Symposium Organizers
Mei Wei, University of Connecticut
Marisha Godek, Medtronic
Shaoqin Gong, University of Wisconsin-Madison
Joerg Jinschek, FEI Company
Symposium Support
Applied Physics Reviews | AIP Publishing, Medtronic, The National Science Foundation
BM3.1: Advances in Biomaterial Design
Session Chairs
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 States
Show Abstract3D 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 Japan
Show AbstractReplication 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 Qatar
Show AbstractBiomaterials 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 States
Show AbstractWhile 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 Kong
Show AbstractThree-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 AbstractBioprinting 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 States
Show AbstractTough 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 States
Show AbstractMesenchymal stem cell, which is derived from bone marrow, has been clinically used for treatment of graft-versus-host disease [1], sepsis [2], paralysis [3], stroke [4] and arterial disease [5] 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 [6]. 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.
[1] Le Blanc K, et al., The Lancet 363, 1439-1441 (2004).
[2] Tyndall A, Pistoia V., Nature medicine 15, 18-20 (2009).
[3] Deshpande DM, et al., Annals of neurology 60, 32-44 (2006).
[4] Lindvall O, Kokaia Z., Nature 441, 1094-1096 (2006).
[5] Rafii S, Lyden D., Nature medicine 9, 702-712 (2003).
[6] 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 States
Show AbstractCurrently 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
Session Chairs
Shaoqin Gong
Krishanu Saha
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 States
Show AbstractStandard 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 reprogramming progression and to determine in what stage of reprogramming cells reside. Second, by controlling the micropattern geometry of our substrate to physically-constrain the process of reprogramming, we activate mechanotransduction pathways (e.g., YAP/Taz), and directly impact chromatin mobility to promote reprogramming. This work provides new evidence that some aspects of the biophysical microenvironment, using biomaterials of defined properties, can be rationally controlled to promote reprogramming.
3:00 PM - BM3.2.02
Fabrication and Investigation of Large Area 3D Nanostructures for In Vitro
Cell Adhesion Studies
Andreea Belu 1 , Dirk Mayer 1 , Andreas Offenhausser 1
1 Peter Grünberg Institute/Institute of Complex Systems Bioelectronics Forschungszentrum Jülich GmbH Juelich Germany
Show AbstractThe behavior of cells which encounters multiple molecular and mechanical cues during their development, represent a vital phenomenon for tissue engineering and regenerative medicine. One way to modulate the cellular responses is to vary surface topographies. Smart or instructive biomaterials with different surface topography can regulate cellular behavior from initial attachment and can further dictate the response of surrounding tissue by presenting optimal surface characteristics. With these advancements, the topography of implantable biomaterials, such as multi-electrode arrays (MEAs), is critical for optimizing the electrical coupling between cells and device’s surface [1].
In this work, we introduce a large area screening of cellular interactions with surface topographies using 3D nanostructured substrates. For this purpose we studied in a systematic and quantitative manner the response of primary cortical neurons with respect to the spatial dimensions such as size, distance, and pitch of symmetric structures. The fabrication technique is based on a top down electron lithography process used to produce Si/SiO2 molds, followed by nanoimprint lithography for polymer stamp replication. The obtained polymer substrates are replicated from the maser structure with high patter fidelity and possess similar feature sizes over large distances. The dimensions and distances between the structures are ranging from 250 nm to 4 µm. These polymer samples are directly employed for cell culture experiments. Using the systematic design of our surface topographies, we studied the geometric limits of neuron adhesion and neurite development by means of immunofluorescence staining, SEM, and FIB-SEM. Furthermore, we investigated cell polarization and adhesion by means of a linear gradient. A special attention was also given to cell’s contact with the artificial solid surfaces, since the interface is the part that determines success of a neural implant [2]. Cells display a wide range of interactions with the nanostructures. We observed that cells could form contact only with the tops of the posts, or it enhances the ECM contact by penetrating into spaces separating these features. By this systematic study pave the way to understand the wide variety of individual cells interaction with a structured surface, especially for the interface between the cell membrane and nanostructured surfaces of solids.
[1] M. E Spira & A. Hai, Nature Nanotechnology, 8, 83–94 (2013).
[2] A. Belu, J. Schnitker, S. Bertazzo, E. Neumann, D. Mayer, A. Offenhäusser & F. Santoro, Journal of Microscopy, 263, 78-86 (2016).
3:15 PM - BM3.2.03
Using Elastomeric Substrata to Modulate the Human Mesenchymal Stem Cell Secretome for Hematopoietic Recovery
Frances Liu 2 1 , Novalia Pishesha 3 2 , Zhiyong Poon 1 , Hidde Ploegh 3 5 , Harvey Lodish 3 5 , Krystyn Van Vliet 2 4 1
2 Department of Biological Engineering Massachusetts Institute of Technology Cambridge United States, 1 Biosystems and Micromechanics Interdisciplinary Research Group Singapore-MIT Alliance for Research and Technology Singapore Singapore, 3 Whitehead Institute Massachusetts Institute of Technology Cambridge United States, 5 Department of Biology Massachusetts Institute of Technology Cambridge United States, 4 Department of Materials Science and Engineering Massachusetts Institute of Technology Cambridge United States
Show AbstractBone marrow ablation is necessary for bone marrow or hematopoietic stem cell transplantation as a treatment for diseases such as lymphomas, leukemias, and sarcomas. Patient recovery depends highly upon cell engraftment and subsequent regeneration of the bone marrow compartment. We have shown recently that an enriched subpopulation of osteoprogenitors isolated from human mesenchymal stem cells (hMSCs) can promote engraftment and regeneration of the bone marrow compartment in vivo through the cell secretome [1]. However, these osteoprogenitors constitute a small fraction of the heterogeneous hMSC population. Here we leverage cell-material interactions to induce a heterogenous population of hMSCs to secrete more factors that are beneficial to hematopoietic recovery. We created polydimethylsiloxane (PDMS) substrata that are cell-culture compatible with varying viscoelastic properties. We observe up to a threefold increase in osteopontin expression on more compliant PDMS substrata prior to changes in terminal differentiation capacity. Osteopontin is a secreted phosphoprotein implicated in pro-angiogenic tissue regeneration and a highly-correlative marker for the osteoprogenitors isolated in our previous studies of bone marrow regeneration. In addition to osteopontin, we observe mechanosensitive regulation of at least six other secreted proteins on our PDMS substrata. This increase in osteopontin expression suggests a population-wide shift towards our desirable osteoprogenitor phenotype and their secretome that is most effective in bone marrow regeneration. To validate that our mechanically modulated hMSCs may play a beneficial role in hematopoietic recovery required of rapid and functional tissue regeneration, we considered whether co-culture with human hematopoietic stem cells (hHSCs) affected hHSC repopulation and blood cell lineage commitment in the the hematopoietic compartment. Our hMSCs grown on compliant PDMS substrata appear to promote proliferation of the hHSCs. In contrast, the stiffest PDMS substrata of the hMSCs maximally primed the hHSCs for myeloid differentiation, with mechanosensitive trends in expression on all PDMS substrata. In summary, we have developed a synthetic polymer-based cell-culture system to modulate the secretome of the hMSCs, with applications including hHSC priming that have the potential to aid in regenerative clinical applications such as hematopoietic recovery.
[1] Poon, Z., Lee, W.C., Guo, G., Lim, C.T., Han, J. and Van Vliet, K.J., “Bone marrow regeneration promoted by biophysically sorted osteoprogenitors from mesenchymal stromal cells,” Stem Cells Translational Medicine 4 1-10, 2015.
3:30 PM - BM3.2.04
Instructing Cells with Programmable Peptide-DNA Hybrids
Ronit Freeman 1 , Nicholas Stephanopoulos 2 , Samuel Stupp 1
1 Simpson Querrey Institute of BioNanotechnology Northwestern University Chicago United States, 2 Biodesign Institute and Department of Chemistry and Biochemistry Arizona State University Tempe United States
Show AbstractPeptides and DNA represent two of the most attractive categories of molecules for the construction of nanomaterials for biology and medicine. Peptides provide a rich palette of biological functionality and self-assembly behavior, and DNA can be used to construct complex nanostructures with programmable, dynamic properties. We sought to merge the advantages of these two molecular platforms through the use of peptide-DNA (P-DNA) hybrid biomaterials and utilize them to mimic the extracellular environment.
The native extracellular matrix is a space in which signals can be displayed dynamically and reversibly, positioned with nanoscale precision, and combined synergistically to control cell function. Artificial forms of this matrix for tissue regeneration need to recapitulate these three characteristics in a single system. Most efforts in this area have effectively addressed only one of these three key phenomena, and focused mainly on static cell adhesion or irreversible switching of bioactivity. Here we describe a unified molecular platform that can be programmed to control the dynamics, spatial positioning, and combinatorial synergies of signals in extracellular matrices. In this approach, a peptide-DNA (P-DNA) molecule is immobilized on a surface through DNA tethers. By engineering a series of tethers responsive to different stimuli, we show that cells adhered and spread on the surface reversibly. The use of P-DNA in cell signaling allowed multiple cycles of reversibility by simply adding soluble biologically compatible molecules such as DNA and enzymes, without the need for external stimuli like photons or electrochemical potentials. The DNA was also used as a molecular ruler to control the distance-dependent synergy between two adhesion peptides. Finally, orthogonal DNA handles were designed to allow for the selective presentation of different signals, with the ability to independently up- or down-regulate each over time.
4:15 PM - *BM3.2.05
Hierarchical Hydrogels to Recruit and Control Cell Behavior In Vivo
Jason Burdick 1
1 University of Pennsylvania Philadelphia United States
Show AbstractHydrogels represent a class of biomaterials that have great promise for the repair of tissues, particularly due to our ability to engineer their biophysical and biochemical properties. Hydrogels can provide instructive signals through material properties alone (e.g., mechanics, degradation, structure) or through the delivery of therapeutics that can influence tissue morphogenesis and repair. Importantly, hydrogel design should reflect both the clinical context and the natural healing cascades of the damaged tissue. Here, I will give examples of the design of hydrogels based on hyaluronic acid (HA) for the repair of musculoskeletal tissues that have limited natural repair processes.
Towards application in cartilage repair, we have developed multi-polymer fibrous hydrogels that permit control over scaffold porosity and therapeutic release via the engineering of specific fiber populations. Fibers are formed through an electrospinning and photocrosslinking process, where individual fiber degradation is controlled through macromer chemistry. We have investigated these scaffolds towards cartilage repair when combined with microfracture in a large animal (i.e., minipig) model. Composite scaffolds were formulated from a combination of HA fibers and control poly(ε-caprolactone) (PCL) fibers, either with or without transforming growth factor-b3 (TGFβ3) delivery. Material choice and TGFβ3 delivery had significant impacts on outcomes; specifically, PCL scaffolds without TGFβ3 had inferior gross appearance and reduced mechanical properties, whereas HA scaffolds that released TGFβ3 resulted in improved histological scores and increased type 2 collagen content. Towards meniscus repair, multi-polymer fibers were designed with fibers that selectively release collagenase to provide an environment permissive to cell recruitment, PDGF to actually recruit cells, and PCL for stability. When investigated in tissue repair, each fiber population was important to the success of repair tissues. Further engineering of the fibers is used to alter cell interactions and the overall material interface with the biological environment. This study highlights the importance of scaffold properties on in vivo cartilage repair.
4:45 PM - BM3.2.06
A Novel Bionanoconjugate to Enable Human Mesenchymal Stem Cell Homing In Vivo
Wenjin Xiao 1 , Tom Green 1 , Paul Race 1 , Adam Perriman 1
1 University of Bristol Bristol United Kingdom
Show AbstractMesenchymal stem cell (MSC) therapy represents a promising alternative in helping to repair the damage caused by cardiovascular disease due to their multipotency, self-renewal and proliferation capability. However, a main obstacle is the targeting of cells to the injury site, a process termed “homing”. Significantly, intracoronary or intramyocardial injection leads to microvascular occlusions with the majority of infused MSCs unable to localize to the infarcted myocardium, which increases the likelihood of producing lethal microemboli and reduces the efficiency of delivery. Accordingly, the synthesis of a new class of artificial cell membrane-binding proteins with chemotrophic homing properties will have far-reaching implications for applying MSCs in regenerative medicine.
In this work, a protein-polymer surfactant bionanoconjugate for MSC membrane binding is designed and constructed to adhere specifically to human cardiac fibronectin for in vivo heart-directed tissue repair. The bionanoconjugate structure has been developed by engineering protein surfaces with synthetic polymer surfactants displaying high membrane binding affinities. It consists of the human heart fibronectin binding domain of a bacterial protein (bp) fused to a supercharged fluorescent protein (scf), which is conjugated to surfactant molecules [1].
Our results have shown that the protein construct comprising scf-bp can be expressed and subsequently purified by immobilised metal affinity chromatography and size exclusion chromatography. scf-bp has been characterized by a variety of biophysical techniques to show that the dual properties of scf and bp have been maintained. Significantly, both the scf-bp precursor and the protein-polymer surfactant bionanoconjugate spontaneously associated with MSC membrane with no visual changes in cell morphology as observed by live-cell confocal microscopy. Notably, the bionanoconjugate displayed enhanced effective and persistent cell membrane insertion. Moreover, these surface-modified MSCs retained their ability to proliferate and to undergo multi-lineage differentiation. Static fibronectin adhesion assays showed high affinity binding of MSCs functionalized with either scf-bp or the bionanoconjugate, and flow adhesion assays performed directly on cardiac tissue samples in a biomimetic bioreactor system are currently being planned.
In conclusion, we report on a novel cell homing bionanoconjugate comprising protein-polymer surfactant complexes that imparts the homing characteristics of a bacterial protein to human MSCs. This new methodology is expected to contribute considerably to the rapidly emerging field of regenerative medicine.
[1] Armstrong, J.P; et al., Nat Commun. 2015. 6. 7405.
5:00 PM - BM3.2.07
Time-Dependent Effects of Confinement on Stem Cell Differentiation Using Degradable Protein Patterned Surfaces
Bethany Almeida 1 , Anita Shukla 1
1 Brown University Providence United States
Show AbstractStem cell geometry is known to guide differentiation (Kilian 2010, Shukla 2015). Previous studies exploring the effects of geometry have largely ignored the dynamic nature of differentiation (Lee 2016) by confining individual cells in specific geometries for a set period of time, after which the study is concluded, eliminating the beneficial role of cell-cell contacts. We hypothesize that the effect of geometry on differentiation is time dependent; thus, stem cells require specific durations of confinement for lineage specification. To investigate this hypothesis, we have developed intrinsically degradable protein patterns using 1-hexadecanethiol (HT) and 2-{2-[2-(2-{2-[2-(1-mercaptoundec-11-yloxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethanol (OEG6) self-assembled monolayers (SAMs) on nanometer scale gold (Au) surfaces.
We determined that altering Au thickness (4, 8, or 10 nm) affects the SAM degradation rate, with 8 nm Au leading to the most stable SAMs. X-ray photoelectron spectroscopy of OEG6 SAMs showed a significant decrease in the C-S bond peak (indicative of thiols) over time at 5 days for 4 nm Au (approximately 23% from day 1 to day 5) and 10 nm Au (approximately 22% decrease from day 0 to day 5), while 8 nm Au showed a significant decrease at 7 days (approximately 7% from day 0 to day 7). We suggest this variability is due to the metal roughness (Strulson 2013), which was determined to be 1.08 ± 0.25 nm, 1.15 ± 0.07 nm, and 1.57 ± 0.39 nm for 4, 8, and 10 nm Au, respectively. Although not statistically different, 4 and 10 nm Au show about 4-fold larger standard deviations than 8 nm Au, suggesting a very heterogeneous surface, while 8 nm Au surfaces were homogeneously smooth. Human mesenchymal stem cells (HMSCs) seeded on bare fibronectin-coated glass surrounded by bioinert OEG6 regions depicted migration behavior that correlated with XPS results. Migration was seen from the protein regions of initial confinement to the original OEG6 regions at 5 days for 4 and 10 nm Au and 7 days for 8 nm Au (53 ± 11.7%, 2.5 ± 0.04%, and 16.0 ± 12.5%, respectively). HMSCs seeded on SAMs with patterned HT surrounded by OEG6 showed similar time-dependent migration.
HMSCs were cultured on fibronectin-SAM patterns on 8 nm Au in populations and tested for adipogenesis with oil red O upon initial pattern degradation (day 7) and 1 week later. Cells on patterns demonstrated no statistically significant difference in oil red O staining at day 7 as compared to non-confined cells (p=0.83). However, at day 14, cells that were initially confined to fibronectin patterns exhibited a 3-fold increase in adipogenesis from day 7 (p=0.03) compared to no increase in non-patterned controls (p=0.40), suggesting that the initial confinement may have influenced adipogenesis. We are currently investigating adipogenic response of geometrically confined individual cells. We have demonstrated that geometric confinement primes differentiation of HMSCs even after SAM degradation.
5:15 PM - BM3.2.08
Theranostic Nanoparticles for Long-Term Cell Tracking and RNA Interference Therapy in Regenerative Medicine
Kai Li 1 2 , Bin Liu 1 , Ben Zhong Tang 3 , Heike E. Daldrup-Link 2
1 Institute of Materials Research and Engineering, A*STAR Singapore Singapore, 2 Department of Radiology and Molecular Imaging Program Stanford University Palo Alto United States, 3 Department of Chemistry, Institute of Molecular Functional Materials, Division of Biomedical Engineering, Division of Life Science, State Key Laboratory of Molecular Neuroscience Hong Kong University of Science and Technology Hong Kong Hong Kong
Show AbstractStem cell-based therapies have emerged as an important technique in regenerative medicine. To maximize the therapeutic outcomes, it has been increasingly important to understand the cell fate (e.g., migration, interaction, differentiation and location) of transplanted cells through advanced labeling and imaging techniques. In this work, we developed novel fluorescent probes that can outperform currently available quantum dot (QD) based probes in practice. Using conjugated polymer or conjugated oligomer with aggregation-induced emission characteristics as the fluorescent domain and biocompatible lipid-PEG derivatives as the encapsulation matrix, the obtained organic nanoparticles (NPs) have shown higher brightness, better stability in biological medium and comparable size and photostability as compared to their counterparts of inorganic QDs. Upon surface functionalization with a cell-penetrating peptide (Tat peptide), the organic NPs greatly outperform inorganic QDs in both in vitro and in vivo long-term cell tracing studies, which will be beneficial to answer crucial questions in stem cell/immune cell therapies. Additionally, the cell tracking study using human mesenchymal stem cell (hMSC) as a model suggests that the internalization of organic NPs shows negligible effect on th emigration, proliferation, differentiation, and secretomes of hMSCs. Thus, the Tat-functionalized organic NPs are ideal for labeling the cells that are not able to be feasibly transfected by green fluorescent protein or luciferase, for instance, the cardiomyocyte. Based on these results, we further developed a new miRNA-decorated Tat-organic NPs for imaging and delivery of miRNA for reverse remodeling of myocardial infarction (MI). In vitro results suggest that the delivery of miRNA into cardiomyocyte could be efficiently induced to re-enter cell cycle to promote the remodeling progress. The in vivo investigation is still ongoing to evaluate the therapeutic outcome of MI treatment in a mouse model. Besides the fluorescent probes, we also designed and demonstrated the application of iron oxide based stem cell trackers for tracking cells through magnetic resonance imaging.
5:30 PM - *BM3.2.09
'Self' Recognition by Macrophages—From Colloids and Materials to Tumors and Differentiation
Dennis Discher 1
1 University of Pennsylvania Philadelphia United States
Show AbstractParticles, implants, and cells of any type interact in vivo with the innate immune system, especially phagocytes that try to ‘eat’ everything. At the same time, ‘Self’ cells are spared due to a polypeptide found on all cells that marks cells as ‘Self’ and limits phagocytic clearance in vitro and in vivo – even when displayed on nanoparticles (1). These findings have been extended to engineered viruses and implants, and based on these studies, an alternative approach to tumor therapy has also emerged. We use the innate immune cell’s ability/tendency to migrate into injury sites (such as tumors) and find three features are required for therapy: 1) highly phagocytic cells that are 2) blinded to ‘Self’ and 3) activated to ‘eat’ cancer cells. Solid tumors can thus be safely and effectively shrunk by phagocytes for at least 1-2 weeks, even after chemotherapy has failed, but profiling of macrophages pulled from tumors provide evidence of differentiation, at least some of which is consistent with differentiation directed by matrix elasticity as shown with hydrogels.
