The Application of Organs-on-a-Chip in Dental, Oral, and Craniofacial Research.

The current development of microfluidics-based microphysiological systems (MPSs) will rapidly lead to a paradigm shift from traditional static 2-dimensional cell cultivation towards organized tissue culture within a dynamic cellular milieu. Especially organs-on-a-chip (OoCs) can very precisely re-create the mechanical and unique anatomical structures of the oral environment. This review provides an introduction to such technology, from commonly used chip materials and fabrication methods to the application of OoC in in vitro culture. OoCs are advantageous because of their small-scaled culture environment, the highly controlled dynamic experimental conditions, and the likeness to the in vivo structure. We specifically focus on current chip designs in dental, oral, and craniofacial (DOC) research. Also, future perspectives are discussed, like model standardization and the development of integrated platforms with advanced read-out functionality. By doing so, it will be possible for OoCs to serve as an alternative for animal testing and to develop highly predictive human models for clinical experiments and even personalized medicine.

Third-generation modular mandible endoprosthesis in Macaca fascicularis.

The aim of this study was to develop a third-generation modular mandible endoprosthesis that would experience less stress concentration at its stems compared to earlier generations, thereby minimizing micromotion and achieving long-term stability. In this three-piece modular design, different degrees of movement were incorporated between the endoprosthesis module interfaces. It was hypothesized that this unique feature would minimize stress concentration at the stems and hence promote osseointegration during the early phase of implantation. The endoprosthesis system was made of commercially pure grade 4 titanium, machined and surface-treated, then sterilized and implanted in segmental mandible defects of nine Macaca fascicularis. Clinical, radiological, histological, and histomorphometric evaluations were performed 4 months post-implantation. The endoprosthesis systems with a degree of movement incorporated, exhibited superior performance compared to the rigid system: 30.9-34.8 times higher percentage bone-implant contact (P< 0.0001) and 3.4-4.1 times higher percentage bone area (P<0.0001), with osseointegration noted at the posterior stems. However, fibrous tissue encapsulation was noted around the majority of the anterior stems in all groups. Although the degree of movement was favourable for improving bone healing and stability of the endoprosthesis system, more work needs to be done to investigate other strategies to further reduce loading on the endoprosthesis to achieve predictable osseointegration at the stems.

A Novel Thermoresponsive Gel as a Potential Delivery System for Lipoxin.

The objective of this study was to evaluate a novel thermoresponsive polyisocyanopeptide (PIC)-based hydrogel as an injectable carrier for local drug delivery for periodontal applications. Three formulations of PIC gels, 0.2%, 0.5%, and 1% w/w, were prepared. As controls, commercially available poloxamer 407 (P407) gels of 20% and 26% w/w were used. Lipoxin A4 (LXA4), a proresolving drug, was suspended into the gel solutions. The systems were evaluated regarding dynamic mechanical properties, injectability and stability, release and bioactivity of LXA4, and cytocompatibility. Results showed that the gelation temperatures of PIC and P407 gels were around 13°C to 23°C. PIC gels were less viscous and mechanically weaker than P407 gels due to the low polymer concentrations. However, PIC gels kept gel integrity for at least 2 wk when incubated with phosphate-buffered saline, whereas P407 gels were disintegrated totally within 1 wk. LXA4 was chemically stable in both neutral and alkaline medium for over 1 mo. The release of LXA4 from either 1% PIC or 26% P407 gels depicted an initial burst release followed by a sustained release for around 4 d. The extent of burst release was negatively correlated to the polymer concentration. LXA4 remained bioactive after release from PIC gels. No cytotoxicity was observed for 1% PIC gel. However, 26% P407 inhibited periodontal ligament cell and gingival epithelial cell growth. In conclusion, the thermoresponsive PIC gel is a potential candidate for periodontal drug delivery.

A theranostic dental pulp capping agent with improved MRI and CT contrast and biological properties.

