Mei Wei, University of Connecticut
Marisha Godek, Medtronic
Shaoqin Gong, University of Wisconsin-Madison
Joerg Jinschek, FEI Company
Applied Physics Reviews | AIP Publishing, Medtronic, The National Science Foundation
BM3.1: Advances in Biomaterial Design
Monday PM, November 28, 2016
Hynes, Level 1, Room 101
9:45 AM - *BM3.1.01
3D Bioprinting for In Vitro Tissue Models
Wei Sun 2 1
2 Mechanical Engineering Tsinghua University Beijing China, 1 Drexel University Philadelphia United StatesShow Abstract
3D Bio-Printing uses cells and biomaterials as building blocks to fabricate personalized 3D structures or functional in vitro biological models. The technology has been widely applied to regenerative medicine, disease study and drug discovery. This presentation will report our recent research on printing cells for construction of micro-organ chips and for building in vitro 3D tumor models. An overview of advances of 3D Bio-Printing will be given. Enabling methods for cell printing will be described. Examples for 3D Printing of tissue engineering model, drug metabolism model and disease model will be reported, along with results of printing parameters on cell viability and 3D tumor structural formation, characterization of cell morphologies, proliferations, protein expressions and chemoresistances. Comparison of biological data derived from 3D printed models with 2D planar petri-dishes models will be conducted. Discussions on challenges and opportunities of 3D Bio-Printing will also be presented.
10:15 AM - BM3.1.03
Roll-to-Roll Fabrication of Porous Polymer Nanosheets for Engineering Multilayered Cellular Organization
Toshinori Fujie 1 2 , Shoichiro Suzuki 3 , Keisuke Nishiwaki 3 , Shinji Takeoka 3
1 Waseda Institute for Advanced Study Waseda University Tokyo Japan, 2 Japan Science and Technology Agency Tokyo Japan, 3 Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering Waseda University Tokyo JapanShow Abstract
Replication of intricate biological tissues using engineered scaffolds is important for tissue engineering in the application of regenerative medicine, drug screening and bio-hybrid actuators. Basement membrane (BM) associated with extracellular matrix (ECM) is an ultra-thin supramolecular assembly composed of biopolymers such as type-IV collagen, laminin, entactin and perlecan associated with mesh-like connective tissues. The BM has an ultra-thin structure with thickness of tens to hundreds of nanometers and porosity of submicrometers to tens of micrometers. In this regard, we focus on free-standing polymer ultra-thin films (nanosheets) as synthetic mimics of the BM. Polymer nanosheets have unique structural features, including a thickness of tens to hundreds of nanometers and a surface area of several tens of square centimeters. Such nanosheets are obtained by exfoliating polymeric ultra-thin films from substrates by dissolving the underlying sacrificial layer. As a result of their ultra-thin structure, these nanosheets are flexible and physically adhesive to variety of surfaces including biological tissues, that are fabricated from polysaccharides, proteins, and biodegradable polyesters. In this study, we envisaged replication of the ultra-thin, flexible and permeable structure of BMs by integrating a macroporous structure in a nanosheet (referred to as “porous nanosheet”) using a combination of gravure coating and polymer-based phase separation. The porous nanosheet with the thickness of 150 nm and average pore diameter of 4 µm was applied for muscle tissue engineering, where it allows for the proliferation and differentiation of muscle cells and the further formation of hierarchical tubular structures through the process of multilayering, enabled by a sheet-like cellular organization. The porous nanosheet possesses unique properties such as flexibility, permeability and also biodegradability to function as artificial BMs as well as scalability adapting to the large-scale production by roll-to-roll process. As a proof of concept, we demonstrated the ultra-thin structure served as a platform for muscle tissue engineering by employing skeletal muscle cells (C2C12 myoblasts). The porous nanosheet realized deposition and permeation of ECM components (e.g., fibronectin, collagen IV and laminin), and also generated multilayered and anisotropic muscular structures. Such structural properties may also contribute to the recapitulation of intricate hierarchical structures such as skeletal muscle myofibers, small intestine and arterial walls.
