Contact Guidance of Connective Tissue Fibroblasts on Submicrometer Anisotropic Topographical Cues Is Dependent on Tissue of Origin, ß1 Integrins, and Tensin-1 Recruitment.

The substratum topography of both natural and synthetic materials is a prominent regulator of cell behaviors including adhesion, migration, matrix fibrillogenesis, and cell phenotype. Connective tissue fibroblasts are known to respond to repeating groove topographical modifications by aligning and exhibiting directed migration, a phenomenon termed contact guidance. Although both reside in collagen rich connective tissues, dermal and gingival fibroblasts are known to exhibit differences in phenotype during wound healing, with gingival tissue showing a fetal-like scarless response. Differences in adhesion formation and maturation are known to underlie both a scarring phenotype and cell response to topographical features. Utilizing repeating groove substrates with periodicities of 600, 900, and 1200 nm (depth, 100 nm), we investigated the roles of integrins αvß3 and ß1 associated adhesions on contact guidance of human gingival (HGFs) and dermal fibroblasts (HDFs). HGFs showed a higher degree of orientation with the groove long axis than HDFs, with alignment of both vinculin and tensin-1 evident on 600 and 900 nm periodicities in both cell types. Orientation with grooves of any periodicity in HGFs and HDFs did not alter the adhesion number or area compared to smooth control surfaces. Growth of both cell types on all periodicities reduced fibronectin fibrillogenesis compared to control surfaces. Independent inhibition of integrin αvß3 and ß1 in both cell types induced changes in spreading up to 6 h and reduced alignment with the groove long axis. At 24 h post-seeding with blocking antibodies, HGFs recovered orientation, but in HDFs, blocking of ß1, but not αvß3 integrins, inhibited alignment. Blocking of ß1 and αvß3 in HDFs, but not HGFs, inhibited tensin-1-associated fibrillar adhesion formation. Furthermore, inhibition of ß1 integrins in HDFs, but not HGFs, resulted in recruitment of tensin-1 to αvß3 focal adhesions, preventing HDFs from aligning with the groove long axis. Our work demonstrates that tensin-1 localization with specific integrins in adhesion sites is an important determinant of contact guidance. This work emphasizes further the need for tissue-specific biomaterials, when integration into host tissues is required.

Reversible Self-Assembled Monolayers with Tunable Surface Dynamics for Controlling Cell Adhesion Behavior.

Cells adhering onto surfaces sense and respond to chemical and physical surface features. The control over cell adhesion behavior influences cell migration, proliferation, and differentiation, which are important considerations in biomaterial design for cell culture, tissue engineering, and regenerative medicine. Here, we report on a supramolecular-based approach to prepare reversible self-assembled monolayers (rSAMs) with tunable lateral mobility and dynamic control over surface composition to regulate cell adhesion behavior. These layers were prepared by incubating oxoacid-terminated thiol SAMs on gold in a pH 8 HEPES buffer solution containing different mole fractions of ω-(ethylene glycol)2-4- and ω-(GRGDS)-, α-benzamidino bolaamphiphiles. Cell shape and morphology were influenced by the strength of the interactions between the amidine-functionalized amphiphiles and the oxoacid of the underlying SAMs. Dynamic control over surface composition, achieved by the addition of inert filler amphiphiles to the RGD-functionalized rSAMs, reversed the cell adhesion process. In summary, rSAMs featuring mobile bioactive ligands offer unique capabilities to influence and control cell adhesion behavior, suggesting a broad use in biomaterial design, tissue engineering, and regenerative medicine.

Advanced non-fluoride approaches to dental enamel remineralization: The next level in enamel repair management.

In modern dentistry, a minimally invasive management of early caries lesions or early-stage erosive tooth wear (ETW) with synthetic remineralization systems has become indispensable. In addition to fluoride, which is still the non-plus-ultra in these early caries/ETW treatments, a number of new developments are in the test phase or have already been commercialized. Some of these systems claim that they are comparable or even superior to fluoride in terms of their ability to remineralize enamel. Besides, their use can help avoid some of the risks associated with fluoride and support treatments of patients with a high risk of caries. Two individual non-fluoride systems can be distinguished; intrinsic and extrinsic remineralization approaches. Intrinsic (protein/peptide) systems adsorb to hydroxyapatite crystals/organics located within enamel prisms and accumulate endogenous calcium and phosphate ions from saliva, which ultimately leads to the re-growth of enamel crystals. Extrinsic remineralization systems function on the basis of the external (non-saliva) supply of calcium and phosphate to the crystals to be re-grown. This article, following an introduction into enamel (re)mineralization and fluoride-assisted remineralization, discusses the requirements for non-fluoride remineralization systems, particularly their mechanisms and challenges, and summarizes the findings that underpin the most promising advances in enamel remineralization therapy.

