Assessment of the biocompatibility, stability, and suitability of novel thermoresponsive films for the rapid generation of cellular constructs

Stimuli responsive polymers (SRP) are of great interest in the bioengineering community due to their use in applications such as drug delivery and tissue engineering. One example of an SRP is poly(N-isopropyl acrylamide) or pNIPAM. This SRP has the capability of changing its conformation with a slight temperature change: adherent mammalian cells spontaneously release as a confluent cell sheet, which can be harvested for cell sheet engineering purposes. Since its initial use in 1968, many researchers have used pNIPAM to obtain a cell sheet composed of their cell type of interest. The differing protocols used for these diverse cell types, such as the conditions used for cell detachment, and the varying methods used for derivatizing substrates with pNIPAM have all led to conflicting reports on the utility of pNIPAM for cell sheet engineering purposes, as well as the relative cytotoxicity of the polymer. In this work, some of the key inconsistencies in the literature and previously unaddressed challenges when utilizing pNIPAM films are overcome for the purpose of rapid generation of cellular constructs, specifically spheroids. Pertinent characteristics of low temperature detachment are investigated for their effect on the kinetics of cell detachment. In addition, a novel, inexpensive method for obtaining pNIPAM films for mammalian cell detachment, combining pNIPAM with a sol-gel, was optimized and compared to plasma polymerization deposition. Furthermore, proper storage conditions (e.g. temperature and relative humidity) for these films were investigated to increase stability of the films for using tissue culture conditions. To increase the speed of generation of cell sheets, electrospun mats and hydrogels with a high surface area-to-volume ratio were developed. The result is a platform appropriate for the rapid formation of cellular constructs, such as engineered tissues and spheroids for cancer cell research

Biomechanics of aligned cell sheets for arterial tissue engineering

Cardiovascular disease remains the leading cause of death in the United States and oftentimes damaged or occluded arteries need to be replaced. Current surgical materials are unsuitable for small diameter, high pressure vessels such as the carotid and coronary artery because they cannot match the mechanical properties of native tissue. Compliance mismatch between the two materials leads to complications such as anastomotic intimal hyperplasia, thrombosis, and aneurysm. Therefore there is significant clinical need for a tissue engineered arterial graft suitable for small diameter, high pressure vessels. An artery is composed of three layers, but the middle layer, the tunica media, is responsible for bearing normal physiological loads. The medial layer features alternating layers of smooth muscle cells and extracellular matrix where each layer has smooth muscle cells and collagen aligned helically along the length of the artery. This structural feature coupled with the composition of the extracellular matrix cause arterial tissue to have a non-linear mechanical response. A tissue engineered vascular graft that recapitulates the native arterial structure may overcome the limitations of current tissue engineered strategies. We hypothesize that a tissue engineered arterial graft can be built by stacking aligned, single layer cell sheets to better mimic native mechanical properties. Cell sheets are confluent layer of cells and extracellular matrix. In the first aim of this work we developed an inexpensive, novel force sensor design capable of measuring small forces (< 1 mN) that we incorporated into a custom-built uniaxial tensile tester. For the second aim we developed a technique for culturing single layer, aligned, bovine vascular smooth cell sheets. We accomplished this by developing a technique for grafting N-isopropylacrylamide, a thermo-responsive polymer, onto the surface of flat and micropatterned poly-dimethylsiloxane (PDMS) substrates. These substrates allow cell sheets to be cultured and then be detached by decreasing the temperature. In the third aim we experimentally characterized the mechanical and structural properties of aligned vascular smooth muscle cell sheets to show that cell orientation can be controlled by the micropattern and that cellular alignment leads mechanical anisotropy. We also successfully modeled the cell sheet mechanical behavior using existing phenomenological models. The results from this work suggest that aligned cell sheets are capable of recapturing the non-linear stress-strain response of native arterial tissue, making them suitable for arterial tissue engineering. The results from this work provide an experimental and computational foundation for future efforts towards building a multi-layered cell sheet based arterial tissue

Active Stimuli-Responsive Polymer Surfaces and Thin Films: Design, Properties and Applications: Active Stimuli-Responsive Polymer Surfaces and Thin Films: Design, Properties and Applications