10:30 AM - BM3.1.04
Improving the Conversion and Kinetic Profiles of Open Vessel Free Radical Photopolymerization for Two Biocompatible Polymers Using Glucose Oxidase
Ali A. Mohammed 1 2 , Juan Aviles Milan 1 , Justin J. Chung 1 , Siwei Li 1 , Theoni K. Georgiou 1 , Julian Jones 1
1 Imperial College London London United Kingdom, 2 Qatar Foundation Doha QatarShow Abstract
Biomaterials are often synthesized by different methods of free radical polymerization (FRP). A popular type of FRP is photopolymerization. UV light is commonly used for its low cost, speed and ease of use, to prepare biocompatible materials such as hydrogels for cartilage application. Irgacure 2959 is a type 1 α-cleavage photoinitiator (PI) frequently used for hydrogel synthesis, mainly due to its high efficiency and low cytotoxicity for a broad range of cell types. Higher concentrations are used to overcome the negative effects of oxygen on free radicals. However, over a certain concentration it is cytotoxic. This limits polymer conversion for biomaterial applications, in turn limiting the kinetics of the polymerisation and the properties of the polymer. It also limits free shape forms if synthesis requires a closed system for nitrogen purging to rid of the oxygen. Unreacted monomers can also have cytotoxic effects hence purification steps are required. In this work we show that an oxido-reductase enzyme called glucose oxidase (GOx) is able to create an oxygen free environment for 2 separate polymers. This allows for 100 % monomer to polymer conversion at non cytotoxic PI concentrations.
2 common polymers used for double network hydrogels are poly (2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS) and polyacrylamide (PAAm). Separately, monomer solutions were prepared in deionised water containing small trioxane crystals, Irgacure 2959 and GOx (controls did not have GOx). Trioxane was used as a reference for 1HNMR analysis at the start and end of the FRP, as well specific time points for the kinetics studies. At 0.05 wt% PI, 1H NMR of PAMPS showed 0 % conversion in the absence of GOx. This is due to instant and rapid inhibition of PI radicals caused by O2. In contrast, an increase to 100 % conversion was exhibited at 0.05 wt % PI in the presence of GOx. Increasing the PI concentration to 2 wt% (soluble limit of the PI) provides 95 % conversion for the control, leaving behind 5 % of unreacted monomers. PAMPS kinetics is improved with GOx, 100 % conversion is reached at 30 minutes, whereas the control reaction has a lag and the reaction only starts at 35 minutes of exposure to UV light.
PAAm exhibits similar results, at 0.05 wt % PI with GOx a conversion of 78% is achieved, and 0% for the control. At 0.5 wt% PI, 100 % conversion is achieved with GOx, and only 17 % conversion for the control. At 2 wt % PI, 76 % conversion of the control is reached. PAAm kinetics improve with GOx, reaching 70 % conversion at 20 minutes, in comparison with the control that has a lag of 20 minutes before the reaction starts.
Ultimately, these low PI concentrations, shorter UV exposure times and improved kinetics profiles will provide a useful ground for hydrogel synthesis that require cell seeding and no purification to remove unreacted monomers. Open vessel synthesis of custom shape biomaterials unlocks potential improvements in technical and industrial techniques.
11:15 AM - *BM3.1.05
Toward a Light-Activated Dynamically Controllable Hydrogel for 3D Cell Culturing
Lydia Sohn 1
1 University of California, Berkeley Berkeley United StatesShow Abstract
While there are many types of gels that are currently being utilized to study how cells interact with their environment (e.g. collagen, alginate, matrigel, hyaluronate, and polyacrylamide), their mechanical properties are often determined during gel synthesis. Once synthesized, these gels have mechanical properties that either are static, i.e. cannot be changed, or have a very limited capacity to change. Recognizing this limitation, researchers have begun to develop strategies for creating gels with varying degrees of stiffness, as the interplay between cells and their physical and heterogeneous microenvironment are highly dynamic. For example, Kloxin et al. (Nature Protocols 5, 1867-1887, 2010) have created a hydrogel that is photodegradable via a crosslinker that degrades when exposed to 365-420 nm light.