References
(1) Rodriguez, P.L.; Harada, T.; Christian, D.A.; Pantano, D.A.; Tsai, R.K.; and Discher, D.E. Minimal 'Self' peptides that inhibit phagocytic clearance and enhance delivery of nanoparticles. Science 2013 339: 971-975
BM3.3: Poster Session I
Session Chairs
Tuesday AM, November 29, 2016
Hynes, Level 1, Hall B
9:00 PM - BM3.3.01
Functional Nucleic Acid Nanotechnology for Programmable Gene Regulation
Boyoung Kim 1 , Minjeong Kim 1 , Bora Jang 1 , Hyokyoung Kwon 1 , Hyukjin Lee 1
1 Ewha Womans University Seoul Korea (the Republic of)
Show Abstract
Nucleic acid nanotechnology has drawn tremendous attention, since it can provide a precise tool for building multi-dimensional structures with a defined size, shape, and surface property. This is particularly of interest to the field of gene delivery. For targeted delivery of nanoparticles, conventional nanoparticles such as liposomes and polymeric micelles are heterogeneous in size, composition, and surface charge leading to suboptimal performance, lack of tissue specificity and potential toxicity. Here, we show that self-assembled functional nucleic nanostructures with a well-defined size and structure can serve as a multi-functional platform to induce efficient RNA interference, mRNA expression, and CRISPR/Cas9 mediated gene editing. For the synthesis of functional RNA structures, we have utilized the rolling circle transcription of pre-designed DNA template to produce a large quantity of functional RNAs. For programmable and simultaneous gene silencing, we have design the dicer substrate RNA nanostructures to regulate three different fluorescent proteins (GFP, RFP, and BFP) in the target cells. Using three arm junction RNA nanostructures, we were able to show programmable regulation of fluorescent protein expression to generate 8 different fluorescent colored cells. For mRNA expression, in vitro transcription (IVT) of mRNA has been carried out to express three distinct fluorescent proteins (GFP, RFP, and BFP) in target cells. Long term expression can be obtained with the programmable assembly of mRNA into defined RNA structures. Finally, delivery of CRISPR/Cas9 with RNA nanostructures was attempted. Three different gRNAs can be co-delivered with Cas9 to show simultaneous gene editing, gene regression, gene induction.
9:00 PM - BM3.3.03
Nanofibrous Nerve Conduits with Pre-Seeded with Bone Marrow Stromal Cells and Pre-Cultured in Bioreactors for Enhancing Peripheral Nerve Regeneration
Xiaojun Yu 1 , Gan Zhou 1
1 Steven Institute of Technology Hoboken United States
Show AbstractPeripheral nerve injury(PNI) can cause dysfunction in movement and sensation. Synthesized nerve guide conduit with Schwann Cells(SCs) can help peripheral nerve regeneration, however poor accessibility of SCs and lack of full coverage on the conduit can lead to failure of nerve regeneration across long gaps. In order to overcome these limitations, we have seeded bone marrow stromal cells(BMSCs) onto nanofibrous nerve conduits in rotating bioreactors for peripheral nerve regeneration.
We have designed a novel polycaprolactone spiral conduit with electrospun aligned nano-fiber to support cell attachment and guide cell migration and neurite extension. Instead of SCs, the BMSCs were isolated from Rat femur and tibia as the cell source. The BMSCs can be obtained with standardize medical procedure and have the potential to differentiate into SCs-like cells for promoting nerve regeneration. The BMSCs were seeded onto nanofibrous nerve conduits with different seeding densities to determine the preferable seeding density for cell proliferation. Pre-seeded BMSCs were cultured with differentiation medium which allowed BMSCs to differentiate into Bone Marrow Stromal Cells derived-Schwann Cells-like Cells (BMSC-SCs). To overcome the drawback associate with traditional static cultures, the rotating bioreactors were adopted to promote cell proliferation and differentiation on nerve conduits. The nerve conduits were pre-seeded with BMSCs inside a bioreactor with different rotating speeds to determine the preferable rotating speed for cell proliferation and differentiation. In order to investigate the ability of the nerve conduits in promoting neurite extension, PC12 cells were cultured onto the nerve conduits, and neurite extension was characterized by immunostaining with anti-neurofilament and imaging with confocal microscopy.
The results indicated that the preferable seeding density was 5x104 cells/cm2 through MTS assay and the immunostaining with anti-S-100. The immunostaining showed that cells formed much thicker layers in bioreactor group as compared to the static control. qRT-PCR showed increase in Neuregulin-1 (Nrg1) expression level in bioreactor group, whereas 16rpm group showed the highest expression level, which confirmed bioreactor’s role in facilitating cell differentiation and the preferable rotating speed for cell differentiation was 16 rpm. Therefore, the bioreactors were helpful to promote the differentiation of BMSCs into BMSC-SCs. The best neurite extension was observed in the bioreactor group, which confirmed the ability of the nerve conduit to promote neurite outgrowth through pre-seeding BMSCs and pre-culturing in bioreactors. The nanofibrous nerve conduits pre-seeded with BMSCs and pre-cultured in bioreactors have the potential to repair PNI.
Acknowledgement: This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Orthopaedic Research Program under Award No. (W81XWH-13-1-0320).
9:00 PM - BM3.3.04
The Effects of Multi-Scales Surface Mechanics Stimuli on Dental Pulp Stem Cell Behaviors
Linxi Zhang 3 , Chung-chueh Chang 1 , Miriam Rafailovich 3 , Marcia Simon 2
3 Department of Materials Science and Engineering Stony Brook University Stony Brook United States, 1 Advanced Energy Research and Technology Center Stony Brook University Stony Brook United States, 2 Department of Oral Biology and Pathology School of Dental Medicine Stony Brook United States
Show AbstractAdult dental pulp stem cells (DPSCs) has been shown the capabilities to be induced to differentiate into either bone, dentine, or nerve tissue through different environment signals, which is mostly accomplished by the addition of chemical inducer in the growing media. In the past few years, another signal, the mechanical stimuli from substrates, has been drawing a lot of attentions. Our group has previously showed that DPSCs can be induced to differentiate into biomineralized bone, simply by altering the mechanics of the substrates on which they are cultured. The principal of entangled polymer surface confinement (EPSC) was applied to obtain varied surface mechanics by spin-casting different thicknesses of monodispersed polybutadiene (PB). However, cells, in this case, are grown on the surface with homogeneous mechanics and it hardly represents the real conditions in vivo, which is heterogeneous. We therefore, investigate the cell behaviors on surfaces with heterogeneous mechanics. In brief, imprinted silicon wafers with multi-scales pattern, from nano- to micro- sizes, were fabricated by Focused Ion Beam (FIB) milling and PB was then spun cast on them. The polymer flows and covers the pattern, consequently forming areas with varying thickness and providing different mechanics. DPSCs were then plated on those substrates and were incubated up to 28 days. Cell behaviors including cell mechanics, cell morphology and biomineralization were characterized by shear modulation force microscopy (SMFM), confocal microscopy, and scanning electron microscopy with energy dispersive x-ray spectrum (SEM/ EDS). The results show that depending on the scale sizes, cells have ability to sense, follow and reflect the underlying substrate mechanics. With the increasing of the scale sizes, cell mechanics began to split into two readings and biomineralized deposits were ceased to observe, suggesting the pattern sizes and fluctuations of substrate mechanics might have great impacts on DPSC behaviors.
9:00 PM - BM3.3.05
Protein Surface Modifications for Tissue Engineering and Regenerative Medicine
Robert Deller 1 , James Armstrong 1 , Adam Perriman 1
1 Cellular and Molecular Medicine University of Bristol Bristol United Kingdom
Show AbstractOvercoming hypoxia remains a significant challenge for the engineering of large 3D osteocyte and chondrocyte tissues for autologous transplantation. This is due to the inherently low diffusion coefficient of oxygen in water in conjunction with an inability to successfully mimic in vivo vascularization. This in turn limits the size of viable engineered constructs and subsequent integration with host tissues. To address this problem we have developed surfactant conjugated haem-protein nanoconstructs capable of binding to the surface of human mesenchymal stem cells (hMSCs). These nanoconstructs provide an additional reservoir of oxygen and thereby limit the onset of central zone necrosis typically observed in large scale tissue engineering.[1] Moreover, chemical modification (cationization) of the protein surface has been shown to increase the equilibrium oxygen affinity of myoglobin, meaning oxygen is only released in response to the onset of hypoxia. Subsequent conjugation of the cationized myoglobin with an amphiphilic polymer surfactant instills high affinity of the construct for the cytoplasmic membrane of hMSCs in 2D cultures without impacting differentiation or inciting cytotoxicity. Significantly, the primed hMSCs display the construct over several days, and osteogenic and chondrogenic based tissue engineering using fibronectin-coated PGA scaffolds show a reduction in the extent of central zone necrosis, yielding larger and higher quality constructs. This new approach to cell functionalization is facile, and accordingly, new research involving the rational reengineering of surfaces of relevant proteins such as growth factors is currently underway.
[1] Armstrong, J. P. K., Shakur, R., Horne, J. P., Dickinson, S. C., Armstrong, C. T., Lau, K., Kadiwala, J., Lowe, R., Seddon, A., Mann, S., Anderson, J. L. R., Perriman, A. W., and Hollander, A. P. (2015) Artificial membrane-binding proteins stimulate oxygenation of stem cells during engineering of large cartilage tissue. Nat Comms 6, 7405.
9:00 PM - BM3.3.06
Enzyme–Polymer Surfactant Nanoconstructs for the Detoxification of Organophosphorous Compounds
Ben Carter 1 , Eleanor Campbell 2 , Colin Jackson 2 , Adam Perriman 1
1 University of Bristol Bristol United Kingdom, 2 Australian National University Canberra Australia
Show AbstractWe report on the synthesis and characterisation of a new class of artificial cell membrane binding enzyme bioconjugates for the detoxification of membrane-solubilised organophosphorous compounds (OPs). These have been developed in response to the failure of a single-dose enzyme therapy for OP poisoning, which has been shown to be effective for only a limited time after treatment. This lack of efficacy is thought to be related to the lipophilic nature of OPs, resulting in partitioning into cell membranes and fat deposits, causing persistence and leaching after the enzyme has been cleared from the system. The constructs have been developed using our new methodology to produce protein–polymer surfactant hybrids, and our recent study has shown significant cell membrane persistence. Here, we exploit electrostatic interactions between charged protein residues and headgroups of ionic polymer surfactants to produce bioconjugates with an amphiphilic corona that both stabilises the protein structure and allows insertion into cell membranes. We modify OpdA from Agrobacterium radiobacter, as it has demonstrated detoxifying activity against a broad range of OP-based pesticides and nerve agents, and hydrolyses paraoxon near the diffusion limited rate. Significantly, the constructs interact with cell membranes and adipocyte lipid-droplets whilst maintaining near-native structure and activity. Neutron reflectometry data show the bioconjugate partitioning into a model bilayer, forming layers at the membrane surface. Confocal fluorescence microscopy with dye-labelled constructs and fluorescent substrates demonstrates construct–membrane binding with continuous substrate turnover.
9:00 PM - BM3.3.07
Mitochondria-Targeted Ceria Nanoparticles as Antioxidants for Alzheimer’s Disease
Hyek Jin Kwon 1 2 , Moon-Yong Cha 3 , Dokyoon Kim 2 , Dong Kyu Kim 3 , Min Soh 1 2 , Kwangsoo Shin 1 2 , Inhee Mook-Jung 3 , Taeghwan Hyeon 1 2
1 School of Chemical and Biological Engineering and Institute of Chemical Processes Seoul National University Seoul Korea (the Republic of), 2 Center for Nanoparticle Research Institute for Basic Science Seoul Korea (the Republic of), 3 Department of Biochemistry and Biomedical Sciences Seoul National University College of Medicine Seoul Korea (the Republic of)
Show AbstractMitochondrial oxidative stress is a fundamental pathologic factor in neurodegenerative diseases, including Alzheimer’s disease. Extraordinary production of reactive oxygen species (ROS), resulting from mitochondrial dysfunction, can induce neuronal cell death. Ceria (CeO2) nanoparticles are known to perform as strong and recyclable ROS scavengers by shuttling between Ce3+ and Ce4+ oxidation states. Consequently, targeting ceria nanoparticles selectively to mitochondria might be a promising therapeutic approach for neurodegenerative diseases. Here, we report the design and synthesis of triphenylphosphonium-conjugated ceria nanoparticles that localize to mitochondria, reduce mitochondrial oxidative stress and suppress neuronal death in a 5XFAD transgenic Alzheimer’s disease mouse model. The triphenylphosphonium-conjugated ceria nanoparticles mitigate reactive gliosis and morphological mitochondria damage observed in these mice. Altogether, our data indicate that the triphenylphosphonium-conjugated ceria nanoparticles are a potential therapeutic candidate for mitochondrial oxidative stress in Alzheimer’s disease.
9:00 PM - BM3.3.08
Carbonaceous Wires for Neural Interfacing
Nicholas Apollo 1 2 , Jonathan Jiang 2 , Azadehsadat Mirabedini 3 , Javad Foroughi 3 , Chi Lik Warwick Cheung 2 1 , Sebastien bauquier 2 1 , Gordon Wallace 3 , Steven Prawer 1 , Mark Cook 1 2 , David Nayagam 2 , David Garrett 1 2 , Richard Williams 4 , Shou Chen 4
1 University of Melbourne Parkville Australia, 2 Bionics Institute Melbourne Australia, 3 Intelligent Polymer Research Institute University of Wollongong Wollongong Australia, 4 Department of Anatomical Pathology St. Vincent's Hospital Fitzroy Australia
Show AbstractTo realize the full clinical and research potential of invasive, direct communication with the nervous system, a new generation of neural interfacing devices is required. Such devices will need at least the following qualities: durability, biocompatibility, and sensitivity throughout a chronic implantation period. Carbonaceous materials possess a wide variety of physical and chemical properties. Increased interest in carbon nanomaterials such as carbon nanotubes (CNT) and graphene have led to several manufacturing techniques and composite materials. Within this family of composite materials, soft, flexible, and electrically-conductive papers, yarns, and fibres possess several properties that make them attractive options as the electrode component of neural interfacing devices. In this work, we investigate the ability of CNT/graphene hybrid yarns and reduced graphene oxide (rGO) fibers to achieve bi-directional communication with the nervous system. The aforementioned materials are used to fabricate freestanding neural probes that are inserted into deep brain areas of an epilepsy animal model using a novel drawing lithography technique. Continuous seizure monitoring is achieved with these materials, along with in vivo electrochemical characterization. Finally, histological techniques are utilized to assess the safety of both the surgical insertion and the health of the electrode-tissue interface.
9:00 PM - BM3.3.09
Promoting Cell Adhesion and Antibacterial Properties via Layer-by-Layer Films
Shanshan Guo 1 , Xian Jun Loh 2 , Dominik Janczewski 3 , Koon Gee Neoh 1
1 National University of Singapore Singapore Singapore, 2 Institute of Materials Research and Engineering Singapore Singapore, 3 Warsaw University of Technology Warsaw Poland
Show AbstractThe control of cell adhesion to an implant surface is fundamental in many processes such as tissue regeneration, wound healing and immune responses. For example, enhancing fibroblast cell adhesion would promote connective tissue integration to the implant and improving vascularity. However, many biomaterials used for tissue regeneration lack cell-adhesiveness and, in addition, are prone to bacterial colonization. Therefore in this study, we develop surface modification strategies that can promote cellular growth while simultaneously resisting bacterial adhesion and biofilm formation. To this goal, the layer-by-layer (LbL) technique, consisting of self-assembled, sequential adsorption of oppositely charged polymers is adopted to engineer surface properties. Our study shows that by tuning surface charge, wettability and crosslinking degrees using LbL films built from commercial polyethyleneimine and derivatives from polymaleic acid, fibroblast and bacterial cell adhesion can be controlled. Therefore, the optimum combination of surface properties can be evaluated in terms of promoting fibroblast cell adhesion and anti-bacterial adhesion/biofilm formation.
9:00 PM - BM3.3.10
Recovery of Physiological Impulse Propagation Across Myocardial Infarction via SiC Semiconducting Nanowires
Paola Lagonegro 1 , Stefano Rossi 2 , Francesca Rossi 1 , Silvana Pinelli 2 , Rossella Alinovi 2 , Michele Miragoli 2 , Giancarlo Salviati 1
1 IMEM-CNR Parma Italy, 2 Department of Clinical and Experimental Medicine University of Parma Parma Italy
Show AbstractMyocardial infarction is a result of a structural remodelling of a prolonged ischemic region, where reparative fibrosis generated by (myo)fibroblasts (MFBs) are the main substrates for initiating cardiac arrhythmia (1,2). Fibrosis, in turn, favours the initiation and perpetuation of arrhythmias by producing collagenous septa which separate bundles of cardiomyocytes (CMs) over distances up to several millimetres, thus inducing structural discontinuities at cellular level (3,4). Fibrotic laminae act as electrical insulators inducing interstitial resistive discontinuities, impeding the spread of electrical activity and resulting in non-uniform conduction (4,5) or zig-zag activation of the myocardium (6) with consequent generation of arrythmogenesis. Here we present an innovative approach to re-instate impulse propagation throughout an infarcted area by using biocompatible (9) and hemocompatible (10) cubic SiC semiconducting nanowires (NWs) (7,8) able to provide a proper conduction path, so resolving the conduction defects. To test whether SiC NWs networks can electrically couple two separate CMs bundles, we seeded HL-1 CMs on glass coverslips on which a drop of collagen containing 80 μg/ml of SiC-NWs was positioned. Cells were stained with voltage sensitive dye Di-8-ANEPPS and impulse propagation where assessed optically. Fascinatingly, separated cells (480 μm distance) seeded on the top of the area covered with NWs beat synchronously, with an action potential that propagate via NWs in the range of 400 mm/sec with only a minimal delay (1.2 ms). It must be stressed that the normal impulse propagation in this cell line is about 80-100 mm/s, while in-vivo is about 400-500 mm/s, thus we are confident that this pilot experiment can be easily repeated in primary excitable cardiac cells. Further we also tested whether SiC NWs can support contraction machinery in the bundles of CMs once electrically synchronized. Cells were loaded with Ca2+ indicator Fluo-4 AM and the calcium transient parameters were also optically monitored, revealing a synchronicity of Ca2+ in the CMs bundles, over distance. SEM analysis confirmed that the CMs bundles were physically connected with SiC-NWs. Our results suggest that coating infarct regions with semiconducting SiC NWs (which cannot be influenced by MFBs), instead of an excitable cell graft, may avoid both conduction blocks and discontinuities exerted by a heterocellular coupling. In vivo tests on rats underwent cardiac cryoinjury treated with SiC-NWs are currently under investigation.
References
1 Heart Rhythm 2, 650, (2005).
2 J Am Coll Cardiol 35, 569-582, (2000).
3 Circulation 122, 1258-1264, (2010).
4 Am J Physiol 269, H571-H582, (1995).
5 PACE 20, 397-413, (1997).
6 Circ Res 58, 356-371, (1986).
7 Biomedical microdevices 15, 353-368, (2013).
8 Nano letters 14, 4368-4375, (2014).
9 Scientific reports 5, 7606, (2015).
10 Silicon Carbide Biotechnology; Saddow, S. E., Ed.; Elsevier Science 209 (2012)
9:00 PM - BM3.3.11
Silicon Oxycarbide Nanowires as a Viable Cell Scaffold for Biomedical Applications
Paola Lagonegro 1 , Francesca Rossi 1 , Carlo Galli 2 1 , Arianna Smerieri 2 , Rossella Alinovi 3 , Silvana Pinelli 3 , Tiziano Rimoldi 4 , Giovanni Attolini 1 , Claudio Macaluso 2 1 , Guido Macaluso 2 1 , Steven Saddow 5 , Giancarlo Salviati 1
1 IMEM-CNR Parma Italy, 2 Department of Biomedical, Biotechnological, and Translational Sciences University of Parma Parma Italy, 3 Department of Clinical and Experimental Medicine University of Parma Parma Italy, 4 Physics and Earth Science Department University of Parma Parma Italy, 5 Engineering Department University of South Florida Tampa United States
Show AbstractTissue regeneration requires biomaterials capable to provide a scaffold for ingrowing cells and replace the missing extracellular matrix (ECM) (1). Silicon oxycarbide (SiOxCy) films were proven to increase platelet aggregation and activation, thereby promoting the creation of an adequate provisional matrix and subsequent wound healing (2). Moreover, SiOxCy can be easily engineered through functionalization and decoration with macro-molecules and nanoparticles (3-5), which make it an ideal platform for bioactive approaches.
Nanowires (NWs) are 1D structures that can be arranged in 3D bundles, which strikingly resemble the organization of ECM fibrils and could therefore be a promising candidate for artificial matrices in different clinical situations (6).
The aim of the present work is to show that CVD-grown SiOxCy NWs are a viable scaffold candidate for the regeneration of connective tissue by assessing the responses of a fibroblastic cell line to NWs-coated substrates and the effects of such NW coating on platelet activation.