Design of 2D and 3D micropatterned materials is highly important for printing technology, microfluidics, microanalytics, information storage, microelectronics and biotechnology. Biotechnology deserves particular interest among the diversity of possible applications because its opens perspectives for regeneration of tissues and organs that can considerably improve our life. In fact, biotechnology is in constant need for development of microstructured materials with controlled architecture. Such materials can serve either as scaffolds or as microanalytical platforms, where cells are able to self-organize in a programmed manner. Microstructured materials, for example, allow in vitro investigation of complex cell-cell interactions, interactions between cells and engineered materials. With the help of patterned surfaces it was demonstrated that cell adhesion and viability as well as differentiation of stem cells1 depend of on the character of nano- and micro- structures 2 as well as their size. There are number of methods based on optical lithography, atomic force microscopy, printing techniques, chemical vapor deposition, which have been developed and successfully applied for 2D patterning. While each of these methods provides particular advantages, a general trade-off between spatial resolution, throughput, “biocompatibility of method” and usability of fabricated patterned surfaces exists. For example, AFM-based techniques allow very high nanometer resolution and can be used to place small numbers of functional proteins with nanometer lateral resolution, but are limited to low writing speeds and small pattern sizes. Albeit, the resolution of photolithography is lower, while it is much faster and cheaper. Therefore, it is highly desirable to develop methods for high-resolution patterning at reasonably low cost and high throughput. Although many approaches to fabricate sophisticated surface patterns exist, they are almost entirely limited to producing fixed patterns that cannot be intentionally modified or switched on the fly in physiologic environment. This limits the usability of a patterned surface to a single specific application and new microstructures have to be fabricated for new applications. Therefore, it is desirable to develop methods for design of switchable and rewritable patterns. Next, the high-energy of the ultraviolet radiation, which is typically used for photolithography, can be harmful for biological species. It is also highly important to develop an approach for photopatterning where visible light is used instead of UV light. Therefore, it is very important for biotechnological applications to achieve good resolution at low costs, create surface with switchable and reconfigurable patterns, perform patterning in mild physiologic conditions and avoid use of harmful UV light. 3D patterning is experimentally more complicated than 2D one and the applicability of available techniques is substantially limited. For example, interference photolithography allows fabrication of 3D structures with limited thickness. Two-photon photolithography, which allows nanoscale resolution, is very slow and highly expensive. Assembling of 3D structures by stacking of 2D ones is time consuming and does not allow fabrication of fine hollow structures. At the same time, nature offers an enormous arsenal of ideas for the design of novel materials with superior properties. In particular, self-assembly and self-organization being the driving principles of structure formation in nature attract significant interest as promising concepts for the design of intelligent materials 3. Self-folding films are the examples of biomimetic materials4. Such films mimic movement mechanisms of plants 5-7 and are able to self-organize and form complex 3D structures. The self-folding films consist of two materials with different properties. At least one of these materials, active one, can change its volume. Because of non-equal expansion of the materials, the self-folding films are able to form a tubes, capsules or more complex structure. Similar to origami, the self-folding films provide unique possibilities for the straightforward fabrication of highly complex 3D micro-structures with patterned inner and outer walls that cannot be achieved using other currently available technologies. The self-folded micro-objects can be assembled into sophisticated, hierarchically-organized 3D super-constructs with structural anisotropy and highly complex surface patterns. Till now most of the research in the field of self-folding films was focused on inorganic materials. Due to their rigidity, limited biocompatibility and non-biodegradability, application of inorganic self-folding materials for biomedical purposes is limited. Polymers are more suitable for these purposes. There are many factors, which make polymer-based self-folding films particularly attractive. There is a variety of polymers sensitive to different stimuli that allows design of self-folding films, which are able to fold in response to various external signals. There are many polymers changing their properties in physiological ranges of pH and temperature as well as polymers sensitive to biochemical processes. There is a variety of biocompatible and biodegradable polymers. These properties make self-folding polymer highly attractive for biological applications. Polymers undergo considerable and reversible changes of volume that allows design of systems with reversible folding. Fabrication of 3D structures with the size ranging from hundreds of nanometers to centimeters is possible. In spite of their attractive properties, the polymer-based systems remained almost out of focus – ca 15 papers including own ones were published on this topic (see own review 8, state October 2011). Thereby the development of biomimetic materials based on self-folding polymer films is highly desired and can open new horizons for the design of unique 3D materials with advanced properties for lab-on-chip applications, smart materials for everyday life and regenerative medicine

Encapsulation of particles and cells using stimuli-responsive self-rolling polymer films

