NanoTopoChip: High-throughput nanotopographical cell instruction.

Surface topography is able to influence cell phenotype in numerous ways and offers opportunities to manipulate cells and tissues. In this work, we develop the Nano-TopoChip and study the cell instructive effects of nanoscale topographies. A combination of deep UV projection lithography and conventional lithography was used to fabricate a library of more than 1200 different defined nanotopographies. To illustrate the cell instructive effects of nanotopography, actin-RFP labeled U2OS osteosarcoma cells were cultured and imaged on the Nano-TopoChip. Automated image analysis shows that of many cell morphological parameters, cell spreading, cell orientation and actin morphology are mostly affected by the nanotopographies. Additionally, by using modeling, the changes of cell morphological parameters could by predicted by several feature shape parameters such as lateral size and spacing. This work overcomes the technological challenges of fabricating high quality defined nanoscale features on unprecedented large surface areas of a material relevant for tissue culture such as PS and the screening system is able to infer nanotopography - cell morphological parameter relationships. Our screening platform provides opportunities to identify and study the effect of nanotopography with beneficial properties for the culture of various cell types. STATEMENT OF SIGNIFICANCE: The nanotopography of biomaterial surfaces can be modified to influence adhering cells with the aim to improve the performance of medical implants and tissue culture substrates. However, the necessary knowledge of the underlying mechanisms remains incomplete. One reason for this is the limited availability of high-resolution nanotopographies on relevant biomaterials, suitable to conduct systematic biological studies. The present study shows the fabrication of a library of nano-sized surface topographies with high fidelity. The potential of this library, called the 'NanoTopoChip' is shown in a proof of principle HTS study which demonstrates how cells are affected by nanotopographies. The large dataset, acquired by quantitative high-content imaging, allowed us to use predictive modeling to describe how feature dimensions affect cell morphology.

TopoWellPlate: A Well-Plate-Based Screening Platform to Study Cell-Surface Topography Interactions.

The field of biomaterial engineering is increasingly using high-throughput approaches to investigate cell-material interactions. Because most material libraries are prepared as chips, immunofluorescence-based read-outs are used to uniquely image individual materials. This paper proposes to produce libraries of materials using a well-based strategy in which each material is physically separated, and thus compatible with standard biochemical assays. In this work, the TopoWellPlate, a novel system to study cell-surface topography interaction in high-throughput is presented. From a larger library of topographies, 87 uniquely defined bioactive surface topographies are identified, which induce a wide variety of cellular morphologies. Topographically enhanced polystyrene films are fabricated in a multistep cleanroom process and served as base for the TopoWellPlate. Thermal bonding of the films to bottomless 96-well plates results in a cell culture ready, topographically enhanced, 96-well plate. The overall metabolic activity of bone marrow-derived human mesenchymal stem cells is measured to show the functionality of the TopoWellPlate as a screening tool, which showed a 2.5-fold difference range in metabolic activity per cell. TopoWellPlates of this and other topographical designs can be used to analyze cells using the wealth of standardized molecular assays available and thus disclose the mechanisms of biomaterials-induced mechanotransduction.

Analysis of high-throughput screening reveals the effect of surface topographies on cellular morphology.

Surface topographies of materials considerably impact cellular behavior as they have been shown to affect cell growth, provide cell guidance, and even induce cell differentiation. Consequently, for successful application in tissue engineering, the contact interface of biomaterials needs to be optimized to induce the required cell behavior. However, a rational design of biomaterial surfaces is severely hampered because knowledge is lacking on the underlying biological mechanisms. Therefore, we previously developed a high-throughput screening device (TopoChip) that measures cell responses to large libraries of parameterized topographical material surfaces. Here, we introduce a computational analysis of high-throughput materiome data to capture the relationship between the surface topographies of materials and cellular morphology. We apply robust statistical techniques to find surface topographies that best promote a certain specified cellular response. By augmenting surface screening with data-driven modeling, we determine which properties of the surface topographies influence the morphological properties of the cells. With this information, we build models that predict the cellular response to surface topographies that have not yet been measured. We analyze cellular morphology on 2176 surfaces, and find that the surface topography significantly affects various cellular properties, including the roundness and size of the nucleus, as well as the perimeter and orientation of the cells. Our learned models capture and accurately predict these relationships and reveal a spectrum of topographies that induce various levels of cellular morphologies. Taken together, this novel approach of high-throughput screening of materials and subsequent analysis opens up possibilities for a rational design of biomaterial surfaces.

