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Stem cells
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Proceedings Papers
Proc. ASME. MSEC2019, Volume 1: Additive Manufacturing; Manufacturing Equipment and Systems; Bio and Sustainable Manufacturing, V001T05A002, June 10–14, 2019
Paper No: MSEC2019-2816
Abstract
In attempts to engineer human tissues in the lab, bio-mimicking the cellular arrangement of natural tissues is critical to achieve the required biological and mechanical form and function. Although biofabrication employing cellular bioinks continues to evolve as a promising solution over polymer scaffold based techniques in creating complex multi-cellular tissues, the ability of most current biofabrication processes to mimic the requisite cellular arrangement is limited. In this study, we propose a novel biofabrication approach that uses forces generated by bulk standing acoustic waves (BSAW) to non-deleteriously align cells within viscous bioinks. We computationally determine the acoustic pressure pattern generated by BSAW and experimentally map the effects of BSAW frequency (0.71, 1, 1.5, 2 MHz) on the linear arrangement of two types of human cells (adipose-derived stem cells and MG63) in alginate. Computational results indicate a non-linear relationship between frequency and acoustic pressure amplitude. Experimental results demonstrate that the spacing between adjacent strands of aligned cells is affected by frequency (p < 0.0001), and this effect is independent of the cell type. Lastly, we demonstrate a synergistic technique of gradual crosslinking in tandem with the BSAW-induced alignment to entrap cells within crosslinked hydrogels. This study represents an advancement in engineered tissue biofabrication aimed at bio-mimicry.
Proceedings Papers
Effect of Needle Diameter on Scaffold Morphology and Strength in E-Jetted Polycaprolactone Scaffolds
Proc. ASME. MSEC2017, Volume 4: Bio and Sustainable Manufacturing, V004T05A008, June 4–8, 2017
Paper No: MSEC2017-2989
Abstract
Electro-hydrodynamic Jetting or E-Jetting is a process in which a polymer, dissolved in a solvent and extruded through a needle onto a substrate. A potential difference is applied between the needle and the substrate to facilitate the homogeneous extrusion of the fiber. This process is used to fabricate two dimensional scaffolds with porous mesh surfaces which act as a template for cell growth. As cells are very minute and are required to attach to the surface of the scaffold, it is essential to for the scaffold to have an adequate pore size that allows for nutrient transfer while preventing the penetration of cells through the scaffold. The fiber dimensions of the scaffold may be modified by varying the diameter of the needle through which the fiber is extruded. The change in fiber diameter subsequently results in the change in the bulk mechanical characteristics of the scaffold. It also causes a change in the net porosity of the scaffold. This paper aims to study the effect of the needle diameter on the bulk mechanical properties of the scaffold such as Young’s modulus, Tensile strength and Breaking Strength as well as morphological properties (porosity and pore size) of the Scaffolds are dependent on the cell type, as each type of cell has a different set of requirements depending on the functionality. Bone cells are smaller than soft tissue cells, hence a common scaffold design may not be suit either of the applications. Thus, a one size fits all approach is not suitable for the scaffold [9]. As seen in Figure 1, the Red Blood Cells are a fraction of the size of the fibroblasts and bone marrow stem cells [20–22]. Similarly, the stiffness of the cells is also different. Electro Hydrodynamic Jetting (E-jetting) is a process that is used to fabricate such 2D scaffolds by extruding a polymer solution through a needle and forming a fiber by applying a scaffold. For this study, twelve scaffolds belonging to three study groups were synthesized using e-jetting. By studying the effect of needle diameter on scaffold morphology and strength, we aim to develop a co-relation between the scaffold parameters, which will ultimately help in the creation of a knowledge database. The purpose of creating this database is to choose a select needle for a selected biomedical application.
Proceedings Papers
Investigation of Cellular Confinement in 3D Microscale Fibrous Substrates: Fabrication and Metrology
Proc. ASME. MSEC2017, Volume 4: Bio and Sustainable Manufacturing, V004T05A018, June 4–8, 2017
Paper No: MSEC2017-3020
Abstract
Engineered microenvironments along with robust quantitative models of cell shape metrology that can decouple the effect of various well-defined cues on a stem cell’s phenotypic response would serve as an illuminating tool for testing mechanistic hypotheses on how stem cell fate is fundamentally regulated. As an experimental testbed to probe the effect of geometrical confinement on cell morphology, poly(ε-caprolactone) (PCL) layered fibrous meshes are fabricated with an in-house melt electrospinning writing system. Gradual confinement states of fibroblasts are demonstrated by seeding primary fibroblasts on defined substrates, including a classical two-dimensional (2D) petri dish and porous 3D fibrous substrates with microarchitectures tunable within a tight cellular dimensional scale window (1–50 μm). To characterize fibroblast confinement, a quantitative 3D confocal fluorescence imaging workflow for 3D cell shape representation is presented. The methodology advanced allows the extraction of cellular and subcellular morphometric features including the number, location, and 3D distance distribution metrics of the shape-bearing focal adhesion proteins.
