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.     

Nano- and sub-micron porous polyelectrolyte multilayer assemblies: Biomimetic surfaces for human corneal epithelial cells

In vivo, corneal epithelial cells adhere on basement membranes that exhibit porosity on the nanoscale with the diameters of pores and fibers ranging from 20 to 200 nm. Polyelectrolyte multilayers with porosity ranging from the nano to the microscale were assembled to mimic the pore sizes of corneal membranes in vivo. The average pore diameter was found to be 100 nm and 600 nm for the nanoporous and sub-micron porous films respectively. In this study, a purely physical feature, specifically, porosity, provided cues to human corneal epithelial cells. Porous surfaces that exhibited either 100 nm or 600 nm pore diameters supported corneal cell adhesion, however, nanoscale porosity significantly enhanced corneal epithelial cellular response. Corneal epithelial cell proliferation and migration speeds were significantly higher on nanoporous topographies. The actin cytoskeletal organization was well defined and vinculin focal adhesions were found in cells presented with a nanoscale environment. These trends prevailed for fibronectin-coated surfaces as well suggesting that for human corneal epithelial cells, the physical environment plays a defining role in guiding cell behavior.     

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.     

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.     

Peptide-immobilized nanoporous alumina membranes for enhanced osteoblast adhesion

Bone tissue engineering requires the ability to regulate cell behavior through precise control over substrate topography and surface chemistry. Understanding of the cellular response to micro-environment is essential for biomaterials and tissue engineering research. This research employed alumina with porous features on the nanoscale. These nanoporous alumina surfaces were modified by physically adsorbing vitronectin and covalently immobilizing RGDC peptide to enhance adhesion of osteoblasts, bone-forming cells. X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) were used to characterize the modified nanoporous alumina surface. Survey and high-resolution C1s scans suggested the presence of RGDC and vitronectin on the surface and SEM images confirmed the pores were not clogged after modification. Cell adhesion on both unmodified and modified nanoporous alumina was compared using fluorescence microscopy and SEM. RGDC was found to enhance osteoblast adhesion after 1 day of culture and matrix production was visible after 2 days. Cell secreted matrix was absent on unmodified membranes for the same duration. Vitronectin-adsorbed surfaces did not show significant improvement in adhesion over unmodified membranes.     

Influence of nanoporous alumina membranes on long-term osteoblast response

A major goal of bone tissue engineering is to design better scaffold configuration and materials to better control osteoblast behavior. Nanoporous architecture has been shown to significantly affect cellular response. In this work, nanoporous alumina membranes were fabricated by a two-step anodization method to investigate bone cell response. Osteoblasts were seeded on nanoporous alumina membranes to investigate both short-term adhesion and proliferation and long-term functionality and matrix production. Cell adhesion and proliferation were characterized using a standard MTT assay and cell counting. The total protein content was measured after cell lysis using the BCA assay. Matrix production was characterized in terms of surface concentrations of calcium and phosphorous, components of bone matrix, using X-ray photoelectron spectroscopy (XPS). The results from nanoporous alumina membranes were compared with those of amorphous alumina, aluminum, commercially available ANOPORE and glass. Results indicate improved osteoblast adhesion and proliferation and increased matrix production after 4 weeks of study.     

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.     

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.     

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.     

Nanoporesize affects complement activation

In the present study, we have shown the vast importance of biomaterial nanotexture when evaluating inflammatory response. For the first time in an in vitro whole blood system, we have proven that a small increase in nanoporesize, specifically 180 nm (from 20 to 200 nm), has a huge effect on the complement system. The study was done using nanoporous aluminiumoxide, a material that previously has been evaluated for potential implant use, showing good biocompatibility. This material can easily be manufactured with different pore sizes making it an excellent candidate to govern specific protein and cellular events at the tissue-material interface. We performed whole blood studies, looking at complement activation after blood contact with two pore size alumina membranes (pore diameters, 20 and 200 nm). The fluid phase was analyzed for complement soluble components, C3a and sC5b-9. In addition, surface adsorbed proteins were eluted and dot blots were performed to detect IgG, IgM, C1q, and C3. All results point to the fact that 200 nm pore size membranes are more complement activating. Significantly, higher values of complement soluble components were found after whole blood contact with 200 nm alumina and all studied proteins adsorbed more readily to this membrane than to the 20 nm pore size membrane. We hypothesize that the difference in complement activation between our two test materials is caused by the type and the amount of adsorbed proteins, as well as their conformation and orientation. The different protein patterns created on the two alumina membranes are most likely a consequence of the material topography.     

