in contact with microneedles and cells which are penetrated by microneedles can be clearly distinguished. Target preparation of biological samples by the FIB technique especially offers the possibility of preparing not only soft materials but also hybrid samples (soft/hard materials). Followed by high resolution imaging by SEM, new insights into cell surface interactions can be obtained.