In this talk, we will discuss our path toward developing a dynamically controllable 3D cell-culture matrix consisting of a hyaluronic acid (HyA) hydrogel whose polymer crosslinking can be achieved via a light-dependent protein-protein interaction. Light would enable an exogenous and rapid method of control on hydrogel properties that is unavailable to diffusion-based methods. A tightly controlled light-stimulated cell-culture platform could illuminate the influence of time-dependent mechanical stimuli on stem-cell fate decision.
11:45 AM - BM3.1.06
Nanofibrous Three-Dimensional Vascularized Cell-Laden Constructs—Fabrication and Evaluation
Qilong Zhao 1 , Min Wang 1
1 Mechanical Engineering University of Hong Kong Hong Kong Hong KongShow Abstract
Three-dimensional (3D) cell-laden constructs with tissue-like structures are desirable for human body tissue regeneration and different techniques including 3D bio-printing have been investigated for making such constructs. However, despite some success, 3D bio-printing of cell-laden constructs still faces major challenges such as structural dimension, vascularization and cellular fate control. Cell-laden constructs should not only have biomimetic cell arrangement but also possess cell-matrix organization similar to that of native tissue. In native tissues, nanofibrous extracellular matrix together with various bioactive molecules directs cell behavior. Electrospinning, a facile technique for fabricating nanofibrous scaffolds and incorporating bioactive molecules, is therefore very attractive. But electrospun scaffolds have shortcomings of small pore size and poor cell infiltration, leading to difficulties to form 3D vascularized cell-laden constructs. In this investigation, we developed a novel technique to simultaneously deposit cell-encapsulated microspheres and growth factor-incorporated nanofibers on a collector in a concurrent electrospinning and electrospray process, aiming to directly make nanofibrous 3D cell-laden constructs. In these constructs, cell behavior would be guided by combinational (structural, biological, etc.) cues. PLGA, sodium alginate and human vein umbilical endothelial cells (HUVEC) were used. During concurrent electrospinning and electrospray, a mono-spinneret was used for electrospinning, which was fed with a water-in-oil emulsion consisting of PLGA solution and vascular endothelial growth factor (VEGF)-containing phosphate buffer saline, while a coaxial spinneret was used for electrospray with inner nozzle and outer nozzle being fed respectively with HUVEC cell suspension and sodium alginate solution. Products of electrospinning and electrospray were simultaneously collected in a bath filled with CaCl2-containing cell culture medium for crosslinking alginate, resulting in VEGF-incorporated nanofibrous PLGA scaffolds embedded with HUVEC-encapsulated hydrogel microspheres. Cells in microspheres were released after breakdown of alginate shell, forming eventually 3D cell-laden constructs. These constructs were studied using different techniques. They possessed biomimetic nanofibrous architecture, adequate mechanical properties and suitable biodegradation rate. VEGF in nanofibers exhibited high encapsulation efficiency and sustained release. Owing to the protection by microspheres, cells in the final cell-laden constructs had high cell viability. Cells were randomly distributed in the fibrous matrix in 3D and showed free stretch and spreading in constructs. With the combination of structurally stable nanofibrous structure and locally delivered VEGF, HUVEC cells in the constructs displayed induced cell morphogenesis, enhanced cytoskeleton development, increased cell proliferation, and improved vascularization potential.
