Adipose-Derived Stem Cells for Tissue Engineering and Regenerative Medicine Applications

Adipose-derived stem cells (ASCs) are a mesenchymal stem cell source with properties of self-renewal and multipotential differentiation. Compared to bone marrow-derived stem cells (BMSCs), ASCs can be derived from more sources and are harvested more easily. Three-dimensional (3D) tissue engineering scaffolds are better able to mimic the in vivo cellular microenvironment, which benefits the localization, attachment, proliferation, and differentiation of ASCs. Therefore, tissue-engineered ASCs are recognized as an attractive substitute for tissue and organ transplantation. In this paper, we review the characteristics of ASCs, as well as the biomaterials and tissue engineering methods used to proliferate and differentiate ASCs in a 3D environment. Clinical applications of tissue-engineered ASCs are also discussed to reveal the potential and feasibility of using tissue-engineered ASCs in regenerative medicine

Embryoid body size-mediated differential endodermal and mesodermal differentiation using polyethylene glycol (PEG) microwell array

Embryoid bodies have a number of similarities with cells in gastrulation, which provides useful biological information about embryonic stem cell differentiation. Extensive research has been done to study the control of embryoid body-mediated embryonic stem cell differentiation in various research fields. Recently, microengineering technology has been used to control the size of embryoid bodies and to direct lineage specific differentiation of embryonic stem cells. However, the underlying biology of developmental events in the embryoid bodies of different sizes has not been well elucidated. In this study, embryoid bodies with different sizes were generated within microfabricated PEG microwell arrays, and a series of gene and molecular expressions related to early developmental events was investigated to further elucidate the size-mediated differentiation. The gene and molecular expression profile suggested preferential visceral endoderm formation in 450 μm embryoid bodies and preferential lateral plate mesoderm formation in 150 μm embryoid bodies. These aggregates resulted in higher cardiac differentiation in 450 μm embryoid bodies and higher endothelial differentiation in 150 μm embryoid bodies, respectively. Our findings may provide further insight for understanding embryoid body size-mediated developmental progress.National Science Foundation (U.S.) (CAREER Award DMR0847287)United States. Office of Naval Research (Naval Research Young National Investigator Award)National Institutes of Health (U.S.) (HL092836, EB02597, AR057837

Carbon-Nanotube-Embedded Hydrogel Sheets for Engineering Cardiac Constructs and Bioactuators

We engineered functional cardiac patches by seeding neonatal rat cardiomyocytes onto carbon nanotube (CNT)-incorporated photo-cross-linkable gelatin methacrylate (GelMA) hydrogels. The resulting cardiac constructs showed excellent mechanical integrity and advanced electrophysiological functions. Specifically, myocardial tissues cultured on 50 μm thick CNT-GelMA showed 3 times higher spontaneous synchronous beating rates and 85% lower excitation threshold, compared to those cultured on pristine GelMA hydrogels. Our results indicate that the electrically conductive and nanofibrous networks formed by CNTs within a porous gelatin framework are the key characteristics of CNT-GelMA leading to improved cardiac cell adhesion, organization, and cell–cell coupling. Centimeter-scale patches were released from glass substrates to form 3D biohybrid actuators, which showed controllable linear cyclic contraction/extension, pumping, and swimming actuations. In addition, we demonstrate for the first time that cardiac tissues cultured on CNT-GelMA resist damage by a model cardiac inhibitor as well as a cytotoxic compound. Therefore, incorporation of CNTs into gelatin, and potentially other biomaterials, could be useful in creating multifunctional cardiac scaffolds for both therapeutic purposes and in vitro studies. These hybrid materials could also be used for neuron and other muscle cells to create tissue constructs with improved organization, electroactivity, and mechanical integrity.United States. Army Research Office. Institute for Soldier NanotechnologiesNational Institutes of Health (U.S.) (HL092836)National Institutes of Health (U.S.) (EB02597)National Institutes of Health (U.S.) (AR057837)National Institutes of Health (U.S.) (HL099073)National Science Foundation (U.S.) (DMR0847287)United States. Office of Naval Research (ONR PECASE Award)United States. Office of Naval Research (Young Investigator award)National Research Foundation of Korea (grant (NRF-2010-220-D00014)