This thesis is focused on the design and development of an approach, allowing the fabrication of biocompatible/biodegradable self-rolled polymer tubes, which are sensitive to stimuli at physiological conditions, can be homogenously filled with cells and are able to self-assemble into a complex 3D construct with uniaxially aligned pores. These constructs are aimed to recreate the microstructure of tissues with structural anisotropy, such as of muscles and bones. The approach consists of two steps of self-assembly. As a first step, cells are adsorbed on the top of an unfolded bilayer; triggered rolling results in a parallel encapsulation of cells inside the tubes. As a second step, the formed self-rolled tubes with encapsulated cells can be assembled in a uniaxial tubular scaffold. Three polymer systems were designed and investigated in the present work in order to allow triggered folding of the bilayer. These systems allow either reversible or irreversible tube formation. The possibility to encapsulate microobjects inside self-rolled polymer tubes was demonstrated on the example of silica particles, yeast cells and mammalian cells. At conditions when bilayer film is unfolded, particles or cells were deposited from their aqueous dispersion on the top of bilayer. An appropriate change of conditions triggers folding of the bilayer and results in encapsulation of particles or cells inside the tubes. One way swelling of an active polymer allows irreversible encapsulation of cells in a way that tubes do not unroll and cells cannot escape. It was demonstrated that encapsulated cells can proliferate and divide inside the tubes for a long period of time. Since used polymers are optically transparent, encapsulated cells can be easily observed using optical and fluorescent microscopy. Reversible swelling of an active polymer provides the possibility to release encapsulated objects. It was demonstrated that in aqueous media microtubes possessing small amount of negatively charged groups on external walls self-assemble in the presence of oppositely charged microparticles that results in a formation of 3D constructs. In obtained aggregates tubes and therefore pores were well-aligned and the orientation degree was extremely high. Moreover, the approach allows the design of porous materials with complex architectures formed by tubes of different sorts. The assembly of cell-laden microtubes results in a formation of uniaxial tubular scaffold homogeneously filled with cells. The results presented in this work demonstrate that the proposed approach is of practical interest for biotechnological applications. Self-rolled tubes can be filled with cells during their folding providing the desired homogeneity of filling. Individual tubes of different diameters could be used to investigate cell behaviour in confinement in conditions of structural anisotropy as well as to mimic blood vessels. Due to their directionality tubes could be used to guide the growth of cells that is of interest for regeneration of neuronal tissue. Reversibly foldable films allow triggered capture and release of the cells that could be implemented for controlled cell delivery. In perspective, self-assembled 3D constructs with aligned pores could be used for bottom-up engineering of the scaffolds, mimicking such tissues as cortical bone and skeletal muscle, which are characterized by repeating longitudinal units. Such constructs can be also considered as a good alternative of traditional 2D flat cell culture

Recent Advances in Hybrid Biomimetic Polymer-Based Films: from Assembly to Applications

Biological membranes, in addition to being a cell boundary, can host a variety of proteins that are involved in different biological functions, including selective nutrient transport, signal transduction, inter- and intra-cellular communication, and cell-cell recognition. Due to their extreme complexity, there has been an increasing interest in developing model membrane systems of controlled properties based on combinations of polymers and different biomacromolecules, i.e., polymer-based hybrid films. In this review, we have highlighted recent advances in the development and applications of hybrid biomimetic planar systems based on different polymeric species. We have focused in particular on hybrid films based on (i) polyelectrolytes, (ii) polymer brushes, as well as (iii) tethers and cushions formed from synthetic polymers, and (iv) block copolymers and their combinations with biomacromolecules, such as lipids, proteins, enzymes, biopolymers, and chosen nanoparticles. In this respect, multiple approaches to the synthesis, characterization, and processing of such hybrid films have been presented. The review has further exemplified their bioengineering, biomedical, and environmental applications, in dependence on the composition and properties of the respective hybrids. We believed that this comprehensive review would be of interest to both the specialists in the field of biomimicry as well as persons entering the field

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

Micropatterned cell sheets as structural building blocks for biomimetic vascular patch application