Fabrication and Characterization of Waterborne Biodegradable Polyurethanes 3-Dimensional Porous Scaffolds for Vascular Tissue Engineering

In this study, a series of 3-D interconnected porous scaffolds with various pore diameters and porosities was fabricated by freeze-drying with non-toxic biodegradable waterborne polyurethane (WBPU) emulsions of different concentration. The structures of these porous scaffolds were characterized by scanning electron microscopy (SEM), and the pore diameters were calculated using CIAS 3.0 software. The pores obtained were 3-D interconnected in the scaffolds. The scaffolds obtained at different pre-freeze temperatures showed a pore diameter ranging from 2.8 to 99.9 μm with a pre-freezing temperature of ?60°C and from 13.1 to 229.1 μm with a pre-freezing temperature of ?25°C. The scaffolds fabricated with WBPU emulsions of different concentration at the same pre-freezing temperature (?25°C) had pores with mean pore diameter between 90.8 and 39.6 μm and porosity between 92.0 and 80.0%, depending on the emulsion concentration. The effect of porous structure of the scaffolds on adhesion and proliferation of human umbilical vein endothelial cells (HUVECs) cultured in vitro was evaluated using the MTT assay and environmental scanning electron microscopy (ESEM). It was found that the better adhesion and proliferation of HUVECs on 3-D scaffolds of WBPU with relative smaller pore diameter and lower porosity than those on scaffolds with larger pore and higher porosity and film. Our work suggests that fabricating a scaffold with controllable pore diameter and porosity could be a good method to be used in tissue-engineering applications to obtain carriers for cell culture in vitro.     

Porous and single-skinned polyethersulfone membranes support the growth of HepG2 cells: a potential biomaterial for bioartificial liver systems

In this study, we evaluated a porous and single-layer skin polyethersulfone (PES) membrane as a material for use in hybrid bioartificial liver support systems. The PES membrane has been characterized as a single-layer skin structure, with a rough porous surface. Specifically, we studied the ability of the human hepatoblastoma cell lines (HepG2) to adhere, grow, and spread on the PES membrane. Furthermore, we examined albumin secretion, low-density lipoprotein uptake, and CYP450 activity of HepG2 cells that grew on the membrane. HepG2 cells readily adhered onto the outer surfaces of PES membranes. Over time, HepG2 cells proliferated actively, and confluent monolayer of cells covered the available surface area of the membrane, eventually forming cell clusters and three-dimensional aggregates. Furthermore, HepG2 cells grown on PES membranes maintained highly specific functions, including uptake capability, biosynthesis and biotransformation. These results indicate that PES membranes are potential substrates for the growth of human liver cells and may be useful in the construction of hollow fiber bioreactors. Porous and single-layer skin PES membranes and HepG2 cells may be potential biomaterials for the development of biohybrid liver devices.     

Control of hepatocyte function on collagen foams: sizing matrix pores toward selective induction of 2-D and 3-D cellular morphogenesis

While microporous biopolymer matrices are being widely tested as cell culture substrates in hepatic tissue engineering, the microstructural basis for their control of cell differentiation is not well understood. In this paper, we studied the role of void size of collagen foams in directing the induction of liver-specific differentiated morphology and secretory activities of cultured rat hepatocytes. Hepatocytes cultured on collagen foams with subcellular sized pore diameters of 10 microm assumed a compact, cuboidal cell morphology, rapidly achieving monolayer coverage, and secreted albumin at the rate of 40 +/- 8 pg/cell/d. Increasing the pore size to 18 microm elicited a distinctly spread cellular phenotype, with discontinuous surface coverage, and albumin secretion rates declined precipitiously to 3.6 +/- 0.8 pg/cell/d. However, when collagen foams with an even higher average void size of 82 microm were used, hepatocytes exhibited high degree of spreading within an extensive three-dimensional cellular network, and exhibited high albumin secretory activity (26 +/- 0.6 pg/cell/d). The effect of void geometry on cellular ultrastructral polarity was further analyzed for the three void size configurations employed. The distribution of the cell-cell adhesion protein, E-cadherin, was primarily restricted to cell-cell contacts on the 10 microm foams, but was found to be depolarized to all membrane regions in cells cultured on the 18 and 82 microm foams. Vinculin-enriched focal adhesions were found to be peripherally clustered on cells cultured on 10 microm foams, but were found to redistribute to the entire ventral surface of cells cultured on the 18 and 82 microm foams. Overall, we demonstrate the significance of designing pore sizes of highly adhesive substrates like collagen toward selective cell morphogenesis in 2-D or 3-D. Subcellular and supercellular ranges of pore size promote hepatocellular differentiation by limiting 2-D cell spreading or effecting 3-D intercellular contacts, while intermediate range of pore sizes repress differentiation by promoting 2-D cell spreading.     