We evaluate the cytotoxicity of the SiOxCy NWs networks and their effect on cell adhesion and proliferation, by indirect and direct contact tests according to ISO 10993-5 guidelines, and on platelet activation. In-vitro tests on L929 mouse fibroblasts cells show that NWs do not release cytotoxic species and may represent a suitable platform for cell growth. In addition we demonstrate the capability of fibroblasts to reorganize the NWs network, adapting it to their needs. Blood tests with platelet-rich plasma and dynamic blood coagulation tests show that SiOxCy NWs induce platelet activation and consequently activate metabolic cascades that are conductive to tissue repair. These results indicate that SiOxCy NWs network are a promising biomaterial for implantable scaffolds for tissue regeneration. In vivo tests are in progress.
References
(1) Journal of cell science 2010; 123: 4195-4200.
(2) Acta biomaterialia 2005; 1: 583-589.
(3) Angewandte Chemie 2005; 44: 6282-6304.
(4) Nanoscale research letters 2012; 7: 680.
(5) Scientific reports 2015; 5: 7606.
(6) Biomaterials 2012; 33: 5013-5022.
9:00 PM - BM3.3.12
PEDOT:PSS Substrates Interface the Development of Cultured Neuronal Networks with Reduced Neuroglia Response
Giada Cellot 2 , Paola Lagonegro 1 , Giuseppe Tarabella 1 , Denis Scaini 3 2 , Filippo Fabbri 1 , Francesca Rossi 1 , Salvatore Iannotta 1 , Maurizio Prato 4 , Roberto Mosca 1 , Claudio Macaluso 5 1 , Laura Ballerini 2 , Giancarlo Salviati 1
2 Department of Neuroscience International School for Advanced Studies Trieste Italy, 1 IMEM-CNR Parma Italy, 3 ELETTRA Trieste Italy, 4 Department of Chemistry and Pharmaceutical Sciences University of Trieste Trieste Italy, 5 Department of Biomedical, Biotechnological and Translational Sciences University of Parma Parma Italy
Show AbstractElectrodes used for brain-machine interface technology should minimize tissue damage and reduce glial reactions, frequent inflammatory responses to chronically implanted neural electrodes. Conductive polymers offer the valuable opportunity to tune material properties to reduce gliosis (Lempka et al., 2009 J. Neural Eng. 6:046001 doi:10.1088/1741-2560/6/4/046001). Here we investigate the biocompatibility of poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) doped with different amounts of ethylene glycol (EG) when interfaced with primary cells from rat hippocampus.
To assess neuronal network viability and synaptic activity cells were cultured in vitro on PEDOT:PSS films and subsequently their morphology characterised by immunofluorescence and scanning electron microscopy (SEM) and in their function by single cell electrophysiology. Glial reactivity to PEDOT:PSS substrates was investigated by immunofluorescence as well. SEM analysis revealed that neurons grown on PEDOT:PSS were similar to controls (Poly-L-ornithine treated glass substrate), showing healthy morphology, characterised by many neurites extending from the soma and forming arborisations. Immunofluorescence experiments show that cell density on PEDOT:PSS substrates was similar to that of controls. Using single cell patch clamp technique, we characterized neuronal features. Membrane passive properties, conventional indicators of neuronal health (Carp, 1992 J.Neurophysiol. 68,1121–1132; Djuric et al., 2015 Neurobiol.Dis. 76,37–45.doi: 10.1016/j.nbd.2015.01.001; Gao et al., 2015 Neurosci.Lett. 591, 138–143.doi:10.1016/j.neulet.2015.02.043) and the spontaneous activity of the network, measured in terms of amplitude and frequency of spontaneous post synaptic currents (PSC), were similar in PEDOT:PSS and control. Different ages (1 and 3 weeks) and doping condition (1% and 3% of EG) gave similar results.
Notably, PEDOT:PSS substrates down-regulate glial cell proliferation, with a partial reduction detectable starting from 1 week of culturing. GFAP-positive glial cells shows a clear reduction in cell density after being interfaced for 3 weeks on PEDOT:PSS, with respect to controls (Cellot et al., 2016 Frontiers in Neuroscience 9 521 doi: 10.3399/fnins.2015.00521). Glial cells sizes appeared similar among all tested groups. Interestingly, an increased number of glial cells and enhanced glial cellular size are usually associated with pathological states thus, the fact PEDOT:PSS apparently reduces glial response is a relevant feature for exploiting use of this polymer to interface nerve cells. In conclusion, our findings suggest that PEDOT:PSS is an attractive candidate for the design of new implantable electrodes, controlling the extent of glial reactivity without affecting neuronal viability and function.
9:00 PM - BM3.3.13
A Novel Low Temperature PCL Based 3D Printing Resin for Craniofacial Repair
Max Lerman 1 , Jesse Placone 2 , Anjana Jeyaram 2 , Anthony Chiu 3 , Madelyn Golding 2 , Gregory Laslo 2 , Steven Jay 2 , John Gillen 4 , John Fisher 2
1 Materials Science and Engineering University of Maryland College Park United States, 2 Fischell Department of Bioengineering University of Maryland College Park United States, 3 Chemical and Biomolecular Engineering University of Maryland College Park United States, 4 Materials Measurement Science National Institutes of Standards and Technology Gaithersburg United States
Show AbstractCraniofacial bone is a particularly difficult tissue to engineer due to the demanding geometric complexity, mechanical stiffness, and cellular infiltration requirements for the biomaterial scaffold. An appealing solution for the fabrication of craniofacial bone scaffolds is 3D printing. Although 3D printing allows for the rapid manufacture of complex custom scaffolds, a suitable biomaterial must first be identified. Polycaprolactone (PCL) remains one of the most widely utilized 3D printing materials for bone tissue engineering, driven by the relative ease of fabrication, non-cytotoxic hydrolytic degradation mechanism, and low material cost. However, while structurally sound constructs can be fabricated, unmodified PCL requires a high (>70°C) extrusion temperature, degrades slower than natural remodeling, and, due to its solid surface architecture, prevents significant cell infiltration. Therefore, in order to realize complex craniofacial bone tissue engineering scaffolds, we have developed a novel biocompatible, low temperature, PCL based resin.
Results indicate PCL viscosity (and printing temperature reduction) was effectively lowered by solubilizing PCL in dimethyl sulfoxide (DMSO). Extrusion 3D printing yields a material with similar mechanical and cytotoxic properties as unmodified PCL. Under identical tensile conditions, the novel printed and processed resin has a Young’s modulus of 170.4±11.5 MPa (n = 7) compared to unmodified PCL at 263.1±9.7 MPa (n = 10). XTT analysis of L929 fibroblast cells demonstrates via a one-way ANOVA at the 95% confidence interval a lack of statistical difference between the printed and processed resin, a known non-cytotoxic control (high density polyethylene), and control cultures after 24 hours. Printed objects are processed post-printing to remove the cytotoxic solvent under freeze-drying conditions, yielding a polymer substrate with microtopology, presenting a unique surface with significant similarities to trabecular bone. Compounding with hydroxyapatite, β-tricalcium phosphate, and/or alginate microparticles containing growth factors yields a dispersed suspension. Rather than traditional surface coating, compounding creates a resin with sustained bioactive release accompanying material degradation. In this manner, additives become distributed across the printed object and allow for continued release, encouraging the development of a biomimetic craniofacial bone environment within the printed construct. This resin demonstrates the first possibility to print temperature-sensitive bioactive molecules embedded within PCL, greatly enhancing the clinical potential of bioactive PCL for craniofacial bone tissue engineering.
9:00 PM - BM3.3.14
Fish Gelatin—Layered Silicates Micro/Nano Composite Hydrogels for Bioadhesive and Sealant Application
Oded Pinkas 1 , Meital Zilberman 1 2
1 Department of Materials Science and Engineering, Faculty of Engineering Tel Aviv University Tel Aviv Israel, 2 Tel Aviv University Tel Aviv Israel
Show AbstractBioadhesives are polymeric hydrogels that can stick to a tissue after crosslinking and are an essential element of nearly all surgeries performed worldwide. They are divided into three groups of devices: (a) Hemostats- formed blood clot (b) Adhesive-attached tissue together. (c) Sealant- creates a sealing barrier that prevents the leakage of gas or liquid from a tissues or suture lines. Although several bioadhesives are commercially available, none of them are ideal. The main limitation in current tissue adhesives is the trade-off between biocompatibility and mechanical strength in particular, wet environments in presence of blood.
Our novel bioadhesive is based on the natural polymers gelatin (cold-water fish) and alginate, crosslinked by carbodiimide (EDC). Two types of hemostatic agents with a layer silicates structure, montmorillonite (MMT) and kaolin were loaded in order to improve the sealing ability in a hemorrhagic environment. The effect of the sealant's components on the mechanical strength was studied in three different methods - burst strength, lap-shear and compression. The bioadhesive's viscosity and gelatin time were evaluated.
The sealant formulation which contains 400 mg/ml gelatin, 10 mg/ml alginate and 20 mg/ml EDC was found as an optimal formulation, enabling burst strength of 387 mmHg. While incorporation of kaolin increased the burst strengths in 25%, the MMT increased the burst strength in 50% even though the concentration of MMT was half that of kaolin. The XRD of MMT loaded bioadhesives indicate the formation of nanocomposite hydrogel compared to microcomposite for kaolin bioadhesive. A qualitative model that describes the effect of the bioadhesive’s parameters on the cohesive and adhesive strength was developed. This research clearly shows that the incorporation of kaolin and MMT in gelatin-alginate surgical sealants is a very promising novel approach for improving the bonding strength and physical properties of surgical sealants for use in hemorrhagic environments.
9:00 PM - BM3.3.15
Blood Compatibility Assessments of Biopolymers for Layer-by-Layer Assembled Multilayer Nano-Film
Hyejoong Jeong 1 , Jangsun Hwang 2 , Hwankyu Lee 3 , Paula Hammond 4 , Jonghoon Choi 2 , Jinkee Hong 1
1 Chemical Engineering and Material Science Chung-Ang University Seoul Korea (the Republic of), 2 Integrative Engineering Chung-Ang University Seoul Korea (the Republic of), 3 Chemical Engineering Dankook University Seoul Korea (the Republic of), 4 Koch Institute for Integrative Cancer Research Massachusetts Institute of Technology Cambridge United States
Show AbstractLayer-by-layer (LbL) assembly is a promising technique manipulates biomaterials as nanometer level of thin films for a wide range of biomedical applications such as drug release and surface energy control [1-3]. Basically, LbL assembly technique fabricates nano- to micro- multilayer films on a substrate in the way of alternating adsorption of polymers via electrostatic interaction, hydrogen bonding, and other interactions [4]. Moreover, since films are assembled in the water in the ambient condition, it is a useful biomedical platform preserving activities of functional species including drugs, proteins, and nucleic acids. For the purpose of multilayer nanofilms for biomedical use, a majority of studies have been reported cytotoxicity results using a few kinds of cells including uncharacterized, targeted, and derived from animals. However, those approaches overlooked ‘early stage of toxic effects of films on human cells’[5-7].
The purpose of LbL films for biomedical use is in vivo system. In the beginning of in vivo test, early stage of blood compatibility of films should be considered. Otherwise, films could have harmful effect on the blood cells or be removed by immune system before arriving at target site. Here we investigated cytotoxicity of extensively used biopolymers by red blood cells(RBC) and peripheral mononuclear cells(PBMCs) originated from human blood. A hemolysis assay refers to leaking of intracellular contents from the RBCs caused by acute cytotoxicity damaging the cell membrane. Using PBMCs which are a group of immune cells including T cells, B cells, NK cells, and monocytes, we implemented cell viability and AnnexinV-Fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay to determine cellular death mechanism indirectly.
We evaluated cytotoxicity of twenty kinds of biopolymers and ten of combinations used for building blocks of LbL film according to the above three assays. The overall results showed that most polycations are less blood compatible than the other polymers. Positive charge could have harmful effect on the cells due to electrostatic attractive force with cell membrane. Even though several polycations caused cell death at certain concentration, they didn’t induce necrosis to cells within 48 h. Moreover, we determined cytotoxic effects of polymers depending on the ionic charges by molecular dynamics simulations. In addition, combinations and multilayer nano-films indicated less cytotoxicity than individual polymers. The reason is that charges of the polymers are offset in the agglomerates or films. We would like to provide cytotoxicity and safety information of biopolymers for researchers who consider in vivo test in the polymer society as well as LbL.
[1] Adv. Mater. 2006, 3203-3224.
[2] Chem. Soc. Rev. 2011, 19-29.
[3] Materials Today 2012, 196-206.
[4] Chem. Commun. 2007, 1395-1405.
[5] Carbohydr. Polym. 2014, 298-304.
[6] Acta Biomater. 2014, 5116-5127.
[7] Biomaterials 2014, 7929-7939.
9:00 PM - BM3.3.16
Synthesis and Characterization of Amino Acid-Functionalized Calcium Phosphate Nanoparticles for siRNA Delivery
Feray Bakan 1 , Goknur Kara 2 , Melike Cokol Cakmak 3 , Meltem Sezen 1 , Murat Cokol 3 , Emir Baki Denkbas 2
1 Sabanci University Nanotechnology Research and Application Center Istanbul Turkey, 2 Hacettepe University, Department of Biochemistry Ankara Turkey, 3 Sabanci University, Faculty of Engineering and Natural Sciences Istanbul Turkey
Show AbstractRecently, targeted therapies in spite of conventional approaches in cancer therapy are becoming more attractive due to the fact that they do not harm healthy cells and have high selectivity. Therefore, siRNA therapy is promising which aims effecting at mRNA level and which is expected to have high selectivity properties. However, a major limitation in the therapeutic use of siRNA is its rapid degradation in plasma and cellular cytoplasm, resulting in short half-life. In addition, as siRNA molecules cannot penetrate into the cell efficiently, it is required to use a carrier system for its delivery. Most alternative methods make use of the fact that nanoparticles are taken up by living cells, and can therefore be used as carriers for biomolecules like DNA or RNA oligonucleotides. Compared to other nanoparticles, calcium phosphates can be used for siRNA delivery due to their advantages, such as; better biodegradability and biocompatibility in biological systems, easy synthesis, high chemical affinity to DNA and RNA.
In this study, three calcium phosphate nanoparticles having different chemical and morphological properties synthesized in a controlled manner and the effect of particle characteristics on siRNA loading efficiency was analyzed particularly and comparatively. For increasing the affinity of negatively charged siRNA with nanoparticles, different amino acids were used for functionalization. For structural characterization, X-ray diffraction (XRD) and Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) techniques were used. Surface charge measurements were conducted using a Zetasizer at physiological pH. The morphology of the particles were characterized by Scanning Electron Microscopy (SEM). According to results, decorating the nanoparticles with various amino acid structures had a drastic impact on siRNA binding. Besides, the nanoparticles without any functionalization also exhibited considerable affinity to the siRNA molecules.
9:00 PM - BM3.3.17
Meta-Compliance and Energy Dissipation in Cactus-Based Solids for Defence and Bone Tissue Engineering Applications.
Ioannis Zampetakis 1 2 , Fabrizio Scarpa 1 , Adam Perriman 2 , Alistair Hetherington 3
1 Advanced Composites Centre for Innovation and Science University of Bristol Bristol United Kingdom, 2 School of Cellular and Molecular Medicine University of Bristol Bristol United Kingdom, 3 School of Biological Sciences University of Bristol Bristol United Kingdom
Show AbstractNatural fibre biocomposites have attracted significant research and industrial interest due to their high specific properties, low density, biodegradability and cost effectiveness1. Cactus fibres, specifically, demonstrate unique deformation mechanisms and energy dissipation properties under cyclic flexural loading as a fibre reinforcement of a polyester matrix yielding a significantly high flexural to axial stiffness ratio, a 7 fold increase in the flexural stiffness of the material and a 4.2 fold increase in the energy dissipation per volume of the matrix2.
A multiscale materials characterization methodology was developed to assess the morphological and mechanical properties of the cacti fibres at a nano, micro, meso, and macro scale through X-ray CT, AFM, quasi state mechanical and flexural tests among others. The analysis revealed that the cacti fibres have a unique fractal-like morphology with a fractal order of 1.8 and self-similarity across scales, both assessed via the fractal box-counting method, which explains their unique properties as composite reinforcements. The natural fibres were also processed into a powder format through a ball milling process and their morphology was assessed following the same multiscale characterization protocol. The fractal order was maintained at 1.8 which supports the prospect of using this powder format as a novel composite reinforcement. Additionally, the morphological information obtained over multiple length scales provides invaluable inputs for the development of modelling simulations of the mechanical behaviour of the cacti fibres. This in turn creates the potential for the production of a new synthetic paradigm of structural reinforcement in a new class of composite materials with unique energy dissipation properties for bone tissue engineering and impact absorption applications.
Initial prototype composite manufacturing methods using polymer matrices including the bio-polymer Polylactic acid (PLA) and Polypropylene (PP) were developed to assess the reinforcement potential of the natural fibre powder format versus the natural fibre sheets. Lastly, cacti fibre implementation as a reinforcement on a recently developed bioink [In Press] for regenerative medicine allows the assessment of the potential of the cacti fibres as a reinforcement for bone tissue engineering scaffolds.
1. O. Faruk, A. K. Bledzki, H.-P. Fink, and M. Sain: Biocomposites reinforced with natural fibers: 2000–2010. Progress in Polymer Science. 37, 1552-1596 (2012).
2. M. Bouakba, A. Bezazi, K. Boba, F. Scarpa, and S. Bellamy: Cactus fibre/polyester biocomposites: Manufacturing, quasi-static mechanical and fatigue characterisation. Composites Science and Technology. 74, 150-159 (2013).
9:00 PM - BM3.3.18
Labeling and Long-Term Tracking of Bone Marrow Mesenchymal Stem Cells In Vitro using NaYF4:Yb3+,Er3+ Upconversion Nanoparticles
Yufei Ma 1 2
1 VA Medical Center, West Roxbury Boston United States, 2 Xi'an Jiaotong University Xi'an China
Show AbstractStem cell–based therapy has held great promise in providing desirable solutions for diseases. However, the main reason for its limited application in clinical trials is the lack of effective long-term cell tracking approaches that are able to promote comprehensive understanding of the fate of transplanted stem cells without impairing their intrinsic properties. Here, we successfully synthesized and utilized NaYF4:Yb3+,Er3+ upconversion nanoparticles (UCNPs) to label and track rabbit bone marrow mesenchymal stem cells (rBMSCs) during the osteogenic differentiation in vitro. To improve their biocompatibility and cellular uptake, we modified the UCNPs with the positively-charged polymers. Long-term effect of cellular uptake of these surface modified UCNPs during the osteogenic differentiation process was systematically evaluated, and no significant difference in cell viability and differentiation capacity in a certain range of nanoparticle concentrations (0~50 µg/mL). Moreover, the surface modified UCNPs at a concentration of 50 µg/mL exhibited the highest biocompatibility and stability, which could well track rBMSCs during the osteogenesis process in vitro. Hence, this study provided the necessary data for the application of these lanthanide-based UCNPs in stem cell labeling and tracking to better understand the mechanism of stem cell fate in tissue engineering, stem cell therapy, etc.
9:00 PM - BM3.3.19
Photothermal Therapy via Chlorophyll-Based Composites
Yuan Zhao 1 , Donglu Shi 1
1 Mechanical and Materials Engineering University of Cincinnati Cincinnati United States
Show AbstractPhotothermal therapy has proven to be very effective among several therapeutic strategies for cancer treatment. Photothermal nanomaterials can be directly injected into tumors and can locally generate sufficient heat for ablation when irradiated by near-infrared (NIR) light. Ablation occurs when the local temperature rises above 42C and will significantly inhibit tumor growth if a sufficient thermal dose is given. However, inorganic photothermal nanomaterials, such as gold, iron oxides, and graphene are not biodegradable; some even have high cytotoxicity. Natural chlorophyll (Chl) has been found to be an effective photothermal material for cancer therapy due to its non-toxicity. Due to its hydrophobic nature, Chl cannot disperse well in aqueous systems such as human body. Therefore, polymer-encapsulation was applied to enhance Chl’s solubility and stability in aqueous systems. Our preliminary data has shown that Chl has very high photothermal efficiency. Chl needs only one tenth of the power density to raise the solution temperature to 42C when compared to same mass of iron oxide nanoparticles. Thus, Chl-based nanocomposites may serve as an ideal and promising material for photothermal therapy.
9:00 PM - BM3.3.20
Combating
Candida Albicans—Aspartic Protease-Triggered Antifungal Hydrogels
Noel Vera-Gonzalez 1 , Anita Shukla 1
1 Brown University Providence United States
Show AbstractCandida is the most common cause of fungal infections, responsible for 46,000 infections per year in the United States (U.S. CDC, 2013). Antimicrobial resistance of Candida is a growing threat that increases the severity of these infections. There is a need for smart, triggered drug delivery systems that limit exposure to antimicrobials, helping prevent resistance. We have developed responsive hydrogel systems to combat skin wound fungal infections by incorporating a degradable peptide sequence that responds specifically to aspartic proteases (Saps) secreted by virulent Candida (M. Fusek, 1994). The bulk hydrogel is made of poly(ethylene glycol) (PEG), which provides superior biocompatibility, water solubility, and enhanced permeation of water-dissolved oxygen, which is important for wound healing (S.M. Grist, 2010).
