Roman Truckenmüller

Tissue assembly and organization: Developmental mechanisms in microfabricated tissues

In vitro-generated tissues hold significant promise in modern biology since they can potentially mimic physiological and pathological tissues. However, these are currently structurally and functionally of limited complexity and necessitate self-organization and recapitulation of tissue development mechanisms in vitro. Tools derived from nano- and microfabrications along with bottom-up strategies are emerging to allow the fabrication of primitive tissues structures that can remodel overtime. Subsequently, clues are accumulating to show that, beyond genetic material, both intrinsic tissue architectures and microenvironmental cues can lead to morphogenesis related mechanisms in vitro. The question arises, however, as how we may design and assemble structures prone to adequate tissue remodeling, predict and manipulate those developmental mechanisms in vitro? Systems integrating architectural, physical and molecular cues will allow more systematic investigation of basic principles of tissue morphogenesis, differentiation or maintenance and will feedback to reproduce the dynamic of tissue development in vitro and form more complex tissues.

Sonic Hedgehog-activated engineered blood vessels enhance bone tissue formation

Large bone defects naturally regenerate via a highly vascularized tissue which progressively remodels into cartilage and bone. Current approaches in bone tissue engineering are restricted by delayed vascularization and fail to recapitulate this stepwise differentiation toward bone tissue. Here, we use the morphogen Sonic Hedgehog (Shh) to induce the in vitro organization of an endothelial capillary network in an artificial tissue. We show that endogenous Hedgehog activity regulates angiogenic genes and the formation of vascular lumens. Exogenous Shh further induces the in vitro development of the vasculature (vascular lumen formation, size, distribution). Upon implantation, the in vitro development of the vasculature improves the in vivo perfusion of the artificial tissue and is necessary to contribute to, and enhance, the formation of de novo mature bone tissue. Similar to the regenerating callus, the artificial tissue undergoes intramembranous and endochondral ossification and forms a trabecular-like bone organ including bone-marrow-like cavities. These findings open the door for new strategies to treat large bone defects by closely mimicking natural endochondral bone repair.

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.

Rapid prototyping of microstructures in polydimethylsiloxane (PDMS) by direct UV-lithography

Microstructuring of polydimethylsiloxane (PDMS) is a key step for many lab-on-a-chip (LOC) applications. In general, the structure is generated by casting the liquid prepolymer against a master. The production of the master in turn calls for special equipment and know how. Furthermore, a given master only allows the reproduction of the defined structure. We report on a simple, cheap and practical method to produce microstructures in already cured PDMS by direct UV-lithography followed by chemical development. Due to the available options during the lithographic process like multiple exposures, the method offers a high design flexibility granting easy access to complex and stepped structures. Furthermore, no master is needed and the use of pre-cured PDMS allows processing at ambient (light) conditions. Features down to approximately 5 µm and a depth of 10 µm can be realised. As a proof of principle, we demonstrate the feasibility of the process by applying the structures to various established soft lithography techniques.

Fabrication of cell container arrays with overlaid surface topographies.

This paper presents cell culture substrates in the form of microcontainer arrays with overlaid surface topographies, and a technology for their fabrication. The new fabrication technology is based on microscale thermoforming of thin polymer films whose surfaces are topographically prepatterned on a micro- or nanoscale. For microthermoforming, we apply a new process on the basis of temporary back moulding of polymer films and use the novel concept of a perforated-sheet-like mould. Thermal micro- or nanoimprinting is applied for prepatterning. The novel cell container arrays are fabricated from polylactic acid (PLA) films. The thin-walled microcontainer structures have the shape of a spherical calotte merging into a hexagonal shape at their upper circumferential edges. In the arrays, the cell containers are arranged densely packed in honeycomb fashion. The inner surfaces of the highly curved container walls are provided with various topographical micro- and nanopatterns. For a first validation of the microcontainer arrays as in vitro cell culture substrates, C2C12 mouse premyoblasts are cultured in containers with microgrooved surfaces and shown to align along the grooves in the three-dimensional film substrates. In future stem-cell-biological and tissue engineering applications, microcontainers fabricated using the proposed technology may act as geometrically defined artificial microenvironments or niches.

Surface modifications by gas plasma control osteogenic differentiation of MC3T3-E1 cells.

