Post-fabrication QAC-functionalized thermoplastic polyurethane for contact-killing catheter applications.

The use of catheters is ubiquitous in medicine and the incidence of infection remains unacceptably high despite numerous advances in functional surfaces and drug elution. Herein we report the use of a thermoplastic polyurethane containing an allyl ether side-chain functionality (allyl-TPU) that allows for rapid and convenient surface modification with antimicrobial reagents, post-processing. This post-processing functionalization affords the ability to target appropriate TPU properties and maintain the functional groups on the surface of the device where they do not affect bulk properties. A series of quaternary ammonium thiol compounds (Qx-SH) possessing various hydrocarbon tail lengths (8-14 carbons) were synthesized and attached to the surface using thiol-ene "click" chemistry. A quantitative assessment of the amount of Qx-SH available on the surface was determined using fluorescence spectroscopy and X-ray photoelectron spectroscopy (XPS). Contact-killing assays note the Q8-SH composition has the highest antimicrobial activity, and a live/dead fluorescence assay reveals rapid contact-killing of Staphylococcus aureus (>75% in 5 min) and Escherichia coli (90% in 10 min) inocula. Scale-up and extrusion of allyl-TPU provides catheter prototypes for biofilm formation testing with Pseudomonas aeruginosa, and surface-functionalized catheters modified with Q8-SH demonstrate their ability to reduce biofilm formation.

Effects of cell type and configuration on anabolic and catabolic activity in 3D co-culture of mesenchymal stem cells and nucleus pulposus cells.

Tissue engineering constructs to treat intervertebral disc degeneration must adapt to the hypoxic and inflammatory degenerative disc microenvironment. The objective of this study was to determine the effects of two key design factors, cell type and cell configuration, on the regenerative potential of nucleus pulposus cell (NPC) and mesenchymal stem cell (MSC) constructs. Anabolic and catabolic activity was quantified in constructs of varying cell type (NPCs, MSCs, and a 50:50 co-culture) and varying configuration (individual cells and micropellets). Anabolic and catabolic outcomes were both dependent on cell type. Gene expression of Agg and Col2A1, glycosaminoglycan (GAG) content, and aggrecan immunohistochemistry (IHC), were significantly higher in NPC-only and co-culture groups than in MSC-only groups, with NPC-only groups exhibiting the highest anabolic gene expression levels. However, NPC-only constructs also responded to inflammation and hypoxia with significant upregulation of catabolic genes (MMP-1, MMP-9, MMP-13, and ADAMTS-5). MSC-only groups were unaffected by degenerative media conditions, and co-culture with MSCs modulated catabolic induction of the NPCs. Culturing cells in a micropellet configuration dramatically reduced catabolic induction in co-culture and NPC-only groups. Co-culture micropellets, which take advantage of both cell type and configuration effects, had the most immunomodulatory response, with a significant decrease in MMP-13 and ADAMTS-5 expression in hypoxic and inflammatory media conditions. Co-culture micropellets were also found to self-organize into bilaminar formations with an MSC core and NPC outer layer. Further understanding of these cell type and configuration effects can improve tissue engineering designs. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 35:61-73, 2017.

Probing the luminal microenvironment of reconstituted epithelial microtissues.

Polymeric microparticles can serve as carriers or sensors to instruct or characterize tissue biology. However, incorporating microparticles into tissues for in vitro assays remains a challenge. We exploit three-dimensional cell-patterning technologies and directed epithelial self-organization to deliver microparticles to the lumen of reconstituted human intestinal microtissues. We also develop a novel pH-sensitive microsensor that can measure the luminal pH of reconstituted epithelial microtissues. These studies offer a novel approach for investigating luminal microenvironments and drug-delivery across epithelial barriers.

Fabrication of 3-D Reconstituted Organoid Arrays by DNA-Programmed Assembly of Cells (DPAC).

Tissues are the organizational units of function in metazoan organisms. Tissues comprise an assortment of cellular building blocks, soluble factors, and extracellular matrix (ECM) composed into specific three-dimensional (3-D) structures. The capacity to reconstitute tissues in vitro with the structural complexity observed in vivo is key to understanding processes such as morphogenesis, homeostasis, and disease. In this article, we describe DNA-programmed assembly of cells (DPAC), a method to fabricate viable, functional arrays of organoid-like tissues within 3-D ECM gels. In DPAC, dissociated cells are chemically functionalized with degradable oligonucleotide "Velcro," allowing rapid, specific, and reversible cell adhesion to a two-dimensional (2-D) template patterned with complementary DNA. An iterative assembly process builds up organoids, layer-by-layer, from this initial 2-D template and into the third dimension. Cleavage of the DNA releases the completed array of tissues that are captured and fully embedded in ECM gels for culture and observation. DPAC controls the size, shape, composition, and spatial heterogeneity of organoids and permits positioning of constituent cells with single-cell resolution even within cultures several centimeters long. © 2016 by John Wiley & Sons, Inc.

