Micropatterning of Poly (N-isopropylacrylamide) (PNIPAAm) Hydrogels: Effects on Thermosensitivity and Cell Release Behavior

The thermally driven, reversible change in the surface properties of poly (N-isopropylacrylamide) (PNIPAAm) hydrogels from a hydrophilic (water-swollen) state to a hydrophobic (deswollen) state when heated above the volume phase transition temperature (VPTT, ~35 oC) makes them useful in inducing controlled cell release. To improve the kinetics of swelling and deswelling, we have prepared microstructured (i.e., micropillared) thermoresponsive surfaces comprising pure PNIPAAm hydrogel and nanocomposite PNIPAAm hydrogel embedded with polysiloxane colloidal nanoparticles (~220 nm diameter, 1 wt%) via photopolymerization. The thermosensitivity (i.e., degree and rate of swelling/deswelling) of these surfaces and how it can be regulated using different micropillar sizes and densities were characterized by measuring the dynamic size changes in micropillar dimensions in response to thermal activation. Our results show that the dynamic thermal response rate can be increased by more than twofold when the micropillar size is reduced from 200 to 100 μm. The temperature-controlled cell release behaviors of pure PNIPAAm and nanocomposite PNIPAAm micropatterned surfaces were successfully characterized using mesenchymal progenitor cells (10T1/2). This study demonstrates that the thermosensitivity of PNIPAAm surfaces can be regulated by introducing micropillars of different sizes and densities, while maintaining good temperature-controlled cell release behavior

Smart thermoresponsive coatings and surfaces for tissue engineering : switching cell-material boundaries

The smart thermoresponsive coatings and surfaces that have been explicitly designed for cell culture are mostly based on poly(N-isopropylacrylamide) (PNIPAAm). This polymer is characterized by a sudden precipitation on heating, switching from a hydrophilic to a hydrophobic state. Mammalian cells cultured on such thermoresponsive substrates can be recovered as confluent cell sheets, while keeping the newly deposited extracellular matrix intact, simply by lowering the temperature and thereby avoiding the use of deleterious proteases. Thermoresponsive materials and surfaces are powerful tools for creating tissue-like constructs that imitate native tissue geometry and mimic its spatial cellular organization. Here we review and compare the most representative methods of producing thermoresponsive substrates for cell sheet engineering

Glycidyl Ether-Based Coatings on Polystyrene Culture Substrates for Temperature-Triggered Cell Sheet Fabrication

Within this work, thermoresponsive coatings based on poly(glycidyl ether)s (PGEs) were developed for applied polystyrene (PS) tissue culture substrates. Following the “grafting to“ approach, block copolymers comprising a random, high molecular weight, thermoresponsive block and a short, hydrophobic benzophenone (BP) block were synthesized via the sequential, monomer-activated, oxy-anionic ring-opening polymerization (ROP). Ultrathin layers in the sub-nanometer range were immobilized on PS by physical adsorption and UV-induced C, H-insertion of PGE block copolymers via their photo-reactive BP anchor block. The coatings mediated the adhesion of human dermal fibroblasts (HDFs) and allowed the temperature triggered detachment of confluent cell sheets. HDF sheet detachment was found to be induced by the cooperative effects between the partial rehydration of the PGE chains and the cell repellant PS substrate background. In order to improve the performance of PGE monolayers, block copolymers were subsequently self-assembled on PS substrates from dilute aqueous solution under selective solvent conditions. UV immobilization yielded thermoresponsive polymer brushes, which undergo a “pancake-to-brush” transition upon temperature reduction. The improved structure and thermal response of the brush-like PGE coatings as well as the optimization of cell culture parameters facilitated the fabrication of confluent HDF, human aortic smooth muscle cell (HAoSMC) and human umbilical vein endothelial cell (HUVEC) sheets, which constitute the main building blocks of blood vessels. To functionalize PS culture substrates via the “grafting from” approach, a solvent-free, microwave-assisted synthesis of well-defined oligo(glycidyl ether)s (OGEs) was developed. Fast reaction rates could be solely attributed to the high reaction temperatures reached during microwave heating and the obtained oligomers exhibited highly molecular weight- and concentration-dependent CPTs in water. Further, end-functional oligo(glycidyl ether) acrylate (OGEA) macromonomers were synthesized by in situ quenching of the oxy-anionic ROP. Subsequently, a photopolymerization process was developed to graft OGEA macromonomers from PS culture substrates. Surfaceinitiated photografting from bulk macromonomer films yielded porous, rigid, gel-like OGEA coatings with unique bottlebrush properties. Bottlebrushes with optimized structure proved to be functional coatings for the fabrication of HDF sheets. The controlled detachment of cell sheets was found to be triggered by the rehydration of OGEA bottlebrush side chains rather than a macroscopic swelling of the gel-like coatings upon temperature reduction. In summary, this work introduces facile methods for the functionalization of applied PS tissue culture surfaces with thermoresponsive, PGE-based coatings and demonstrates their high potential as functional substrates for cell sheet fabrication