Different materials have been used for vital dental pulp treatment. Preferably a pulp capping agent should show appropriate biological performance, excellent handling properties, and a good imaging contrast. These features can be delivered into a single material through the combination of therapeutic and diagnostic agents (i.e. theranostic). Calcium phosphate based composites (CPCs) are potentially ideal candidate for pulp treatment, although poor imaging contrast and poor dentino-inductive properties are limiting their clinical use. In this study, a theranostic dental pulp capping agent was developed. First, imaging properties of the CPC were improved by using a core-shell structured dual contrast agent (csDCA) consisting of superparamagnetic iron oxide (SPIO) and colloidal gold, as MRI and CT contrast agent respectively. Second, biological properties were implemented by using a dentinogenic factor (i.e. bone morphogenetic protein 2, BMP-2). The obtained CPC/csDCA/BMP-2 composite was tested in vivo, as direct pulp capping agent, in a male Habsi goat incisor model. Our outcomes showed no relevant alteration of the handling and mechanical properties (e.g. setting time, injectability, and compressive strength) by the incorporation of csDCA particles. In vivo results proved MRI contrast enhancement up to 7weeks. Incisors treated with BMP-2 showed improved tertiary dentin deposition as well as faster cement degradation as measured by µCT assessment. In conclusion, the presented theranostic agent matches the imaging and regenerative requirements for pulp capping applications. STATEMENT OF SIGNIFICANCE: In this study, we combined diagnostic and therapeutic agents in order to developed a theranostic pulp capping agent with enhanced MRI and CT contrast and improved dentin regeneration ability. In our study we cover all the steps from material preparation, mechanical and in vitro characterization, to in vivo study in a goat dental model. To the best of our knowledge, this is the first time that a theranostic pulp capping material have been developed and tested in an in vivo animal model. Our promising results in term of imaging contrast enhancement and of induction of new dentin formation, open a new scenario in the development of innovative dental materials.

Blood and fibroblast responses to thermoset BisGMA-TEGDMA/glass fiber-reinforced composite implants in vitro.

OBJECTIVES: This in vitro study was designed to evaluate both blood and human gingival fibroblast responses on fiber-reinforced composite (FRC) aimed to be used as oral implant abutment material. MATERIAL AND METHODS: Two different types of substrates were investigated: (a) Plain polymer (BisGMA 50%-TEGDMA 50%) and (b) FRC. The average surface roughness (Ra) was measured using spinning-disk confocal microscope. The phase composition was identified using X-ray diffraction analyzer. The degree of monomer conversion (DC%) was determined using FTIR spectrometry. The blood response, including the blood-clotting ability and platelet adhesion morphology, was evaluated. Fibroblast cell responses were studied in cell culture environment using routine test conditions. RESULTS: The Ra of the substrates investigated was less than 0.1 µm with no signs of surface crystallization. The DC% was 89.1 ± 0.5%. The FRC substrates had a shorter clotting time and higher platelets activation state than plain polymer substrates. The FRC substrates showed higher (P < 0.01-0.001) amount of adhered cells than plain polymer substrates at all time points investigated. The strength of attachment was evaluated using serial trypsinization, the number of cells detached from FRC substrates was 59 ± 5%, whereas those detached from the plane polymer substrates was 70 ± 5%, indicating a stronger (P < 0.01) cell attachment on the FRC surfaces. Fibroblasts grew more efficiently on FRC than on plain polymer substrates, showing significantly higher (P < 0.01) cell metabolic activities throughout the experiment. CONCLUSIONS: The presence of E-glass fibers enhances blood and fibroblast responses on composite surfaces in vitro.

The effect of enamel matrix derivative (Emdogain®) on gene expression profiles of human primary alveolar bone cells.

Emdogain® is frequently used in regenerative periodontal treatment. Understanding its effect on gene expression of bone cells would enable new products and pathways promoting bone formation to be established. The aim of the study was to analyse the effect of Emdogain® on expression profiles of human-derived bone cells with the help of the micro-array, and subsequent validation. Bone was harvested from non-smoking patients during dental implant surgery. After outgrowth, cells were cultured until subconfluence, treated for 24 h with either Emdogain® (100 µg/ml) or control medium, and subsequently RNA was isolated and micro-array was performed. The most important genes demonstrated by micro-array data were confirmed by qPCR and ELISA tests. Emdogain tipped the balance between genes expressed for bone formation and bone resorption towards a more anabolic effect, by interaction of the PGE2 pathway and inhibition of IL-7 production. In addition the results of the present study indicate that Emdogain possibly has an effect on gene expression for extracellular matrix formation of human bone cells, in particular on bone matrix formation and on proliferation and differentiation. With the micro-array and the subsequent validation, the genes possibly involved in Emdogain action on bone cells were identified. These results can contribute to establishing new products and pathways promoting bone formation.

Influence of nanostructural environment and fluid flow on osteoblast-like cell behavior: a model for cell-mechanics studies.