The responsive eight amino acid peptide, LRF(p-NO2)↓FLAPK (LFFK), was synthesized using solid phase peptide synthesis with Fmoc chemistry; high performance liquid chromatography with mass spectroscopy (HPLC-MS) confirmed synthesis. The peptide was conjugated to PEG-acrylate functionalized with succinimidyl valerate under basic conditions. Mass spectrometry confirmed conjugation. We formulated responsive hydrogels containing anidulafungin (an active antifungal drug) via free radical photopolymerization of 10% (w/v) PEG-LFFK-PEG (Acryl-PEG-LRF(p-NO2)↓FLAPK-PEG-Acryl). This photopolymerization process allows for controllable hydrogel mechanical parameters (geometry, stiffness, and pore size), leading to tunable drug release kinetics. To test hydrogel degradation, pepsin, a mammalian aspartic protease, was used at concentrations that mimic physiologically relevant Sap proteolytic activity (approximately 8,000 units/mL). The peptide was preferentially cleaved by pepsin between the two hydrophobic phenylalanines or between leucine and phenylalanine, confirmed by HPLC-MS, resulting in hydrogel degradation. No hydrogel degradation was observed in HEPES buffered saline, indicating that these hydrogels are specifically responsive to aspartic proteases. These gels were stable for up to 4 weeks under acidic conditions (pH 2) with only approximately 0.4% of the total loaded drug being released. In the presence of pepsin, the hydrogels released 2.1±1.3% of the total loaded drug within one hour, 64.9±10% after two hours, and 88.4±0.02% after twenty-four hours. Non-peptide containing hydrogels (i.e. pure PEG hydrogels) remained intact in pepsin for at least 24 hours, releasing just 0.05±0.003% of the total loaded drug. In conclusion, we have developed a responsive target-triggered hydrogel system that releases an antifungal only in the presence of aspartic proteases. This approach allows us to locally delivery controlled doses of a drug in a way that may delay drug resistance.
9:00 PM - BM3.3.21
3D Graphene Scaffold for Tissue Engineering and Regenerative Medicine
Manuela Loeblein 3 4 , Guillaume Perry 2 1 , Siu Hon Tsang 6 , Wenjin Xiao 5 7 , Dominique Collard 2 , Philippe Coquet 4 8 , Yasuyuki Sakai 5 , Edwin Hang Tong Teo 3 4
3 School of Electrical and Electronic Engineering Nanyang Technological University Singapore Singapore, 4 Nanyang Technological University Singapore Singapore, 2 LIMMS CNRS/IIS University of Tokyo Tokyo Japan, 1 School of Electronic Engineering, Bangor University Bangor, Gwynedd United Kingdom, 6 Nanyang Technological University Singapore Singapore, 5 Institute of Industrial Science University of Tokyo Tokyo Japan, 7 School of Cellular and Molecular Medicine University of Bristol Bristol United Kingdom, 8 Institut d'Electronique, de Microélectronique et de Nanotechnologie Université Lille 1 Lille France
Show AbstractThe development of 3D tissue is a critical issue in regenerative medicine to avoid relying on donor organs. Many methods including engineered polymer scaffolds have been proposed for 3D culture. However, there are still two major limitations with these scaffolds due to the in-vivo high cell density and the cell oxygenation. In this work, we propose to use a novel 3D graphene scaffold (3D-C), to overcome these limitations.
The first 3D-C was fabricated by chemical vapour deposition a few years ago and exhibited higher porosity (99.7%), specific surface area (850 m2 g-1) and lower density (5 mg cm-3) than polymer scaffolds [1]. These properties make it extremely interesting for 3D tissue culture. HepG2 cells (liver cell line) were thus cultured on the collagen-coated 3D-C over two weeks.
One day after inoculation, the cells were attached on the scaffold. Both single cell and cell aggregates were observed with optical microscope. During the culture period, the cells gradually formed more aggregates. On day 14, a scanning electron microscope was used to characterize the culture and we observed that 3D-C was covered by the cells, which formed a large piece of tissue. However some areas of the scaffold were still uncovered.
In order to assess the cell proliferation on 3D-C, the DNA amount per 3D-C was measured. The DNA amount increased over the two weeks’ culture from 5.59 μg per 3D-C on day 1, 11.61 μg on day 7 to 26.143 μg on day 14, proving that the cell growth was exponential. Furthermore, the high cell viability on 3D-C was confirmed by the presence of cell nucleus with haematoxylin & eosin staining.
To produce energy and perform their functionalities, well-oxygenated cells consume only glucose. However, cells consume also pyruvate and produce lactate in case of a lack of oxygen. By measuring the ratio between the lactate production and the glucose consumption, the oxygenation status was determined. On 3D-C, this ratio decreased over the culture period showing that glucose was the main source of energy and cells were well oxygenated. The oxygenation status was also investigated at the cellular level by immunostaining of a hypoxia-induced factor (HIF-1α). The cells on 3D-C did not expressed HIF-1α in contrary to the control group (limited oxygenation).
To confirm the 3D-C potential for regenerative medicine, its biodegradation was investigated using an in-vitro testing assay based on enzymatic process. With oxidized 3D-C, larges part of 3D-C form flakes, which were detached due to the enzymatic activity on the oxygen containing chemical groups. The flakes were subsequently degraded to small particles, to single layer of graphene and finally to CO2 molecules.
These different results showed that 3D-C is an excellent candidate for applications in regenerative medicine [2].
[1] Chen et al., Nat. Mater. 2011, 10, 424.
[2] Loeblein, Perry et al., Adv. Healthcare Mater. 2016, 5, 1177.
9:00 PM - BM3.3.22
Fabrication of Bioactive Glass-Based Scaffolds by Stereolithograhy—Effect of Partial Pre-Sintering on Structural and Mechanical Properties of Scaffolds
Boonlom Thavornyutikarn 1 , Terence Turney 1 , Passakorn Tesavibul 2 , Kriskrai Sitthiseripratip 2 , Nattapon Chatarapanich 3 , Bryce Feltis 4
1 Monash University Clayton Australia, 2 National Metal and Materials Technology Center Pathumthani Thailand, 3 Kasetsart University Chonburi Thailand, 4 RMIT University Bundoora Australia
Show AbstractScaffolds made from 45S5 Bioglass® ceramic (BG) show clinical potential in bone regeneration due to their excellent bioactivity and ability to bond to natural bone tissue. However, porous BG scaffolds are limited by their mechanical integrity and by the substantial volume contractions occurring upon sintering. This study examines stereolithographic (SLA) methods to fabricate mechanically robust and porous Bioglass®-based ceramic scaffolds, with regular and interconnected pore networks and with various computer-aided design architectures. It was found that a diamond-like (DM) architecture gave scaffolds the most controllable results without any observable closed porosity in the final scaffolds. When the pore dimensions of the DM scaffolds of the same porosity (~60 %vol) were decreased from 700 to 400 µm, the compressive strength values increased from 3.5 to 6.7 MPa. In addition, smaller dimensional shrinkage could be obtained by employing partially pre-sintered bioglass, compared to standard 45S5 Bioglass®. Scaffolds derived from pre-sintered bioglass also showed slightly greater compressive strengths.
9:00 PM - BM3.3.23
Porcine Skin-Derived ECM Nanofibers Regulate Activities of Human Dermal Fibroblasts
Meng Xu 1 , Hongjun Wang 1
1 Stevens Institute of Technology Hoboken United States
Show AbstractIntroduction: Tissue-engineered skin grafts have proved to be promising in treating extensive and large wound defects. In recognition of the essential role of scaffold-defined microenvironment in regulating the phenotype of skin cells, efforts have been made to design biomimetic nanofibrous matrices for fabrication of skin grafts. Despite the morphological and dimensional advantages of such fibrous matrices, chemical composition can be a key factor to induce the corresponding cellular responses. [1] Thus, the current study is aimed to explore the use of porcine skin-derived extracellular matrix (ECM) nanofibers for formation of skin grafts with a particular interest in wound healing-related signal transduction in fibroblasts by ECM molecules.
Materials and Methods: Porcine skin-derived ECM was obtained via homogenization, centrifugation, and freeze-drying. Polycaprolactone (PCL)/porcine skin-derived ECM (PSE) nanofibers and PCL/collagen nanofibers were fabricated using the established electrospinning method [2]. Nanofibers was characterized using scanning electron microscope (SEM), hydroxyproline assay and GAG assay. Proliferation of human primary foreskin fibroblasts on both matrices was determined by MTT assay and DNA assay. Cell migration on various nanofibrous matrices was studied by using the “0.9-mm wound gaps” model. Fibroblast-to-myofibroblast differentiation was determined by immunofluorescent staining for α-smooth muscle actin. Expression of relevant integrin and signaling transduction genes were determined by q-PCR. Key signaling transduction proteins were also detected via western blot.
Results and Discussion: Both PCL/PSE and PCL/collagen nanofibers supported the growth of human foreskin fibroblasts. Compared to PCL/collagen nanofibers, PCL/PSE nanofibers could better regulate wound healing-related behaviors of fibroblasts, including facilitated migration and elevated differentiation. Furthermore, PCL/PSE nanofibers up-regulated expressions of several integrin genes that are involved in ECM-regulated signal transduction. The protein level of integrin-mediated focal adhesion kinase and downstream vinculin was also investigated and both were upregulated.
Conclusions:The bioactive components of porcine skin-derived ECM nanofibers regulate the wound healing-related activities of fibroblasts via ECM-induced signal transduction. Taken together, PSE nanofibers have great potentials for facilitated skin regeneration.
References:
[1] Denise C. Hocking. Therapeutic Applications of Extracellular Matrix Denise. Advances in Wound Care. 2015; 4(8): 441-443.
[2] Bhardwaj N, Kundu SC. Electrospinning: A fascinating fiber fabrication technique. Biotechnol Adv. 2010; 28:325-47.
9:00 PM - BM3.3.24
Layer-by-Layer Assembled Multilayer Film Composed of Polycation/Polyanion/bFGF Tri-Layers—Enhanced Encapsulation and Sustained Release of bFGF for iPS Cell Culture
Uiyoung Han 1 , Hee Ho Park 2 , Juhyun Park 3 , Tai Hyun Park 2 , Jinkee Hong 1
1 Chungang University Seoul Korea (the Republic of), 2 Seoul National University Seoul Korea (the Republic of), 3 Kangwon National University Gangwon Korea (the Republic of)
Show AbstractBasic fibroblast growth factor (bFGF) is a potent mitogen for normal diploid fibroblast cell and established cell line and it also help to the stem cell proliferation and differentiation1. bFGF is therefore required for the successful maintenance of stem cell cultures to reduce spontaneous differentiation into other cell. Induced pluripotent stem cell (iPSc) have been hailed as a potential therapeutic applications for diseases from the individual patient2. In iPSc culture, however, frequent replacing media containing bFGF is necessary to maintain undifferentiated iPSc since bFGF is easily denatured and lose its activity at cell culture condition. To increase the stability of bFGF and protect bFGF against denaturation, bFGF should be handled within mild condition and introduce novel container such as nanoparticles, hydrogel or nanofilms3.
Here, we designed the LbL deposited multilayer nanofilm which can release active bFGF during 3 days for biweekly replacing media in iPSc culture. The nanofilm was created by alternately dipping a substrate into polyelectrolyte, polysaccharide and bFGF solution. Blending of poly(beta-amino ester) (PBAE) and collagen type 1 (COL) was used for polycation layers and polyanion layers consisted of poly(acrylic acid) (PAA) or heparin (HEP). bFGF was deposited on polyanion layer by electrostatic interaction with PAA or binding of heparin to the high affinity site. We investigated the encapsulation efficiency of bFGF into nanofilm by comparing tri-layer and tetra-layer system which means (polycation/polyanion/bFGF)n and (polycation/polyanion/bFGF/polyanion)n. (n = number of tri-layers or tetra-layers) As a result, the maximum bFGF loading amount into nanofilm was 50.7 ng/cm2 (n = 12) in tri-layer structure and 8.4 ng/cm2 (n = 12) in tetra-layer structure. In cell culture, the release of bFGF from the multilayer film was maintained 3 days, up to 3 weeks. Finally, human iPSCs that were grown in the presence of the nanofilm maintained their undifferentiated morphology and expression levels of pluripotency marker proteins such as SSEA-4 and OCT4 activity. Furthermore, it will be expected to be a great tool not only to maintain the undifferentiated state of pluripotent stem cells but also in other developmental studies such as the differentiation of iPSCs to neuronal cells, cardiomyocytes, or hepatocytes by conjugating specific differentiation-stimulating factors.
(1) Pittelkow, M. R.; Shipley, G. D. J. Cell. Physiol. 1989, 140, 565.
(2) Robinton, D. A.; Daley, G. Q. Nature 2012, 481, 295.
(3) Macdonald, M. L.; Rodriguez, N. M.; Shah, N. J.; Hammond, P. T. Biomacromolecules 2010, 11, 2053.
9:00 PM - BM3.3.25
Magnetically Activated Scaffolds for Stem Cell Stimulation
Darina Gilroy 1 3 , Conor Buckley 3 , Fergal O'Brien 1 2 3 , Cathal Kearney 1 2
1 Royal College of Surgeons in Ireland Dublin Ireland, 3 Trinity College Centre for Bioengineering Dublin Ireland, 2 Advanced Materials and Bioengineering Research Center Dublin Ireland
Show AbstractThe proliferation, differentiation and matrix production of mesenchymal stem cells (MSCs) can be regulated by mechanical signalling. This motivates the design of bioreactor systems that dynamically load cells to direct cell fate and biomaterials with specific (visco)elastic properties. Despite the success of bioreactor systems, they can be expensive, complex and are often only able to stimulate cells prior to implantation. Therefore, the aim of this work was to develop a biomaterial that (1) is intrinsically responsive to a non-contact external stimulus and (2) can house stem cells and mechanically stimulate them. This was achieved by incorporating iron oxide microparticles into collagen and alginate-based tissue engineering scaffolds to produce a magnetically responsive biomaterial. These self-contained bioreactors can apply compressive forces to cells through non-contact magnetic fields. The first phase of the work focused on developing and characterising candidate biomaterials. The second phase demonstrated the viability and proliferation of rat MSCs seeded onto the materials.
In the first phase, two candidate scaffolds were fabricated. First, ferrous collagen scaffolds were fabricated by incorporating iron-oxide laden microparticles into a 5mg/mL collagen slurry and freeze drying the mixture to form a porous scaffold. The second scaffold consisted of alginate (ProNova), which was directly mixed with iron oxide microparticles (7%, Sigma) and crosslinked using carbodiimide chemistry. With the aiming of maximising magnetic deformation, 5 variables were investigated in these alginate scaffolds: alginate concentration (0.75-1%), crosslinking density (2.5-1.25mM AAD), iron oxide distribution, crosslinking temperature, effect of lyophilisation. Alginate scaffolds were soak-loaded with collagen type I to facilitate cell binding. Stress-strain behaviour (Zwick machine), scaffold deformation in a magnetic field (6.2kGs) and pore morphology (histology and SEM) were assessed. In phase two, candidate scaffolds were seeded with 1x106 rat MSCs/mL and cell survival was determined on day 7 using Live/Dead stain.
Maximum deformations of 30% and 50% in response to magnetic stimulation were observed for the collagen- and alginate-based scaffolds respectively. Both scaffolds demonstrated an open and inter-connected pore structure. Young’s modulus, which was inversely related to magnetically-induced deformation, was most sensitive to cross-linking in the alginate-based scaffolds. In phase 2, MSCs seeded on both magnetically-activated alginate and collagen scaffolds exhibited enhanced, albeit non-significant, cell survival on day 7.
These data present an exciting approach to magnetically adaptable biomaterials that can be manipulated to provide a range of stimuli and properties, and are supportive of cell survival. Ongoing work is investigating the full potential of these systems to direct cell behaviour in vitro and in vivo.
9:00 PM - BM3.3.26
Developing Composite Bone Scaffolds by a Hybrid 3D-Bioplotting/Thermally-Induced Phase Separation Technique
Junyi Liu 1 , Riley Sheppard 1 , Conor Flavin 1 , Brian Malerick 1 , Yao Ntifafa 1 , Nicholas Uth 1 , Paul F. James 2 , Jing Zhang 3 , Amy Yousefi 1
1 Chemical, Paper and Biomedical Engineering Miami University Oxford United States, 2 Biology Miami University Oxford United States, 3 Statistics Miami University Oxford United States
Show AbstractHigh metabolic activity of bone cells imposes a challenge in the repair/regeneration of critical size bone defects using 3D scaffolds, as the diffusional constraints of oxygen and nutrient supply can limit the size of scaffolds that can be used for in vivo implantation. Hence, scaffold design has a key role in the outcome of bioengineered bone of clinically-relevant sizes. We have recently shown that hierarchical polymeric scaffolds, produced by a hybrid 3D-Bioplotting (3DB)/thermally induced phase separation (TIPS), can support the growth of MC3T3-E1 osteoblastic cells in vitro. The scaffold matrix was made of microporous ploy(lactic-co-glycolic acid) (PLGA) produced by the TIPS method (pore size < 50 mm). Interconnected macrochannels (> 300 mm in diameter) were also generated within the matrix by embedding a 3D-Bioplotted polyethylene glycol (PEG) into the PLGA/1,4-dioxane solution prior to the TIPS process, and then leaching it out after dioxane extraction. Macrochannels can promote cell migration into and vascularization of the graft upon implantation, while further enhancing the transport of oxygen and nutrients.
Nanohydroxyapatite (nHA) has been shown to promote mineralization and osteogenic differentiation of mesenchymal stem cells (MSCs). In an ongoing study funded by the National Institute of Health (NIH), we have produced hierarchical composite scaffolds made of PLGA and nHA particles (< 200 nm in size). We are currently using a fractional factorial design of experiments (DoE) to study the effect of PLGA concentration (7–13%), nHA content (0–30%), diameter of macrochannels (250–450 mm), as well as the process temperature (TIPS) (-20–0°C) on the 3D architecture and mechanical properties of the scaffolds. Although the primary goal is to enhance bone formation, scaffold modulus will serve as a constraint in our design. Three levels (low, medium, and high) have been chosen for each of the four factors. While a full factorial design would lead to 3^4=81 runs, the fractional factorial design used reduces the number of runs to 18; and the I-Optimal design criterion has been used to randomize the runs so as to minimize the average prediction variance.
This presentation will focus on the scaffold design and characterization. This set of 18 scaffolds will eventually be seeded with human MSCs and then cultured for up to 8 weeks. The response surface methodology will be used to find the optimal scaffold architecture and composition that maximizes bone formation within these scaffolds. We anticipate that fine-tuning the size of macrochannels (3DB) and micropores (TIPS), along with modulating the composition of PLGA/nHA matrix, will lead to 3D constructs as potential bone graft substitutes. To validate this hypothesis, bone formation within the optimized scaffold will be compared to a negative control (TIPS-only scaffolds, no macrochannels) inside a mechanically-loaded bioreactor. The commercial CellCeram® scaffold will be used as a positive control.
9:00 PM - BM3.3.27
In Vitro Evaluation of 3D Bioprinted Alginate-Hydroxyapatite Hydrogel Scaffolds for Bone Tissue Regeneration
Stephanie Bendtsen 1 , Mei Wei 1 2
1 Institute of Materials Science University of Connecticut Storrs United States, 2 Materials Science and Engineering University of Connecticut Storrs United States
Show Abstract3D bioprinting, or 3D printing of living cells incorporated within a printable biomaterial, has gained recent popularity in the field of tissue regeneration. This additive manufacturing technique has been applied to engineering of all types of tissues and provides a potential solution to limitations in availability and success rates posed by the current organ transplant waiting list for patients in need of an organ or tissue. Furthermore, the possibility of incorporating the patient's own cells into the engineered tissue construct may provide the patient with enhanced healing and acceptance in vivo. Specifically for bone tissue engineering, materials capable of encapsulating cells to produce a scaffold of uniform cell distribution is of particular interest as current scaffolds may suffer from limited cell incorporation and uniform infiltration. Additionally, these methods most often produce brittle scaffolds with limited capability to treat irregular shaped defects that bone tends to experience as a result of damage. To address these limitations posed by rigid bone regenerative scaffolds, our group has recently developed an alginate-hydroxyapatite hydrogel formulation with optimal rheological properties for 3D printing soft scaffolds for bone tissue regeneration. We have successfully encapsulated living MC3T3-E1 cells within the hydrogel system and bioprinted them into cylindrical, porous scaffolds with an initial 95% viability rate.
