Microscale Strategies for Generating Cell-Encapsulating Hydrogels

Hydrogels in which cells are encapsulated are of great potential interest for tissue engineering applications. These gels provide a structure inside which cells can spread and proliferate. Such structures benefit from controlled microarchitectures that can affect the behavior of the enclosed cells. Microfabrication-based techniques are emerging as powerful approaches to generate such cell-encapsulating hydrogel structures. In this paper we introduce common hydrogels and their crosslinking methods and review the latest microscale approaches for generation of cell containing gel particles. We specifically focus on microfluidics-based methods and on techniques such as micromolding and electrospinning.National Science Foundation (U.S.) (DMR0847287)National Institutes of Health (U.S.) (EB008392)National Institutes of Health (U.S.) (DE019024)National Institutes of Health (U.S.) (HL099073)National Institutes of Health (U.S.) (AR057837)National Institutes of Health (U.S.) (HL092836)United States. Army Research Office (contract W911NF-07-D-0004)United States. Army Research Office (Institute for Soldier Nanotechnology)United States. Army. Corps of EngineersInnovative Med Tech (Postdoctoral fellowship

Microfabrication of spatially-patterned, polymer scaffolds for applications in stem cell and tissue engineering

textTissue engineering is a recently developed field that combines material science, cell biology, and engineering to create or improve functional tissues/organs. The field of tissue engineering has progressed from a fledgling science to an emerging technology, in large part due to parallel advances in the application of biomaterials and understanding stem cell behavior. Current studies have evaluated certain types of natural and synthetic biomaterials for feasibility of replicating the physio-chemical microenvironments of stem cells. Furthermore, technologies derived from micro-machining and solid free-form fabrication industries have utilized these biomaterials to create scaffolds that resemble tissue-like structures. Recent scaffold fabrication methods have attempted to overcome certain challenges in engineering tissues and organs. One of the fundamental limitations in current tissue engineering efforts has been the inability to develop multiple tissue types (i.e. bone, cartilage, muscles, ligaments) within a single scaffold structure in a predesigned manner. The differentiation of multiple cells within a three-dimensional (3D) scaffold using a single stem cell population has yet to be developed due to challenges in integrating various biochemical factors in a spatially-patterned method. This dissertation discusses scaffold micro-fabrication techniques that use layerby-layer, ultraviolet-based (UV) stereolithography systems. These approaches in microfabricating scaffolds provide an optimal, biomimetic environment for the pre-patterned differentiation of mesenchymal stem cells into skeletal-type tissues. We demonstrated both laser-based and digital micromirror device-based stereolithography systems for creating intricate scaffold architectures with multiple bio-factors encapsulated in predetermined regions. We showed that micro-stereolithography has the powerful capability of building 3D complex scaffolds with specific pore sizes and shapes in a layer-by-layer fashion using photo-crosslinkable monomers. These polymer-based scaffolds were functionalized with specific signaling proteins to create a biomimetic niche in which stem cells can respond, attach, and differentiate. The ultimate goal of this project is to integrate novel concepts of micro-manufacturing along with polymer-controlled release kinetics and stem cell biology to attain pre-designed architectures of tissue structures.Biomedical Engineerin

Fabrication of 3D cell-laden hydrogel microstructures through photo-mold patterning