Establishment of dynamic culture conditions for the fabrication of advanced in vitro tissue models.

In this thesis, a system for the application of flow-induced, physiologically relevant shear stress on mammalian cells was established. The circulation of cell culture medium in culture circuits was generated by peristaltic pumps, which can be operated with a self-built control unit. Culture chambers for different applications were computer-assisted designed, characterized by computational fluid dynamics and subsequently manufactured either by CNC milling or 3D printing. Using this system, shear-induced changes on the cellular functionality of in vitro barrier models were investigated. The application of low, physiologically relevant shear forces to an established intestinal epithelial cell line, HT29-MTX, resulted in increased mucus production, as well as structural reorganization of the confluent cell layer towards 3-dimensional villi-like structures. The results could be transferred from solid cell culture substrates to commercially available cell culture inserts bearing membranes, allowing us to develop an improved in vitro model of the intestinal barrier with a physiologically relevant mucus layer. We then proceeded with the endothelial barrier. Here, we investigated the effects of flow-induced stress comparatively for HUVECs and iPSC-ECs. Both cell types showed the characteristic alignment upon application of flow, as well as increased layer thicknesses and improved functionality of the endothelial glycocalyx. In addition, for the first time, we were able to detach isotropic (HUVECs) and anisotropic (iPSC-ECs) cell monolayers from thermoresponsive surfaces and wrapped them around a 3D-printed scaffold while maintaining alignment, which represents an important step toward the development of blood vessels in vitro. Finally, we implemented a similar tubular construct into dynamic culture. The indirect coculture of HUVECs with fibroblasts allowed intercellular communication and resulted in the formation of stable vascular networks, while a second independent circuit allowed perfusion of the tubular blood vessel mimick. In summary, we established a versatile and reliable platform for the application of physiological shear forces to flat and lumenized tissues, enabling the development of improved in vitro barrier models.In dieser Arbeit wurde ein System zur Applikation fluss-induzierter, physiologisch relevanter Scherkräfte auf Säugerzellen etabliert. Die Zirkulation von Zellkulturmedium in Kulturkreisläufen wurde dabei über Peristaltikpumpen generiert, welche mit einer selbstgebauten Steuerung betrieben werden können. Kulturkammern für unterschiedliche Einsatzgebiete wurden computergestützt entworfen, mithilfe numerischer Strömungsmechanik charakterisiert und anschließend entweder durch CNC-Fräsen oder 3D-Druck hergestellt. Mithilfe dieses Systems wurde der Einfluss scherinduzierten Änderungen auf die zelluläre Funktionalität von in vitro Barrieremodellen untersucht. Die Applikation geringer, physiologisch relevanter Scherkräfte auf eine etablierte intestinale epitheliale Zelllinie, HT29-MTX, führte zu einer erhöhten Mucusproduktion, sowie der strukturellen Reorganisation der konfluenten Zellschicht hin zu 3-dimenionalen, Darmzotten-artigen Gebilden. Die Ergebnisse konnten von festen Zellkultursubstraten auf kommerziell erhältliche Zellkultureinsätze mit mikroporösen Membranen übertragen werden, sodass wir ein verbessertes in vitro Modell der intestinalen Barriere mit physiologisch relevanten Mucusschichten entwickeln konnten. Im Anschluss widmeten wir uns dem Studium der Barrierefunktion von Endothelzellen. Dabei untersuchten wir die Auswirkungen von fluss-induziertem Stress vergleichend für HUVECs und iPSCs-ECs. Beide Zellarten zeigten die charakteristische Ausrichtung mit dem Fluss, sowie erhöhte Schichtdicken und verbesserte Funktionalität der endothelialen Glykokalix. Zudem konnten wir erstmalig intakte isotrope (HUVECs) und anisotrope (iPSC-ECs) Zellmonolagen temperatur-gesteuert von thermoresponsiven Oberflächen ablösen und um ein 3D-gedrucktes Gerüst rollen, wobei die Zellausrichtung erhalten blieb, was einen wichtigen Schritt zur Entwicklung von Blutgefäßen in vitro darstellt. Abschließend implementierten wir ein ähnliches Konstrukt in die dynamische Kultur. Die Erweiterung des Systems durch eine indirekte Kokultur der HUVECs mit Fibroblasten ermöglichte die Ausbildung stabiler, vaskularer Netzwerke und ein zweiter unabhängiger Kreislauf erlaubte die Perfusion der künstlichen tubulären Blutgefäße. Zusammenfassend konnten wir eine vielseitige und verlässliche Plattform zur Applikation von physiologischen Scherkräften auf adhärenten Säugerzellen in 2D oder in 3D etablieren, die den Aufbau von verbesserten in vitro Barrieremodellen ermöglicht

