A photolabile hydrogel for guided three-dimensional cell growth and migration

Tissue engineering aims to replace, repair or regenerate tissue/organ function, by delivering signalling molecules and cells on a three-dimensional (3D) biomaterials scaffold that supports cell infiltration and tissue organization. To control cell behaviour and ultimately induce structural and functional tissue formation on surfaces, planar substrates have been patterned with adhesion signals that mimic the spatial cues to guide cell attachment and function. The objective of this study is to create biochemical channels in 3D hydrogel matrices for guided axonal growth. An agarose hydrogel modified with a cysteine compound containing a sulphydryl protecting group provides a photolabile substrate that can be patterned with biochemical cues. In this transparent hydrogel we immobilized the adhesive fibronectin peptide fragment, glycine-arginine-glycine-aspartic acid-serine (GRGDS), in selected volumes of the matrix using a focused laser. We verified in vitro the guidance effects of GRGDS oligopeptide-modified channels on the 3D cell migration and neurite outgrowth. This method for immobilizing biomolecules in 3D matrices can generally be applied to any optically clear hydrogel, offering a solution to construct scaffolds with programmed spatial features for tissue engineering applications.     

Shear assay measurements of cell adhesion on biomaterials surfaces

The paper examines the adhesion of human osterosarcoma (HOS) cells to selected biomaterials surfaces that are relevant to implantable biomedical systems and bio-micro-electro-mechanical systems (BioMEMS). The four biomaterials that were explored include: silicon, silicon coated with a nanoscale layer of titanium, Ti–6Al–4V, and poly-di-methy-siloxane (PDMS). The interfacial strengths between the HOS cells and the biomaterials surfaces were determined using a shear assay technique. The adhesion forces were determined using a combination of confocal microscopy images of the three-dimensional cell structure, and computational fluid dynamics (CFD) simulations that coupled actual cell morphologies and non-Newtonian fluid properties in the computation of the adhesion forces. After cell detachment by the shear assay, immunofluorescence staining of the biomedical surfaces was used to reveal the proteins associated with cell detachment. These revealed that the nano-scale Ti coating increases the cell/surface adhesion strength. Silicon with Ti coating has the strongest adhesion strength, while the other surfaces had similar adhesion strength. The measured strengths are shown to be largely associated with the detachment of focal adhesion proteins from extra-cellular matrix (ECM) proteins.     

Plant surfaces with cuticular folds are slippery for beetles

Plant surfaces covered with three-dimensional (3D) waxes are known to strongly reduce insect adhesion, leading to slippery surfaces. Besides 3D epicuticular waxes, cuticular folds are a common microstructure found on plant surfaces, which have not been quantitatively investigated with regard to their influence on insect adhesion. We performed traction experiments with Colorado potato beetles on five plant surfaces with cuticular folds of different magnitude. For comparison, we also tested (i) smooth plant surfaces and (ii) plant surfaces possessing 3D epicuticular waxes. Traction forces on surfaces with medium cuticular folds, of about 0.5 µm in both height and thickness and a spacing of 0.5–1.5 µm, were reduced by an average of 88 per cent in comparison to smooth plant surfaces. Traction forces were reduced by the same order of magnitude as on plant surfaces covered with 3D epicuticular waxes. For surface characterization, we performed static contact angle measurements, which proved a strong effect of cuticular folds also on surface wettability. Surfaces possessing cuticular folds of greater magnitude showed higher contact angles up to superhydrophobicity. We hypothesize that cuticular folds reduce insect adhesion mainly due to a critical roughness, reducing the real contact area between the surface and the insect''s adhesive devices.     