Proceedings Papers
Proc. ASME. MSEC2016, Volume 2: Materials; Biomanufacturing; Properties, Applications and Systems; Sustainable Manufacturing, V002T03A006, June 27–July 1, 2016
Paper No: MSEC2016-8787
Abstract
Stem cells are critical components of regenerative medicine therapy. However, the therapy will require millions to billions of therapeutic stem cells. To address the need, we have recently cultured stem cells in 3D microgels and used them as a vehicle for cell expansion within a low shear stress rotating wheel type bioreactor within a 500ml volumetric setting. This study specifically highlights the cell encapsulation in microbead process, harvesting and operation of microbeads within a dynamic bioreactor environment. We have specifically encapsulated stem cells (human adipose derived) into microbeads prepared from alginate hydrogels via an electrostatic jetting process. This study highlights the effect of fabrication process parameters on end-point biological quality measures such as stem cell count and viability. We were able to maintain a >80% viability during the 21 day static culture period. We have also measured the concentration of metabolites produced during the expansion, specifically lactate production measured during specific time points within culture inside the rotating wheel bioreactor Future work will need to address predicting yields in higher volume settings, efficiency of harvest and a more detailed description of the hydrodynamics affecting stem cell growth.
Proceedings Papers
Proc. ASME. MSEC2013, Volume 1: Processing, V001T01A005, June 10–14, 2013
Paper No: MSEC2013-1025
Abstract
This paper highlights the development of a multi-arm bioprinter (MABP) capable of concurrent deposition of multiple materials with independent dispensing parameters including deposition speed, material dispensing rate and frequency for functional zonal-stratified articular cartilage tissue fabrication. The MABP consists of two Cartesian robots mounted in parallel on the same mechanical frame. This platform is used for concurrent filament fabrication and cell spheroid deposition. A single-layer structure is fabricated and concurrently deposited with spheroids to validate this system. Preliminary results showed that the MABP was able to produce filaments and spheroids with well-defined geometry and high cell viability. The resulting filament width has a variation of +/-170 μm and the center-to-center filament distance was within 100 μm of the specified distance. This fabrication system is aimed to be further refined for printing structures with varying porosities to mimic the natural cartilage structure in order to produce functional tissue-engineered articular cartilage using cell spheroids containing cartilage progenitor cells (CPCs).
Proceedings Papers
Proc. ASME. MSEC2013, Volume 1: Processing, V001T01A006, June 10–14, 2013
Paper No: MSEC2013-1081
Abstract
Bioprinting, or layer by layer additive tissue fabrication, is a revolutionary concept recently emerged as an interdisciplinary effort to produce three-dimensional living organ for clinical application. Among many challenges, it was agreed that inclusion of vascular system is critical for maintaining the viability and functionality of relatively thick 3D bioprinted tissue constructs. Our previous research addressed the printability of novel vessel-like micro-fluidic channels with alginate hydrogel and co-axial nozzles. Here, we further investigated the influence of bioprinting parameters on cartilage progenitor cells (CPCs) survival during and post printing. The results of this study revealed that quantifiable cell death could be induced by varying dispensing pressure, co-axial nozzle geometry, biomaterial concentration. However, damaged cells were able to recover during incubation, as well as undergo proliferation to certain extend. These findings may serve as a guideline for optimizing our system as well as predict cell damage in future studies.