Native tissues are characterized by spatially organized three-dimensional (3D) microscaled units which functionally define cells–cells and cells–extracellular matrix interactions. The ability to engineer biomimetic constructs mimicking these 3D microarchitectures is subject to the control over cell distribution and organization. In the present study we introduce a novel protocol to generate 3D cell laden hydrogel micropatterns with defined size and shape. The method, named photo-mold patterning (PMP), combines hydrogel micromolding within polydimethylsiloxane (PDMS) stamps and photopolymerization through a recently introduced biocompatible ultraviolet (UVA) activated photoinitiator (VA-086). Exploiting PDMS micromolds as geometrical constraints for two methacrylated prepolymers (polyethylene glycol diacrylate and gelatin methacrylate), micrometrically resolved structures were obtained within a 3 min exposure to a low cost and commercially available UVA LED. The PMP was validated both on a continuous cell line (human umbilical vein endothelial cells expressing green fluorescent protein, HUVEC GFP) and on primary human bone marrow stromal cells (BMSCs). HUVEC GFP and BMSCs were exposed to 1.5% w/v VA-086 and UVA light (1 W, 385 nm, distance from sample = 5 cm). Photocrosslinking conditions applied during the PMP did not negatively affect cells viability or specific metabolic activity. Quantitative analyses demonstrated the potentiality of PMP to uniformly embed viable cells within 3D microgels, creating biocompatible and favorable environments for cell proliferation and spreading during a seven days' culture. PMP can thus be considered as a promising and cost effective tool for designing spatially accurate in vitro models and, in perspective, functional constructs

Microstructure Bio-Material for Behavioral Analysis

Biological applications have a limitation of creating tissue like structures in order to mimic the advanced real like structures, such as human tissues in a small scale. Conventional methods of using lab mice for cancer behavior have limitations due to observation complications. Fabricating an artificial human tissue which can behave similar to a human body tissue consists of components, such as Laminin and Collagen. Collagen in human tissue has elements, such as integrin and serum. Creating serum based proteins are somewhat challenging due to the conditional requirements. This particular approach will address the primary state of the art technique of observing the interaction with cells by mimicking the organs on a chip with blood circulation using a micro-fluidic pump. Bio-material hydrogel structures implanted on a silicon polymer based chip described in this thesis will overcome the limitations of in-vitro analysis. Water purification has become a vital issue in developing countries of the world. Water pollution due to Ammonia has been one of the major concerns with industrial revolution. Purifications were mainly done by chemical methods that can cause human health concerns. The analytically demonstrated method in this thesis using bio-compatible hydrogel will address a new dimension to the water conservation method without causing health issues and eliminating the environmental pollution due to complicated degradable structures. Filtration and efficiency are among the main concerns of using bacteria types such as AOB/NOB directly without encapsulating. Application of using bio-compatible hydrogel based dual encapsulated single pallet structure described in this thesis will address the issue of filtering capability. Pallets can be removed once nitrified, without letting it grow inside the water contaminating aqua based living breads and plants. The process will improve the efficiency of Ammonia removal due to encapsulation. Drug delivery using micro locomotives in neuro-surgery has become one of the future concerns with the development of science. Conventional delivery systems such as vaccines and open surgeries take longer response time once surgeries become more complex. Moreover there is a risk factor of injuring healthy nerves in the organ. Drug delivery approaches of drug encapsulated microspheres and drug embedded nematodes described in this thesis become more applicable to complex scenarios. Nematodes become useful in the future of microsurgeries, as many biologists are focusing on using their healthy nerves to implant in humans. Therefore, such applications like magnetizing nematodes help move locomotives to targeted locations and capture scan images for future medical approaches

4D Printing at the Microscale

3D printing of adaptive and dynamic structures, also known as 4D printing, is one of the key challenges in contemporary materials science. The additional dimension refers to the ability of 3D printed structures to change their properties—for example, shape—over time in a controlled fashion as the result of external stimulation. Within the last years, significant efforts have been undertaken in the development of new responsive materials for printing at the macroscale. However, 4D printing at the microscale is still in its early stages. Thus, this progress report will focus on emerging materials for 4D printing at the microscale as well as their challenges and potential applications. Hydrogels and liquid crystalline and composite materials have been identified as the main classes of materials representing the state of the art of the growing field. For each type of material, the challenges and critical barriers in the material design and their performance in 4D microprinting are discussed. Importantly, further necessary strategies are proposed to overcome the limitations of the current approaches and move toward their application in fields such as biomedicine, microrobotics, or optics

Multicomponent patterning of nanocomposite polymer and nanoparticle films using photolithography and layer-by-layer self -assembly