Introducing nanoroughness on various biomaterials has been shown to profoundly effect cell-material interactions. Similarly, physical forces act on a diverse array of cells and tissues. Particularly in bone, the tissue experiences compressive or tensile forces resulting in fluid shear stress. The current study aimed to develop an experimental setup for bone cell behavior, combining a nanometrically grooved substrate (200 nm wide, 50 nm deep) mimicking the collagen fibrils of the extracellular matrix, with mechanical stimulation by pulsatile fluid flow (PFF). MC3T3-E1 osteoblast-like cells were assessed for morphology, expression of genes involved in cell attachment and osteoblastogenesis and nitric oxide (NO) release. The results showed that both nanotexture and PFF did affect cellular morphology. Cells aligned on nanotexture substrate in a direction parallel to the groove orientation. PFF at a magnitude of 0.7 Pa was sufficient to induce alignment of cells on a smooth surface in a direction perpendicular to the applied flow. When environmental cues texture and flow were interacting, PFF of 1.4 Pa applied parallel to the nanogrooves initiated significant cellular realignment. PFF increased NO synthesis 15-fold in cells attached to both smooth and nanotextured substrates. Increased collagen and alkaline phosphatase mRNA expression was observed on the nanotextured substrate, but not on the smooth substrate. Furthermore, vinculin and bone sialoprotein were up-regulated after 1 h of PFF stimulation. In conclusion, the data show that interstitial fluid forces and structural cues mimicking extracellular matrix contribute to the final bone cell morphology and behavior, which might have potential application in tissue engineering.

Dynamic cell adhesion and migration on nanoscale grooved substrates.

Organised nanotopography mimicking the natural extracellular matrix can be used to control morphology, cell motility, and differentiation. However, it is still unknown how specific cell types react with specific patterns. Both initial adhesion and preferential cell migration may be important to initiate and increase cell locomotion and coverage with cells, and thus achieve an enhanced wound healing response around an implantable material. Therefore, the aim of this study was to evaluate how MC3T3-E1 osteoblast initial adhesion and directional migration are influenced by nanogrooves with pitches ranging from 150 nm up to 1000 nm. In this study, we used a multi-patterned substrate with five different groove patterns and a smooth area with either a concentric or radial orientation. Initial cell adhesion measurements after 10 s were performed using atomic force spectroscopy-assisted single-cell force spectroscopy, and demonstrated that nascent cell adhesion was highly induced by a 600 nm pitch and reduced by a 150 nm pitch. Addition of RGD peptide significantly reduced adhesion, indicating that integrins and cell adhesive proteins (e.g. fibronectin or vitronectin) are key factors in specific cell adhesion on nanogrooved substrates. Also, cell migration was highly dependent on the groove pitch; the highest directional migration parallel to the grooves was observed on a 600 nm pitch, whereas a 150 nm pitch restrained directional cell migration. From this study, we conclude that grooves with a pitch of 600 nm may be favourable to enhance fast wound closure, thereby promoting tissue regeneration.

Submicron-patterning of bulk titanium by nanoimprint lithography and reactive ion etching.

Nanopatterns on titanium may enhance endosseous implant biofunctionality. To enable biological studies to prove this hypothesis, we developed a scalable method of fabricating nanogrooved titanium substrates. We defined nanogrooves by nanoimprint lithography (NIL) and a subsequent pattern transfer to the surface of ASTM grade 2 bulk titanium applying a soft-mask for chlorine-based reactive ion etching (RIE). With respect to direct write lithographic techniques the method introduced here is fast and capable of delivering uniformly patterned areas of at least 4 cm(2). A dedicated silicon nanostamp process has been designed to generate the required thickness of the soft-mask for the NIL-RIE pattern transfer. Stamps with pitch sizes from 1000 nm down to 300 nm were fabricated using laser interference lithography (LIL) and deep cryogenic silicon RIE. Although silicon nanomachining was proven to produce smaller pitch sizes of 200 nm and 150 nm respectively, successful pattern transfer to titanium was only possible down to a pitch of 300 nm. Hence, the smallest nanogrooves have a width of 140 nm. An x-ray photoelectron spectroscopy study showed that only very few contaminations arise from the fabrication process and a cytotoxicity assay on the nanopatterned surfaces confirmed that the obtained nanogrooved titanium specimens are suitable for in vivo studies in implantology research.

The influence of nanoscale topographical cues on initial osteoblast morphology and migration.