12:00 PM - BM3.1.07
Fine Cell-Laden Droplet Formation under DOD Inkjet Printing with Nozzle of 30 μm Diameter for Highly Precise 3D Biostructure
Young Kwon Kim 1 , Sungjune Jung 2 , Joonwon Kim 1
1 Department of Mechanical Engineering Pohang University of Science and Technology Pohang Korea (the Republic of), 2 Pohang University of Science and Technology Pohang Korea (the Republic of)Show Abstract
Bioprinting has great potential as an innovative alternative for tissue engineering and regenerative medicine, and the growing interests in bioprinting have led to many physiological and clinical studies. Among the bioprinting methods, drop-on-demand (DOD) inkjet printing has abundant advantages of high resolution, high throughput, high reliability, and so on. However, since many studies on cell printing utilized larger diameter of nozzle, ≥48 μm, than that of cell, ~20 μm owing to cell damage and nozzle blockage, there is a performance limit of resolution or drop size. Therefore, for the sake of highly precise 3D biofabrication, it is crucial to solidly investigate the jetting formation from a nozzle of fine diameter corresponding to cell diameter. During DOD inkjet printing with nozzle of 30 μm diameter, we observed the cell-laden droplet formation process in morphological view using mouse fibroblasts with cell concentrations of 2×10^6 cells/mL. In order to understand the effect of cell on the droplet formation, the morphology of cell-laden droplet is compared with that of a serum-free media without cells under the almost identical operating conditions. Finally, we confirmed that cell viabilities from 30 and 80 μm diameters are 92 % and 94 % compared to that of control, respectively.
This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant No. HI15C0001).
12:15 PM - BM3.1.08
Self-Healing, Injectable and Cytocompatible Double Network Hydrogels
Christopher Rodell 1 , Neville Dusaj 1 , Christopher Highley 1 , Jason Burdick 1
1 Department of Bioengineering University of Pennsylvania Philadelphia United StatesShow Abstract
Tough hydrogels have gained interest in recent years, as their resilience toward mechanical failure perpetuates their use in load bearing biomedical applications (e.g., cartilage, intervertebral disc). While double network (DN) hydrogels enable toughness, DNs amenable to both injection and cell encapsulation have not been realized. Here, these features were developed through a combination of extensible covalent networks and self-healing supramolecular networks. Covalently crosslinked hyaluronic acid (HA) networks were formed from methacrylated HA (MeHA, 100% modified, 3.0wt%) by Michael addition with dithiothreitol (DTT; ratio thiol/methacrylate = 0.2; pH 8 overnight) and exhibited failure at >80% compressive strain. Supramolecular networks were formed by guest-host (GH) assembly of adamantane and cyclodextrin HA (Ad-HA & CD-HA, 30% modified, 5.0wt%) and exhibited rapid self-healing (>95% shear modulus, <6 sec following 500% strain at 1.0 Hz). DNs were then formed by physical interpenetration (GH DN) or with covalent crosslinking between these two networks (MethGH DN; 20% methacrylated Ad-HA and CD-HA).
The mechanical properties of both DNs were investigated relative to MeHA covalent networks. Compressive analysis to 90% strain demonstrated different modes of failure: brittle (MeHA), ductile (GH DN), and recovery (MethGH DN). MethGH DNs exhibited increased failure stress (335±30 kPa vs 163±30 kPa) and moduli (11.0±0.2 kPa vs 2.2±0.3 kPa) compared to MeHA alone. At increased DTT concentration, moduli >250 kPa were achieved for MethGH DNs, and toughening (>eightfold increase) was demonstrated in tension. Under repeated compressive loading (5 cycles, 80% strain), MethGH DNs underwent rapid internal self-healing (i.e., immediate recovery of moduli and strain energy) in contrast to irrecoverable damage for MeHA and GH DN systems. Self-healing was likewise demonstrated macroscopically between gel fragments. Thus, supramolecular GH bonds allowed toughening and self-healing of DNs — with improved outcomes with crosslinking between the networks to improve stress transfer.