Numerous studies have shown that the physicochemical properties of biomaterials can control cell activity. Cell adhesion, proliferation, differentiation as well as tissue formation in vivo can be tuned by properties such as the porosity, surface micro- and nanoscale topography and chemical composition of biomaterials. This concept is very appealing for tissue engineering since instructive properties in bioactive materials can be more economical and time efficient than traditional strategies of cell pre-differentiation in vitro prior to implantation. The biomaterial surface, which is easy to modify due to its accessibility, may provide the necessary signals to elicit a certain cellular behavior. Here, we used gas plasma technology at atmospheric pressure to modify the physicochemical properties of polylactic acid and analyzed how this influenced pre-osteoblast proliferation and differentiation. Tetramethylsilane and 3-aminopropyl-trimethoxysilane with helium as a carrier gas or a mixture of nitrogen and hydrogen were discharged to polylactic acid discs to create different surface chemical compositions, hydrophobicity and microscale topographies. Such modifications influenced protein adsorption and pre-osteoblast cell adhesion, proliferation and osteogenic differentiation. Furthermore polylactic acid treated with tetramethylsilane enhanced osteogenic differentiation compared to the other surfaces. This promising surface modification could be further explored for potential development of bone graft substitutes.

A fast process for imprinting micro and nano patterns on electrospun fiber meshes at physiological temperatures.

Electrospun fiber meshes are patterned at length scales comparable to or lower than their fiber diameter. Simple nano- and microgrooves and closed geometric shapes are imprinted in different tones using a fast imprint process at physiological temperatures. Human mesenchymal stromal cells cultured on patterned scaffolds show differences in cellular morphology and cytoskeleton organization. Microgrooved electrospun fibers support upregulation of alkaline phosphatase and bone morphogenetic protein-2 gene expression when cells are cultured in osteogenic medium.

Microwell Scaffolds for the Extrahepatic Transplantation of Islets of Langerhans

Allogeneic islet transplantation into the liver has the potential to restore normoglycemia in patients with type 1 diabetes. However, the suboptimal microenvironment for islets in the liver is likely to be involved in the progressive islet dysfunction that is often observed post-transplantation. This study validates a novel microwell scaffold platform to be used for the extrahepatic transplantation of islet of Langerhans. Scaffolds were fabricated from either a thin polymer film or an electrospun mesh of poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) block copolymer (composition: 4000PEOT30PBT70) and were imprinted with microwells, ∼400 µm in diameter and ∼350 µm in depth. The water contact angle and water uptake were 39±2° and 52.1±4.0 wt%, respectively. The glucose flux through electrospun scaffolds was three times higher than for thin film scaffolds, indicating enhanced nutrient diffusion. Human islets cultured in microwell scaffolds for seven days showed insulin release and insulin content comparable to those of free-floating control islets. Islet morphology and insulin and glucagon expression were maintained during culture in the microwell scaffolds. Our results indicate that the microwell scaffold platform prevents islet aggregation by confinement of individual islets in separate microwells, preserves the islet’s native rounded morphology, and provides a protective environment without impairing islet functionality, making it a promising platform for use in extrahepatic islet transplantation.

Regeneration of the lung: Lung stem cells and the development of lung mimicking devices.

Inspired by the increasing burden of lung associated diseases in society and an growing demand to accommodate patients, great efforts by the scientific community produce an increasing stream of data that are focused on delineating the basic principles of lung development and growth, as well as understanding the biomechanical properties to build artificial lung devices. In addition, the continuing efforts to better define the disease origin, progression and pathology by basic scientists and clinicians contributes to insights in the basic principles of lung biology. However, the use of different model systems, experimental approaches and readout systems may generate somewhat conflicting or contradictory results. In an effort to summarize the latest developments in the lung epithelial stem cell biology, we provide an overview of the current status of the field. We first describe the different stem cells, or progenitor cells, residing in the homeostatic lung. Next, we focus on the plasticity of the different cell types upon several injury-induced activation or repair models, and highlight the regenerative capacity of lung cells. Lastly, we summarize the generation of lung mimics, such as air-liquid interface cultures, organoids and lung on a chip, that are required to test emerging hypotheses. Moreover, the increasing collaboration between distinct specializations will contribute to the eventual development of an artificial lung device capable of assisting reduced lung function and capacity in human patients.

3D Nanofabrication of Fluidic Components by Corner Lithography

A reproducible wafer-scale method to obtain 3D nanostructures is investigated. This method, called corner lithography, explores the conformal deposition and the subsequent timed isotropic etching of a thin film in a 3D shaped silicon template. The technique leaves a residue of the thin film in sharp concave corners which can be used as structural material or as an inversion mask in subsequent steps. The potential of corner lithography is studied by fabrication of functional 3D microfluidic components, in particular i) novel tips containing nano-apertures at or near the apex for AFM-based liquid deposition devices, and ii) a novel particle or cell trapping device using an array of nanowire frames. The use of these arrays of nanowire cages for capturing single primary bovine chondrocytes by a droplet seeding method is successfully demonstrated, and changes in phenotype are observed over time, while retaining them in a well-defined pattern and 3D microenvironment in a flat array.