An investigation of the initial attachment and orientation of osteoblast-like cells on laser grooved Ti-6Al-4V surfaces

This paper presents the results of an experimental study of the initial cell spreading and adhesion on longitudinally- and transversally-oriented micro-grooves produced by the laser irradiation of laser grooved Ti-6Al-4V surfaces. The initial spreading and orientations of human osteosarcoma (HOS) cells were observed and quantified after 15-min, 1-hour, 4-hour and 24-hour cell culture periods. Immuno-fluorescence staining of adhesion proteins (actin and vinculin) was then used to study the spreading and adhesion of HOS cells in 1 hour and 4 hour culture experiments. The initial cell adhesion was also quantified using enzymatic detachment tests. The results showed that cell spreading and adhesion were enhanced by longitudinally- and transversally-oriented micro-grooves. The effects, which increase with time, were not remarkable after 1 hour, but obvious after 4 hours. Contact guidance was found to promote cell adhesion due to the increase in interactions between the focal adhesions and the patterned extra-cellular matrix (ECM) proteins on the laser micro-grooved surfaces.     

Fibrin-mediated endothelial cell adhesion to vascular biomaterials resists shear stress due to flow

In vitro endothelial cell (EC) seeding onto biomaterials for blood-contacting applications can improve the blood compatibility of materials. Adhesive proteins adsorbed from serum that is supplemented with the culture medium intercede the initial cell adhesion and subsequent spreading on material surface during culture. Nevertheless, physical and chemical properties of vascular biomaterial surface fluctuate widely between materials resulting in dissimilarity in protein adsorption characteristics. Thus, a variation is expected in cell adhesion, growth and the ability of cell to resist shear stress when tissue engineering on to vascular biomaterials is attempted. This study was carried out with an objective to determine the significance of a matrix coating on cell adhesion and shear stress resistance when cells are cultured on materials such as polytetrafluoroethylene (PTFE, Teflon) and polyethyleneterephthalate (Dacron), ultra high molecular weight polyethylene (UHMWPE) and titanium (Ti), that are used for prosthetic devices. The study illustrates the distinction of EC attachment and proliferation between uncoated and matrix-coated surfaces. The cell attachment and proliferation on uncoated UHMWPE and titanium surfaces were not significantly different from matrix-coated surfaces. However, shear stress resistance of the cells grown on composite coated surfaces appeared superior compared to the cells grown on uncoated surface. On uncoated vascular graft materials, the cell adhesion was not supported by serum alone and proliferation was scanty as compared to matrix-coated surface. Therefore, coating of implant devices with a composite of adhesive proteins and growth factors can improve EC attachment and resistance of the cells to the forces of flow.     

Osteoblast attachment to a textured surface in the absence of exogenous adhesion proteins

The present study investigated whether osteoblasts could attach to a culture substratum through a surface texture-dependent mechanism. Four test groups were used: (A) untextured, and three texture groups with maximum feature sizes of (B) <0.5 /spl mu/m, (C) 2 /spl mu/m, and (D) 4 /spl mu/m, respectively. All surfaces were coated with the nonadhesive protein bovine serum albumin (BSA). Osteoblasts were allowed to adhere in serum-free medium for either 1 or 4 h, at which time nonadherent cells were removed. At 4 h, untextured surface A exhibited no cell attachment, while textured surfaces B, C, and D exhibited 9%, 32%, and 16% cell adhesion, respectively. At 16 h of incubation, adherent osteoblasts on textured surface C exhibited focal adhesion contacts and microfilament stress-fiber bundles. These results indicate that microtextured surfaces in the absence of exogenous adhesive proteins can facilitate osteoblast adhesion.     

Biocompatibility of TiO2 nanotubes fabricated on Ti using different surfactant additives in electrolyte

This study examined the in vitro cell-material interactions on four different types of titanium surfaces: a polished Ti surface, TiO₂ nanotube surfaces fabricated in a fluorinated glycerol solution (TN), fluorinated glycerol solution with 1 wt% anionic surfactant sodium dodecyl sulphate (TN-SDS), and fluorinated glycerol solution with 1 wt% cationic surfactant cetyl trimethyl ammonium bromide (TN-CTAB), respectively. The surfaces exhibited distinct surface morphologies and geometrical features. Surface energy calculation shows that TN surface enhances the hydrophilic character by significantly increasing the surface energy. The osteoblast cell growth behavior on the four different surfaces was examined using the MC3T3-E1 cell line for 1 day. When the anodized surfaces were compared for the cell-materials interaction, each of the surfaces showed different properties that affected the cell–material interactions. Proliferation of the cells was noticed with distinctive cell-to-cell attachment on the TN surfaces. Good cellular adhesion with extracellular matrix extensions between the cells was noticed in the TN samples. The TiO₂ nanotubes grown in the surfactant-assisted fluorinated electrolyte did not show significant cell growth on the surface and some cell death was observed. The cell adhesion, differentiation and alkaline phosphatase activity were more pronounced on the TN surface. The MTT assays also revealed an increase in living cell density and proliferation on the TN surfaces. Overall, a rough surface morphology and surface energy are important factors for better cell material interactions.     

