Regulation of Cell Behavior at the Cell-Surface Interface

The growth and morphology of fibroblasts cultured on a physically and chemically modified surface was investigated. The need to understand cellular relationships with surface topography and chemistry is essential in the fields of biomedical engineering and biotechnology. It is well documented that mammalian cell behavior senses and responds to the surrounding micro- and nano- scale environment, but the research defining the chemistry, surface architecture, and material properties for control of this behavior is still in its infancy. The cell response to a substrate is complex, involving membrane proteins, extracellular matrix (ECM), cytoskeletal rearrangement, and changes in gene expression. Conventional cell culture is carried out on two-dimensional (2-D) cell culture platforms, such as polystyrene (PS) or glass, and forces cell behavior to adapt and adhere to an unnatural, planar environment. The biological behavior of these cells is used as a starting point for drug screening, implant design, and metabolic processes, but this is a misrepresentation of cells in their native environment. This discrepancy may be hampering biological research or initiating experimental efforts that are invalid. This body of work seeks to address these issues and contains established protocols for inexpensive, pseudo three-dimensional (3-D) culture scaffolds. The research described offers a multi-disciplinary approach for fabrication of biomaterials to achieve user defined or in vivo cell behavior using human fibroblasts. To provide insight into the design of alternative cell culture templates we have analyzed cell-surface interactions and characterized the surface properties. The substrates fabricated utilized micro-roughened surface topography with 2 – 6 µm wide features and surface chemistry as a method for controlling cell behavior. Surface roughness was templated onto polydimethylsiloxane (PDMS) and PS. The fabricated polymer surfaces have been characterized by atomic force microscopy (AFM), contact angle goniometry, fluorescence microscopy, and infrared (IR) spectroscopy. Initial studies of the textured surface yielded a super-hydrophobic surface with a 154° contact angle and high surface adhesion that was investigated using surface free energy calculations. This was followed by modification of the micro-roughness with self-assembled monolayers (SAMs), proteins, or thin films of polymer for use as a culture platform for cells. Cell behavior on the modified polymers was compared and analyzed against unmodified surfaces and tissue culture PS dishes. Cell morphology on rough PDMS surface was altered by the surface topography decreasing the average cell area to 1760 µm2 compared to an average cell area of 3410 µm2 on smooth PDMS. Gene expression changes were also noted with a 2.3 fold increase in the matrix metalloproteinase, MMP14, in cells on the rough surface compared to cells cultured on Petri dishes. Surface roughness was also combined with other surface modification methods for cell culture, including cell alignment and cell sheet engineering. 50 µm wide lines of fibronectin (FN) patterned on the rough PDMS induced cell directionality while still maintaining a pseudo 3-D culture system creating the first cell culture surface of its kind. The micro-roughness was also templated onto PS and chemically modified with a thermo-responsive polymer. This novel surface produced confluent cell sheets that detached from the surface when cooled below 32°C. Cell sheets cultured on the modified PS surfaces had an increase in FN fibril formation stimulated by the surface roughness when compared to cell sheets detached from a smooth, control surface. The minor alterations to surface topology were proven to be effective in modifying cell biochemical response compared to cells cultured on flat substrates. Differences in surface topography and chemistry stimulated changes in cell adhesion, cytoskeletal arrangement, ECM composition, and gene expression. These cell properties were used as markers for comparison to native cell systems and other reports of 3 D culture scaffolds. The mechanism of altering cell response is discussed in each chapter with respect to the specific type of surface used and compared to cell response and behavior on planar culture systems. New fabrication procedures are described that include the incorporation of other surface modification techniques such as SAMs, surface patterning, and thermo-responsive polymer grafting with surface roughness for original cell culture platforms to mimic an in vivo environment. The research presented here demonstrates that micro- and nano- changes to surface topography have large impacts on the cell-surface relationship which have important implications for research and medical applications involving adherent cells

Chapter Fabrication Methodologies of Biomimetic and Bioactive Scaffolds for Tissue Engineering Applications

Tissue engineering has offered wide technologies for developing functional biomaterials substitutes for repair and regeneration of damaged tissue and organs. Biomimetic materials with their inherent nature to mimic natural materials are possible through the recent advances in the fabrication technology. With the help of porous or dense implants made with biodegradable materials, it is possible to incorporate different vital growth factors, genes, drugs, stem cells and proteins. In this review, we presented various fabrication methodologies of biomimetic and bioactive scaffolds for tissue engineering applications. An overview of the nanocomposites of biomaterials is presented. Further an example of one of the hybrid nanocomposite material is given for additive manufacturing