The solubility of calcium oxalates explains some aspects of their underrepresentation in the oral cavity.

OBJECTIVE: Clarifying the discrepancy between frequently high oxalate concentrations present in saliva, but negligible amounts of calcium oxalate deposits found on oral surfaces. METHODS: Studying the calcium oxalate concentration range that can lead to heterogeneous crystallization in the oral cavity. a) Minimum: calcium oxalate monohydrate (COM) seed crystals were pre-grown ([Ca2+] = [C2O42-] = 1 mM, 30 min, 37 °C), and then re-immersed for ≥6 h to find the solubility equilibrium concentration (no growth, no dissolution). The concentrations tested were [Ca2+]/[C2O42-] : 0.055/0.050, 0.060/0.055, 0.070/0.065 and 0.080/0.075 mM. Supersaturations were calculated via the Debye-Hückel-theory and COM morphologies examined by scanning electron microscopy (SEM). b) Maximum (at the heterogeneous/homogeneous crystallization equilibrium): hydroxyapatite (HA) seed crystals were used to heterogeneously crystallize COM (37 °C, 24 h), using oxalate concentrations between 0.2 and 0.5 mM and calcium concentrations of 0.5 mM. COM-forming oxalate consumption was spectroscopically examined; COM precipitates were investigated by SEM; and HA identity was confirmed by X-ray analysis. RESULTS: Within the concentration range of [Ca2+]/[C2O42-]:0.060/0.055 mM (minimum) and [Ca2+]/[C2O42-]:0.50/0.25 mM (maximum) COM precipitates heterogeneously. In terms of mass, this corresponds to a range of 8.04-36.53 mg/l (daily) or an average of 14.32 mg COM (mimicking e.g. plaque mineralization). Higher concentrations react homogeneously (mimicking precipitation within saliva). CONCLUSION: In vivo, only ∼0.05 % oxalate present in saliva reacts with oral surfaces daily, corresponding to ∼0.0665 µmol/l or ∼9.72 µg COM per day. Calcium-consuming calcium phosphate formation and phosphoproteins such as statherin obviously hinder intraoral COM formation.

Incorporation of osteopontin peptide into kidney stone-related calcium oxalate monohydrate crystals: a quantitative study.

Polyelectrolyte-crystal interactions regulate many aspects of biomineralization, including the shape, phase, and aggregation of crystals. Here, we quantitatively investigate the role of phosphorylation in interactions with calcium oxalate monohydrate crystals (COM), using synthetic peptides corresponding to the sequence 220-235 in osteopontin, a major inhibitor of kidney stone-related COM formation. COM formation is induced in the absence or presence of fluorescent-labeled peptides containing either no (P0), one (P1) or three (P3) phosphates and their adsorption to and incorporation into crystals determined using quantitative fluorimetry (also to determine maximum adsorption/incorporation), confocal/scanning electron microscopy and X-ray/Raman spectroscopy. Results demonstrate that higher phosphorylated peptides show stronger irreversible adsorption to COM crystals (P3: K0 ~ 66.4 × 106 M-1; P1: K0 ~ 29.4 × 106 M-1) and higher rates of peptide incorporation into crystals (maximum: P3: ~ 58.8 ng and P1: ~ 8.9 ng per µg of COM) than peptides containing less phosphate groups. However, crystals grown at that level of incorporable P3 show crystal-cleavage. Therefore, extrapolation of maximum incorporable P3 was carried out for crystals that are still intact, resulting in ~ 49.1 ng P3 µg-1 COM (or ~ 4.70 wt%). Both processes, adsorption and incorporation, proceed via the crystal faces {100} > {121} > {010} (from strongest to weakest), with X-ray and Raman spectroscopy indicating no significant effect on the crystal structure. This suggests a process in which the peptide is surrounded by growing crystal matrix and then incorporated. In general, knowing the quantity of impurities in crystalline/ceramic matrices (e.g., kidney stones) provides more control over stress/strain or solubilities, and helps to categorize such composites.