In this study, we assessed the capability of our alginate-hydroxyapatite 3D printed scaffolds to support proliferation of encapsulated MC3T3-E1 cells and early signs of bone regeneration. Scaffolds of differing infill geometries were 3D printed using a HyRel System 30 3D printer to assess the effect of varying scaffold degrees of interconnectivity on cell proliferation. After 3D printing, scaffolds were immersed for 1 hour in a calcium chloride bath and then incubated in α-MEM cell culture media for 14 days. Cell proliferation was assessed over the incubation period using an AlamarBlue assay. Early signs of bone deposition were also evaluated by quantification of ALP activity and osteocalcin and osteopontin assays. Thus, the 3D printing alginate-hydroxyapatite scaffolds have great potential for bone regeneration in vivo for non-load bearing applications.
Acknowledgements: The authors would like to thank the NSF GK-12 program (0947869) and additional NSF grants (CBET-1347130, CBET-1339536 and CBET 1226018) for their support.
9:00 PM - BM3.3.28
Electrospun Calcified Gelatin Nanofibers—An Outstanding Approach for Guided Tissue Regeneration
Nihal Hassan 1 , Suher Zada 1 , Nageh Allam 2
1 Biotechnology American University in Cairo Cairo Egypt, 2 Physics American University in Cairo Cairo Egypt
Show AbstractBioscaffolds play a vital role in different fields of biomedical applications as they are used to improve or replace a biological function. The key aspect of these scaffolds is to mimic the physical and the functional characteristics of the natural extra cellular matrix (ECM). They should be porous structures with biocompatible, biodegradable and non-immunogenic characteristics to enhance cell attachment and ensure cell proliferation. Various techniques were used for scaffolds fabrication such as particulate leaching, lypholization, melt molding and gas foaming and on top of those is the electrospinning technique. In our study different gelatin concentrations have been investigated to obtain smooth nanofibers using diluted acetic acid. We succeeded in electrospinning of high concentration of gelatin dissolved in 40% diluted acetic acid where nanofibers fabricated had diameter ranging from 140 to 260 nm. In a new approach calcium carbonate crystals (CaCO3) were co-electrospun with gelatin at different concentrations. Smooth fibers were obtained at lower concentrations of CaCO3 where beaded broken fibers were obtained at higher concentrations. The diameter of nanofibers was found to increase with the increase of calcium carbonate concentration reaching up to 400 nm. Crosslinking of fiber mats using glutaraldehyde (GTA) vapors was considered a mandatory step as gelatin nanofibers have poor mechanical properties and low water resistance. Different crosslinking time intervals were investigated for better stability where the 20-hours-crosslinked mats showed the best rigidity with no cytotoxic effect on human fibroblast cell line. Although the stability of nanofibers is elevated with prolonged crosslinking time, the pore size distribution among different mats was found to be closely the same up to 250 nm. Crosslinked mats showed distinguished mass increase during both swelling and biodegradability tests especially with the decrease of calcium carbonate concentration among the mats. Not only calcified gelatin fibers showed promising results in MTT assay but also an overall improved functional and structural properties for guided tissue regeneration.
9:00 PM - BM3.3.29
Biocompatibility of DC Magnetron-Sputtered TiO2 Coatings with Nano-Scale Morphology and Controlled Phase Composition on Glass Substrates
Flora Imrie 1 , Klaus Almtoft 2 , Christian Jeppesen 2 , Sascha Louring 2 , Erik Ortel 3 , Vasile-Dan Hodoroaba 3 , Iain Gibson 1
1 Institute of Medical Sciences University of Aberdeen Aberdeen United Kingdom, 2 Tribology Centre Danish Technological Institute Aarhus Denmark, 3 Federal Institute for Materials Research and Testing Berlin Germany
Show AbstractSETNanoMetro, a European Seventh Framework project, seeks to develop standard synthetic routes and metrological characterisation methods for the development and production of TiO2 nanoparticles and nano-sized coatings with highly-defined, homogeneous and reproducible characteristics. These materials are being tested for their potential in selected technological applications, including as biomaterials, specifically as coatings on dental or orthopaedic metallic prostheses. This study aimed to assess how variations in nano-scale morphology and phase composition of TiO2 coatings affect their biocompatibility in vitro.
Pulsed DC magnetron sputtering was used to deposit a layer of Ti metal followed by a layer of TiO2 on standard glass microscope slides. The substrate bias voltage was varied during deposition to control the morphology and phase composition of the TiO2 layers. In order of increasing substrate bias voltage, the phase compositions of the TiO2 layers were: predominantly anatase, mixed anatase/rutile, and predominantly rutile, as confirmed by XRD. Examination of the coating cross-sections by SEM revealed feather-like columnar structures in the thin (950 nm thick) and thick (1550 nm thick) anatase coatings and in the mixed anatase/rutile coating (900 nm thick). In the rutile coating (730 nm thick), the columns were denser and less feather-like. Top-view SEM showed square-pyramidal morphology of the columns in the anatase coatings, with columns generally 100 nm or smaller in size (thin coating) or up to 200 nm across (thick coating). In the mixed anatase/rutile coating, the top-view showed less regular columnar morphology with more elongated columns (up to approx. 100 nm by 200 nm). In the rutile coating, the top-view showed a less ordered, pebble-like morphology.
MG-63 human osteoblast-like cells and RAW 264.7 murine macrophage cells were cultured on the TiO2-coated substrates for 24 or 72 h before quantification of cell proliferation using the WST-1 cell proliferation assay. A toxic response was defined as a reduction in cell viability (as shown by proliferation capacity) of greater than 30%. After 24 h culture, proliferation of MG-63 cells was significantly greater than the control (p < 0.05) on the thin anatase and mixed anatase/rutile coatings. After 72 h of culture no significant difference to the control was observed. For RAW 264.7 cells, proliferation was non-significantly decreased compared to the control on all coatings except the rutile coating after 24 h. After 72 h, RAW 264.7 proliferation was significantly decreased (p < 0.01) to 68% and 61% of the control for the thick and thin anatase coatings, respectively, indicating a toxic response. The results indicate that nano-sized TiO2 coatings show different biocompatibility to different test cell types, with a dependence upon coating phase composition and morphology.
9:00 PM - BM3.3.30
Scaffolds for Bone Regeneration—Bioglass-Hydroxyapatite and Polymer-Hydroxyapatite Composites
Zivile Stankeviciute 1 , Laurynas Alinauskas 1 , Linas Jonusauskas 2 , Sima Rekstyte 2 , Mangirdas Malinauskas 2 , Milda Alksne 3 , Virginija Bukelskiene 3 , Ieva Gendviliene 4 , Vygandas Rutkunas 4 , Edita Garskaite 1
1 Department of Applied Chemistry Vilnius University Vilnius Lithuania, 2 Department of Quantum Electronics Vilnius University Vilnius Lithuania, 3 Department of Biological Models Institute of Biochemistry Vilnius Lithuania, 4 Institute of Odontology, Faculty of Medicine Vilnius University Vilnius Lithuania
Show AbstractBone-graft substitutes have been developed as alternatives to autologous or allogeneic bone grafts. They consist of scaffolds made of synthetic or natural biomaterials that promote the migration, proliferation and differentiation of bone cells for bone regeneration. In order to guide cell behaviour through cell-material interactions, scaffolds with precisely tunable chemical and physical properties are crucial.
Herein we demonstrate a preparation of the porous scaffolds composed of bioactive glass (BG)-hydroxyapatite (HAP) and polylactic acid (PLA)-HAP. Porous BG-HAP scaffolds were prepared by pressing the HAP and BG powders produced via wet chemistry approach into the pellets, while PLA-HAP scaffolds were produced mechanically mixing synthesised nanocrystalline HAP with melted PLA. Various processing conditions are examined and the effect of solution concentration, pH, sintering temperature and doping composition on the final material morphological and structural properties will be presented and discussed. The calcination temperature, morphological and structural features were studied by TG/DTA, XRD and FE-SEM techniques.
Additionally, the topographical patterning of PLA-HAP composite surfaces was performed by femtosecond laser direct writing. Microstructured surfaces and the dopant distribution within the composite and formed grooves were evaluated by optical confocal profilometer and FE-SEM. The influence of the HAP concentration as well as the laser power (intensity) and translation velocity (energy dose) on the composite surface morphology is discussed. The initial results proves the method to be applicable for surface modification at super-/sub-cellular spatial dimensions which offers study of cell-to-substrate specific response.
Furthermore, biocompatibility of PLA-HAP scaffolds were studied using two different primary rat stem cell lines – dental pulp (rDPSC) and periosteal (rPSC) derived. Both cell lines grown on the tested scaffolds were alive. The viability of grown cells was recognized after staining with acridine orange and ethidium bromide. The initial results need further coherent investigation in detail.
9:00 PM - BM3.3.31
The Use of 3D Correlative and Multi-Modal Imaging Techniques to Investigate Multiscale Structural Properties of Interconnected Porous Biomaterial for Tissue Engineering
Ali Chirazi 1 , Mythili Prakasam 2 , Alain Largeteau 2 , Grzegorz Pyka 3 , Anna Prokhodtseva 3 , Daniel Lichau 1
1 FEI Application Software Group Merignac Cedex France, 2 Institut de Chimie de la Matière Condensée de Bordeaux PESSAC France, 3 FEI Czech Republic s.r.o. Brno Czech Republic
Show AbstractBiomaterial for tissue engineering is a topic of huge progress with recent surge in fabrication and characterization advances. Biomaterials for tissue engineering applications or as scaffolds depends on various parameters such as fabrication technology, porosity, pore size, mechanical strength and surface available for cell attachment. To serve the function of the scaffold, the porous biomaterial should have enough mechanical strength to aid in tissue engineering. Conventionally high temperatures are used as a mean of fabrication technology to increase the mechanical strength of biomaterial. Employment of high temperature impedes the application of this type of material to lose its biocompatibility and inability to load the drug/ protein carriers for tissue regeneration/ healing.
ICMCB laboratory in France, part of French National Research Labs (CNRS), has developed a new and innovative manufacturing process which is based on low temperature consolidation processing method which does not use any kind of high temperature sintering technology. This new manufacturing technology could help in obtaining high strength materials by optimizing few processing parameters such as pressure, temperature and dwell time.
The aforesaid method of consolidation yielded the monolith with porosity in the range of 80-93%. The porous monolith consisted of high porous body with fine thin pore walls and interconnected spaces. The presence of the interconnected pores will assist in cell proliferation. The multiscale porous media interconnectivity and its influence on wall’s density variation and morphology needs to be studied thoroughly in order to be able to relate unique microstructural aspects of the biomaterial to the final thermos-mechanical and thermos-diffusive properties.
The three dimensional interconnectivity of the porous media through scales for the newly manufactured biomaterial has been investigated using newly developed 3D correlative and multi-modal imaging techniques. Multiscale X-ray tomography, FIB-SEM Slice & View stacking and high resolution STEM-EDS electronic tomography observations have been combined allowing quantification of morphological and geometrical spatial distributions of the multiscale porous network through length scales spanning from tens of microns to less than a nanometer. The spatial distribution of the wall thickness has also been investigated and its possible relationship with pore connectivity and size distribution has been studied.
9:00 PM - BM3.3.32
Gradient Aligned Magnetic Nanocarriers in Gelatin-Silk Nerve Conduit
Chun-Chang Lin 1 , San-Yuan Chen 1
1 Material Science and Engineering Hsinchu Taiwan
Show AbstractGuidance cue, an important issue for nerve cell regrowth, which greatly affect axon elongation behavior. Here, we report the gelatin-silk nerve conduit (GSNC) integrating aligned NGF-encapsulated amphiphilic gelatin nanocapsules (N-AGNCs) that can guide nerve cell regrowth and continuously release nutrition. The AGNCs comprising superparamagnetic nanoparticles (SPIOs) and amphiphilic gelatin were synthesized by double emulsion, which exhibited excellent saturation magnetization (Ms) and a diameter of 250 nm. The N-AGNCs were incorporated into the GSNC as a precursor, and aligned via applying an external magnetic field before starting gelation in low temperature. To note, a gradient distribution of N-AGNCs can be constructed by adding AGNCs with different SPIOs concentration (e.g., 3-6 mg ml-1, which yields the Ms from 40 to 60 emu g-1) in GSNC. This gradient distribution of N-AGNCs can not only provide nutrition but guide the cell growth by arranging the distribution of NGF. Moreover, the degradation rate of the GSNC can be manipulated by adding different content of silk (i.e., 4.5 mg to 13.5 mg), and the gelatin (15 mg to 25 mg) can improve cell adhesion. Combining these features, the novel GSNC serves as an excellent cell culture platform for long-term cell observation, proliferation and guidance.
9:00 PM - BM3.3.33
Rapid Interconnected Porous Membranes by Blending Chitosan and Polyurethan Diol as Extracellular Matrix Surrogate
Kamakshi Bankoti 1 , Santanu Dhara 1
1 Indian Institute of Technology Kharagpur India
Show AbstractBiodegradable porous polymer membranes are of interest to wound healing and it act as matrix for supporting tissue repair and regeneration.The membranes mimic extracellular matrix (ECM) for supporting cell adhesion, proliferation and differentiation in wound bed accelerating tissue regeneration. Native ECM is an interpenetrating network (IPN) like structure of crosslinked proteins interlaced with high molecular weight biomacromolecules and is vital, dynamic macromolecular mesh work providing cues for integrin mediated cell adhesion, efficient transport of nutrients, oxygen and removal of cell excreted materials. Interconnected porous membrane was prepared through blending of aqueous polyurethane diol-chitosan in different proportion. SEM and AFM micrographs revealed the macroporous structure of the prepared membrane. Microscopic phase analysis at different time point evidenced phase separation in the blend. Effect of phase separation on the mechanical and in-vitro different mode of degradation (hydrolytic, enzymatic and pH dependant) properties of membrane were studied. FTIR spectra of the membranes formed by blending revealed Ionic interactions. The improved stability in aqueous and enzymatic environment revealed the ability of the membrane to sustain in in-vivo biological surroundings. Bleding of polymers resulted in introduction of macropososity (6 to 20 miron pores), hydrophobicity (70 to 90 degree), swelling (70 to 150 %) and higher protein adsorbtion.Higher protein adsorption is essential for both attracting cells towards the scaffold and also biological integration of scaffold to host tissue. In-vitro cytocompatibility was demonstrated by enhanced proliferation of MG63 cells on membranes. In-vivo studies by subcutaneous implantation in rat model revealed the nontoxic, biodegradable nature of macroporous membrane. the membrane was intact with the host tissue and showed minimal inflammatory response. No capsular layer/fibrosis was found around the surrounding tissue of blend membrane C7P3. The hydrophobicity and slower degradation reveals the potential of these membranes to be used as controlled release drug delivery system. Good cytocompatibility, hemocompatibily and preliminary biocompatibility expose its utility as biomaterial for tissue regeneration applications.
9:00 PM - BM3.3.34
Evaluation of the Bacteriostatic Effect of Chitosan Spheres Containing Silver Nanoparticles
Eliana Rigo 1 , Luci Vercik 1 , Yasmin Bortoletto 2 , Isadora Oliveira 1 , Ricardo Sousa 2 , Flavia Munin 2 , Andres Vercik 1 , Renata Doria 2
1 Department Basic Science University of São Paulo Pirassununga Brazil, 2 Department Veterinary Medicine University of São Paulo Pirassununga Brazil
Show AbstractThe search for alternative routes that enable the application of drugs with certain specificities, such as high toxicity, quick metabolism, narrow therapeutic range and the drugs used in long-term therapeutic treatments considerably broadened the studies of the pharmaceutical area, highlighting the controlled delivery systems. These systems exhibit advantages compared to conventional treatments, with higher effectiveness, acceptance and low toxicity. The usage of polymeric materials such as chitosan greatly enhances the controlled drug delivery system due to its biocompatibility and biodegradability. This work aimed to prepare chitosan spheres (CS) with silver nanoparticles (AgNps), using the coagulation method in two conditions: sodium hydroxide solution (NaOH) and sodium tripolyphosphate solution (TPP). The resulting spheres were immersed in the silver nanoparticles solution and then dried at 30°C in a forced circulation oven during 24h. The antimicrobial evaluation was carried out by sensibility test with the agar diffusion disk method using Staphylococcus aureus ATTCC 25922 and Escherichia coli ATTCC 25923 strains. The results indicate that the chitosan spheres immersed in the solutions containing the AgNPs showed an inhibition indicating effect bacteriostatic. Among the conditions the spheres obtained in the TPP solution has the highest inhibition halos.
9:00 PM - BM3.3.35
Citotoxicity Assay of Gelatin/Chitosan and Hydroxyapatite Membranes Obtained by In Situ Precipitation
Eliana Rigo 1 , Filipe Habitzreuter 1 , Luci Vercik 1
1 Department Basic Science University of São Paulo Pirassununga Brazil
Show AbstractGuided Tissue Regeneration (GTR) uses physical barriers typically constituted of polymeric membranes. Among them, gelatin (G) and chitosan (CS) are interesting for this purpose, due to their biodegradability and high similarity with adjacent tissues. The incorporation of hydroxyapatite (HA) in the polymeric matrix aims to induce osteogenesis on the damaged region, in addition to grant mechanical resistance to the formed composites. In this work, the membranes were produced by an in situ precipitation technique of Ca(OH)2 and H3PO4 to form HA directly in an aqueous solution of gelatin. Thus, the formation of HA in the polymeric matrix is homogeneous, lowering possible inflammations or surgical failure. The amount of CS was varied, forming composites with 20/80, 50/50 and 80/20 of G/CS and 0,3M of HA. Lastly, crosslinking steps with sodium tripolyphosphate (TPP) and glutaraldehyde (GTA) were performed. The composites were characterized by Fourier Transform Infrared Spectroscopy (FTIR) and X-ray Diffraction (XRD) and subjected to swelling essay in a phosphate buffer saline solution (PBS). Furthermore, cytotoxicity assays were carried out using the Neutral Red (NR) incorporation method. The FTIR spectra show good agreement amongst all membranes and through the XRD analysis, HA and b-TCP (tricalcium phosphate) peaks were identified. The swelling essay pointed out that the crosslinked membranes with GTA exhibited higher resistance to the aqueous medium; however the cytotoxicity assay showed that the membranes crosslinked with TPP had better cellular viability. In conclusion, the in situ precipitation method was efficient to produce membranes of G/CS with HA with reasonable results concerning cellular viability to the membranes crosslinked with TPP, evincing its use in GTR.
9:00 PM - BM3.3.36
Controlled Released of Antibacterial Ag/Poly (L-Lactic Acid)/Poly(Vinyl Alcohol) (Ag/PLLA/PVA) Core–Shell Nanofibers Prepared by Cold Atmospheric Plasma (CAP) Treatment and Electrospinning
Mian Wang 1 , Michael Keidar 2 , Thomas J. Webster 1
1 Northeastern University Boston United States, 2 George Washington University DC United States
Show AbstractThe most commonly used method for incorporating antibacterial silver nanoparticles into electrospun nanofibers is by suspending silver nanoparticles into polymer solutions. However, nanofibers produced by this method have reduced antibacterial properties due to silver nanoparticle aggregation. In order to avoid this, the objective of the present study was to utilize cold atmospheric plasma (CAP) [1], which is an ionized gas with ions, electrons, and excited atoms, as a reducing agent for photosensitive silver salts. Highly porous core-shell nanofibers were generated with 10% PLLA containing 1% silver nitrate (w/w) and 6% PVA solution by rapid phase separation during the electrospinning process. After fabrication, samples were then treated by cold atmospheric plasma with different times (30s, 60s, 90s, and 180s). Treated and untreated samples were observed by scanning electron microscopy (Hitachi, S-3700N). Anti-bacterial experiments and cell cytotoxicity studies will be conducted and reported later. SEM images of untreated and CAP treated nanofibers showed that the diameter of electrospun nanofibers ranged from 400 nm to 900 nm. Nanopores could be observed on the nanofiber surface, with the diameter of nanopores on the untreated surface ranging from 5 nm to 20 nm. However, the nanopores became larger as CAP treated time increased. For 30s of CAP treatment, nanopores on the surface enlarged to about 30 nm in diameter, and 40 nm with 60s of CAP treatment. In this study, a nanoporous core-shell nanofiber matrix was fabricated by an electrospinning method. Anti-bacterial silver nanoparticle reduction was successfully prepared by CAP treatment of the electrospun scaffold.
Acknowledgement: This study was supported by Northeastern University.
References: [1] M. Wang, X. Cheng, W. Zhu, et al. Tissue engineering: Part A, 2014, 5(20), 1060-1071.