Through use of a phosphine catalyst, Michael addition crosslinking was rapidly achieved (<30 min) under physiological conditions. Encapsulated mesenchymal stem cells within MeHA and DNs exhibited high viability (>95%, day 0, live/dead) with increasing metabolic activity (through day 14, Alamar blue) and maintained viability (>98%, day 14, live/dead) with culture. Upon injection into tissue, MeHA rapidly diffused prior to crosslinking while DNs remained localized by supramolecular bonds, allowing subsequent covalent crosslinking. Neither cell inclusion nor injection reduced DN mechanical properties, relative to controls. Owing to their injectable behavior, ease of cell encapsulation, and capacity for repetitive loading without detriment to mechanical strength, supramolecular DN hydrogels are a promising platform for regenerative medicine applications.
12:30 PM - BM3.1.09
Cytoprotective Effects of RGD Peptide Incorporated Multilayer Nanofilms on Mesenchymal Stem Cells In Vivo System
Daheui Choi 1 , Younsun Won 2 , Miso Yang 1 , Jiwoong Heo 1 , Hwankyu Lee 3 , Seung Soon Jang 4 , Hyun-Bum Kim 2 , EunAh Lee 2 , Jinkee Hong 1
1 Chung-Ang University Seoul Korea (the Republic of), 2 Kyung Hee University Yongin-si Korea (the Republic of), 3 Dankook University Yongin-si Korea (the Republic of), 4 Georgia Institute of Technology Atlanta United StatesShow Abstract
Mesenchymal stem cell, which is derived from bone marrow, has been clinically used for treatment of graft-versus-host disease , sepsis , paralysis , stroke  and arterial disease  by direct intravenous injection. However, once the MSCs are administrated in blood vessel, the cells are subjected to high intensity of shear stress from blood stream. In addition, the MSCs are transplanted in single cell state, which is the situation that cell-extracellular matrix (ECM) interaction is disrupted for a prolonged period, leading to cell death because apoptotic signal pathways are activated. Therefore, in these reason, current trial method using directly injection of MSCs to blood has resulted in extremely poor retention of MSC in blood and low targeting efficiency to injured area.
To overcome this difficulties of clinical trial for MSCs, in this report, we prepared elaborate Layer-by-Layer (LbL) assembled films on MSCs for increase stability in harsh environments like vessel condition. The LbL assembly is well-known film preparation method by repetitive adsorption of oppositely charged polymers . It is also feasible to precisely make nano- to micro-sized film using various interactions such as electrostatic interaction, hydrogen bonding, and covalent bonding and so on. By taking full advantages of LbL assembly, we tried to prepare a serious of nanofilms assembled using arginyl-glycyl-aspartic acid (RGD peptide), poly (l-lysine) (PLL), and hyaluronic acid (HA) by varying the film composition, structure, and function. The RGD peptide is kind a small peptide that interact with integrin that is located on cell plasma membrane which has a function of cell attachment, proliferation and survival. PLL and HA have been mainly used due to low cytotoxicity. Here, we chose PLL as a positively charged material and RGD peptide and HA as negatively charged materials to make nanofims.
By providing LbL film on MSCs, we found that the film coated MSCs showed significantly increase cell survival during agitation culture which is mimicked blood vessel that has high intensity of shear stress and non-attachable condition. And also the integrin was activated by RGD peptide incorporated film, leading to activation of survival related protein, Akt. Due to properties of LbL film showing high flexibility and looseness, the coated MSCs could retain their own stem cell properties and differentiation properties. In this report, we concluded that the LbL film could enhance cell functions such as viability, stability and stem-ness and this approach could provide tool for increase the efficacy of MSCs for cell therapy.
 Le Blanc K, et al., The Lancet 363, 1439-1441 (2004).
 Tyndall A, Pistoia V., Nature medicine 15, 18-20 (2009).
 Deshpande DM, et al., Annals of neurology 60, 32-44 (2006).
 Lindvall O, Kokaia Z., Nature 441, 1094-1096 (2006).
 Rafii S, Lyden D., Nature medicine 9, 702-712 (2003).