Effects of imperfect bonding on three-phase inclusion-crack interactions

Bibliography: p. 75-81The solution for the elastic three-phase circular inclusion problem plays a fundamental role in many practical and theoretical applications. In particular, it offers the fundamental solution for the generalized self-consistent method in the mechanics of composites materials. In this thesis, a general method is presented for evaluating the interaction between a pre-existing radial matrix crack and a three-phase circular inclusion. The bonding at the inclusion-interphase interface is considered to be imperfect with the assumption that the interface imperfections are constant. On the remaining boundary, that being the interphase-matrix interface, the bonding is considered to be perfect. Using complex variable techniques, we derive series representations for the corresponding stress functions inside the inclusion, in the interphase layer and in the surrounding matrix. The governing boundary value problem is then formulated in such a way that these stress distributions simultaneously satisfy the traction free condition along the crack face, the imperfect interface condition and the prescribed asymptotic loading conditions. Stress intensity factor (SIF) calculations are performed at the crack tips for different material property combinations and crack positions. The results illustrate convincingly the role of an interphase layer as well as the effects of an imperfect interface on crack behavior. Moreover, the conclusions reached in this dissertation provide a quantitative description of the interaction problem between a three-phase circular inclusion with interface imperfections and a radial matrix crack

MEMS-based Mechanical Characterization of Micrometer-sized Biomaterials

The mechanical properties of biomaterials play important roles in performing their specialized functions: synthesizing, storing, and transporting biomolecules; maintaining internal structures; and responding to external environments. Besides biological cells, there are also many other biomaterials that are highly deformable and have a diameter between 1μm and 100μm, comparable to that of most biological cells. For example, many polymeric microcapsules for drug delivery use are spherical particles of micrometers size. In order to mechanically characterize individual micrometer-sized biomaterials, the capability of capturing high-resolution and low-magnitude force feedback is required. This research focuses on the development of micro devices and experimental techniques for quantifying the mechanical properties of alginate-chitosan microcapsules. The micro devices include microelectromechanical systems (MEMS) capacitive force sensors and force-feedback microgrippers, capable of measuring sub-μN forces. Employing the MEMS devices, systems were constructed to perform the micro-scale compression testing of microcapsules. The force sensors are capable of resolving forces up to 110μN with a resolution of 33.2nN along two independent axes. The force sensors were applied to characterizing the mechanical properties of hydrogel microparticles without assembling additional end-effectors. The microcapsules were immobilized by a PDMS holding device and compressed between the sensor probe and holding device. Young's modulus values of individual microcapsules with 1%, 2%, and 3% chitosan coating were determined through the micro-scale compression testing in both distilled deionized (DDI) water and pH 7.4 phosphate buffered saline (PBS). The Young's modulus values were also correlated to protein release rates. Instead of compressing the microcapsule against the wall of the holding device, a force-feedback MEMS microgripper with the capability of directly compressing the microcapsule between two gripping arms has been used for characterizing both the elastic and viscoelastic properties of the microcapsules during micromanipulation. The single-chip microgripper integrates an electrothermal microactuator and two capacitive force sensors, one for contact detection (force resolution: 38.5nN) and the other for gripping force measurements (force resolution: 19.9nN). Through nanoNewton force measurements, closed-loop force control, and visual tracking, the system quantified the Young's modulus values and viscoelastic parameters of alginate microcapsules, demonstrating an easy-to-operate, accurate compression testing technique for characterizing soft, micrometer-sized biomaterials.Ph

High-throughput three-dimensional cellular platforms for screening biophysical microenvironmental signals

Cells in vivo are subjected continuously to multiple biochemical and biophysical stimuli from their microenvironment that regulate cell fate and function. Although two-dimensional (2D) platforms to check cell responses to various microenvironmental factors have been established, these methods lack physiological relevance. Macroscale three-dimensional (3D) cell culture platforms were developed to provide physiologically relevant environments. However, most of these systems failed to analyze the effects of biophysical stimuli on high throughput. Screening microenvironmental factors is essential to mimic an in vitro model of physiological conditions. Multiple trial-and-error methods with a number of experimental conditions are required to analyze the appropriate microenvironmental factors, making the screening process complex, laborious, and expensive. High-throughput (HT) microscale cell culture platforms offer a cost-effective alternative to macroscale 3D cell cultures and allow efficient microenvironmental screening. This chapter introduces a variety of techniques to build microscale 3D cell culture platforms. The different HT and combinatorial platforms investigating the effects of various biophysical cues on cells are discussed in detail. Creating microscale tissue arrays on an HT platform is extremely useful for drug screening when recreating normal or diseased state tissues. These systems could also be extrapolated to incorporate combinations of multiple microenvironmental factors to analyze their synergistic effects on cell behaviors