To successfully develop a functional tissue-engineered vascular patch, recapitulating the hierarchical structure of vessel is critical to mimic mechanical properties. Here, we use a cell sheet engineering strategy with micropatterning technique to control structural organization of bovine aortic vascular smooth muscle cell (VSMC) sheets. Actin filament staining and image analysis showed clear cellular alignment of VSMC sheets cultured on patterned substrates. Viability of harvested VSMC sheets was confirmed by Live/Dead® cell viability assay after 24 and 48 hours of transfer. VSMC sheets stacked to generate bilayer VSMC patches exhibited strong inter-layer bonding as shown by lap shear test. Uniaxial tensile testing of monolayer VSMC sheets and bilayer VSMC patches displayed nonlinear, anisotropic stress-stretch response similar to the biomechanical characteristic of a native arterial wall. Collagen content and structure were characterized to determine the effects of patterning and stacking on extracellular matrix of VSMC sheets. Using finite-element modeling to simulate uniaxial tensile testing of bilayer VSMC patches, we found the stress-stretch response of bilayer patterned VSMC patches under uniaxial tension to be predicted using an anisotropic hyperelastic constitutive model. Thus, our cell sheet harvesting system combined with biomechanical modeling is a promising approach to generate building blocks for tissue-engineered vascular patches with structure and mechanical behavior mimicking native tissue

Designing Stimuli-Responsive Nanocomposites to Investigate Interface Dynamics

Inspired by nature, this research focuses on designing multifunctional renewable nanocomposites with high toughness and stimuli-responsiveness. In recent years, cellulose nanocrystals (CNCs) have been explored due to their abundance, renewable resource, and unique mechanical strength and structural coloration. CNCs naturally self-assemble into the helicoidal (Bouligand) structure that effectively endure high impacts but is brittle without an attendant soft phase. A thermoresponsive polymer, poly(diethylene glycol methyl ether methacrylate) (PMEO2MA), was incorporated into CNCs via evaporation-induced self-assembly to improve toughness of the resulting nanocomposites and to study responses in polymer dynamics under varying temperature and humidity conditions. To study microscopic scale responses of PMEO2MA, a water-sensitive dye was implemented to probe the chains dynamics via fluorescence lifetimes. Fluorescence lifetime imaging microscopy (FLIM) generated a fluorescence lifetime distribution within the film that probed the different polymer dynamics. Two-component exponential fitting was needed where τ1 had shorter lifetimes that associated with more hydrated domains while τ2 had longer fluorescence lifetimes likely related to polymer confined to the CNC interfaces. There are three postulated environments the polymer chains were experiencing in the CNCs structure: i) PMEO2MA confined to CNCs via physical absorption, ii) PMEO2MA in-between CNCs structures, iii) PMEO2MA in hydrated regions. We also examined how anchoring PMEO2MARhB to CNCs would affect polymer dynamics and mechanical properties. The grafting-from technique was utilized to covalently bond the polymer chains to CNCs surface to form CNC-g-PMEO2MARhB. Due to one chain-end being restricted to CNCs, the Grafted-CNC films had longer fluorescence lifetimes when compared to their blended PMEO2MARhB-CNC counterpart. The Grafted-CNC films showed improvement in tensile strength and modulus, indicating better stress-transfer in comparison to the blended PMEO2MARhB-CNC. This observation illustrated a correlation between the measured fluorescence lifetime and mechanical properties. From these studies, CNCs nanocomposites can be tuned by environmental stimuli and different chain confinement resulted in optical responses and mechanical performances. Fluorescence lifetime imaging microscopy was co-opted from biological applications to capture sensitive events in nanoseconds at high resolution to experimentally study polymer dynamics. The technique provided insights at the microscopic scale to improve material design for CNCs nanocomposites

The toolbox of porous anodic aluminum oxide–based nanocomposites: from preparation to application

Anodic aluminum oxide (AAO) templates have been intensively investigated during the past decades and have meanwhile been widely applied through both sacrificial and non-sacrificial pathways. In numerous non-sacrificial applications, the AAO membrane is maintained as part of the obtained composite materials; hence, the template structure and topography determine to a great extent the potential applications. Through-hole isotropic AAO features nanochannels that promote transfer of matter, while anisotropic AAO with barrier layer exhibits nanocavities suitable as independent and homogenous containers. By combining the two kinds of AAO membranes with diverse organic and inorganic materials through physical interactions or chemical bonds, AAO composites are designed and applied in versatile fields such as catalysis, drug release platform, separation membrane, optical appliances, sensors, cell culture, energy, and electronic devices. Therefore, within this review, a perspective on exhilarating prospect for complementary advancement on AAO composites both in preparation and application is provided