In this dissertation, the fabrication, characterization, and application examples of 3D multicomponent nanocomposite micropatterns (MNMs) with precise spatial arrangements are described. The ability to produce such small-scale 3D structures with versatility in composition and structure is a new development based on the integration of nanoscale layer-by-layer (LbL) self-assembly and microscale photolithographic patterning, enabling construction of surfaces with microscale patterns that possess nanotopographies. The techniques used here are analogous to surface micromachining, except that the deposition materials are polymers, biological materials, and colloidal nanoparticles used to produce 3D MNMs. A key feature of the resulting 3D MNMs is that the physical and chemical properties of the multilayer nanofilms may be finely tuned using the versatile LbL assembly process, which makes them attractive for many applications requiring polymeric structures with small features. The work presented here involves development of techniques for the fabrication, characterization, and applications of 3D MNMs, and evaluation of the process parameters involved in the developed techniques. These results clearly demonstrate the feasibility of the polymer 3D MNMs for biotechnological applications; specifically, they have potential as tailored surfaces for direct comparison of cell-material interactions on a single substrate, and for co-culture systems. In reality, the approach described here may enable study of and control over cell-biomaterial and cell-cell interactions in a whole new fashion. The techniques developed in this work represent a major advancement of nanoscale engineering through the integration of nanoscale LbL self-assembly and microscale photolithographic patterning for constructing 3D MNMs with varying physical and chemical properties in precise spatial arrangements. A major finding of this work, related to the applicability of the developed techniques, is that most of the seemingly harsh processes involved in constructing the 3D MNMs have minimal or no deleterious effects on the biological models used here. The exception is the resist developer (MF319), which due to its highly basic nature, results in disintegration of nanofilms exposed to it directly. Nevertheless, the methods developed here are not limited by the photoresists and resist developers used here; biocompatible photoresists and aqueous base developers could potentially be used. This work has pursued the development of organic and inorganic nanofilm scaffolds which can eventually be combined to achieve functionality desired for specific applications. It is anticipated that the 3D MNMs developed in this work will provide general platforms for studying biological processes, which will not only impact stem cell research in general but also provide useful information in support of biomedical device development, and tissue engineering. Although the intended purpose for developing 3D MNMs is to produce novel bioactive systems, their applicability is more general and may find use in a broad range of applications including electronics, photonics, optoelectronics, and chemical and biochemical sensors

NANOSCALE FUNCTIONALIZATION AND CHARACTERIZATION OF SURFACES WITH HYDROGEL PATTERNS AND BIOMOLECULES

The advent of numerous tools, ease of techniques, and concepts related to nanotechnology, in combination with functionalization via simple chemistry has made gold important for various biomedical applications. In this dissertation, the development and characterization of planar gold surfaces with responsive hydrogel patterns for rapid point of care sensing and the functionalization of gold nanoparticles for drug delivery are highlighted. Biomedical micro- and nanoscale devices that are spatially functionalized with intelligent hydrogels are typically fabricated using conventional UV-lithography. Herein, precise 3-D hydrogel patterns made up of temperature responsive crosslinked poly(N-isopropylacrylamide) over gold were synthesized. The XY control of the hydrogel was achieved using microcontact printing, while thickness control was achieved using atom transfer radical polymerization (ATRP). Atomic force microscopy analysis showed that to the ATRP reaction time governed the pattern growth. The temperature dependent swelling ratio was tailored by tuning the mesh size of the hydrogel. While nanopatterns exhibited a broad lower critical solution temperature (LCST) transition, surface roughness showed a sharp LCST transition. Quartz crystal microbalance with dissipation showed rapid response behavior of the thin films, which makes them applicable as functional components in biomedical devices. The easy synthesis, relative biocompatibility, inertness, and easy functionalization of gold nanoparticles (GNPs) have made them useful for various biomedical applications. Although ATRP can be successfully carried out over GNPs, the yield of stable solution based GNPs for biomedical applications prove to be low. As an alternative approach, a novel method of ISOlating, FUnctionalizing, and REleasing nanoparticles (ISOFURE) was proposed. Biodegradable poly(β-amino ester) hydrogels were used to synthesize ISOFURE-GNP composites. ATRP was performed inside the composite, and the final hydrogel coated GNPs were released via matrix degradation. Response analysis confirmed that the ISOFURE method led to the increased stability and yield of the hydrogel coated ISOFURE-GNPs. The ISOFURE protocol was also utilized in functionalizing GNPs with enzyme catalase in the absence of a stabilizing reagent. Biotin-streptavidin affinity was used as the bioconjugation method. Activity analysis of the conjugated enzyme showed that the ISOFURE-GNPs showed enhanced biomolecular loading relative to solution based stabilizing reagent passivated GNPs