9:00 PM - BM3.3.37
Selectively Inhibiting Cancer Cells by Intracellular Enzyme-Instructed Assembly (EIA) of Derivatized Dipeptides#xD;
Jie Li 1 , Bing Xu 1
1 Brandeis University Waltham United States
Show AbstractTo address the problem that tight ligand-receptor binding, paradoxically, is a major root of drug resistance in cancer chemotherapy, we focus on the development of a novel process—enzyme-instructed assembly (EIA)—to kill cancer cells selectively. We design and synthesize the small peptide precursors as the substrates of carboxylesterase (CES). CES cleaves the ester bond pre-installed on the precursors to form the peptides that self-assemble in water to form molecular nanofibers. As the process that turns the non-self-assembling precursors into the self-assembling peptides upon the catalysis of CES, EIA occurs intracellularly to selectively inhibit a range of cancer cells including drug resistant cancer cells that exhibit relatively high CES activities. While it is innocuous to normal cells which have lower level of CES. In addition, in vivo toxicity evaluation confirms that EIA is innocuous to mice, agreeing with the exceptional selectivity of EIA in cell assays. This work illustrates a new approach to amplify the enzymatic difference between cancer and normal cells and to expand the pool of drug candidates for potentially overcoming drug resistance in cancer therapy.
9:00 PM - BM3.3.38
Nanofibril Reinforced Gelatin Hydrogels for 3-D Cell Cultivation
Sol Lee 1 , Hyuk Sang Yoo 1
1 Kangwon National University Chuncheon Korea (the Republic of)
Show AbstractBiodegradable poly Biodegradable poly e-caprolactone (PCL) with ester linkages is widely used for the materials of implantable biomedical devices. Easily electrospinable PCL were developed to various type of nanofibrous mesh and its extracellular mimicking structure of the mesh was the advantage to apply to tissue engineering. Therefore, some of previous studies focused on 3D culture system using nanofiber mesh with various biocompatible polymers. PCL nanofiber is densely packing during electrospinning process, so it is hard to offer 3D environment in cell cultivation.
In this study, we modified electrospun PCL mesh to be suitable for 3D cell culture scaffold by fragmentation of PCL nanofiber and surface modification of the gelatin type A and B. PCL dissolved in chloroform/methanol 3/1 (v/v) mixture was electrospun on ethanol bath by applying high voltage (15kV) with 1ml/h of flow rate. PCL nanofiber was hydrolyzed by 1M NaOH for 3h after milling for 30sec. Fragmented PCL nanofibers were observed using optical microscopes.
Fragmented PCL nanofiber (fibril) was coated with gelatin type A and B by electrostatic interaction. Gelatin type A has a positive charge and type B has a negative charge in phosphate buffer at pH 6.0. Fibril was coated with gelatin in the solution with 4 concentrations, and it was formed that the higher gelatin concentration was the higher amount of gelatin that the fibril could be coated. Five gelatin layers were coated on fibrils with a coating sequence of type A, type B, type A, type B, type A. Gel was formed by alginate solution (4%) and gelatin solution (10%) mixture ( 2 : 1 (v/v)). Fibril (10mg/ml, 50mg/ml, 100mg/ml, 200mg/ml) was mixed with Alginate-gelatin mixture. In order to induce hydrogel property CaCl2 was used to crosslink for 3h. To characterize of gel, rheology was measured. We further applied alginate-gelatin hydrogels mixed with gelatin coated fibril to 3D cell cultivation and investigated the effect of gelatin coated fibril in the gel to cell proliferation and differentiation.
Symposium Organizers
Mei Wei, University of Connecticut
Marisha Godek, Medtronic
Shaoqin Gong, University of Wisconsin-Madison
Joerg Jinschek, FEI Company
Symposium Support
Applied Physics Reviews | AIP Publishing, Medtronic, The National Science Foundation
BM3.4: Biomaterials for Soft Tissue Regeneration
Session Chairs
Tuesday AM, November 29, 2016
Hynes, Level 1, Room 101
9:30 AM - *BM3.4.01
Silk Biomaterials for Soft Tissue Regeneration
David Kaplan 1
1 Tufts University Medford United States
Show AbstractLarge soft tissue defects remain a challenge to restore in terms of structure and function, as options for tissue regeneration with retention of tissue volume and function remain very limited. To address this challenge, we have been pursuing silk protein-based biomaterial systems. These systems have been pursued in various formats, from injectable gels to implantable sponges. The design, characterization and utility of these biomaterials will be reviewed in the context of tuning mechanics, degradation lifetime and variations in design to match clinical needs. The systems are all based on the unique slow degrading silk materials which offer good compatibility in vivo, are based on approved silk-based medical devices, and offer tremendous versatility in terms of processing, properties and utility in vivo. Issues of material format, large volume tissue regeneration and acellular vs cellular options will be discussed.
10:00 AM - BM3.4.02
Engineering Artificial Axons for Myelin Research
Daniela Espinosa-Hoyos 1 , Anna Jagielska 1 , Huifeng Du 1 , Nicholas Fang 1 , Krystyn Van Vliet 1
1 Massachusetts Institute of Technology Cambridge United States
Show AbstractNerve function regeneration can be limited directly by poor remyelination, or development of a myelin protein sheath around axons to promote signal conduction. Clinical trials for neurodegenerative disorders exhibit some of the highest attrition rates, inhibiting progress against neuro-regeneration options. Disorders of myelin formation and degeneration are particularly challenging due to a lack of credible preclinical models to enable the understanding of myelin biology and pathology. Oligodendrocytes generate and repair myelin around axons in the central nervous system through a series of tightly regulated processes that may involve a myriad of cell-cell interactions. Emerging research including our own suggests that mechanosensitivity of the oligodendrocyte lineage, and physical and mechanical features of axons, may impact key features of myelination such as the onset of oligodendrocyte differentiation, thickness and length of the myelin segments, and the structure of nodes of Ranvier. The standard coculture system of neurons and oligodendrocytes on polystyrene is deficient. Off-target, cell cross-talk and cell-autonomous phenomena are unresolved; the mechanical and biophysical complexity of the tissue matrix are ignored; there are conflicting effects of necessary medium factors and cues that complicate experiments and analysis; and it involves significant time, cost and irreproducibility. Here we discuss the design of materials for artificial axons, which will enable the study myelination in vitro in minimally-permissive environments, using a combination of materials engineering and high resolution 3D microfabrication. The flexibility and robustness of our methods allow us to engineer mechanical properties and biochemical functionality of individual axons and their environment with high degree of control at the micrometer level. Using conventional microscopy techniques and high-throughput analysis methods, we show cell-material interactions in these artificial axons is maintained. These materials and devices now facilitate answers to basic questions about myelin regeneration, and applied studies including in vitro drug screening.
10:15 AM - BM3.4.03
Hybrid Nano-Scaffolds for Neuroengineering
Shreyas Shah 1
1 Physiological Communications Nokia Bell Labs New Providence United States
Show AbstractThe mammalian brain is a phenomenal piece of ‘organic machinery’. While remarkable advances have been made to better understand how the brain works and how to overcome neural deficiencies, its inherent complexity makes it difficult to fully comprehend, repair and manipulate. This is partly due to the lack of adequate tools to effectively probe the central nervous system (CNS). Yet, given that the biomolecular interactions and chemical communication occur at the nanoscale, there is great potential in leveraging advanced nanomaterials for neuroscience research. Herein, we demonstrate rationally-designed approaches, based on the assembly of hybrids comprised of synthetic nanomaterials and biomaterial scaffolds, to address challenges in neural regeneration and neural modulation.
In the first approach, a hybrid nano-scaffold was developed to guide the selective differentiation of stem cells for neural regeneration. Establishing a controlled methodology to guide differentiation into specific cells of interest is a prevailing challenge. In neural regeneration, the selective differentiation into oligodendrocytes is desirable, yet difficult due to the overwhelming tendency of neural stem cells to become astrocytes. Most studies have focused on employing specific biomolecules or introducing genetic modifications to guide this process. In contrast, we combined a graphene-based nanomaterial with an electrospun nanofiber mesh to achieve remarkable differentiation into mature oligodendrocytes. By combining the unique properties of graphene (e.g. enhanced protein adsorption, high electrical conductivity) with the morphological features of nanofibers (e.g. size, topography, transplantability), our hybrid nano-scaffold has great scope for treating CNS damage.
In the second approach, a hybrid nano-scaffold was developed to mediate near-infrared light (NIR)-triggerable neural modulation. Optogenetics is a revolutionary technology that has transformed neuroscience, allowing for the control of neuronal activity by using light and genetically-encoded photosensitive proteins. However, the efficient modulation of neuronal activity is contingent on delivering a sufficient dose of visible light to the target cells. Since light in the visible range is highly scattered and lacks deep tissue penetration, current approaches require invasive procedures to implant fiber optics in animal models. We overcame this barrier by employing upconverting nanoparticles (UCNPs), which have the capability to emit high energy visible light upon excitation with deep penetrating NIR light. By further embedding UCNPs within injectable polymers, we provide an early demonstration employing a scaffold to achieve modulation of neuronal activity. This approach takes us one step closer to a potentially wireless optogenetic solution.
Overall, our innovative hybrid nanomaterial-based scaffolding approaches hold immense potential for advancing neuroscience research.
10:30 AM - BM3.4.04
Smart Phenytoin Loaded Nanofibrous Wound Dressing for Wound Healing and Detection of Healing Progression
Isra Ali 1 , Islam Khalil 1 2 , Ibrahim El-Shebiny 1
1 Nanomaterials Laboratory, Center for Material Science Zewail City of Science and Technology Giza Governorate Egypt, 2 Department of Pharmaceutics and Industrial Pharmacy Misr University of Science and Technology Governorate Egypt
Show AbstractSkin, the soft organ that protects the human internal body from surrounding environment, could be lost due to accidental injuries and burns. Skin tissues could heal naturally after injuries and burns, however normal reorganization of new tissues still represent a challenge especially in complicated wounds such as the diabetic ones. Diabetic wounds possess high alkalinity that obstructs wound healing rapidly. Topical application of phenytoin shows promising wound healing activity. However, Phenytoin is available in the market in a spray form which is not convenient for the patient to get his/her dose accurately. Therefore, we propose to fabricate a smart responsive highly porous electrospun nanofibrous matrix loaded with the appropriate dose for wound healing to act as: (a) wound dressing, (b) scaffolding material for regeneration of damaged skin and (c) sensor for detecting the progress of healing underneath the dressing.
The wound dressing would be fabricated in the form of a three-layer sandwich structure, where each layer has a specific role. First, a fabricated thin layer of electrospun carbapol-based nanofibers would be in direct contact with the diabetic wound. This layer is highly dissolvable, with adhesive property to wound and neutralize wound pH in order to facilitate wound healing. The second layer is composed of Phenytoin-loaded PLLA based nanofibers that could deliver the required dose in a well-controlled and sustained manner. Finally, a sensor layer would be added containing a humidity sensor dye that could respond to change in humidity during wound healing through changing its color. This would help the patient to monitor healing progression and remove the dressing when healing is complete. The three layers were prepared successfully through electrospinning technique after tailoring different parameters to produce highly porous wound dressing mats in order to facilitate oxygen and nutrient diffusion for cell. Scanning electron microscope revealed that diameter of carbapol based nanofibers and PLLA based nanofibers were around 124 ± 19 nm and 267 ± 37 nm respectively. FTIR, DSC and TGA instruments were used to confirm the chemical and thermal stability of the fabricated nanofibers. Physicochemical characterization showed that PLLA based nanofibers showed swelling maximum after 6 hours, where their initial weight increased by 50-60%. In addition, biodegedability test showed that the nanofibers were capable of losing more than 25% of their initial weights in 14 days. Finally in vitro drug release profile showed that the nanofibers could deliver Phenytoin in a well-controlled manner and prolonged period along more than 10 days. Hence, the fabricated system could be a smart wound dressing material for controlled treatment of wounds as well as detecting the healing progress through monitoring loss of humidity upon wound closure.
11:15 AM - *BM3.4.05
Tissue-Inspired Niches for HPSC Pancreatic Lineage Specification
Sha Jin 1 , Kaiming Ye 1
1 Department of Biomedical Engineering Binghamton University Binghamton United States
Show AbstractDiabetes mellitus has become a global epidemic with striking impacts on society and economy. While islet transplantation is a promising treatment for diabetes, it has not been widely available to majority of patients due to the scarcity of donors. Here, we report a new biofabrication technique for creating islet organoids from human pluripotent stem cells. We developed a unique collagen 3D scaffold and discovered that islet organoids can be formed from pancreatically differentiated human induced pluripotent stem cells (hPSCs). Immunohistochemistry assay revealed that these cell clusters formed from hPSCs exhibited a typical tissue structure of the islets. The real-time PCR and glucose challenging experiments divulged that these cells can secrete insulin and C-peptide upon glucose challenging. Furthermore, TEM indicated the existence of insulin-secretion granules in beta cells consisting of islet organoids, suggesting a high degree of maturation of these cells. To further improve the maturity of the islet organoids from hPSCs, we developed a decellularized ECM scaffolds and show the significant elevation of maturity of islet cells within tissue-inspired environment. The augment of this technology to other stem cell differentiations will bring cell replacement therapy one step closer to treating many diseases such as diabetes in more controllable clinical settings.
11:45 AM - BM3.4.06
A Cytocompatible Elastomeric Composite
Eric Finkelstein 2 1 , Ashlee Bucco 2 1 , Melodie Lawton 2 1 , Emily Mihalko 2 , Patrick Mather 2 1
2 Syracuse Biomaterials Institute Syracuse University Syracuse United States, 1 Biomedical and Chemical Engineering Syracuse University Syracuse United States
Show AbstractSilicone elastomers are utilized for many biomaterials applications. The goal of our research is to develop a novel elastomeric composite for use as a biomimetic synthetic vascular graft material. Cardiovascular disease is a major health problem throughout the world, with patients often requiring bypass surgery. When autologous blood vessels are not suitable for the bypass procedure due to an underlying pathology (ie. diabetes), a synthetic vascular graft is utilized. Synthetic grafts are also utilized for vascular access for dialysis or when the bypass graft is in an extremity. Synthetic grafts often fail for mechanical and biological reasons. Our material, a composite of polycaprolactone (PCL) and polydimethylsiloxane (PDMS), is biomimetic, having mechanical properties similar to native vessels, and is mechanically strong. A vascular graft material should be biocompatible and cytocompatible as it will be implanted in the body and cells will migrate onto the material from the native blood vessel. To fabricate our material, PCL is electrospun, and then this is imbibed with silanol-terminated PDMS. The PDMS is catalyzed with dibutyl tin dilauryate (DBTBL). By testing extracts from out composites in a CCK-8 cytotoxicity assay, we found that composites made using 1.0wt% DBTDL resulted in poor viability for vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs). Reducing the level of catalyst to 0.5wt% dramatically improved cell viability as assessed using L929 mouse fibroblasts (a cell line commonly utilized for cytotoxicity assays) as well as VECs and VSMCs. In addition, reducing the catalyst had no effect on PDMS polymerization as assessed by gel fraction. Tensile strength of the material was also assessed. From our studies, we conclude that reducing the level of an organo-metallic catalyst used in material synthesis can dramatically improve cytocompatibility of the resultant material, while having no effect on material properties. This lays the groundwork for further studies of direct cell-material interactions with our material, and has a potential impact on the silicone medical device industry.
12:00 PM - BM3.4.07
Engineering Gelatin/Tropoelastin Hydrogel Constructs for Neural Tissue Repair
Jonathan Soucy 1 , Iman Noshadi 1 2 , Suzanne Mithieux 3 , Abigail Koppes 1 , Anthony Weiss 3 , Nasim Annabi 1 2 , Ryan Koppes 1
1 Chemical Engineering Northeastern University Boston United States, 2 Harvard-MIT Division of Health Sciences and Technology Massachusetts Institute of Technology Cambridge United States, 3 School of Life and Environmental Sciences, Charles Perkins Centre University of Sydney Sydney Australia
Show AbstractNeurons’ limited capacity to spontaneously regenerate and the formation of scar tissue accompanying spinal cord injuries often leads to permanent neural dysfunction. In recent years, Schwann cells (SCs) have been transplanted into the spine to promote neural tissue regeneration[1]. However, due to low cell viability of delivered cells at the injury sites, cell-based therapies often fail to be effective treatments[2]. Hydrogel-based scaffolds are advantageous for cell delivery in vivo by supporting cell viability while possessing phase changing properties to allow minimally invasive injection. Hydrogels are hydrophilic polymeric networks that can be made to mimic the structural, biological, and mechanical properties of the extracellular matrix of the tissues[3].
In this work, we propose to synthesize a naturally-derived, novel copolymer hydrogel to promote SC adhesion, differentiation, and proliferation. Composite hydrogels of gelatin methacrylate (GelMA) and methacrylated tropoelastin (MeTro) prepolymers have been prepared by free radical chemistry at varying ratios, concentrations, and irradiation time. The mechanical properties of the engineered hydrogel were finely controlled by tuning these parameters.
GelMA/MeTro hybrid hydrogels were prepared via a photopolymerization reaction as explained previously[4,5]. Briefly, prepolymer solutions containing different concentrations of GelMA, MeTro, and free radical initiator (Irgacure 2959), were irradiated with UV light (300-400nm, intensity 6.9mW/cm2) for varying amounts of time. Tensile and compressive modulus of the hybrid hydrogel were assessed on an Instron®5944 mechanical tester and found to exhibit characteristics in-between or superior to that of pure GelMA and MeTro hydrogels. Results from varying the prepolymer compositions and crosslinking times indicate a number of potential soft tissue engineering applications from the nervous system to muscle and tendon.
Encapsulated SC viability, structure, and phenotype will be assessed within each of previously assessed conditions to determine the hydrogel construct that promotes the best in vitro growth characteristics. Viability and structure will be visualized with a calcein-AM/ethidium homodimer Live/Dead assay, while phenotype will be examined by anti-S100, phalloidin (F-Actin), and DAPI to identify potential hydrogel compositions for future in vivo investigations.
1. Assunção-Silva, R. C., et al. (2015). Stem cells international 2015: 948040.
2. Harting, M. T., et al. (2008). Neurosurgical Focus 24(3-4).
3. Drury, J. L. and D. J. Mooney (2003). Biomaterials 24(24): 4337-4351.
4. Nichol, J. W., et al. (2010). Biomaterials 31(21): 5536-5544.
5. Annabi, N., et al. (2013). Biomaterials 34(22): 5496-5505.
12:15 PM - BM3.4.08
Injectable Hydrogels to Deliver and Engraft Schwann Cells in Spinal Cord Lesion
Karen Dubbin 1 , Laura Marquardt 1 , Vanessa Doulames 1 , Lei Cai 1 , Giles Plant 1 , Sarah Heilshorn 1
1 Stanford University Stanford United States
Show AbstractApproximately 12,000 new spinal cord injuries (SCI) occur in the US each year, primarily affecting young adults. Schwann cells are a promising therapy currently being explored in clinical trials to treat SCI; however, significant limitations in cell delivery and long-term survival decrease their therapeutic potential. The low cell retention post-transplantation is partly attributed to (i) mechanical forces during injection that damage the cell membrane and (ii) the lack of a three-dimensional (3D) matrix to support cell viability post-injection. We hypothesize that the development of a shear thinning, injectable hydrogel will improve cell viability, engraftment, and regenerative capacity after transplantation. This hydrogel, produced from a blend of engineered, recombinant protein and peptide-modified, synthetic polymer is termed SHIELD: Shear-thinning Hydrogel for Injectable Encapsulation and Long-term Delivery. In vitro evaluation of SHIELD versions of different stiffness was explored for proliferation, caspase activity, and metabolic activity to determine the optimal formulation for Schwann cells. One version of SHIELD was used to encapsulate adult rat Schwann cells and injected into a rat cervical (C5) contusion model. Schwann cells transplanted within the hydrogel reduced lesion volumes in the injured spinal cord when compared to cells delivered in saline and injury only control groups. Even a minimal functional recovery would result in a vast quality-of-life improvement for SCI patients; therefore, developing a regenerative therapy for SCI would be extremely significant clinically.
12:30 PM - *BM3.4.09
Redox Active Multivalent Nano-Rare Earth Particle for Wound Healing Application
Soumen Das 2 , Srinu Chigurupati 3 , William Self 2 , Mark Mattson 4 , Kenneth Liechty 5 , Sudipta Seal 1
2 University of Central Florida Orlando United States, 3 FDA DC United States, 4 National Institutes of Health Baltimore United States, 5 University of Colorado School of Medicine Aurora United States, 1 NanoScience and Technology Center, Material Science and Engineering, Advance Material and Processing Center, College of Medicine University of Central Florida Orlando United States
Show AbstractWound repair and tissue regeneration is mediated through complex interactions of extracellular matrix, various residential cells, soluble mediators and infiltrating leukocyte subtypes. Impaired normal/diabetic wound healing can result in multiple adverse health outcomes and, although antibiotics can forestall infection, treatments that accelerate wound healing are lacking. Cerium oxide nanoparticles (CNPs), a novel nanomaterial with oxygen buffering capacity and can regulate intracellular oxygen environment. This redox activity of the CNPs is facilitated by the ability of CNPs to mediate its oxidation state between 3+ and 4+ oxidation states. In this study, we have report pro-angiogenic property of CNPs and application which accelerates the healing of full-thickness dermal wounds in mice. Interestingly, we have also found that increase in surface 3+ oxidation state facilitate the angiogenesis process. Not only in normal mice, CNPs also accelerated diabetic wound closing and reduced oxidative stress by suppressing NF-kB- pathway. The ability of topical application of Nanoceria to accelerate wound healing in an animal model may provide a rationale to develop this technology for use in humans affected by traumatic injury, diabetes and/or burns.