SYNTHESIS AND CHARACTERIZATION OF THERMALLY RESPONSIVE POLYMER LAYERS

Future devices such as biomedical and microfluidic devices, to a large extent, will depend on the interactions between the device surfaces and the contacting liquid. Further, biological liquids containing proteins call for controllable interactions between devices and such proteins, however the bulk material must retain the inherent mechanical properties from which the device was fabricated from. It is well known that surface modification is a suitable technique to tune the surface properties without sacrificing the bulk properties of the substrate. In the present study, surface properties were modified through temperature responsive polymer layers. After the modification, the surfaces gained switchability toward protein interaction as well as surface wettability properties. Poly(N-isopropylacrylamide) (PNIPAM), a well studied thermo-responsive polymer was utilized in the subsequent work. Firstly, thermally responsive brushes made from well defined block copolymers incorporating NIPAM and the surface reactive monomer, glycidyl methacrylate (GMA) were fabricated in a single step process. Reaction of the PGMA block with surface hydroxyl groups anchors the polymers to the surface yet allows PNIPAM to assemble at the interface at high enough concentration to exhibit thermally responsive properties in aqueous solutions. Surface properties of the resulting brushes prepared the 1-step process are compared to characteristics of PNIPAM brushes synthesized by already established methods. The thickness, swelling, and protein adsorption of the PNIPAM films were studied by ellipsometry. Chemical composition of the layer was studied by angle-resolved x-ray photoelectron spectroscopy. Film morphologies and forces of adhesion to fibrinogen were examined using atomic force microscopy (AFM) tapping mode and colloidal probe technique. Block copolymer (BCP) and conventional brush films were abraded and subsequently examined for changes in thermally responsive behavior. The results show that deposition of PNIPAM-b-PGMA provides an effective route to create thermally responsive brushes via a 1-step process, with properties equaling and surpassing that of traditional brushes obtained in multiple steps. Further, the 1-step deposition of reactive BCPs can be extended to fabricate mixed block copolymer films. Well defined BCP containing ethylene glycol and GMA were deposited from a joint solution with PNIPAM-b-PGMA. Mixed brush films were also fabricated via a 2-step process for comparison of the resulting properties. PNIPAM BCP layers were utilized as the grafted primary layer with which end-functionalized PEG was grafted in a second step. Protein adhesion and adsorption of the resulting mixed brush films were studied by AFM colloidal probe technique and ellipsometry. In the next part of the work reported, monolayers of PNIPAM containing nanogels were anchored to the surface of silicon wafers, glass slides, polyvinylidene fluoride (PVDF) fibers, and tungsten wires using a \u27grafting to\u27 approach. The particles of were synthesized with different diameters by free radical precipitation polymerization and reversible addition chain transfer polymerization (RAFT) techniques. The behavior of the synthesized grafted layers with the behavior of PNIPAM brushes (densely end-grafted) is compared. Indeed, the grafted monolayer swells and collapses reversibly at temperatures below and above the transition temperature of PNIPAM. AFM in aqueous environment was utilized to study the actuation behavior of the nanogel films. Wettability studies of the grafted layers were performed using various contact angle measurement methods to determine the contact angle changes on different substrates. New methods for the development of thermally responsive polymer films are described. The methods enable the grafting of films with tunable film thickness, temperature response, and well defined biological interaction. The complete grafting of the responsive polymer films require no organic rinsing after grafting step