Towards the design of 3D multiscale instructive tissue engineering constructs: Current approaches and trends

The design of 3D constructs with adequate properties to instruct and guide cells both in vitro and in vivo is one of the major focuses of tissue engineering. Successful tissue regeneration depends on the favorable crosstalk between the supporting structure, the cells and the host tissue so that a balanced matrix production and degradation is achieved. Herein, the major occurring events and players in normal and regenerative tissue are overviewed. These have been inspiring the selection or synthesis of instructive cues to include into the 3D constructs. We further highlight the importance of a multiscale perception of the range of features that can be included on the biomimetic structures. Lastly, we focus on the current and developing tissue-engineering approaches for the preparation of such 3D constructs: top-down, bottom-up and integrative. Bottom-up and integrative approaches present a higher potential for the design of tissue engineering devices with multiscale features and higher biochemichal control than top-down strategies, and are the main focus of this review.The research leading to these results has received funding from the European Research Council grant agreement ERC-2012-ADG-20120216-321266 for the project ComplexiTE. Portuguese Foundation for Science and Technology is gratefully acknowledged for the fellowship of Sara M. Oliveira (SFRH/BD/70107/2010)

Design of Metallic Nanostructures for Wavelength and Angle Selective Light Management.

Sub-wavelength metal nanoparticles demonstrate a resonant coupling to incident optical fields known as the localized surface plasmon resonance, enabling enhanced absorption, scattering, and nano-focusing of light. In this work, plasmonic properties of metal nanoparticles and nanorods are studied and engineered to realize selective management of incident light as a function of wavelength, angle, and polarization, for application to photovoltaics and selectively transmissive / absorptive systems. For photovoltaics (PV) applications, metal nanoparticle scattering is exploited to realize a wavelength selective backscattering layer. Placed behind a thin film PV absorbing layer, an array of silver nanoparticles backscatters light on resonance while off-resonance light is transmitted, allowing engineering of selective transparency vs. absorption and modulation of photocurrent. Further tuning the array by considering anisotropic particle shape (increasing the aspect ratio), the plasmonic resonance becomes a function of both wavelength and incident angle. We propose employing such a nanorod array to realize an angle selective photovoltaic window for building integration: light normal to the window is off resonance, retaining high transmission and window quality visibility, while angled light, including direct sunlight, is resonantly scattered and harvested for conversion to photocurrent. Optical analysis indicates 20 - 30% improvement in direct sunlight absorption and photocurrent is possible without sacrificing window transparency in the viewing direction. Beyond photovoltaics, we consider integrating angle selective metal nanorods with actuating micro-origami structures to control their orientation with respect to incident light. By tuning the plasmonic and angular properties of the system, we propose a novel method to realize balanced 0 - 90+% transmission modulation of the full visible spectrum for application to adjustable smart glass window coatings, potentially significantly improving on current implementations. Large area patterning of deeply sub-wavelength (10's of nm) metal nanorods remains a challenge for traditional nanofabrication techniques. We investigate and describe ways to realize the structures of interest based on the electrochemical synthesis of high aspect ratio self-assembled nanoporous anodized aluminum oxide (AAO) films, including both bottom-up (electroplating) and top-down (reactive ion etching) approaches. Finally, the anisotropic and angle dependent scattering properties of high aspect ratio AAO itself are considered for similar light management applications.PHDElectrical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113523/1/brobrts_1.pd

Production and in vitro evaluation of macroporous, cell-encapsulating alginate fibres for nerve repair