The natural environment of a living cell is not only organized on a micrometer, but also on a nanometer scale. Mimicking such a nanoscale topography in implantable biomaterials is critical to guide cellular behavior. Also, a correct positioning of cells on biomaterials is supposed to be very important for promoting wound healing and tissue regeneration. The exact mechanism by which nanotextures can control cellular behavior are thus far not well understood and it is thus far unknown how cells recognize and respond to certain surface patterns, whereas a directed response appears to be absent on other pattern types. Focal adhesions (FAs) are known to be involved in the process of specific pattern recognition and subsequent response by cells. In this study, we used a high throughput screening "Biochip" containing 40 different nanopatterns to evaluate the influence of several nanotopographical cues like depth, width, (an)isotropy and spacing (ridge-groove ratio) on osteoblast behavior. Microscopical analysis and time lapse imaging revealed that an isotropic topography did not alter cell morphology, but it highly induced cell motility. Cells cultured on anisotropic topographies on the other hand, were highly elongated and aligned. Time-lapse imaging revealed that cell motility is highly dependent on the ridge-groove ratio of anisotropic patterns. The highest motility was observed on grooves with a ratio of 1:3, whereas the lowest motility was observed on ratios of 1:1 and 3:1. FA measurements demonstrated that FA-length decreased with increasing motility. From the study it can be concluded that osteoblast behavior is tightly controlled by nanometer surface features.

Influence of nuclear geometry on the formation of genetic rearrangements in human cells.

Interphase chromosomes are divided into discrete domains, with limited overlapping and movement. We explored the role of nuclear topology in the formation of chromosome aberrations by irradiating normal human fibroblasts with high-energy heavy ions from different directions. Cells with elliptical nuclei were grown in an aligned manner onto micrometer grooved culturing substrates to have a predetermined orientation with respect to the accelerated iron ions. Particles were directed either perpendicular to the cell layer or along the major or minor axis of the nucleus. Analysis of chromosome aberrations by mFISH showed that, at the same radiation dose, the yield of chromosomal damage and its complexity are largely modified by the irradiation geometry. The results demonstrate that the architecture of the cell nucleus determines the formation of chromosomal rearrangements.

Bone particles and the undersized surgical technique.

During the installation of implants, osteogenic bone particles are translocated along the surface. These particles may contribute to peri-implant bone healing. Based on this phenomenon, it is hypothesized that implants placed with undersized drilling, besides showing higher primary-implant stability, also enhance the amount and osteogenic responses of these bone particles. Biocomp implants were inserted into bicortical fresh-cadaver bone by a press-fit or an undersized surgical technique, and peak-insertion torque values (ITV) were measured. After explantation, the implants were incubated in culture medium up to 24 days. Histology, bone-implant contact (BIC), micro-CT, scanning electron microscopy (SEM), and calcium (Ca) measurements were performed. ITV were significantly higher for implants placed with an undersized technique. Moreover, histology, BIC, micro-CT, SEM, and Ca measurements confirmed the presence of more bone-like tissue on implants inserted with an undersized technique. The undersized surgical technique not only results in higher primary-implant stability, but also induces more translocated bone particles, thus having a positive influence on the osteogenic response.

Healing of bone defects in the goat mandible, using COLLOSS E and beta-tricalciumphosphate.

COLLOSS E, an equine extracellular matrix product containing native transforming growth factor beta1 and several bone morphogenetic proteins, has shown osteoinductive properties in ectopic sites. This study was set up to examine its properties in an orthotoptic site in conjunction with a beta-tricalciumphosphate (beta-TCP) scaffolding material. Thirty-two 17-mm circular defects in goat mandibles were filled with COLLOSS E, beta-TCP, COLLOSS E + beta-TCP, or left empty. After 9 weeks the results were quantified by micro-computed tomography and histology. The empty defects contained the highest percentage of new bone (62%). The beta-TCP scaffold resulted in 38% (p = 0.0029), the mixture of beta-TCP/COLLOSS E resulted in 36% (p = 0.0057), while the use of COLLOSS E alone resulted in 55% (not significant p = 0.34). These results show that addition of TCP did not result in the expected synergy with regard to the healing of the defect and seemed even to inhibit the healing process. On the other hand, the addition of COLLOSS E induced the formation of small islands of new bone, not connected to the defect edges. This was not observed in the specimens not containing COLLOSS E (4.61% of bone formation centrally in the defect vs. 0.56%; p = 0.042). In conclusion, the results of the present study are somewhat unexpected in that the empty defects showed the most bone ingrowth; however, this ingrowth was always connected to the defect edges. In contrast, the application of COLLOSS E with or without beta-TCP induced bone formation in the center of the defects also.