Photocatalytic and antibacterial activities of silver and iron doped titania nanoparticles in solution and polyaspartic coatings.

Visible region active photocatalytic coatings are of interest for antimicrobial activity in low light applications or those employing LED lights with limited UV content. This work examined Ag and Fe doped titania nanoparticles (nTiO2) with varying dopant ranges in polyaspartic polymer coatings for potential light and dark activity. First, the Ag and Fe doped nTiO2 were synthesized by sol-gel chemistry with varying dopant concentrations, then characterized with respect to their size and aggregate size distribution, crystallinity, and surface and band gap features. The photocatalytic activity was then tested with methylene blue under both AM 1.5 G and visible light. From both sample sets (Ag and Fe doped nTiO2), the best photo catalytically active sample materials were chosen for antibacterial tests with gram-negative Escherichia coli (E. coli) and gram-positive Bacillus subtilis (B. subtilis) in (a) solution and (b) polyaspartic nanocomposites under UV and visible irradiation. The results showed that Ag doped nTiO2 samples delivered the best and excellent antibacterial action, even in the dark, attributed to both an enhanced band gap and surface area, as well as a combination of photocatalytic activity and Ag being present at the nanoparticle's surface. No leaching of Ag at room temperature was observed from the nTiO2 structure, giving potential for next generation coatings that are both light and dark active.

A Mass-Producible and Versatile Sensing System: Localized Surface Plasmon Resonance Excited by Individual Waveguide Modes.

A plasmonic sensing system that allows the excitation of localized surface plasmon resonance (LSPR) by individual waveguide modes is presented conceptually and experimentally. Any change in the local environment of the gold nanoparticles (AuNPs) alters the degree of coupling between LSPR and a polymer slab waveguide, which then modulates the transmission-output signal. In comparison to conventional LSPR sensors, this system is less susceptible to optical noise and positional variation of signals. Moreover, it enables more freedom in the exploitation of plasmonic hot spots with both transverse electric (TE) and transverse magnetic (TM) modes. Through real-time measurement, it is demonstrated that the current sensing system is more sensitive than comparable optical fiber plasmonic sensors. The highest normalized bulk sensitivity (7.744 RIU-1) is found in the TM1 mode. Biosensing with the biotin-streptavidin system shows that the detection limit is on the order of 10-14 M of streptavidin. With further optimization, this sensing system can easily be mass-produced and incorporated into high throughput screening devices, detecting a variety of chemical and biological analytes via immobilization of the appropriate recognition sites.

Dye distance mapping using waveguide evanescent field fluorescence microscopy and its application to cell biology.

Previous studies have measured the distance between cells and the substratum at sites of adhesion via the emission of a fluorescent dye and waveguide methods. Here, we demonstrate a novel approach to measure the position of fluorescent dyes above a waveguide surface in the 10-200 nm distance range throughout an entire area, yielding a 2D dye distance map or a 3D contour plot. The dye is located in a multilayered Langmuir Blodgett (LB) film or in the plasma membrane of a cell. Waveguide evanescent field fluorescence (WEFF) images obtained using two different waveguide modes are employed allowing, with a simple mathematical approach, the calculation of dye distance maps. Ultra-thin steps made using LB technology, adhesion distances and the bending of the plasma membrane between focal adhesions of osteoblastic cells are shown as examples. The errors are discussed. False color representation of a dye distance map with four osteoblasts. The inset represents an overexposed WEFF image of the same field of view.

Surface plasmon resonance sensing properties of a 3D nanostructure consisting of aligned nanohole and nanocone arrays.

Molecular surface plasmon resonance (SPR) sensing is one of the most common applications of an array of periodic nanoholes in a metal film. However, metallic nanohole arrays (NHAs) with low-hole count have lower resolution and SPR sensing performance compared to NHAs with high-hole count. In this paper, we present a compact three-dimensional (3D) plasmonic nanostructure with extraordinary optical transmission properties benefiting from surface plasmon matching and enhanced localized surface plasmon coupling. The 3D nanostructure consisted of a gold film containing a NHA with an underlying cavity and a gold nanocone array (NCA) at the bottom of the cavity. Each nanocone was aligned with the nanohole above and the truncated apex of each nanocone was in close proximity (100 nm) to the gold film. The NHA-NCA structures outperformed conventional NHA structures in terms of bulk sensitivity and Figure of Merit (FOM). Furthermore, the NHA-NCA structure with 525 nm periodicity was capable of sensing streptavidin down to 2 nM exhibiting a 10-fold increase in streptavidin sensitivity compared to conventional NHA structures. The sensitivity and performance of the 3D nanostructure can be further improved by exploiting multiplexing methods in combination with stable light sources and detection systems.