The prospects for successful peripheral nerve repair using fibre guides are considered to be enhanced by the use of a scaffold material, which promotes attachment and proliferation of glial cells and axonal regeneration. Macroporous alginate fibres were produced by extraction of gelatin particle porogens from wet spun fibres produced using a suspension of gelatin particles in 1.5% w/v alginate solution. Gelatin loading of the starting suspension of 40.0, 57.0, and 62.5% w/w resulted in gelatin loading of the dried alginate fibres of 16, 21, and 24% w/w respectively. Between 45 and 60% of the gelatin content of hydrated fibres was released in 1 h in distilled water at 37 °C, leading to rapid formation of a macroporous structure. Confocal laser scanning microscopy (CLSM) and image processing provided qualitative and quantitative analysis of mean equivalent macropore diameter (48–69 μm), pore size distribution, estimates of maximum porosity (14.6%) and pore connectivity. CLSM also revealed that gelatin residues lined the macropore cavities and infiltrated into the body of the alginate scaffolds, thus, providing cell adhesion molecules, which are potentially advantageous for promoting growth of glial cells and axonal extension. Macroporous alginate fibres encapsulating nerve cells [primary rat dorsal root ganglia (DRGs)] were produced by wet spinning alginate solution containing dispersed gelatin particles and DRGs. Marked outgrowth was evident over a distance of 150 μm at day 11 in cell culture, indicating that pores and channels created within the alginate hydrogel were providing a favourable environment for neurite development. These findings indicate that macroporous alginate fibres encapsulating nerve cells may provide the basis of a useful strategy for nerve repair

INTEGRATION AND CHARACTERIZATION OF TOBACCO MOSAIC VIRUS BASED NANOSTRUCTURED MATERIALS IN THREE-DIMENSIONAL MICROBATTERY ARCHITECTURES

The realization of next-generation portable electronics, medical implants and miniaturized, autonomous microsystems is directly linked with the development of compact and efficient power sources and energy storage devices with high energy and power density. As the components of these devices are continuously scaled down in size, there is a growing demand for decreasing the size of their power supply as well, while maintaining performance comparable to larger assemblies. This dissertation presents a novel approach for the development of microbattery electrodes that is based on integrating both micro and nano structured components for the formation of hierarchical electrodes. These electrodes combine both high energy density (enabled by the high surface area and mass loading) with high power density (due to the small thickness of the active battery materials). The key building block technologies in this work are the bottom-up self-assembly and metallization of a biological template and the top-down microfabrication processes enabled by Microelectromechanical Systems (MEMS) technology. The biotemplate used is the Tobacco mosaic virus (TMV), a rod-like particle that can be genetically modified to express functional groups with enhanced metal binding properties. In this project, this feature is combined with standard microfabrication techniques for the synthesis of nanostructured energy-related materials as well as their hierarchical patterning in device architectures. Specifically, synthesis of anode (TiO2) and cathode (V2O5) materials for Li-ion batteries in a core/shell configuration is presented, where the TMV biomineralization is combined with atomic layer deposition of the active material. These nanostructured electrodes demonstrate high energy storage capacities, high rate capabilities and superior performance to electrodes with planar geometries. In addition, a toolbox of biofabrication processes for the defined patterning of virus-templated structures has been developed. Finally, the nanocomposite electrodes are integrated with three-dimensional micropillars to form hierarchical electrodes that maintain the high rate performance capabilities of nanomaterials while exhibiting an increase in energy density compared to nanostructures alone. This is in accordance with the increase in surface area added by the microstructures. Investigation of capacity scaling for varying active material thickness reveals underlying limitations in nanostructured electrodes and highlights the importance of this method in controlling both energy and power density with structural hierarchy. These results present a paradigm-shifting technology for the fabrication of next-generation microbatteries for MEMS and microsystems applications