Biofunctionalization strategies on tantalum-based materials for osseointegrative applications

The use of tantalum as biomaterial for orthopedic applications is gaining considerable attention in the clinical practice because it presents an excellent chemical stability, body fluid resistance, biocompatibility, and it is more osteoconductive than titanium or cobalt-chromium alloys. Nonetheless, metallic biomaterials are commonly bioinert and may not provide fast and long-lasting interactions with surrounding tissues. The use of short cell adhesive peptides derived from the extracellular matrix has shown to improve cell adhesion and accelerate the implant’s biointegration in vivo. However, this strategy has been rarely applied to tantalum materials. In this work, we have studied two immobilization strategies (physical adsorption and covalent binding via silanization) to functionalize tantalum surfaces with a cell adhesive RGD peptide. Surfaces were used untreated or activated with either HNO₃ or UV/ozone treatments. The process of biofunctionalization was characterized by means of physicochemical and biological methods. Physisorption of the RGD peptide on control and HNO₃-treated tantalum surfaces significantly enhanced the attachment and spreading of osteoblast-like cells; however, no effect on cell adhesion was observed in ozone-treated samples. This effect was attributed to the inefficient binding of the peptide on these highly hydrophilic surfaces, as evidenced by contact angle measurements and X-ray photoelectron spectroscopy. In contrast, activation of tantalum with UV/ozone proved to be the most efficient method to support silanization and subsequent peptide attachment, displaying the highest values of cell adhesion. This study demonstrates that both physical adsorption and silanization are feasible methods to immobilize peptides onto tantalum-based materials, providing them with superior bioactivity.     

Surface coatings for improving the corrosion resistance and cell adhesion of AZ91D magnesium alloy through environmentally clean methods

Two different coatings, Mg-Al hydrotalcite and aragonite (CaCO₃), were produced on die-cast AZ91D Mg alloy (Mg-9wt.%Al-1wt.%Zn) through environmentally clean methods. The corrosion resistance and human mesenchymal stem cell adhesion properties of the coatings on AZ91D were investigated. Results showed that through environmentally clean methods, both Mg-Al hydrotalcite and aragonite CaCO₃ surface coatings could be produced on AZ91D surfaces. These coatings increased the corrosion potential and polarization resistance and decreased the corrosion rate of AZ91D in simulated body fluid. Both surface coatings also improved stem cell spreading and interaction in culture. Further long-term animal testing is suggested before these surface coatings can be recommended for use in clinical applications.     

Polyelectrolyte Multilayer-Treated Electrodes for Real-Time Electronic Sensing of Cell Proliferation

We report on the use of polyelectrolyte multilayer (PEM) coatings as a non-biological surface preparation to facilitate uniform cell attachment and growth on patterned thin-film gold (Au) electrodes on glass for impedance-based measurements. Extracellular matrix (ECM) proteins are commonly utilized as cell adhesion promoters for electrodes; however, they exhibit degradation over time, thereby imposing limitations on the duration of conductance-based biosensor experiments. The motivation for the use of PEM coatings arises from their long-term surface stability as promoters for cell attachment, patterning, and culture. In this work, a cell proliferation monitoring device was fabricated. It consisted of thin-film Au electrodes deposited with a titanium-tungsten (TiW) adhesion layer that were patterned on a glass substrate and passivated to create active electrode areas. The electrode surfaces were then treated with a poly(ethyleneimine) (PEI) anchoring layer and subsequent bilayers of sodium poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). NIH-3T3 mouse embryonic fibroblast cells were cultured on the device, observed by optical microscopy, and showed uniform growth characteristics similar to those observed on a traditional polystyrene cell culture dish. The optical observations were correlated to electrical measurements on the PEM-treated electrodes, which exhibited a rise in impedance with cell proliferation and stabilized to an approximate 15 % increase as the culture approached confluency. In conclusion, cells proliferate uniformly over gold and glass PEM-treated surfaces, making them useful for continuous impedance-based, real-time monitoring of cell proliferation and for the determination of cell growth rate in cellular assays.     