Waveguide evanescent field scattering microscopy: bacterial biofilms and their sterilization response via UV irradiation.

Waveguide Evanescent Field Scattering (WEFS) microscopy is introduced as a new and simple tool for label-free, high contrast imaging of bacteria and bacteria sensors. Bacterial microcolonies and single bacteria were discriminated both by their bright field images and by their evanescent scattering intensity. By comparing bright field images with WEFS images, the proportion of planktonic: sessile (i.e., "floating": attached) bacteria were measured. Bacteria were irradiated with UV light, which limited their biofilm forming capability. A quantitative decrease in attachment of individual, sessile bacteria and in attached, microcolony occupied areas was easily determined within the apparent biofilms with increasing UV dose. WEFS microscopy is an ideal tool for providing rapid quantitative data on biofilm formation.

Optimization of gold nanoring arrays for biosensing in the fiber-optic communication window.

To improve the limit of detection in a nanoplasmonic sensor system, the optical performance of the metal nanostructures should be optimized according to the best spectral window of the measurement instrument. We propose that the spectral window from 1460 to 1610 nm can potentially provide ultrahigh instrumental resolution for biosensing. We optimized gold nanoring arrays such that the extinction peak position is inside the proposed window, the extinction peak is sharp enough to track the peak shift with high resolution and the figure of merit (sensitivity/linewidth) of the array is optimized at the same time. The peak-sharpening effect of the array caused by coherent interaction plays a central role in the optimization. The optimized array has a lattice constant in the range [1000 nm,1060 nm], a bulk index sensitivity of around 450 nm/RIU and a figure of merit larger than 4. It is an enabling sensor element for a near-infrared sensor chip with ultrahigh resolution.

Orientation distribution of highly oriented type I collagen deposited on flat samples with different geometries.

The structural arrangement of type I collagen in vivo is critical for the normal functioning of tissues, such as bone, cornea, tendons, and blood vessels. At present, there are no established low-cost techniques for fabricating aligned collagen structures for applications in regenerative medicine. Here, we report on a straightforward approach to fabricate collagen films, with defined orientation distributions of collagen fibrillar aggregates within a matrix of oriented collagen molecules on flat sample surfaces. Langmuir-Blodgett (LB) technology was used to deposit thin films of oriented type I collagen onto flat substrates exhibiting various shapes. By varying the shapes of the substrates (e.g., rectangles, squares, circles, parallelograms, and various shaped triangles) as well as their sizes, a systematic study on collagen alignment patterns was conducted. It was found that the orientation and the orientation distribution of collagen along these various shaped substrates are directly depending on the geometry of the substrate and the dipping direction of that sample with respect to the collagen/water subphase. An important factor in tissue engineering is the stability, durability, and endurance of the constructed artificial tissue and thus its functioning in regenerative medicine applications. By testing these criteria, we found that the coated films and their alignments were stable for at least three months under different conditions and, moreover, that these films can withstand temperatures of up to 60 °C for a short time. Therefore, these constructs may have widespread applicability in the engineering of collagen-rich tissues.

Large area periodic, systematically changing, multishape nanostructures by laser interference lithography and cell response to these topographies.

The fabrication details to form large area systematically changing multishape nanoscale structures on a chip by laser interference lithography (LIL) are described. The feasibility of fabricating different geometries including dots, ellipses, holes, and elliptical holes in both x- and y- directions on a single substrate is shown by implementing a Lloyd's interferometer. The fabricated structures at different substrate positions with respect to exposure time, exposure angle and associated light intensity profile are analyzed. Experimental details related to the fabrication of symmetric and biaxial periodic nanostructures on photoresist, silicon surfaces, and ion milled glass substrates are presented. Primary rat calvarial osteoblasts were grown on ion-milled glass substrates with nanotopography with a periodicity of 1200 nm. Fluorescent microscopy revealed that cells formed adhesions sites coincident with the nanotopography after 24 h of growth on the substrates. The results suggest that laser LIL is an easy and inexpensive method to fabricate systematically changing nanostructures for cell adhesion studies. The effect of the different periodicities and transition structures can be studied on a single substrate to reduce the number of samples significantly.