DEVELOPMENT OF PROTEIN-IMPRINTED POLYSILOXANE BIOMATERIALS: PROTEIN SELECTIVITY AND CELLULAR RESPONSES

Surface modification is an extensively researched approach in order to overcomethe limitations, and improve the performance of orthopedic and dental implants. It is atthe surface of the implant materials that the initial interactions of tissues or body fluidstake place. Therefore, surface properties of biomaterials are the important factors that cancontrol these biological responses. Molecular imprinting is a surface modificationtechnique that creates specific recognition sites on the surface of biomaterials. Todevelop the recognition sites, a functional monomer is assembled with templatebiomolecule and then crosslinked. After removal of the template, the surface can rebindthe molecules. Therefore, desired reactions can be initiated at the interface between tissueand implants by modifying surfaces to selectively bind certain types of biomolecules,such as proteins. The objective of this project was to observe the potential of molecularimprinting technique for creating biomaterials that can recognize specific biomolecules.Fluorescently labeled lysozyme or RNase A was used as a template biomolecule and theprotein-imprinted scaffolds were fabricated by sol-gel processing. To interpret the densityof binding sites created, the quantity of surface-accessible protein was determined. Theamount of protein available on the surface was proportional to the amount loaded.Protein-imprinted scaffolds were evaluated for their ability to selectively recognize thetemplate biomolecule. Further, for these selectivity studies, a combination of theimprinted protein and a competitor protein were rebound to the polysiloxane scaffolds.The template protein rebound to the surface was measured more than twice as much ascompetitor. These scaffolds were then tested to understand their interaction with cells.The results of DNA and alkaline phosphatase activities indicate that the scaffolds thusdeveloped support growth and adhesion of osteoblastic cells. These initial selectivity andcytocompatibility studies show the potential of molecular-imprinted polysiloxanescaffolds to be used as tissue engineered materials for stable and controlled interactions atthe tissue-implant interface

Fabrication and plasma modification of nanofibrous tissue engineering scaffolds

This paper provides a comprehensive overview of nanofibrous structures for tissue engineering purposes and the role of non-thermal plasma technology (NTP) within this field. Special attention is first given to nanofiber fabrication strategies, including thermally-induced phase separation, molecular self-assembly, and electrospinning, highlighting their strengths, weaknesses, and potentials. The review then continues to discuss the biodegradable polyesters typically employed for nanofiber fabrication, while the primary focus lies on their applicability and limitations. From thereon, the reader is introduced to the concept of NTP and its application in plasma-assisted surface modification of nanofibrous scaffolds. The final part of the review discusses the available literature on NTP-modified nanofibers looking at the impact of plasma activation and polymerization treatments on nanofiber wettability, surface chemistry, cell adhesion/proliferation and protein grafting. As such, this review provides a complete introduction into NTP-modified nanofibers, while aiming to address the current unexplored potentials left within the field

Analysis of Attachment, Proliferation and Maturation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells on Specific Substrata

Most severe degenerative diseases of retina are often due to malfunctions of retinal pigment epithelium (RPE). Absence of effective treatments has led to development of cell-biomaterial constructs with the aim of creating RPE equivalents for transplantation. Presently, the poor biocompatibility of allologous and xenologous culture substrata in addition with limited amount of source tissue poses the major issues. Well-defined synthetic substrata together with utilization of human embryonic stem cell-derived RPE cells (hESC RPE) are suggested to be potential solutions. In addition, need exists for an effective method to determine the developmental status of cells during the culturing period. This need could be addressed with automated image analysis. The aim of this thesis was to examine the capability of a few specific cell culture substrata to enable attachment, proliferation and maturation of hESC RPE cells. Study included total of 17 xeno-free synthetic materials including 12 BioMaDe Gelators, Purecoat amine and carboxyl, poly(D,L-lactic-co-glycolic acid) (75:25), poly(D,L-lactic acid) (96:4) and poly(L-lactic acid-co-?-caprolactone) (70:30). In addition five materials with natural-origin were studied including chitosan, type I collagen, Matrigel and Substrate X. Type IV collagen was used as control. Growth and maturation were monitored by taking images with specific time intervals. At the end point cellular developmental status was determined by assessing the expression of maturation specific mRNAs by PCR techniques and proteins by immunofluorescence microscopy. In addition, images were used to determine the potential of ImageJ-software as user-friendly image analysis tool for RPE cell analysis. Study demonstrated poor attachment and cell survival on every xeno-free synthetic substrate with cells retaining their initial developmental phase throughout the culturing period, which was supported by gene expression analysis. On the contrary, cells on natural materials attached and proliferated readily. Maturity was further confirmed with immunofluorescence labeling. Image analysis with ImageJ, in turn, confronted many problems mainly arising from heterogeneity of the images. As a conclusion, xeno-free synthetic materials tested in this study show low potential as RPE cell substrata. However, means to enhance their performance are suggested. Despite the good results obtained with natural materials, their ill-defined structure prone to alterations in physiological conditions remains an obstacle for entering clinical experiments. Further experiments should concentrate on combining the strengths of both approaches, that is, incorporation of attachment-related functional groups into well-defined xeno-free synthetic body. In order to increase image homogeneity imaging conditions should be more carefully considered. This way the benefits of automated image analysis could be more effectively exploited. /Kir1