Interactions of Fibroblasts with Different Morphologies Made of an Engineered Spider Silk Protein

Spider silk has been investigated for decades due to the intriguing mechanical and also biomedical properties of the silk fibers. Previously, it has been shown that recombinant silk proteins can also be processed into other morphologies. Here, we characterized scaffolds made of the recombinant spider silk protein eADF4(C16) concerning their surface interactions with fibroblasts. Studies of BALB/3T3 cells on hydrogels and films made of eADF4(C16) showed low cell adhesion without observable duplication. Electro‐spun non‐woven scaffolds made of eADF4(C16), however, enabled both their adhesion and proliferation. Since eADF4(C16) lacks specific motifs for cell attachment, fibroblasts cannot generate focal adhesions with the material's surface, and, therefore, other cell–interface interactions such as topographical anchorage or cell attachment mediated by adhesion of extracellular matrix proteins are discussed in this paper. On non‐woven meshes protrusion of filopodia and/or lamellipodia between individual fibers increase the surface contact area, which depends on the diameter of the fibers of the non‐woven meshes. In contrast, at flat (film) or microstructured surfaces (hydrogels) such interactions seem to be precluded.     

CyclinD1, CDK4, and P21 expression by IEC-6 cells in response to NiTi alloy and polymeric biomaterials

In order to investigate how cells recognize biomaterials, mRNA that was expressed in attached Intestinal epithelial cells (IEC-6) on various suture substrates was evaluated. The expressed cell cycle regulators (cyclin D1, CDK4 and p21) mRNA were then isolated and detected using the real time- polymerase chain reaction (PCR) method. As a result, cyclin D1 gene expression was affected by cell-polymer adhesion and was associated with cell proliferation. In addition, CDK4 gene expression was affected by cell proliferation rather than by cell-biomaterial interaction. The p21 mRNA gene expression was higher in cells on more hydrophilic surfaces than on hydrophobic surfaces. Further, the cyclin D1, CDK4 and p21 gene expression were also influenced by the surface chemistry of suture materials. We concluded that the expression of cyclin D1, CDK4 and p21 mRNA was a powerful method for studying cell-biomaterial interactions or the evaluation of the carcinogenic activity of biomaterials.     

Biomimetic coatings functionalized with adhesion peptides for dental implants

A complete biological integration into the surrounding tissues (bone, gingiva) is a critical step for clinical success of a dental implant. In this work biomimetic coatings consisting either of collagen type I (for the gingiva region) and hydroxyapatite (HAP) or mineralized collagen (for the bone interface) have been developed as suitable surfaces regarding the interfaces. Additionally, using these biomimetic coatings as a matrix, adhesion peptides were bound to further increase the specificity of titanium implant surfaces. To enhance cell attachment in the gingiva region, a linear adhesion peptide developed from a laminin sequence (TWYKIAFQRNRK) was bound to collagen, whereas for the bone interface, a cyclic RGD peptide was bound to HAP and mineralized collagen using adequate anchor systems. The biological potential of these coatings deduced from cell attachment experiments with HaCaT human keratinocytes and MC3T3-E1 mouse osteoblasts showed the best results for collagen and laminin sequence coating for the gingiva region and mineralized collagen and RGD peptide coatings for regions with bone contact. Our concept opens promising approaches to improve the biological integration of dental implants.     