Mineralized tissue formation by BMP2-transfected pulp stem cells.

Previously, in vitro differentiation of odontoblasts was shown for dental pulp stem cells (DPSCs) transfected with bone morphogenetic protein-2 (Bmp2). For this study, we hypothesized that such cells also show potential for mineralized tissue formation in vivo. DPSCs were transfected with Bmp2 and seeded onto a ceramic scaffold. These complexes were cultured in medium without dexamethasone, and thereafter placed subcutaneously in nude mice for 1, 4, and 12 weeks. Samples were evaluated by histology and real-time PCR for osteocalcin, bone sialoprotein, dentin sialophosphoprotein, and dentin matrix protein 1. Results indicated that only the transfected DPSCs showed obvious mineralized tissue generation, and 12 weeks of implantation gave the highest percentage of mineralized tissue formation (33 +/- 7.3% of implant pore area). Real-time PCR confirmed these results. In conclusion, Bmp2-transfected DPSCs effectively show mineralized tissue formation upon ectopic implantation.

Analytical assessment of the osteoinductive material COLLOSSE.

In this study, the growth factors in COLLOSSE were analyzed, using ELISA tests, mass spectrometry, western blotting, and a 24-day cell culture experiment using osteoblast-like cells. The results of the ELISA testing, mass spectrometry, and western blotting all confirmed that TGF-beta1 was the main growth factor in COLLOSSE at 55 ng/mg. The results from the culture test showed that the cell proliferation, alkaline phosphatase activity, and matrix calcification were all drastically changed by the addition of COLLOSSE, mirroring the effects of addition of TGF-beta1. We conclude that COLLOSSE is not only a rich source of TGFbeta-1, but also contains the growth factors TGFbeta-2, BMP-2, BMP-3, BMP-7, IGF-1, and possibly VEGF. Other growth factors might be present in COLLOSSE, but were not identified due to inherent detection limits of the used ELISA and mass spectrometry techniques. The number of osteoinductive factors in COLLOSSE causes a synergistic effect, explaining the new bone formation found in previously described in vivo studies, with much lower growth factor concentrations when compared with recombinant BMPs.

[Bone substitutes, growth factors and distraction osteogenesis]. / Botsubstituten, groeifactoren en distractieosteogenese.

Up to the present time, bone transplants are commonly used to reconstruct bone defects. Recently, several bone substitutes have been suggested to overcome the disadvantages of the procedure of bone harvesting. However, research reveals that an autogenous bone graft is still the gold standard. To replicate the structure and function of natural bone, growth factors or even living, bone-forming cells can be added to enhance the formation of new bone. In that case, one speaks of cell-based tissue-substition. As an alternative distraction osteogenesis, a mechanical-based way of tissue engineering is suggested. In this procedure, tissue-generation takes place without the addition of external material. A combination of both tissue-substitution techniques, consisting of the addition of bone-replacement materials or growth factors during distraction osteogenesis, has also been evaluated in research on animals, although not with unequivocal results.

Simulated microgravity activates MAPK pathways in fibroblasts cultured on microgrooved surface topography.

This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and microgrooved substrata (groove depth: 0.5 mum, width: 1 mum), which were subjected to simulated microgravity. The aim of the study was to clarify which of these parameters was more dominant to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell alignment. Expression of collagen type I, and alpha1-, beta1-, beta3-integrin were investigated by QPCR. Finally, immunoblotting was applied to visualise MAPK signalling pathways. Microscopy and image analysis showed that the fibroblasts aligned along the groove direction on all textured surfaces. On the smooth substrata, cells had spread out in a random fashion. The alignment of cells cultured on grooved surfaces under simulated microgravity, after 48 h of culturing appeared similar to those cultured at 1g, although cell shape was different. Analysis of variance proved that all main parameters: topography, gravity force, and time were significant. In addition, gene levels were reduced by simulated microgravity particularly those of beta3-integrin and collagen, however alpha-1 and beta-1 integrin levels were up-regulated. ERK1/2 was reduced in RPM, however, JNK/SAPK and p38 remained active. The members of the small GTPases family were stimulated under microgravity, particularly RhoA and Cdc42. The results are in agreement that application of microgravity to fibroblasts promotes a change in their morphological appearance and their expression of cell-substratum proteins through the MAPK intracellular signalling pathways.

The effect of combined application of TGFbeta-1, BMP-2, and COLLOSS E on the development of bone marrow derived osteoblast-like cells in vitro.