The use of silk-based devices for fracture fixation.

Metallic fixation systems are currently the gold standard for fracture fixation but have problems including stress shielding, palpability and temperature sensitivity. Recently, resorbable systems have gained interest because they avoid removal and may improve bone remodelling due to the lack of stress shielding. However, their use is limited to paediatric craniofacial procedures mainly due to the laborious implantation requirements. Here we prepare and characterize a new family of resorbable screws prepared from silk fibroin for craniofacial fracture repair. In vivo assessment in rat femurs shows the screws to be self-tapping, remain fixed in the bone for 4 and 8 weeks, exhibit biocompatibility and promote bone remodelling. The silk-based devices compare favourably with current poly-lactic-co-glycolic acid fixation systems, however, silk-based devices offer numerous advantages including ease of implantation, conformal fit to the repair site, sterilization by autoclaving and minimal inflammatory response.

An algorithm-based topographical biomaterials library to instruct cell fate.

It is increasingly recognized that material surface topography is able to evoke specific cellular responses, endowing materials with instructive properties that were formerly reserved for growth factors. This opens the window to improve upon, in a cost-effective manner, biological performance of any surface used in the human body. Unfortunately, the interplay between surface topographies and cell behavior is complex and still incompletely understood. Rational approaches to search for bioactive surfaces will therefore omit previously unperceived interactions. Hence, in the present study, we use mathematical algorithms to design nonbiased, random surface features and produce chips of poly(lactic acid) with 2,176 different topographies. With human mesenchymal stromal cells (hMSCs) grown on the chips and using high-content imaging, we reveal unique, formerly unknown, surface topographies that are able to induce MSC proliferation or osteogenic differentiation. Moreover, we correlate parameters of the mathematical algorithms to cellular responses, which yield novel design criteria for these particular parameters. In conclusion, we demonstrate that randomized libraries of surface topographies can be broadly applied to unravel the interplay between cells and surface topography and to find improved material surfaces.

Influence of micro-patterned PLLA membranes on outgrowth and orientation of hippocampal neurites.

In neuronal tissue engineering many efforts are focused on creating biomaterials with physical and chemical pathways for controlling cellular proliferation and orientation. Neurons have the ability to respond to topographical features in their microenvironment causing among others, axons to proliferate along surface features such as substrate grooves in micro-and nanoscales. As a consequence these neuronal elements are able to correctly adhere, migrate and orient within their new environment during growth. Here we explored the polarization and orientation of hippocampal neuronal cells on nonpatterned and micro-patterned biodegradable poly(l-lactic acid) (PLLA) membranes with highly selective permeable properties. Dense and porous nonpatterned and micro-patterned membranes were prepared from PLLA by Phase Separation Micromolding. The micro-patterned membranes have a three-dimensional structure consisting of channels and ridges and of bricks of different widths. Nonpatterned and patterned membranes were used for hippocampal neuronal cultures isolated from postnatal days 1-3 hamsters and the neurite length, orientation and specific functions of cells were investigated up to 12 days of culture. Neurite outgrowth, length plus orientation tightly overlapped the pattern of the membrane surface. Cell distribution occurred only in correspondence to membrane grooves characterized by continuous channels whereas on membranes with interconnected channels, cells not only adhered to and elongated their cellular processes in the grooves but also in the breaking points. High orientation degrees of cells were determined particularly on the patterned porous membranes with channel width of 20 mum and ridges of 17 mum whereas on dense nonpatterned membranes as well as on polystyrene culture dish (PSCD) controls, a larger number of primary developed neurites were distributed. Based on these results, PLLA patterned membranes may directly improve the guidance of neurite extension and thereby enhancing their orientation with a consequently highly ordered neuronal cell matrix, which may have strong bearings on the elucidation of regeneration mechanisms.