Degradable polyester scaffolds with controlled surface chemistry combining minimal protein adsorption with specific bioactivation

Advanced biomaterials and scaffolds for tissue engineering place high demands on materials and exceed the passive biocompatibility requirements previously considered acceptable for biomedical implants. Together with degradability, the activation of specific cell–material interactions and a three-dimensional environment that mimics the extracellular matrix are core challenges and prerequisites for the organization of living cells to functional tissue. Moreover, although bioactive signalling combined with minimization of non-specific protein adsorption is an advanced modification technique for flat surfaces, it is usually not accomplished for three-dimensional fibrous scaffolds used in tissue engineering. Here, we present a one-step preparation of fully synthetic, bioactive and degradable extracellular matrix-mimetic scaffolds by electrospinning, using poly(D,L-lactide-co-glycolide) as the matrix polymer. Addition of a functional, amphiphilic macromolecule based on star-shaped poly(ethylene oxide) transforms current biomedically used degradable polyesters into hydrophilic fibres, which causes the suppression of non-specific protein adsorption on the fibres’ surface. The subsequent covalent attachment of cell-adhesion-mediating peptides to the hydrophilic fibres promotes specific bioactivation and enables adhesion of cells through exclusive recognition of the immobilized binding motifs. This approach permits synthetic materials to directly control cell behaviour, for example, resembling the binding of cells to fibronectin immobilized on collagen fibres in the extracellular matrix of connective tissue.     

Surface wettability of plasma SiOx:H nanocoating-induced endothelial cells' migration and the associated FAK-Rho GTPases signalling pathways

Vascular endothelial cell (EC) adhesion and migration are essential processes in re-endothelialization of implanted biomaterials. There is no clear relationship and mechanism between EC adhesion and migration behaviour on surfaces with varying wettabilities. As model substrates, plasma SiO_x:H nanocoatings with well-controlled surface wettability (with water contact angles in the range of 98.5 ± 2.3° to 26.3 ± 4.0°) were used in this study to investigate the effects of surface wettability on cell adhesion/migration and associated protein expressions in FAK-Rho GTPases signalling pathways. It was found that EC adhesion/migration showed opposite behaviour on the hydrophilic and hydrophobic surfaces (i.e. hydrophobic surfaces promoted EC migration but were anti-adhesions). The number of adherent ECs showed a maximum on hydrophilic surfaces, while cells adhered to hydrophobic surfaces exhibited a tendency for cell migration. The focal adhesion kinase (FAK) inhibitor targeting the Y-397 site of FAK could significantly inhibit cell adhesion/migration, suggesting that EC adhesion and migration on surfaces with different wettabilities involve (p)FAK and its downstream signalling pathways. Western blot results suggested that the FAK-Rho GTPases signalling pathways were correlative to EC migration on hydrophobic plasma SiO_x:H surfaces, but uncertain to hydrophilic surfaces. This work demonstrated that surface wettability could induce cellular behaviours that were associated with different cellular signalling events.     

Investigation of cancer cell behavior on nanofibrous scaffolds

Tissue engineering and the use of nanofibrous biomaterial scaffolds offer a unique perspective for studying cancer development in vitro. Current in vitro models of tumorigenesis are limited by the use of static, two-dimensional (2D) cell culture monolayers that lack the structural architecture necessary for cell-cell interaction and three-dimensional (3D) scaffolds that are too simplistic for studying basic pathological mechanisms. In this study, two nanofibrous biomaterials that mimic the structure of the extracellular matrix, bacterial cellulose and electrospun polycaprolactone (PCL)/collagen I, were investigated as potential 3D scaffolds for an in vitro cancer model. Multiple cancer cell lines were cultured on each scaffold material and monitored for cell viability, proliferation, adhesion, infiltration, and morphology. Both bacterial cellulose and electrospun PCL/collagen I, which have nano-scale structures on the order of 100-500 nm, have been used in many diverse tissue engineering applications. Cancer cell adhesion and growth were limited on bacterial cellulose, while all cellular processes were enhanced on the electrospun scaffolds. This initial analysis has demonstrated the potential of electrospun PCL/collagen I scaffolds toward the development of an improved 3D in vitro cancer model.     