BM3.5: Biomaterials for Musculoskeletal Tissue Regeneration
Session Chairs
Drew Clearfield
Alexandra Porter
Tuesday PM, November 29, 2016
Hynes, Level 1, Room 101
2:30 PM - *BM3.5.01
Cellular and Functional Evaluation of Bony Repair Using GFP Reporter Mice and Fluorescent Cryohistological Techniques
David Rowe 1
1 University of Connecticut Health Center Farmington United States
Show AbstractWe have developed an in vivo approach for evaluating the effectiveness of a scaffold/cell based skeletal repair strategy that is rapid, informative and relatively low cost so that the initial modifications inherent in any developmental process can be tested and the best solution identified. The workflow is performed in immune-compromised mice (NSG) carrying a Col3.6GFPtpz reporter to identify host-derived cells. When a scaffold/donor cell strategy is being developed, the donor cells will carry a distinguishable GFP color that reflects the level of cellular differentiation or cell lineage. The cellular aspects of the repair uses a cryohistological imaging method that is effective in non-decalcified tissues such that fluorescent mineralization lines can be co-localized with the GFP signals of the host and donor cells. The technology allows multiple round of fluorescent histological staining (AP, TRAP, EdU, antibodies) to be performed on the same tissue section to create a stack of superimposed images that relate a specific biological signal to the developing repair process. The imaging utilizes an epifluorescent whole tissue (100x) automated scanning platform (Axioscan Z1) capable of imaging multiples sections per slide. The output allows the user to analyze the histological outcome on their computer from the stacked output file usually within 7-10 days of animal sacrifice.
Using this histological platform, we encourage material scientists to first test their scaffold without cells in a calvarial defect model to assess potential toxicity to the host. Subsequently, the scaffold can be loaded with a growth factor or donor progenitor cells and evaluated in a bilateral calvarial defect in which one of the two holes can serve as a control for the test objective. The outcome is assessed by digital X-ray of the excised calvaria followed by the histological analysis. Once the best conditions for calvarial defect repair is obtained, then a functional test of skeletal repair is performed in a long bone segmental defect model (femur or tibia, either acute or chronic non-union) that utilizes an external pin fixation procedure. The fixation allows for repetitive digital x-rays of the repair site to determine the temporal progression of the healing response, modulation of the loading forces across the defect space and the ability to remove the fixation pins to demonstrate that the regenerated bony tissue is capable of restoring normal ambulation or for direct mechanical testing without obstructing plates or intramedullary pins. Histological evaluation will demonstrate the extent of bone remodeling which in turn will reflect the regenerative potential of the cells (host or donor) that population the repair zone. It is only after the best strategy for obtaining a functional repair is identified in the mouse that it is implemented in a larger animal model to ensure that the strategy can be scaled for an eventual human application.
3:00 PM - BM3.5.02
Biomimetic Multizonal Scaffolds for Osteochondral Tissue Engineering
Drew Clearfield 1 , Mei Wei 1
1 Department of Materials Science and Engineering University of Connecticut Storrs United States
Show AbstractCurrent clinical treatments for osteochondral defects are limited due to the intrinsic poor repair capacity of the tissue. Tissue engineering-based approaches are actively being developed, but often fail to recapitulate the unique zonal organization of osteochondral tissue that is vital for its function. In the current work, two novel lyophilization-based process have been developed that yield monolithic scaffolds with zone-specific collagen fiber anisotropy and chemical composition. In one process, coined sequential freeze casting, an aqueous collagen suspension is unidirectionally frozen at a specific cooling rate to mimic the aligned superficial zone of articular cartilage. The frozen material is flipped 90°, partially melted, and a second collagen suspension is unidirectionally frozen atop the first. The entire construct is then lyophilized to confer porosity. Scaffolds fabricated by this method mimic the superficial, transition, and deep zone morphologies of articular cartilage in a single material. Furthermore, zone-specific pore size can be controlled by the applied cooling rate during the freezing regimes. To mimic additional complexity of osteochondral tissue, a second process coined lyophilization bonding was developed. In this process, aligned collagen-hyaluronic acid and collagen-hydroxyapatite scaffolds are joined together by the lyophilization of an intermediate collagen-hyaluronic acid suspension. The resulting scaffolds mimic the specific fiber directionality and chemical composition of the superficial, transition, calcified cartilage, and osseous zones of osteochondral tissue. Confocal microscopy displayed a depth-dependent increase of hyaluronic acid within the chondral compartment. Elemental mapping confirmed a mineral boundary reminiscent of a native tidemark between calcified and non-calcified cartilage zones. TGA and XRD analyses identified the presence of carbonated hydroxyapatite in the osseous zone. These multizonal scaffolds may serve as excellent candidates as biomaterials for osteochondral tissue repair.
3:15 PM - BM3.5.03
The Nature of Protein Layer Adsorbed on Bioactive Glass Scaffolds and Its Impact on Cell Response
Ukrit Thamma 1 , Roman Golovchak 2 , Tia Kowal 1 , Matthias Falk 1 , Himanshu Jain 1
1 Lehigh University Bethlehem United States, 2 Austin Peay State University Clarksville United States
Show AbstractThere are extensive investigations of how the composition and structure of bioactive glass (BG) may influence cell response, which ultimately dictate its performance as a bioscaffold. The first BG, the 45S5 composition (24.4mol%Na2O–26.9mol%CaO–2.6mol%P2O5–46.1mol%SiO2), was invented over 40 years ago. Since then the ability of BGs to grow hydroxyapatite has been considered to be the most crucial factor for its performance as a bioscaffold. We show that this is an overly simplified consideration, since the surface of glass is almost immediately covered with the proteins that are present in the cell culture medium or in body fluids. Consequently, the cells interact most directly with this proteinated interfacial layer. We hypothesize that the structure and composition of BG affect the cell response by altering the structure of the protein layer. In this work we have characterized the structure of bovine serum albumin, a major blood serum protein, by X-ray photoelectron spectroscopy and Raman spectroscopy. The attachment and proliferation of MC3T3-E1 pre-osteoblast cells correlate with specific features of protein structure, which itself is influenced by BG micro/nano structure. Our results indicate that (i) a protein layer quickly adsorbs to the surface of 45S5 bioglass, (ii) the conformation of adsorbed proteins is guided by the glass nanotopography, (iii) the adsorbed protein film mediates the attachment of cells, and (iv) the secondary conformation of the attached proteins directly impacts cell adhesion.
3:30 PM - BM3.5.04
PEDOT:PSS Polyelectrolyte Incorporated Hydrogels for Muscle Tissue Regeneration
Andrew Spencer 1 , Hicham Fenniri 1 , Nasim Annabi 1 2 3
1 Chemical Engineering Northeastern University Boston United States, 2 Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology Cambridge United States, 3 Wyss Institute for Biologically Inspired Engineering, Harvard University Boston United States
Show AbstractThe function of many tissues, including muscle tissue, is highly dependent upon electrochemical signals between neighboring cells. Naturally, many of these tissues are conductive. For the regeneration of such tissues, scaffolds must be designed with properties that mimic the native tissue, such as mechanical strength, chemical composition, microarchitecture and conductivity. Here we have synthesized a hydrogel scaffold with properties that can be tailored to mirror those of excitable tissues. By dispersing a polyelectrolyte (PE) complex, poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS), within a photo-crosslinkable biopolymer, gelatin methacrylate (GelMA), we have produced a flexible and electroactive hydrogel that is rapidly crosslinked when exposed to light. Live/Dead, Actin/DAPI and PrestoBlue assays on cell-laden 3D constructs of this scaffold demonstrate that viability of C2C12 myoblasts inside the conducting scaffold was not affected by the incorporation of the PE complex. Ex vivo stimulation of rat abdominal tissue showed that the scaffold can act as a conductive bridge, enabling stimulation of severed pieces of tissue connected only by the conductive composite hydrogel. Additionally, it was seen that the minimum voltage required to stimulate both pieces of tissue connected with the hydrogel, termed the threshold voltage, was lower with increasing concentration of the PE complex. The engineered conductive hydrogel has potential to be used as a flexible 3D scaffold for the regeneration of soft excitable tissues.
4:15 PM - *BM3.5.05
The Cellular Origins of Bone Mineralization
Alexandra Porter 2 , Linn Hobbs 1
2 Department of Materials Imperial College London London United Kingdom, 1 Department of Materials Science and Engineering Massachusetts Institute of Technology Cambridge United States
Show AbstractIn bone, mineralization is extensive and forms a bicontinuous composite of mineral (chiefly carbonated hydroxyapatite and precursors) and self-assembling proteins (mostly type I collagen). The process of mineralization and link between how mineral is delivered from the cells (osteoblasts) and crystallizes in the extracellular matrix (i.e. collagen and non-collagenous proteins) is poorly understood. Electron microscopies are extending the understanding of these crucial interfaces—their microstructures and ultrastructures, and their formation routes—beyond that provided by traditional light-microscope-based histology. In situ testing methods in the electron microscopes are also providing links between structure and tissue mechanics at the fibril level of hierarachy. We have provided evidence that the mineral is delivered prepacked via vesicles which emerge from osteoblasts early in mineralization. A series of chemical and morphological transformations of the mineral occur during transportation of the mineral from the cells to the proteins in the extracellular milieu. Analogous sequences and mineral structures are observed on the surface of hydroxyapatite implants very soon after implantation in a canine model. These observations have important implications in deciphering both how normal bone forms and in understanding, and ultimately treating, pathological mineralization.
4:45 PM - BM3.5.06
Evaluation of Particle Size Effects of Hydroxyapatite/Collagen Nanocomposite Powder on Its Bone Paste Properties
Taira Sato 2 1 , Yuki Shirosaki 3 , Mamoru Aizawa 2 , Masanori Kikuchi 1
2 School of Science and Technology Meiji University Kawasaki Japan, 1 National Institute for Materials Science Tsukuba Japan, 3 Frontier Research Academy for Young Researcher Kyushu Institute of Technology Kita-Kyushu Japan
Show AbstractSelf-setting bone pastes is utilized for minimally invasive surgery, fit to the complicated shape of bone defects easily and applied to additive manufacturing to control its structure; however, final phase of practically available pastes is hydroxyapatite which has no or very low biodegradability and has risk of secondary fracture, etc. Thus, surgeons desire biodegradable/bioresorbable self-setting bone paste. A hydroxyapatite/collagen bone-like nanocomposite (HAp/Col) is incorporated into bone remodeling process, and its porous body has been widely used in Japan as an excellent bioresorbable bone filler; therefore, it might be good candidate for bioresorbable bone paste material.
A HAp/Col bone paste with appropriate injectability, anti-washout property, viscoelasticity, in vivo biocompatibility and bioresorbability had been successfully fabricated by mixing of the HAp/Col powder and (3-glycidoxypropyl)trimethoxysilane (GPTMS) aqueous solution. In this study, influences of the HAp/Col powder particle size on physical properties of the paste were investigated by using the HAp/Col particles with three different classified particle sizes.
The HAp/Col at the hydroxyapatite and collagen mass ratio of 80:20 was synthesized by the simultaneous titration method. The HAp/Col powder, a powder phase of HAp/Col self-setting bone pastes, was prepared by a uniaxial compacting dehydration, freeze-drying, crushing and classifying to 25-53, 53-100 and 100-212 µm in sizes by sieving. Distilled water was used as a liquid phase of the paste for a preliminary injectability test, because the previous report revealed that initial viscosity of the HAp/Col-GPTMS paste was independent of GPTMS concentrations. The powder/liquid ratios of the pastes were ranging from 0.33 to 1.50 g/cm3. The paste mixed was filled in a syringe of 1.8 mm in inner diameter of extrusion path. An extrusion force of the paste was measured by application of compressive stress to the paste-filled syringe at a crosshead speed of 5 mm/min at 10 min after the paste mixing started. A paste extrusion force in this study was defined as a mean of stress values in plateau area of the stress-strain curve. Other properties of the HAp/Col powder and paste were also investigated.
Extrusion forces of the pastes prepared with 25-53, 53-100 and 100-212 µm powders were 15.93±1.39 N, 19.32±0.68 N and 19.49±0.33 N, respectively. The extrusion forces showed tendency to increase with increasing of the particle size in the range of 25-212 µm. This result suggested that the pastes with smaller particles would have advantages for the smooth flow through the syringe with small inner diameter and showed better injectability. The results of other properties of the HAp/Col powder and paste will be presented on a podium.
5:00 PM - BM3.5.07
Engineering a Photocrosslinkable and Antibacterial Hyaluronic Acid/Elastin like Polypeptide Hybrid Hydrogel for Cartilage Tissue Regeneration
Ehsan Shirzaei Sani 1 , Nasim Annabi 1 , Thomas J. Webster 1 , Iman Noshadi 2
1 Chemical Engineering Northeastern University Boston United States, 2 Wyss Institute for Biologically Inspired Engineering at Harvard University Boston United States
Show AbstractEhsan Shirzaei Sani1, Iman Noshadi1,2,3, Roberto Portillo Lara1,Wendy Yu1, Benjamin Geilich1, Thomas J. Webster1,4,5, and Nasim Annabi1,2,3
1Department of Chemical Engineering, Northeastern University, Boston, MA, 02115-5000, USA.
2Biomaterials Innovation Research Center, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA.
3Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA.
4Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, Saudi Arabia.
5Wenzhou Institute of Biomaterials and Engineering, Wenzhou Medical University, Wenzhou, China.
Cartilaginous connective tissues play important roles associated with mechanical load support and energy dissipation, in joints of the musculoskeletal system. Cartilage is composed of specialized cells called chondrocytes, which are dispersed within a dense extracellular matrix (ECM) comprised primarily of type II collagen and proteoglycans. Cartilage is characteristically avascular, aneural and alymphatic, and has low cell densities, which limits its ability for self-repair after injury.
In this work, we engineered a hybrid hydrogel composed of methacrylated hyaluronic acid (MeHA) and elastin-like polypeptides (ELPs) for cartilage regeneration, by photocrosslinking different ratios of MeHA and ELP. ELPs are artificial thermoresponsive and elastic polypeptides. The ELPs macromolecular structure can be modified through DNA recombinant techniques, which make these proteins suitable for different biomedical applications. The ELP sequence used in this study contained cysteine residues with thiol groups, which are able to form disulfide bonds upon exposure to visible or UV light. Antimicrobial ZnO nanoparticles were incorporated into the engineered hybrid hydrogels to impart antibacterial properties. Cell viability tests were performed using 3T3 fibroblast cells (ATCC® CRL-1658™). Also, bacterial properties of the hybrid hydrogels were evaluated using colony forming unit (CFU) assay with methicillin-resistant Staphyloccocus aureus (MRSA, ATCC® 43300™). The antimicrobial and physical properties of the engineered hybrid hydrogel could be finely-tuned by modifying the concentration of ZnO nanoparticles, ratio of MeHA/ELP, final polymer concentration, and crosslinking conditions. Based on the in vitro studies, MeHA/ELP hydrogels with 2% w/v MeHA and 10% w/v ELP showed more than 90% cell viability at days 1, 3, and 5 post-seeding (p < 0.05). The engineered hydrogels also prevented bacterial growth at the optimal ZnO concentration of 0.2 % (w/v). In conclusion, the engineered hybrid hydrogels have the potential to be used in biomedical applications especially as an antibacterial hydrogel for cartilage tissue regeneration.
5:15 PM - BM3.5.08
Injectable ADP/Chitosan/Ceramic (ACC) Sponge for Cellular Encapsulation in Bone Repair Applications
Kaushar Jahan 1 , Maryam Tabrizian 1
1 McGill University Montreal Canada
Show AbstractBone defects result from injuries that do not repair without medical intervention. Autologous bone graft, the gold standard for treating bone defects, is challenging due to (a) donor scarcity and (b) donor site morbidity that follows the procedure. Regenerative medicine has shown potential as an alternative intervention; it is based on the use of scaffolds which mimic the structure of the tissue that requires repair and simultaneously supports, reinforces and organizes the regenerating tissue. An injectable adenosine/chitosan/ceramic (ACC) sponge with rapid gelation time has been previously developed in our lab showing to be a biocompatible, biodegradable, and potentially osteoconductive scaffold. Based on these results, the current project is focused on the encapsulation of cells (pre-osteoblasts and other bone cell precursors) within the ACC scaffold. The physico-chemical and in vitro characterization of the cell-laden sponge was done through Scanning Electron Microscopy and micro Computed-Tomography, showing the internal structure of the scaffold. Moreover, proliferative quantification was assessed through Alamar Blue assay. A live/dead analysis was performed to determine the viability of the cells inside the sponges. Furthermore, an alkaline phosphatase assay will be performed to determine mineralization of the cells which can be confirmed by an alizarin red assay. Quantitative polymerase chain reaction will also be performed to determine the expression of some key genes in bone formation. Subsequently, the loaded scaffold can then be implanted in a mice fracture model to study its bone repair potential. Ultimately, this sponge may be a clinical alternative to bone graft by decreasing the burden of complications associated with graft donor sites while simultaneously delivering cells able to deliver therapeutic agents at the site of injury.
5:30 PM - BM3.5.09
Silk and Peptide Amphiphile Based Trilayered Osteochondral Scaffold
Soner Cakmak 1 , Anil Cakmak 1 , David Kaplan 2 , Menemse Gumusderelioglu 3
1 Environmental Engineering Hacettepe University Ankara Turkey, 2 Biomedical Engineering Tufts University Boston United States, 3 Chemical Engineering Hacettepe University Ankara Turkey
Show AbstractNew biomaterials with the properties of both bone and cartilage extracellular matrices (ECM) should be designed and used with co-culture systems to address clinically applicable osteochondral constructs. Herein, a co-culture model is described based on a trilayered silk fibroin-peptide amphiphile (PA) scaffold cultured with human articular chondrocytes (hACs) and human bone marrow mesenchymal stem cells (hBMSCs) in an osteochondral cocktail medium for the cartilage and bone sides, respectively and the trophic effects of these two cells on each other were investigated under co-culture conditions. For this purpose, silk scaffolds from 6% (w/v) solution was used as the bone part and arginine-glycine-aspartic acid-serine (RGDS) containing PA hydrogel (PA-RGDS) was used as the cartilage part, while silk scaffolds from 4% (w/v) solution was designed as the bone-cartilage interface. The pore sizes of the silk scaffolds with 6% (w/v) and 4% (w/v) silk contents were calculated as 416 ± 87 and 194 ± 67 μm, respectively. According to the real time polymerase chain reaction, the trophic effects of hACs cultured in the cocktail medium on hBMSCs were revealed as enhanced expressions of runt related transcription factor, alkaline phosphatase, collagen type I, bone sialoprotein, osteopontin and increased calcium content. When the expression levels of SOX9, aggrecan and collagen type II were evaluated, it was seen that the chondrogenic differentiation of hACs in PA-RGDS hydrogels occurred significantly earlier than pellet cultures. No significant trophic effect of co-culture on hACs was determined and this finding showed that TGF-β1 had stronger influence on the chondrocyte differentiation over the co-culture. However, the hACs in the co-culture still preserved the amount of synthesized chondrogenic ECM with comparable level to single culture even though the culture medium did not contain exogenous TGF-β family. By histology stainings, it was seen that the hBMSCs in co-culture intensely filled the pores of the scaffold and it was seen that these cells also produced higher amounts of denser mineralized structures detected by Alizarin red staining. Besides, densely production of cartilage ECM by the hACs in co-culture on PA-RGDS hydrogels was shown by Alcian blue staining. The findings of this study has shown that hBMSCs could able to form an enabled bone structure by co-culture technique without the use of expensive growth factors and chondrocytes could produce cartilage matrix even in the absence of TGF-β1 and the use of a co-culture strategy including layered scaffolds could be further pursued towards the goal to treat osteochondral defects.