Rapid Organoid Reconstitution by Chemical Micromolding.

Purified populations of cells can be reconstituted into organoids that recapitulate aspects of their in vivo structure and function. These organoids are useful as models of healthy and diseased tissue in the basic sciences, in vitro screens, and regenerative medicine. Existing strategies to reconstitute organoids from purified cells face obstacles with respect to cell-viability, multicellular connectivity, scalability, and compatibility with subsequent experimental or analytical techniques. To address these challenges, we developed a strategy for rapidly casting populations of cells into microtissues of prescribed size and shape. This approach begins by chemically remodeling the adhesive properties of living cells with membrane-anchored ssDNA with modest annealing kinetics. Populations of complementary labeled cells are then combined into microwells that rapidly mold the DNA-adhesive cell populations into 3D aggregates of uniform size and shape. Once formed, aggregates are removed from the molds in the presence of "capping" oligonucleotides that block hybridization of residual surface DNA between aggregates in suspension. Finally, transfer of aggregates to biomimetic gels for 3D culture completes the process of reconstitution. This strategy of chemical micromolding allows for control over aggregate internal topology and does not perturb the natural process of self-organization in primary human mammary epithelial cells.

Coupling between apical tension and basal adhesion allow epithelia to collectively sense and respond to substrate topography over long distances.

Epithelial sheets fold into complex topographies that contribute to their function in vivo. Cells can sense and respond to substrate topography in their immediate vicinity by modulating their interfacial mechanics, but the extent to which these mechanical properties contribute to their ability to sense substrate topography across length scales larger than a single cell has not been explored in detail. To study the relationship between the interfacial mechanics of single cells and their collective behavior as tissues, we grew cell-sheets on substrates engraved with surface features spanning macroscopic length-scales. We found that many epithelial cell-types sense and respond to substrate topography, even when it is locally nearly planar. Cells clear or detach from regions of local negative curvature, but not from regions with positive or no curvature. We investigated this phenomenon using a finite element model where substrate topography is coupled to epithelial response through a balance of tissue contractility and adhesive forces. The model correctly predicts the focal sites of cell-clearing and epithelial detachment. Furthermore, the model predicts that local tissue response to substrate curvature is a function of the surrounding topography of the substrate across long distances. Analysis of cell-cell and cell-substrate contact angles suggests a relationship between these single-cell interfacial properties, epithelial interfacial properties, and collective epithelial response to substrate topography. Finally, we show that contact angles change upon activation of oncogenes or inhibition of cell-contractility, and that these changes correlate with collective epithelial response. Our results demonstrate that in mechanically integrated epithelial sheets, cell contractility can be transmitted through multiple cells and focused by substrate topography to affect a behavioral response at distant sites.

Programmed synthesis of three-dimensional tissues.

Reconstituting tissues from their cellular building blocks facilitates the modeling of morphogenesis, homeostasis and disease in vitro. Here we describe DNA-programmed assembly of cells (DPAC), a method to reconstitute the multicellular organization of organoid-like tissues having programmed size, shape, composition and spatial heterogeneity. DPAC uses dissociated cells that are chemically functionalized with degradable oligonucleotide 'Velcro', allowing rapid, specific and reversible cell adhesion to other surfaces coated with complementary DNA sequences. DNA-patterned substrates function as removable and adhesive templates, and layer-by-layer DNA-programmed assembly builds arrays of tissues into the third dimension above the template. DNase releases completed arrays of organoid-like microtissues from the template concomitant with full embedding in a variety of extracellular matrix (ECM) gels. DPAC positions subpopulations of cells with single-cell spatial resolution and generates cultures several centimeters long. We used DPAC to explore the impact of ECM composition, heterotypic cell-cell interactions and patterns of signaling heterogeneity on collective cell behaviors.

A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity.