Influence of electrostatic forces on particle propulsion in the evanescent field of silver ion-exchanged waveguides.

The effect of electrostatic interaction between carboxylate- and amino-functionalized polystyrene particles and a charged waveguide surface on the propulsion speed in optical tweezers is considered to be a function of the pH and ionic strength. It was shown that with the variation of the pH of the aqueous solution in which the particles were immersed, a systematic change in propulsion speed with a maximum speed could be achieved. The appearance of a maximum speed was ascribed to changes in the particle-waveguide separation as a result of the combination of two forces: Coulomb repulsion/attraction and induced dipole forces. The highest maximum speed at low ionic strength was around 12 µm/s. Changes in the ionic strength of the solution influenced the gradient of the dielectric constant near the involved surfaces and also led to a slightly reduced hydrodynamic radius of the particles. The combination of these effects subsequently increased the maximum speed to about 23 µm/s.

Visualization of the solubilization process of the plasma membrane of a living cell by waveguide evanescent field fluorescence microscopy.

Waveguide evanescent field fluorescence microscopy (WEFF) is a novel microscopy technology that allows imaging of a cell's plasma membrane in the vicinity of a glass substrate with high axial resolution, low background and little photobleaching. Time-lapse imaging can be performed to investigate changes in cell morphology in the presence or absence of chemical agents. WEFF microscopy provides a method to investigate plasma membranes of living cells and allows a comparison to simplified model membranes immobilized on planar substrates. The interaction of the nonionic detergent Triton X-100 with plasma membranes of osteoblasts in an aqueous environment was investigated. Solubilization of the membranes very close to the waveguide surface was visualized and related to the three-stage solubilisation model proposed for liposomes and supported lipid bilayers. Findings for the plasma membranes of cells are in excellent agreement with results reported for these artificial model systems.

Focal contact formation of vascular smooth muscle cells on Langmuir-Blodgett and solvent-cast films of biodegradable poly(ester amide)s.

The ability of biomaterials to support the adhesion of cells is a necessary condition for their use in scaffold-guided tissue engineering. Waveguide evanescent field fluorescence (WEFF) microscopy is a relatively new microscopic technique that allows the number of cell adhesions to a waveguide surface be measured by imaging the interfacial contact region between the cells and their substratum. In this work, the adhesion of human coronary artery smooth muscle cells (HCASMCs) to ultrathin films (20 nm) of poly(ester amide)s (PEAs) prepared by Langmuir-Blodgett (LB) technology on waveguides was investigated and compared with conventional vinculin immunostaining on solvent cast PEA films. Cell culture was conducted both in the presence and absence of serum to evaluate the effect of nonspecific protein adsorption that mediates cell adhesion. WEFF microscopy analyses revealed that the cationic PEA enhanced the number of attachment sites compared with the control waveguides regardless of the culture medium. Although differences in cell adhesions between different PEAs were suggested by the results, no statistically significant differences were found. Similar results were observed with presently and previously reported vinculin immunostaining studies, further validating the use of WEFF microscopy to quantify cell adhesions. Moreover, the focal adhesions of the HCASMCs to the PEA surfaces indicate these PEAs can promote integrin signaling, which is vital in cell survival, migration, and proliferation, and ultimately in scaffold-guided vascular tissue engineering.

Waveguide evanescent field fluorescence microscopy: high contrast imaging of a domain forming mixed lipid Langmuir-Blodgett monolayer mimicking lung surfactant.

Waveguide evanescent field fluorescence (WEFF) microscopy is a new development that allows the imaging of contact regions between biological cells and their substratum, as well as imaging of ultrathin films such as monomolecular Langmuir-Blodgett (LB) films. Mixed-lipid monolayer systems mimicking lung surfactant were fabricated on waveguides using the LB technique and imaged by both WEFF and standard wide field epi-fluorescence microscopy. These two technologies were compared with respect to contrast, photobleaching, and sensitivity. It was found that WEFF microscopy produced images with a much higher contrast, lower photobleaching, and higher sensitivity. In addition, fine structures in the lipidic domains were observed for the first time.