 Decher G.,Science, 29, 1232-1237 (1997)
12:45 PM - BM3.1.10
High-Throughput, High Replicate Screening of Microfabricated Biomaterials Using Flow Cytometry
Kirsten Parratt 1 , Jenny Jeong 1 , Peng Qiu 1 2 , Krishnendu Roy 1 2
1 Georgia Institute of Technology Atlanta United States, 2 Emory University Atlanta United StatesShow Abstract
Currently there is no widely accessible system for screening of biomaterial-encapsulated cells which is simultaneously high replicate, high-throughput, non-destructive, and allows highly multiplexed analyses. This limits our ability to study how material structure can be engineered to control cell function. Flow cytometry, primarily used for the study of cells in suspension, can be modified to address this deficiency. Flow cytometers automate the collection of a large number of unique events in a short time period, which can be combined to characterize a large population and collect data with high statistical power. This is particularly important for the study of cell-biomaterial interactions because inherent heterogeneity in cell characteristics can result in a range of responses from the population. By multiplexing microparticle shape, size, and fluorescence as variables in flow cytometry, a rapid assay system was developed for the study of cells encapsulated in hydrogel materials. These variables were used to “barcode” encapsulating materials such that samples could be pooled for culture and analysis. While size and fluorescence have been previously investigated, shape is a novel variable in flow cytometry and can be used to greatly expand the number of materials which can be tested in a single assay.
This high-throughput biomaterial screening platform was demonstrated using a popular model system; poly(ethylene glycol) diacrylate-based (PEGDA) hydrogels and mesenchymal stem cells (MSCs). Soft lithography was used to fabricate PDMS molds that consist of arrays of > 104 replicates and crosslinked hydrogel microparticles were collected in suspension. Using two commercially available flow cytometers, the ImageStreamX MarkII and the LSR Fortessa, twelve test microparticle barcodes were investigated. Test populations consisted of three sizes (20, 40, and 60 µm cross-sectional length) and four shapes (square, right triangle, circle, and equilateral triangle). On the ImageStream these microparticles were analyzed on the lowest fluidic setting. Next, a data handling method was designed in IDEAS software to separate the different barcodes with high accuracy. Using the collected images, “true” members of each population were selected and features that gave the best separation between groups were identified. The accuracy of the gating scheme was evaluated to be greater than 84% for all experimental groups.
To elucidate the impact of material structure on cellular function, cells were encapsulated in PEGDA-based hydrogels with varying amounts of covalently incorporated glycosaminoglycans (GAGs) that are known to induce chondrogenesis and osteogenesis in MSCs. The cell encapsulation density and viability were quantified for each microparticle, and chondrogenesis or osteogenesis was evaluated to compare the hydrogel formulations.
BM3.2: Smart Biomaterials
Monday PM, November 28, 2016
Hynes, Level 1, Room 101
2:30 PM - *BM3.2.01
Micropatterning of Reprogramming Cultures to Track and Control Nuclear Properties for the Production of High-Quality Stem Cells
Krishanu Saha 1 , Ty Harkness 1 , Nicole Piscopo 1 , Ryan Prestil 1 , Stephanie Seymour 1
1 University of Wisconsin-Madison Madison United StatesShow Abstract
Standard cellular reprogramming methods are noisy, laborious, and poorly understood. To address this key bottleneck, here we bring together two innovations in 1) watching and 2) physically-constraining the process of reprogramming. First, watching reprogramming in action elucidates processes occurring in the middle of reprogramming. Compared to the nuclei of starting fibroblasts, nuclei of the endpoint induced pluripotent stem cells (iPSCs) are smaller, more circular, and contain different ‘open and closed’ chromatin. How these radical changes in nuclear properties occur is still a mystery. We have developed an innovative micropatterned substrate that enables the live, in situ imaging of nuclei within reprogramming populations. The substrates separate cultures into thousands of small adhesive ‘islands’ of 100-800 microns in diameter. Islands are surrounded by polyethylene glycol which prevents cell adhesion. On these islands, our image-based “fingerprints” of nuclei can identify cells that have been fully reprogrammed. They also provide a new method to identify so-called “roadblocks” that stall