Developing tissues contain motile populations of cells that can self-organize into spatially ordered tissues based on differences in their interfacial surface energies. However, it is unclear how self-organization by this mechanism remains robust when interfacial energies become heterogeneous in either time or space. The ducts and acini of the human mammary gland are prototypical heterogeneous and dynamic tissues comprising two concentrically arranged cell types. To investigate the consequences of cellular heterogeneity and plasticity on cell positioning in the mammary gland, we reconstituted its self-organization from aggregates of primary cells in vitro. We find that self-organization is dominated by the interfacial energy of the tissue-ECM boundary, rather than by differential homo- and heterotypic energies of cell-cell interaction. Surprisingly, interactions with the tissue-ECM boundary are binary, in that only one cell type interacts appreciably with the boundary. Using mathematical modeling and cell-type-specific knockdown of key regulators of cell-cell cohesion, we show that this strategy of self-organization is robust to severe perturbations affecting cell-cell contact formation. We also find that this mechanism of self-organization is conserved in the human prostate. Therefore, a binary interfacial interaction with the tissue boundary provides a flexible and generalizable strategy for forming and maintaining the structure of two-component tissues that exhibit abundant heterogeneity and plasticity. Our model also predicts that mutations affecting binary cell-ECM interactions are catastrophic and could contribute to loss of tissue architecture in diseases such as breast cancer.

Formation of spatially and geometrically controlled three-dimensional tissues in soft gels by sacrificial micromolding.

Patterned three-dimensional (3D) cell culture models aim to more accurately represent the in vivo architecture of a tissue for the purposes of testing drugs, studying multicellular biology, or engineering functional tissues. However, patterning 3D multicellular structures within very soft hydrogels (<500 Pa) that mimic the physicochemical environment of many tissues remains a challenge for existing methods. To overcome this challenge, we use a Sacrificial Micromolding technique to temporarily form spatially and geometrically defined 3D cell aggregates in degradable scaffolds before transferring and culturing them in a reconstituted extracellular matrix. Herein, we demonstrate that Sacrificial Micromolding (1) promotes cyst formation and proper polarization of established epithelial cell lines, (2) allows reconstitution of heterotypic cell-cell interactions in multicomponent epithelia, and (3) can be used to control the lumenization-state of epithelial cysts as a function of tissue size. In addition, we discuss the potential of Sacrificial Micromolding as a cell-patterning tool for future studies.

Nitinol-based nanotubular coatings for the modulation of human vascular cell function.

In this study, we describe the synthesis of an upright nanotubular coating with discrete, exposed nanotubes on top of superelastic Nitinol via anodization and characterization of the surface elemental composition and nickel release rates. We demonstrate, for the first time, that this coating could improve re-endothelialization by increasing the cell spreading and migration of primary human aortic endothelial cells on Nitinol. We also show the potential for reducing neointimal hyperplasia by decreasing the proliferation and expression of collagen I and MMP-2 in primary human aortic smooth muscle cells (HASMC). Furthermore, we did not observe the nanotubular surface to induce inflammation through ICAM-1 expression in HASMC as compared to the flat control. This coating could be used to improve Nitinol stents by reducing restenosis rates and, given the extensive use of Nitinol in other implantable devices, act as a generalized coating strategy for other medical devices.

Novel functionalization of discrete polymeric biomaterial microstructures for applications in imaging and three-dimensional manipulation.

Adapting ways to functionalize polymer materials is becoming increasingly important to their implementation in translational biomedical sciences. By tuning the mechanical, chemical, and biological qualities of these materials, their applications can be broadened, opening the door for more advanced integration into modern medical techniques. Here, we report on a method to integrate chemical functionalizations into discrete, microscale polymer structures, which are used for tissue engineering applications, for in vivo localization, and three-dimensional manipulation. Iron oxide nanoparticles were incorporated into the polymer matrix using common photolithographic techniques to create stably functional microstructures with magnetic potential. Using magnetic resonance imaging (MRI), we can promote visualization of microstructures contained in small collections, as well as facilitate the manipulation and alignment of microtopographical cues in a realistic tissue environment. Using similar polymer functionalization techniques, fluorine-containing compounds were also embedded in the polymer matrix of photolithographically fabricated microstructures. The incorporation of fluorine-containing compounds enabled highly sensitive and specific detection of microstructures in physiologic settings using fluorine MRI techniques ((19)F MRI). These functionalization strategies will facilitate more reliable noninvasive tracking and characterization of microstructured polymer implants as well as have implications for remote microstructural scaffolding alignment for three-dimensional tissue engineering applications.