Precision improvement of robotic bioprinting via vision-based tool path compensation

Abstract Robotic 3D bioprinting is a rapidly advancing technology with applications in organ fabrication, tissue restoration, and pharmaceutical testing. While the stepwise generation of organs characterizes bioprinting, challenges such as non-linear material behavior, layer shifting, and trajectory tracking are common in freeform reversible embedding of suspended hydrogels (FRESH) bioprinting, leading to imperfections in complex organ construction. To overcome these limitations, we propose a computer vision-based strategy to identify discrepancies between printed filaments and the reference robot path. Employing error compensation techniques, we generate an adjusted reference path, enhancing robotic 3D bioprinting by adapting the robot path based on vision system data. Experimental assessments confirm the reliability and agility of our vision-based robotic 3D bioprinting approach, showcasing precision in fabricating human blood vessel segments through case studies. Significantly, it minimizes the printing layer width disparity to just 0.15 mm compared to the 0.6 mm in traditional methods, and it decreases the average error for curved filaments to 7.0 mm2 from the previous 12.7 mm2 in conventional printing. While these results underscore the significant potential of our innovation in creating precise biomimetic constructs, further investigation is necessary to tackle challenges such as accurately distinguishing closely stacked layers using a vision system, especially under varying lighting conditions. These limitations, coupled with issues of computational complexity and scalability in larger-scale bioprinting, emphasize the importance of enhancing the reliability of the vision-based approach across various conditions. Nonetheless, our innovation demonstrates substantial promise in creating precise biomimetic constructs and paves the way for future advancements in vision-guided robotic bioprinting, including the integration of multi-material printing techniques to enhance versatility

Visible Light-based Stereolithography Bioprinting of Cell-adhesive Gelatin Hydrogels

Stereolithography-based bioprinting offers advantages in resolution and rapid printing time, and thus has received major attention in recent years. However, traditional stereolithography-based bioprinting utilizes a ultraviolet light which may cause mutagenesis and carcinogenesis of cells. In this paper, we present a new visible light crosslinkable bioink that is based on cell-adhesive gelatin. The bioink consists of Eosin Y (EY) based photoinitiator and gelatin methacrylate (GelMA) pre-polymer solution. We examined the feasibility of using visible light from a commercial beam projector to pattern the EY-GelMA bioink. We measured the absorbance of bioink to characterize its sensitivity to visible light and performed bioprinting to test its ability to promote cell adhesion. It is found that the EY-GelMA bioink has an absorption peak at roughly 522 nm and that it can be successfully crosslinked by visible light from the commercial projector. We performed the bioprinting experiments and visualized the cell morphology using nuclei/F-actin staining. Experimental results show that most of the cells attached to the EY-GelMA bioink after five days’ culturing. Ultimately, the EY-GelMA bioink can support both visible light crosslinking and cell adhesion, offering great potential in bioprinting and tissue engineering.Applied Science, Faculty ofArts and Social Sciences, Irving K. Barber Faculty of (Okanagan)Engineering, School of (Okanagan)ReviewedFacult

A novel, well-resolved direct laser bioprinting system for rapid cell encapsulation and microwell fabrication

A direct laser bioprinting (DLBP) system is introduced in this work. The DLBP system applies visible-laser-induced photocrosslinking at a wavelength of 405 nm using the photoinitiator VA086. It is shown that such a system can fabricate vertical structures with fine features (less than 50 μm), and high cell viability (greater than 95%). Experimental characterizations and theoretical simulations are presented, with good agreement seen between the experiments and theory. The DLBP system is applied to the fabrication of (1) cell-laden hydrogel microgrids, (2) hydrogel microwells, as well as a test of (3) cell encapsulation, and (4) cell seeding. The DLBP system is found to be a promising tool for bioprinting.Applied Science, Faculty ofScience, Irving K. Barber Faculty of (Okanagan)Biomedical Engineering, School ofChemistry, Department of (Okanagan)Engineering, School of (Okanagan)ReviewedFacult