Responsive cell–material interfaces

Major design aspects for novel biomaterials are driven by the desire to mimic more varied and complex properties of a natural cellular environment with man-made materials. The development of stimulus responsive materials makes considerable contributions to the effort to incorporate dynamic and reversible elements into a biomaterial. This is particularly challenging for cell–material interactions that occur at an interface (biointerfaces); however, the design of responsive biointerfaces also presents opportunities in a variety of applications in biomedical research and regenerative medicine. This review will identify the requirements imposed on a responsive biointerface and use recent examples to demonstrate how some of these requirements have been met. Finally, the next steps in the development of more complex biomaterial interfaces, including multiple stimuli-responsive surfaces, surfaces of 3D objects and interactive biointerfaces will be discussed

Temperature-responsive polymer brush coatings for advanced biomedical applications

Modern biomedical technologies predict the application of materials and devices that not only can comply effectively with specific requirements, but also enable remote control of their functions. One of the most prospective materials for these advanced biomedical applications are materials based on temperature-responsive polymer brush coatings (TRPBCs). In this review, methods for the fabrication and characterization of TRPBCs are summarized, and possibilities for their application, as well as the advantages and disadvantages of the TRPBCs, are presented in detail. Special attention is paid to the mechanisms of thermo-responsibility of the TRPBCs. Applications of TRPBCs for temperature-switchable bacteria killing, temperature-controlled protein adsorption, cell culture, and temperature-controlled adhesion/detachment of cells and tissues are considered. The specific criteria required for the desired biomedical applications of TRPBCs are presented and discussed

Self-Cleaning Membranes Based on Thermoresponsive Double Network Hydrogels

By providing continuous glucose monitoring, a subcutaneously implanted glucose sensor would greatly improve the quality of life for diabetics. However, implantation of a sensor triggers the host response in which proteins and cells attach and accumulate onto the sensor membrane surface. This membrane biofouling severely limits sensor lifetime and accuracy by restricting glucose diffusion. Whereas attempts to reduce membrane biofouling have mostly relied on passivation approaches, we have designed “self-cleaning” membranes whose surfaces actively detach adhered proteins and cells upon thermal cycling. Thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) single network (SN) hydrogels deswell and reswell, respectively, when heated above and cooled below their volume phase transition temperature (VPTT). A self-cleaning PNIPAAm membrane would ideally be typically swollen (OFF-state) to facilitate glucose diffusion to the embedded sensor or sensing material. However, when transdermally heated above the VPTT, the membrane would reversibly switch to the deswollen state. This cyclical process would cause the active detachment of proteins and cells, thereby cleaning the surface to restore glucose diffusion. Double network (DN) designs, based on asymmetrically crosslinked, interpenetrating PNIPAAm networks, as well as considerations of membrane geometry and size were utilized to achieve the functional requirements of a self-cleaning membrane. This research was comprised of four major studies. In the first study, thermoresponsive PNIPAAm DN nanocomposite hydrogels containing inorganic polysiloxane nanoparticles were prepared. Inorganic, hydrophobic polysiloxane nanoparticles (~50 nm and ~200 nm average diameters) were introduced during formation of the 1st or 2nd network of the PNIPAAm DN hydrogel. In the second study, thermoresponsive PNIPAAm DN hydrogels were prepared with an electrostatic comonomer (2-acrylamido-2-methylpropanesulfonic acid, AMPS). The negatively charged AMPS was incorporated at varying levels (0-75 wt% based on NIPAAm weight) during formation of the 1st network but was excluded from the 2nd network to retain the thermoresponsive behavior. In the third study, the combined impact of a PNIPAAm DN design and reduction of hydrogel size to the micron-scale on thermosensitivity and cell release efficacy was evaluated. PNIPAAm SN and DN hydrogels were prepared as 1.5 mm-thick planar slabs as well as micropillar arrays. The final aspect of this work was focused on evaluating the charged membrane design in terms of functional requirements essential to a final implanted glucose biosensor. This study paralleled previous efforts to likewise characterize a particular DN nanocomposite membrane