Role of polymers in microfluidic devices

Polymers are sustainable and renewable materials that are in high demand due to their excellent properties. Natural and synthetic polymers with high flexibility, good biocompatibility, good degradation rate, and stiffness are widely used for various applications, such as tissue engineering, drug delivery, and microfluidic chip fabrication. Indeed, recent advances in microfluidic technology allow the fabrication of polymeric matrix to construct microfluidic scaffolds for tissue engineering and to set up a well-controlled microenvironment for manipulating fluids and particles. In this review, polymers as materials for the fabrication of microfluidic chips have been highlighted. Successful models exploiting polymers in microfluidic devices to generate uniform particles as drug vehicles or artificial cells have been also discussed. Additionally, using polymers as bioink for 3D printing or as a matrix to functionalize the sensing surface in microfluidic devices has also been mentioned. The rapid progress made in the combination of polymers and microfluidics presents a low-cost, reproducible, and scalable approach for a promising future in the manufacturing of biomimetic scaffolds for tissue engineering

InVERT molding for scalable control of tissue microarchitecture

Complex tissues contain multiple cell types that are hierarchically organized within morphologically and functionally distinct compartments. Construction of engineered tissues with optimized tissue architecture has been limited by tissue fabrication techniques, which do not enable versatile microscale organization of multiple cell types in tissues of size adequate for physiological studies and tissue therapies. Here we present an ‘Intaglio-Void/Embed-Relief Topographic molding’ method for microscale organization of many cell types, including induced pluripotent stem cell-derived progeny, within a variety of synthetic and natural extracellular matrices and across tissues of sizes appropriate for in vitro, pre-clinical, and clinical studies. We demonstrate that compartmental placement of non-parenchymal cells relative to primary or induced pluripotent stem cell-derived hepatocytes, compartment microstructure, and cellular composition modulate hepatic functions. Configurations found to sustain physiological function in vitro also result in survival and function in mice for at least 4 weeks, demonstrating the importance of architectural optimization before implantation.National Institutes of Health (U.S.) (EB008396)National Institutes of Health (U.S.) (DK56966)National Cancer Institute (U.S.) (Cancer Center Support Core Grant P30-CA14051)National Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (1F32DK091007)National Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (1F32DK095529)National Science Foundation (U.S.). Graduate Research Fellowship Program (1122374

A contactless electrical stimulator: application to fabricate functional skeletal muscle tissue

Engineered skeletal muscle tissues are ideal candidates for applications in drug screening systems, bio-actuators, and as implantable constructs in tissue engineering. Electrical field stimulation considerably improves the differentiation of muscle cells to muscle myofibers. Currently used electrical stimulators often use direct contact of electrodes with tissue constructs or their culture medium, which may cause hydrolysis of the culture medium, joule heating of the medium, contamination of the culture medium due to products of electrodes corrosion, and surface fouling of electrodes. Here, we used an interdigitated array of electrodes combined with an isolator coverslip as a contactless platform to electrically stimulate engineered muscle tissue, which eliminates the aforementioned problems. The effective stimulation of muscle myofibers using this device was demonstrated in terms of contractile activity and higher maturation as compared to muscle tissues without applying the electrical field. Due to the wide array of potential applications of electrical stimulation to two- and three-dimensional (2D and 3D) cell and tissue constructs, this device could be of great interest for a variety of biological applications as a tool to create noninvasive, safe, and highly reproducible electric fields.World Premier International Research Center Initiative (WPI