Migration of periodontal ligament fibroblasts on nanometric topographical patterns: influence of filopodia and focal adhesions on contact guidance.

Considered to be the "holy grail" of dentistry, regeneration of the periodontal ligament in humans remains a major clinical problem. Removal of bacterial biofilms is commonly achieved using EDTA gels or lasers. One side effect of these treatment regimens is the etching of nanotopographies on the surface of the tooth. However, the response of periodontal ligament fibroblasts to such features has received very little attention. Using laser interference lithography, we fabricated precisely defined topographies with continuous or discontinuous nanogrooves to assess the adhesion, spreading and migration of PDL fibroblasts. PDL fibroblasts adhered to and spread on all tested surfaces, with initial spreading and focal adhesion formation slower on discontinuous nanogrooves. Cells had a significantly smaller planar area on both continuous and discontinuous nanogrooves in comparison with cells on non-patterned controls. At 24 h post seeding, cells on both types of nanogrooves were highly elongated parallel to the groove long axis. Time-lapse video microscopy revealed that PDL fibroblast movement was guided on both types of grooves, but migration velocity was not significantly different from cells cultured on non-patterned controls. Analysis of filopodia formation using time-lapse video microscopy and labeling of vinculin and F-actin revealed that on nanogrooves, filopodia were highly aligned at both ends of the cell, but with increasing time filopodia and membrane protrusions developed at the side of the cell perpendicular to the cell long axis. We conclude that periodontal ligament fibroblasts are sensitive to nanotopographical depths of 85-100 µm, which could be utilized in regeneration of the periodontal ligament.

Optical waveguides formed by silver ion exchange in Schott SG11 glass for waveguide evanescent field fluorescence microscopy: evanescent images of HEK293 cells.

Planar glass waveguides with a specific number of modes were fabricated by Ag(+)-Na(+) exchange in Schott SG11 glass. The effective refractive indices were determined using m-line spectroscopy in both s- and p-polarization. By using the reversed Wentzel-Kramers-Brillouin approximation, the index profiles were described by a nonlinear diffusion equation. The diffusion coefficients for Ag(+) were established, as well as the penetration depth of the evanescent field in an aqueous environment for the different modes. The integrals of \E\(2) fields for the evanescent-guided fields were investigated. These are important when evanescent fields are used for illumination in interface microscopy, an alternative method to total internal reflection fluorescence (TIRF) microscopy. The photoluminescent behavior of the waveguides was investigated as a function of ion exchange time and excitation wavelengths. Comparable images were obtained of fluorescently labeled HEK293 cells using TIRF microscopy and waveguide evanescent field fluorescence microscopy. Imaging was performed using HEK293 cells, delivering similar images and information.

Controlled deposition of highly oriented type I collagen mimicking in vivo collagen structures.

The structural arrangement of type I collagen in vivo is critical for the normal functioning of tissues, such as bone, cornea, and blood vessels. At present, there are no low-cost techniques for fabricating aligned collagen structures for applications in regenerative medicine. Here, we report a straightforward approach to fabricate collagen films, with defined orientation of collagen fibrillar aggregates within a matrix of oriented collagen molecules. Langmuir-Blodgett (LB) technology was used to deposit thin films of oriented type I collagen onto substrates. It was found that collagen does not behave like classical LB materials, such as amphiphilic hydrocarbon acids or lipids. The thickness of the deposited collagen films and the area-pressure isotherms were found to depend on the amount of material spread. In addition, no film collapse was detected and the deposited LB films were thicker than the theoretical dimension of a collagen monolayer (1.5 nm) formed by triple helical collagen molecules. Individual LB films with thicknesses of up to 20 nm were obtained, and multiple depositions yielded overall film thicknesses of up to 100 nm. Films consisted of a matrix of collagen molecules containing thicker fibrillar aggregates of collagen (micrometers in length). These fibrillar aggregates were built up of shorter unit molecules forming "spun thread" structures, some of which exhibited a zigzag pattern. In addition to aligning collagen unidirectionally (similar for example to tendon), we performed a two-step deposition procedure, in which the substrate was turned 90 degrees between two consecutive collagen deposition steps. The resulting films showed orthogonally aligned collagen layers, mimicking the structure of cornea. Thus, this technique permits control of the thickness of individual layers, the orientation of successive layers, and the number of layers within the construct. Therefore, it may have widespread applicability for the engineering of collagen-rich tissues.