Improved adhesion of human cultured periosteal sheets to a porous poly(L-lactic acid) membrane scaffold without the aid of exogenous adhesion biomolecules

Human cultured periosteal sheets, which are developed from small excised periosteum tissue segments (PTSs) in culture dishes by simple expansion culture, have been applied as a promising autologous osteogenic grafting material for periodontal regenerative therapy. However, the weak initial adhesion of PTSs to dish surfaces often hampers cellular outgrowth and limits the number of preparations. To correct this weakness and still avoid the use of animal-derived adhesion biomolecules, we have developed a novel, biodegradable, porous poly(L-lactic acid) (pPLLA) membrane. Freshly excised PTSs bound well to the highly porous pPLLA membrane, possibly due to the presence of semihemispheric 20-30 μm diameter openings on the upper surface. Global gene expression analysis demonstrated that periosteal sheets cultured on pPLLA membranes upregulated expression of many adhesion molecules. Osteogenic induction stimulated the production of proteoglycans by these cells and concomitantly enhanced their expansion and penetration into the deep pore regions of the membrane in parallel with the progression of in vitro mineralization. These findings suggest that our pPLLA membranes not only facilitate initial adhesion, primarily mediated by adsorbed proteins, but also enhance biological adhesion by inducing endogenous adhesion molecules in periosteal sheet cultures. Therefore, the efficacy of periosteal sheets in therapy should be greatly enhanced by using this new pPLLA membrane.     

Osteogenic differentiation of marrow stromal cells cultured on nanoporous alumina surfaces

A major goal in orthopedic biomaterials research is to design implant surfaces, which will enhance osseointegration in vivo. Several microscale as well as nanoscale architectures have been shown to significantly affect the functionality of bone cells i.e., osteoblasts. In this work, nanoporous alumina surfaces fabricated by a two-step anodization process were used. The nanostructure of these surfaces can be controlled by varying the voltage used for anodization process. Marrow stromal cells were isolated from mice and seeded on nanoporous and amorphous (control) alumina surfaces. Cell adhesion, proliferation, and viability were investigated for up to 7 days of culture. Furthermore, the cell functionality was investigated by calcein staining. The cells were provided with differentiation media after 7 days of culture. The alkaline phosphatase (ALP) activity and matrix production were quantified using a colorimetric assay and X-ray photoelectron spectroscopy (XPS) for up to 3 weeks of culture (2 weeks after providing differentiation media). Further, scanning electron microscopy (SEM) was used to investigate osteoblast morphology on these nanoporous surfaces. Over the 3-week study, the nanoporous alumina surfaces demonstrated approximately 45% increase in cell adhesion, proliferation, and viability, 35% increase in ALP activity, and 50% increase in matrix production when compared with the control surfaces.     

Human mesenchymal stem cell adhesion and proliferation in response to ceramic chemistry and nanoscale topography

Modification of the chemistry and surface topography of nanophase ceramics was used to provide biomaterial formulations designed to direct the adhesion and proliferation of human mesenchymal stem cells (HMSCs). HMSC adhesion was dependent upon both the substrate chemistry and grain size, but not on surface roughness or crystal phase. Specifically, cell adhesion on alumina and hydroxyapatite was significantly reduced on the 50 and 24 nm surfaces, as compared with the 1500 and 200 nm surfaces, but adhesion on titania substrates was independent of grain size. HMSC proliferation was minimal on the 50 and 24 nm substrates of any chemistry tested, and thus significantly lower than the densities observed on either the 1500 or 200 nm surfaces after 3 or more consecutive days of culture. Furthermore, HMSC proliferation was enhanced on the 200 nm substrates, compared with results obtained on the 1500 nm substrates after 7 or more days of culture. HMSC proliferation was independent of both substrate surface roughness and crystal phase. Rat osteoblast and fibroblast adhesion and proliferation exhibited similar trends to that of HMSCs on all substrates tested. These results demonstrated the potential of nanophase ceramic surfaces to modulate functions of HMSCs, which are pertinent to biomedical applications such as implant materials and devices.     