Monocyte adhesion and adhesion molecule expression on human endothelial cells on plasma-treated PET and PTFE in vitro

The aim of this study was to evaluate in vitro the inflammatory potential of endothelialized surfaces of polyethylene terephthalate (PET) and polytetrafluorethylene (PTFE) after ammonia gas plasma modification. HUVECs grown on polystyrene and HUVECs stimulated with tumor necrosis factor (TNF-alpha) were used as controls. At day 1 and day 7, surfaces were evaluated for U937 cells and HUVECs using flow cytometry and immunohistochemistry. Plasma-treated PET (T-PET) and treated PTFE (T-PTFE) increased U937 cell adhesion compared to the negative control but this was not statistically significant. Maximal adhesion of U937 cells to HUVEC was observed on TNF-alpha stimulated endothelium with significant differences between day 1 and day 7. There was a small increase in U937 cell adhesion to plasma-treated PET compared to PTFE on both day 1 and day 7, but this was not statistically significant. Immunohistochemical staining demonstrated two patterns of distribution for monocyte adhesion on materials. On T-PET the cells were positioned in clusters attached to HUVECs and on T-PTFE the cells were randomly distributed on HUVECs and material. The effects of plasma-treated PET and PTFE on HUVEC adhesion and proliferation were also studied. On day 1 there were slight increases in the growth of HUVECs on both of T-PET and T-PTFE but this was not statistically significant. On day 7, cell number increased significantly on all of surfaces compared to the negative control. The results demonstrate that the plasma treatment of PET and PTFE with ammonia improves the adhesion and growth of endothelial cells and these surfaces do not exhibit a direct inflammatory effect in terms of monocyte adhesion. Plasma-treated PTFE enhances HUVECs growth and was less adhesive for monocytes as compared with treated PET. The monocyte adhesion to endothelial cells on surfaces can be used as a tool for the evaluation of material surface modification and further to study the mechanisms of cell to cell interactions in response to surfaces.     

The influence of surface chemistry and topography on the contact guidance of MG63 osteoblast cells

The purpose of the present study was to determine in vitro the effects of different surface topographies and chemistries of commercially pure titanium (cpTi) and diamond-like carbon (DLC) surfaces on osteoblast growth and attachment. Microgrooves (widths of 2, 4, 8 and 10 μm and a depth of 1.5–2 μm) were patterned onto silicon (Si) substrates using microlithography and reactive ion etching. The Si substrates were subsequently vapor coated with either cpTi or DLC coatings. All surfaces were characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. Using the MG63 Osteoblast-Like cell line, we determined cell viability, adhesion, and morphology on different substrates over a 3 day culture period. The results showed cpTi surfaces to be significantly more hydrophilic than DLC for groove sizes larger than 2 μm. Cell contact guidance was observed for all grooved samples in comparison to the unpatterned controls. The cell viability tests indicated a significantly greater cell number for 8 and 10 μm grooves on cpTi surfaces compared to other groove sizes. The cell adhesion study showed that the smaller groove sizes, as well as the unpatterned control groups, displayed better cell adhesion to the substrate.     

On the post-mortem fracture surface morphology of short fiber reinforced thermoplastics

The fracture surface morphology of short fiber reinforced thermoplastics (SFRTs) has often been used to assess qualitatively the degree of fiber–matrix interfacial adhesion. Mechanical properties such as tensile strength, fracture toughness and failure strain, etc. are then correlated with the morphology. Fracture surfaces showing fibers surrounded by a large amount of matrix material is commonly regarded as indication of strong fiber–matrix interfacial adhesion while smooth fibers are characteristic of weak interfacial adhesion. Many experimental results of SFRTs have been so interpreted. However, it is shown in this paper that strictly speaking, such interpretations are generally incorrect. Moreover, the amount of matrix material does not provide a quantitative measure of the adhesion. Correct implication of the morphology of fracture surfaces is clarified. Short glass fiber reinforced polyamide 6,6/polypropylene (PA 6,6/PP) blends toughened by rubber are employed as examples for SFRTs since the PA 6,6/PP blend system by changing PA 6,6 concentration in the matrix blend represents a wide range of matrix materials. It is demonstrated that the fracture surface morphology of such composites is dependent on both fiber–matrix interfacial adhesion strength and matrix shear yield strength. Consequently, tensile failure strain is well correlated with the post-mortem fracture surface morphology of these SFRTs.