This study investigated the combined application of Transforming Growth Factor beta-1 (TGFbeta-1) and Bone Morphogenetic Protein-2 (BMP-2) to stimulate osteogenic expression in vitro. TGFbeta-1 and BMP-2 fulfill specific roles in the formation of new bone. COLLOSS E, a bone-derived collagen product containing a variety of naturally occurring growth factors, was also used. Growth factors were administered to osteoblast-like cells from rat bone marrow (RBM). Proliferation and differentiation were monitored up to 24 days, by measuring total DNA content, alkaline phosphatase activity, and calcium content. Genetic expression of a set of differentiation markers at day 7 was measured by Q-PCR. Adding BMP-2 alone induced high proliferation rates, compared to the growth factor supplemented groups, and it induced high differentiation rates, compared to the control group. Adding TGFbeta-1 combined with BMP-2, TGFbeta-1 alone, or COLLOSS E resulted in a significant decrease in proliferation rate, but an increase in differentiation rate, compared to the control group. Additive or synergistic effects of application of TGFbeta-1 and BMP-2 were not observed. The observed effects of COLLOSS E mainly resembled those of TGFbeta-1 application alone. It can be concluded that BMP-2 is the most suitable candidate for osteogenic stimulation of RBM cells in these settings.

The behavior of osteoblast-like cells on various substrates with functional blocking of integrin-beta1 and integrin-beta3.

This study was designed to examine the influence of integrin subunit-beta1 and subunit-beta3 on the behavior of primary osteoblast-like cells, cultured on calcium phosphate (CaP)-coated and non coated titanium (Ti). Osteoblast-like cells were incubated with specific monoclonal antibodies against integrin-beta1 and integrin-beta3 to block the integrin function. Subsequently, cells were seeded on Ti discs, either non coated or provided with a 2 microm carbonated hydroxyapatite coating using Electrostatic Spray Deposition. Results showed that on CaP coatings, cellular attachment was decreased after a pre-treatment with either anti-integrin-beta1 or anti-integrin-beta3 antibodies. On Ti, cell adhesion was only slightly affected after a pre-treatment with anti-integrin-beta3 antibodies. Scanning electron microscopy showed that on both types of substrate, cellular morphology was not changed after a pre-treatment with either antibody. With quantitative PCR, it was shown for both substrates that mRNA expression of integrin-beta1 was increased after a pre-treatment with either anti-integrin-beta1 or anti-integrin-beta3 antibodies. Furthermore, after a pre-treatment with either antibody, mRNA expression of integrin-beta3 and ALP was decreased, on both types of substrate. In conclusion, osteoblast-like cells have the ability to compensate to great extent for the blocking strategy as applied here. Still, integrin-beta1 and beta3 seem to play different roles in attachment, proliferation, and differentiation of osteoblast-like cells, and responses on CaP-coated substrates differ to non coated Ti. Furthermore, the influence on ALP expression suggests involvement of both integrin subunits in signal transduction for cellular differentiation.

The threshold at which substrate nanogroove dimensions may influence fibroblast alignment and adhesion.

The differences in morphological behaviour between fibroblasts cultured on smooth and nanogrooved substrata (groove depth: 5-350 nm, width: 20-1000 nm) have been evaluated in vitro. The aim of the study was to clarify to what extent cell guidance occurs on increasingly smaller topographies. Pattern templates were made using electron beam lithography, and were subsequently replicated in polystyrene cell culture material using solvent casting. The replicates were investigated with atomic force microscopy (AFM). After seeding with fibroblasts, morphological characteristics were investigated using scanning electron microscopy (SEM) and light microscopy, in order to obtain qualitative and quantitative information on cell alignment. AFM revealed that the nanogroove/ridge widths were replicated perfectly, although at deeper levels the grooves became more concave. The smooth substrata had no distinguishable pattern other than a roughness amplitude of 1 nm. Interestingly, microscopy and image analysis showed that fibroblast after 4 h had adjusted their shape according to nanotopographical features down to cut-off values of 100 nm width and 75 nm depth. After 24 h culturing time, fibroblasts would even align themselves on groove depths as shallow as 35 nm. It appears depth is the most essential parameter in cellular alignment on groove patterns with a pitch ratio of 1:1. On the smooth substrata, cells always spread out in a random fashion. Analysis of variance (ANOVA) demonstrated that both main parameters, topography and culturing time, were significant. We conclude that fibroblast cells cultured on nanotopography experience a threshold feature size of 35 nm, below this value contact guidance does no longer exist.