Micropatterning of a nanoporous alumina membrane with poly(ethylene glycol) hydrogel to create cellular micropatterns on nanotopographic substrates

In this paper, we describe a simple method for fabricating micropatterned nanoporous substrates that are capable of controlling the spatial positioning of mammalian cells. Micropatterned substrates were prepared by fabricating poly(ethylene glycol) (PEG) hydrogel microstructures on alumina membranes with 200 nm nanopores using photolithography. Because hydrogel precursor solution could infiltrate and become crosslinked within the nanopores, the resultant hydrogel micropatterns were firmly anchored on the substrate without the use of adhesion-promoting monolayers, thereby allow tailoring of the surface properties of unpatterned nanoporous areas. For mammalian cell patterning, arrays of microwells of different dimensions were fabricated. These microwells were composed of hydrophilic PEG hydrogel walls surrounding nanoporous bottoms that were modified with cell-adhesive Arg-Gly-Asp (RGD) peptides. Because the PEG hydrogel was non-adhesive towards proteins and cells, cells adhered selectively and remained viable within the RGD-modified nanoporous regions, thereby creating cellular micropatterns. Although the morphology of cell clusters and the number of cells inside one microwell were dependent on the lateral dimension of the microwells, adhered cells that were in direct contact with nanopores were able to penetrate into the nanopores by small extensions (filopodia) for all the different sizes of microwells evaluated.     

Novel apatite fiber scaffolds can promote three-dimensional proliferation of osteoblasts in rodent bone regeneration models

We have successfully synthesized hydroxyapatite fibers via a homogenous precipitation method. Using these hydroxyapatite fibers, we have produced the apatite fiber scaffolds (AFS) with well-controlled pore sizes (porosity above 95%). The AFS is relatively simple to synthesize, and its porosity and pore size are controllable. The usefulness of AFS as a scaffold for bone regeneration was evaluated by (1) seeding and culturing cells in the AFS in vitro, (2) implanting the AFS seeded with cells inside the subcutaneous tissue of mice. The AFS had biocompatibility to support cell adhesion, proliferation, and differentiation. Ectopic bone formation could be formed in the AFS at 12 weeks after implantation into the subcutaneous tissue. Because of its high interpore connection, pore diameters, and porosity, it was believed that AFS was an effective scaffold that provided a three-dimensional cell culture environment. In both in vitro and in vivo environments, the more porous AFS was more advantageous in cell proliferation, cell adhesion, proliferating capacity, robust cell differentiation, ultimately inducing bone ingrowth inside the scaffolds.     

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.     

Improved bone-forming functionality on diameter-controlled TiO2 nanotube surface

The titanium dioxide (TiO₂) nanotube surface enables significantly accelerated osteoblast adhesion and exhibits strong bonding with bone. We prepared various sizes (30–100 nm diameter) of titanium dioxide (TiO₂) nanotubes on titanium substrates by anodization and investigated the osteoblast cellular behavior in response to these different nanotube sizes. The unique and striking result of this study is that a change in osteoblast behavior is obtained in a relatively narrow range of nanotube dimensions, with small diameter (～30 nm) nanotubes promoting the highest degree of osteoblast adhesion, while larger diameter (70–100 nm) nanotubes elicit a lower population of cells with extremely elongated cellular morphology and much higher alkaline phosphatase levels. Increased elongation of nuclei was also observed with larger diameter nanotubes. By controlling the nanotopography, large diameter nanotubes, in the ～100 nm regime, induced extremely elongated cellular shapes, with an aspect ratio of 11:1, which resulted in substantially enhanced up-regulation of alkaline phosphatase activity, suggesting greater bone-forming ability than nanotubes with smaller diameters. Such nanotube structures, already being a strongly osseointegrating implant material, offer encouraging implications for the development and optimization of novel orthopedics-related treatments with precise control toward desired cell and bone growth behavior.