A facile method to fabricate poly(L-lactide) nano-fibrous morphologies by phase inversion.

Scaffolds with a nano-fibrous morphology are favored for certain tissue engineering applications as this morphology mimics the tissue's natural extracellular matrix secreted by the cells, which consists of mainly collagen fibers with diameters ranging from 50 to 400 nm. Porous poly(L-lactide) (PLLA) scaffolds obtained by phase inversion methods generally have a solid-wall pore morphology. In contrast, this work presents a facile method to fabricate highly porous and highly interconnected nano-fibrous scaffold sheets by phase inversion using PLLA of very high molecular weight (5.7x10(5) g mol(-1)). The scaffold sheets consist of nano-fibers within the desired range of 50-500 nm. When applying phase separation micromolding as a fabrication method besides the porous nano-fibrous morphology, an additional topography can be introduced into these sheets. Culturing of C2C12 pre-myoblasts on these nano-fibrous sheets reveals very good cell adhesion, morphology and proliferation. Excellent alignment of the cells is induced by fabrication of 25 microm wide microchannels in these sheets. These results warrant further evaluation of these sheets as tissue engineering scaffolds.

Development and analysis of multi-layer scaffolds for tissue engineering.

The development of 3D scaffolds consisting of stacked multi-layered porous sheets featuring microchannels is proposed and investigated in this work. In this concept, the inner-porosity of the sheets allows diffusion of nutrients and signalling products between the layers whereas the microchannels facilitate nutrient supply on all layers as they provide space for the culture medium to be perfused throughout the scaffold. Besides the above, these scaffolds have excellent distribution of the cells as seeding and attaching of the cells occurs on individual layers that are subsequently stacked. In addition, these scaffolds enable gaining local data from within the scaffolds as unstacking of the stacked layers allows for determination of various parameters per layer. Here, we show the proof of this concept by culturing C2C12 pre-myoblasts and A4-4 cells on stacked Poly(l-lactic acid) (PLLA) sheets featuring microchannels. The results obtained for culturing under static conditions clearly indicate that despite inhibited cell proliferation due to nutrient limitations, diffusion between the layers takes place and cells on various layers stay viable and also affect each other. Under dynamic conditions, medium flow through the channels improves nutrient availability to the cells on the various layers, drastically increasing cell proliferation on all layers.

Designing porosity and topography of poly(1,3-trimethylene carbonate) scaffolds.

Using phase separation micromolding (PSmicroM) we developed porous micro-patterned sheets from amorphous poly(1,3-trimethylene carbonate) (PTMC). The use of these PTMC sheets can be advantageous in tissue engineering applications requiring highly flexible constructs. Addition of poly(ethylene oxide) (PEO) in various amounts to PTMC casting solutions provides PTMC sheets with tailored porosity and pore sizes in the range 2-20 microm. The pore-forming effect of PEO during the phase separation process is evaluated and glucose transport measurements show that the pores are highly interconnected. Additionally, tailoring the micro-pattern design yields PTMC sheets with various surface topographies. Cell culturing experiments with C2C12 pre-myoblasts revealed that cell attachment and proliferation on these sheets is relatively high and that the micro-pattern topography induces a clearly defined cell organization.

One-step fabrication of porous micropatterned scaffolds to control cell behavior.

This paper reports a one-step method to fabricate highly porous micropatterned 2-D scaffold sheets. The scaffold sheets have high glucose diffusion, indicating that the porosity and pore morphology of the scaffolds are viable with respect to nutrient transport, and a micropattern for cell alignment. HUVEC culturing proved that the scaffold sheets are suitable for cell culturing. More extensive culturing experiments with mouse myoblasts, C2C12, and mouse osteoblasts, MC3T3, showed that tissue organization can be controlled; the micropattern design affects the extent of cell alignment and tissue formation. Cells are favorably settled in the micropattern and even at higher confluence levels, when the cells start to overgrow the ridges of the micropattern, these cells align themselves in the direction of the micropattern. Preliminary multi-layer stacking experiments indicate that the 2-D scaffold sheets are very promising as basis for building 3-D scaffolds.