Proceedings Papers
Proc. ASME. MSEC2008, ASME 2008 International Manufacturing Science and Engineering Conference, Volume 2, 465-474, October 7–10, 2008
Paper No: MSEC_ICM&P2008-72054
Abstract
This work examines the release of a model water-soluble compound from electrospun polymer nanofiber assemblies. Such release attracts attention in relation with biomedical applications, such as controlled drug delivery. It is also important for stem cell attachment and differentiation on biocompatible electrospun nanofiber scaffolds containing growth factors, which have been encapsulated by means of electrospinning. Typically, the release mechanism has been attributed to solid-state diffusion of the encapsulated compound from the fibers into the surrounding aqueous bath. Under this assumption, a 100% release of the encapsulated compound is expected in a certain (long) time. The present work focuses on certain cases where complete release does not happen, which suggests that solid-state diffusion may not be the primary mechanism at play. We show that in such cases the release rate can be explained by desorption of the embedded compound from nanopores in the fibers, or from the outer surface of the fiber in contact with the water bath. After release, the water-soluble compound rapidly diffuses in water, whereas a release rate is determined by the limiting desorption stage. A model system of Rhodamine 610 fluorescent dye embedded in electrospun monolithic Poly(methylmethacrylate) PMMA or Poly(caprolactone) PCL nanofibers, or in nanofibers electrospun from PMMA/PCL blends, or in core/shell PMMA/PCL nanofibers is studied. Both the experimental results and theory point at the above-mentioned desorption-related mechanism and the predicted characteristic time, release rate, and effective diffusion coefficient agree fairly well with the experimental data. A practically important outcome of this surface release mechanism is that only the compound on the fiber and pore surfaces can be released, whereas the material encapsulated in the bulk cannot be freed within the time scales characteristic of the present experiments (days to months). Consequently, in such cases complete release is impossible. We also demonstrate how the release rate can be manipulated by the polymer content and molecular weight affecting nanoporosity and the desorption enthalpy, as well as by the nanofiber structure (monolithic fibers, fibers from polymer blends and core-shell fibers). In particular, it is shown that by manipulating the above parameters, release times from tens of hours to months can be attained.
Proceedings Papers
Proc. ASME. MSEC2007, ASME 2007 International Manufacturing Science and Engineering Conference, 83-90, October 15–18, 2007
Paper No: MSEC2007-31031
Abstract
Currently there is no adequate bone replacement available that combines a long implant life with complete integration and appropriate mechanical properties. This paper reports on the use of human mesenchymal stem cells (MSCs) to populate porous bioceramic scaffolds produced by selective laser sintering (SLS) to create bespoke bioactive bone replacement structures. Apatite-wollastonite glass ceramic was chosen for use in this study because of its combination of excellent mechanical and biological properties, and has been processed using an indirect SLS approach. Process maps have been developed to identify process conditions for the SLS stage of manufacture and an optimised furnace cycle for the material has been developed to ensure that the required material phases for bioactivity are present in the manufactured scaffold. Results from tissue culture with the MSC’s on the scaffolds (using confocal and scanning electron microscopy) show that MSCs adhere, spread and retain viability on the surface, and penetrate into the pores of apatite wollastonite (A-W) glass ceramic scaffolds over a 21 day culture period. The MSC’s also show strong indications of osteogenesis, indicating that the MSC’s are differentiating to osteoblasts. These results indicate good biocompatibility and osteo supportive capacity of SLS generated A-W scaffolds and excellent potential in bone replacement applications.
Proceedings Papers
Proc. ASME. MSEC2006, Manufacturing Science and Engineering, Parts A and B, 423-428, October 8–11, 2006
Paper No: MSEC2006-21053
Abstract
This report is part of a continued effort to evaluate the in vitro osteoblast responses on different phospholipid coatings on Titanium (Ti) implant materials. It has been established that, among analogous phopholipids, the Ti surfaces coated with calcium phosphate (CaP) complex of phosphatidylserine induce the best calcium deposition and osteoblast growth and metabolism. This communication describes an effort to optimize the chemical structure of phosphatidylserine at its position−1 and −2, as Ti surface coating relative to enhancement in osteoblast differentiation and growth in culture. Four synthetic phosphatidylserine analogs with varying fatty acyl chain length and unsaturation were converted to CaP complex, coated on Ti discs, and the osteoblast progenitor cells were cultured on them for up to 14 days to study their differentiation, growth and biochemistry as marked by the expression of alkaline phosphatase specific activity and protein production. In a separate experiment, the topography of the glass surface (glass Petri-dishes) coated the analogous phosphatidylserines, after immersion in simulated body fluid, was examined by scanning electron microscopy (SEM). The presence of calcium and phosphate ions in this deposit was also confirmed. The inclusion of unsaturation in fatty acyl chain in phosphatidylserine enhanced the Total protein production (TPP) as well as the alkaline phosphatase (ALP) specific activity.