5:45 PM - BM3.5.10
Delivery of Kartogenin-Conjugated Hyaluronic Acid for Enhancing
In Vitro Chondrogenic Differentiation
Lauren Levy 3 1 , Bethany Almeida 2 , Nisha Hollingsworth 2 , Anita Shukla 2
3 Department of Chemistry Brown University Providence United States, 1 Department of Medicine, Renal Division Brigham and Women's Hospital Boston United States, 2 School of Engineering, Center for Biomedical Engineering Brown University Providence United States
Show AbstractChondrocytes do not have the potential to regenerate; thus, loss of cartilage loss is an irreversible process. To treat cartilage loss, multipotent bone marrow-derived human mesenchymal stem cells (hMSCs) are commonly used (Mobasheri 2014). However, in vitro chondrogenic differentiation often results in a heterogeneous population of cells, which is suboptimal for therapeutic use. Kartogenin (KGN) is a recently identified small molecule that promotes chondrogenesis and may help improve stem cells' chondrogenic differentiation. KGN’s inherent hydrophobicity hinders its delivery to cells in the aqueous culture environment (Johnson 2012). We hypothesized that conjugating KGN to a hydrophilic polymer would improve its water solubility and increase its bioactivity in promoting chondrogenesis. To study this hypothesis, KGN was conjugated to hyaluronic acid (HA) via Mitsunobu esterification, a novel conjugation method for KGN. HA was selected for its biocompatibility and affinity for CD44, a cell surface receptor targeted by KGN.
Fourier transform infrared spectroscopy (FTIR) showed characteristic ester absorption bands at 1720 cm-1 and 1237-1073 cm-1, indicating successful ester bond formation between one of HA’s alcohols and KGN’s carboxylic acid for the reaction product only. For a representative synthesis, size exclusion chromatography revealed a percent conjugation of 74%. As KGN is hydrophobic, we sought to demonstrate that this conjugation improves its water solubility. A turbidity study showed that the HA-KGN conjugate is soluble up to 1 mM in water and between 100 µM and 1 mM in 1X PBS, whereas unconjugated KGN is insoluble in these solvents.
Next, we integrated the HA-KGN conjugate into 3D pellet culture of hMSCs to investigate if conjugation enhanced chondrogenic differentiation. Immunocytochemistry at 7 and 21 days of culture revealed that HA-KGN significantly increased the collagen II to collagen I ratios (p < 0.05), indicative of the chondrogenic phenotype. Alcian blue staining at 7 days also showed enhanced sulfated glycosaminoglycan extracellular matrix (ECM) composition typical of chondrocytes. To further investigate the chondrogenic effect of HA-KGN, qRT-PCR was performed at 7 days. Relative gene expression analysis showed up-regulation of COL10A1 and down-regulation of COL2A1 and COL1A1 for HA-KGN treated hMSCs compared to KGN controls. This gene expression pattern is consistent with temporal expression of ECM components during chondrogenesis, suggesting that HA-KGN might enhance chondrogenic differentiation with greater efficacy than unconjugated KGN specifically during the early stages of chondrogenesis. Thus, we proposed that the enhanced cellular availability of KGN, via its link to HA and the HA-CD44 interaction, allows for increased activation of the intracellular signaling pathways implicated in chondrogenesis, such that hMSCs more effectively undergo chondrogenesis.
BM3.6: Poster Session II
Session Chairs
Wednesday AM, November 30, 2016
Hynes, Level 1, Hall B
9:00 PM - BM3.6.01
Electrospun Nanofiber-Based Highly Porous Hydrogel for Efficient 3D Cell Culture
Dong-Hee Kang 1 , Sungrok Wang 1 , Dong-Yoon Kim 1 , Myung-Han Yoon 1
1 School of Material Science and Engineering Gwangju Institue Technology and Science Gwangju Korea (the Republic of)
Show AbstractElectrospun nanofibers have been utilized as a culture scaffold to mimic the extracellular matrix (ECM) in a biological tissue. Although these nanofiber scaffolds exhibit nanoscale morphology useful for tissue engineering, it has been very rare to demonstrate that they exhibit hydrogel-like properties such as high water-swelling ratio in combination with nanoscale fibril morphology which is critical for emulating the characteristic of natural ECM. Herein, we developed a new strategy to prepare electrospun nanofiber-based hydrogel via modulating polymer fiber interactions. The degree of water swelling of resultant fibrous networks could be effectively controlled and its feasibility as a 3D cell culture scaffold was demonstrated. Interestingly, it was found that cellular adhesion was much favorable on highly swollen nanofiber scaffolds compared with those with less swollen, which is counter-intuitive to the conventional knowledge that stiff scaffolds enhances cellular adhesion. We expect that our strategy to fabricate highly-swollen nanofiber scaffolds can be generalized as a methodology to prepare artificial ECM-like scaffolds with enhance cell viability using various types of materials such as synthetic polymers, protein- or –carbohydrate-based natural materials, and etc.
9:00 PM - BM3.6.02
Microwave Irradiation-Driven Conformational Diversity of Amyloid Fibrils
Taeyun Kwon 1 , Chang Young Lee 2 , Kilho Eom 3
1 Sungkyunkwan University Suwon Korea (the Republic of), 2 Ulsan National Institute of Science and Technology (UNIST), Ulsan Korea (the Republic of), 3 Sungkyunkwan University Suwon Korea (the Republic of)
Show AbstractAmyloid fibrils have been highlighted due to their pathological role, which is determined by their conformations. For instance, the conformations of prion amyloid fibrils determine the prion infectivity. Despite recent findings regarding the important role of the conformational diversity of amyloid fibrils in their pathological role, the underlying mechanisms in amyloid fibril formation with conformational heterogeneity have remained elusive. In this work, we employed the microwave-based chemical synthesis which allows us to finely tune the thermodynamics of self-assembly process leading to the conformational diversity of amyloid fibrils. It is shown that the length of amyloid fibril and the helical structure of the fibril can be controlled using microwave irradiation. This study sheds light on the microwave-based chemical synthesis for studying the conformational heterogeneity of amyloid fibrils and for developing novel design protocol, which allows for delicately manipulating the molecular structure of protein fibrils.
9:00 PM - BM3.6.03
Investigation of Structural Changes in Polycaprolactone Polymer Scaffolds Obtained with Different Collectors by Synchrotron X-Ray Imaging
Svetlana Gorodzha 1 , Roman Shkarin 2 , Venera Weinhardt 3 , Maria Surmeneva 1 , Tilo Baumbach 2 , Roman Surmenev 1
1 Institute of Physics and Technology Tomsk Russian Federation, 2 Institute for Photon Science and Synchrotron Radiation Eggenstein-Leopoldshafen Germany, 3 The Centre for Organismal Studies Heidelberg Germany
Show AbstractIntroduction
Polymer electrospun 3-D scaffolds are gaining interest as biomaterials for repairing of damaged bone tissues. The structure of the scaffold during the fabrication depends on many factors, including a type of collector used in electrospinning. Collector is a conductive substrate for collecting the charged fibers and could be presented in different shape. In case of 3-D scaffolds applied for bone tissue engineering the uniform distribution of fibers in a whole volume of the scaffold is needed for homogeneous cells adhesion and proliferation. Standard techniques used for investigation biomaterial structure provide information only in 2-D level without accessing in-depth information or with low resolution. In this study, an internal structure of polycaprolactone (PCL) polymer 3-D scaffolds obtained with static plate (SP) and rotating mandrel (RM) collectors were compared using Synchrotron X-ray radiation to determine an internal structure with high resolution. To the best of our knowledge, this is the first report on applying of Synchrotron X-ray imaging technique in comparing of internal structure of 3-D scaffolds obtained with different types of collector.
Experimental methods
Preparation of PCL scaffold were done with the same parameters as described in previous work [1]. Pyramid-like samples after static plate collector (PCL-SP) of height 1 cm and polymer film-like samples with 0.5 mm thickness after rotating collector (PCL-RM) were subjected to X-ray imaging methods respectively with resolution up to 2.5 µm at the Topo-Tomo beamline, ANKA light source facility (KIT, Germany).
Results and discussion
During comparison of two microstructures it was found that in case of PCL-SP the internal structure includes not only the presence of fiber structure, but the solid form of the polymer forming pillar for subsequent formation of fibers. On the other hand, air voids in the solid polymer part was detected. In case of PCL-RM sample the formation of micro-fiber structure was uniform in the whole volume of the sample without appearing solid polymer with no fiber structures.
Conclusion
Synchrotron X-ray radiation is a powerful tool to visualize an internal structure of weekly absorbing materials. Usage of rotating collectors allows obtaining a uniform fiber structure in each level of the sample.
References
[1] S.N. Gorodzha et al. IOP Conf. Ser. Mater. Sci. Eng. 98 (2015) 012024.
Acknowledgments
ANKA light source is thanked for the allocated beamtime and Thorsten Muller for the assistance during the experiments.
9:00 PM - BM3.6.04
Biomaterial Fiber Scaffolds for Nerve Regeneration
Alexander Mitropoulos 1 2 3 , Joseph Loverde 1 , Jonathan Ness 1 , Haana Yu 1 , Grace Yu 1 , Laura Murcin 1 , Luis Alvarez 1 4
1 Department of Chemistry and Life Science United States Military Academy West Point United States, 2 Photonics Research Center United States Military Academy West Point United States, 3 Department of Mathematical Science United States Military Academy West Point United States, 4 Center for Molecular Sciences United States Military Academy West Point United States
Show AbstractPeripheral nerve damage is one of the most debilitating injuries sustained by Warfighters leading to life-long disability. Peripheral nerve injury is often permanently incapacitating because of the complexity of sensory and motor reinnervation. Failure to regain neural function normally results in limb amputation. Large gap nerve injuries must be surgically treated with grafts in order to have good outcomes. However, this remains a clinical problem, as full recovery may never occur. Novel nerve repair technologies such as biological scaffolds and conduits have shown promise. An alternative approach is to use nerve guidance conduits (NGC) made from biomaterials that provide pathways for nerve out-growth and promote nerve regeneration by controlling morphology and structure to guide specific axons to their desired locations. Here we use a fiber based scaffold made from degradable biomaterials to guide the direction and location of regeneration. Further this NGC can guide axon extension leading to spatial separation between sensory and motor axons. Here we show the development of a scalable spool-based manufacturing process to make collagen fibers 20 µm in diameter. These fibers are coated with biomaterials found in native nerve ECM to assist with axon extension over distances exceeding 3 cm. Material characterization of the fibers was performed after individual steps to understand secondary structures that enhance the chemical structure and biocompatibility. Chick sensory axons grow at a rate of 1 cm over seven days as measured by confocal microscopy. Additionally, to promote the specificity of axon extension nerve growth factor (NGF) has been tethered to the fiber surface. Additional growth factors can be tethered to promote separation and growth of sensor and motor axons over extended distances. This work seeks to translate findings to address clinical needs in trauma repair, neuro-degeneration, and neural interfaces.
9:00 PM - BM3.6.05
Sticky Stem Cells—Reengineering the Membrane Using Hybrid Bionanomaterials
Rosalia Cuahtecontzi Delint 1 , Adam Perriman 1 , Andrew Collins 1 , Wael Kafienah 1 , Terrence McMaster 1 , Ross Anderson 1 , Paul Race 1
1 University of Bristol Bristol United Kingdom
Show AbstractMore than 120,000 people in the USA need organ transplants1. However, demand is significantly greater than availability providing a strong driving force for the development of new research areas. Tissue engineering and regenerative medicine involve the integration of scaffolds and stem cells to provide functional tissue for autologous grafts. One of the challenges in this area, specifically when seeding cells in decellularised scaffolds, is the efficiency of strong, localised and rapid adhesion.
To address this issue, we describe the rational design of a novel hybrid nano-construct that spontaneously insert into the membrane of human mesenchymal stems cells (hMSCs) and will provide increased adhesion to scaffolds. Significantly, it has recently been shown that a 21 peptide chain found in placenta growth factor 2 has an extremely high binding activity for proteins found in the extracellular matrix (ECM)2 such as fibronectin, vitronectin, tenascin C, osteopontin, fibrinogen and collagen I. This sequence was fused to a supercharged fluorescent protein that interacts with hMSC membranes. Electrostatic assembly was used to produce a polymer surfactant corona at the surface of the protein which will facilitate the attachment to the cell membrane via hydrophobic interactions between the hydrophobic tail and the bilayer on the cell membrane without the loss of protein fluorescence3. By combining these elements, it will be possible to display multiple copies of the nanoconstruct on the membrane to promote increased adherence to ECM proteins present on decellularised scaffolds. We have expressed and purified the fusion protein prior to conjugation with characterization by a variety of different biophysical methods and have subsequently demonstrated successful membrane insertion.
(1) U.S. Department of Health & HumanServices. Organ Procurement and Transplantation Network https://optn.transplant.hrsa.gov/.
(2) Martino, M. M.; Briquez, P. S.; Güç, E.; Tortelli, F.; Kilarski, W. W.; Metzger, S.; Rice, J. J.; Kuhn, G. A.; Müller, R.; Swartz, M. A.; Hubbell, J. A. 2014, 343, 885–889.
(3) Armstrong, J. P. K.; Shakur, R.; Horne, J. P.; Dickinson, S. C.; Armstrong, C. T.; Lau, K.; Kadiwala, J.; Lowe, R.; Seddon, A.; Mann, S.; Anderson, J. L. R.; Perriman, A. W.; Hollander, A. P. Nat. Commun. 2015, 6, 7405.
9:00 PM - BM3.6.06
Combining Dual Growth Factor Delivery and Contact Guidance in Electrospun Nanofibrous Scaffolds for Peripheral Nerve Regeneration
Chaoyu Liu 1 , Min Wang 1
1 University of Hong Kong Hong Kong Hong Kong
Show AbstractUsing nanofibrous scaffolds capable of providing controlled growth factor delivery and contact guidance are advantageous in tissue engineering. The aim of this investigation was to study the fabrication of nanofibrous bicomponent scaffolds for the dual delivery of glial cell line-derived growth factor (GDNF) and nerve growth factor (NGF) and also the fabrication of aligned-fiber scaffolds providing both biochemical and topographical cues and evaluate their biological performance for peripheral nerve tissue engineering. GDNF and NGF were incorporated into core-shell structured poly(lactic-co-glycolic acid) (PLGA) and poly(D,L-lactic acid) (PDLLA) nanofibers, respectively, through emulsion electrospinning. Using dual-source dual-power electrospinning (DSDP-ES), nonwoven bicomponent scaffolds composed of GDNF/PLGA fibers and NGF/PDLLA fibers with different fiber component ratios were successfully made. The structure and properties, including in vitro release behavior and in vitro degradation, of mono- and bicomponent scaffolds were studied. Subsequently, aligned-fiber bicomponent scaffolds (GDNF/PLGA fibers and NGF/PDLLA fibers) were fabricated using DSDP-ES and high-speed electrospinning (HS-ES). Concurrent and sustained releases of GDNF and NGF from bicomponent scaffolds were achieved and their release profiles could be tuned. The aligned-fiber topography and dual and sustained delivery of growth factors were realized in aligned-fiber bicomponent scaffolds. In vitro biological investigations were conducted for both nonwoven and aligned-fiber bicomponent scaffolds. The bioactivity of GDNF and NGF was preserved to a large extent in scaffolds. Rat pheochromocytoma cells (PC12 cells) were found to attach, spread and proliferate on all scaffolds. Fibrous scaffolds alone only induced limited cell differentiation into neuron-like phenotype characterized by neurite outgrowth. The release of growth factors from scaffolds could induce much improved neurite outgrowth and neural differentiation. GDNF and NGF released from GDNF/PLGA scaffolds and NGF/PDLLA scaffolds, respectively, could induce dose-dependent neural differentiation separately. A synergistic effect of GDNF and NGF released from bicomponent scaffolds of specific component ratio on promoting neural differentiation was found. The fiber alignment in aligned-fiber bicomponent scaffolds could induce neurite outgrowth, neural differentiation and neurite alignment to certain extent. The release of growth factors and aligned-fiber morphology induced neurite alignment and much enhanced neural differentiation in a synergetic manner. The properties of bicomponent scaffolds, including excellent biocompatibility, reasonable mechanical properties, controlled degradation rate, tunable dual release behavior of growth factors and contact guidance, make these scaffolds highly promising for peripheral nerve tissue regeneration.
9:00 PM - BM3.6.07
Targeted Delivery of Biomaterials to Cell Membranes Using Holographic Optical Tweezers for Regenerative Medicine Purposes
Monika Jakimowicz 1 , Adam Perriman 1 , Sean Davis 1 , Avinash Patil 1
1 University of Bristol Bristol United Kingdom
Show AbstractThe application of holographic optical tweezers for the spatially resolved delivery of biomolecules to individual cell cytoplasmic membranes possesses great potential for single cell experiments, with particular utility in the field of regenerative medicine. This novel approach utilises membrane-free, liquid-in-liquid phases known as coacervate microdroplets, which readily sequester a range of biomolecules in a dose-dependent fashion. Employing holographic optical tweezers for the trapping and manipulation of loaded coacervate microdroplets allows for targeted delivery of guest species. We have successfully demonstrated this with green fluorescent protein, dye-tagged oligonucleotides, none of which possess native membrane affinity, as well as Hoechst 33258 dye. The high spatial-precision that optical tweezer technology offers can be exploited to target specific areas of individual cells, and to spontaneously functionalise discrete areas of cell membranes. Successful payload delivery is confirmed using confocal fluorescence microscopy, revealing the painted area to be localized to the site of the interaction.
The results suggest that the absence of an outer membrane around the payload grants easier and more efficient interactions with cells, compared to other microdroplet systems such as liposomes, which require rupturing using external stimuli.(1) The high sequestration efficiency, superficial membrane adhesion of the microdroplets, and high spatial precision of delivery make this “cell paintballing” technology a desirable method for studying the lateral diffusion of membrane-active drugs, measuring membrane receptor-ligand forces, and spatially-controlled cell functionalization, with potential applications in single-cell transfection and differentiation.
(1) Armstrong, J. P. K., Olof, S. N., Jakimowicz, M. D., Hollander, A. P., Mann, S., Davis, S. A., Perriman, A. W. (2015). Cell paintballing using optically targeted coacervate microdroplets. Chem. Sci., 6(11), 6106–6111. http://doi.org/10.1039/C5SC02266E
9:00 PM - BM3.6.08
Designing a Novel Bionanohybrid to Enable In Vivo Stem Cell Homing
Tom Green 1 , Wenjin Xiao 1 , Paul Race 1 , Adam Perriman 1
1 University of Bristol Bristol United Kingdom
Show AbstractHeart disease currently causes more deaths than any other in the developed world, affecting an estimated 38 million people worldwide. Current treatment is palliative and unable to restore the heart to optimum capacity prior to the onset of disease. Moreover, replacements from donors are often in short supply. Regenerative therapy utilizing stem cells is, therefore, an attractive prospect for restoring heart tissue, but this approach is limited by inefficient cell localisation to affected tissue post administration. Here, we propose a method of in vivo homing by re-appropriating a naturally occurring adhesion molecule to enable mesenchymal stem cells (MSCs) to target specific tissue. To enable the molecule to adhere to the MSC surface, we have expressed it as a fusion protein conjugated to a highly cationic variant of another naturally occurring protein, which has previously been shown to possess high membrane affinity. We have shown rapid and effective incorporation of the fusion protein into the MSC membrane as visualised by live cell confocal fluorescence microscopy. Current work is focussed on determining the orientation of the protein at the cell membrane using antibody staining, which will be followed by the development of a binding assay to assess the specificity of the functionalized cells for target tissue.
9:00 PM - BM3.6.09
Rheological Characterisation of Alginate Based Hydrogels for Tissue Engineering
Pengfei Duan 1 , Nehir Kandemir 1 , Jiajun Wang 1 , Jinju Chen 1
1 Newcastle University Newcastle Upon Tyne United Kingdom
Show AbstractAlginate hydrogels have received intensive attentions for tissue engineering and drug delivery systems in the past two decades. For both tissue engineering and drug delivery, the mechanical properties are important because they would affect cell-materials interactions and injectability of drugs encapsulated in hydrogel carriers. To improve the ability of tissue regeneration, alginate gel is often covalently modified with peptides or copolymerised with other protein-based materials (i.e., fibrin, collagen). These modified alginate gels have been demonstrated to enhance cell adhesion and proliferation. However, it remains elusive how these modifications may affect the mechanical properties of alginate hydrogels. Therefore, in this study we adopted oscillatory rheological tests to characterise the viscoelastic properties of alginate gels with various concentrations, RGD (arginine-glycine-aspartic acid) modified alginate with different concentrations, and copolymerised collagen/alginate/fibrin hydrogels with different concentrations. The tests were performed at 37°C using Malvern Kinexus Pro rotational rheometer which is equipped with temperature control stage. A special solvent trap system was used to avoid dehydration of the sample during the tests. The alginate gels have demonstrated shear thinning behaviour which indicates that they are suitable candidates as carriers for cells or drug delivery. Our results also revealed that the addition of RGD has very small effect on the elastic modulus and viscosity of alginate. The copolymerised collagen/alginate/fibrin hydrogels are stiffer than the corresponding collagen gels. At linear viscoelastic region, the elastic modulus and loss modulus for collagen/alginate/fibrin hydrogels exhibit some frequency dependent behaviour which did not occur for alginates and RGD modified alginates. This may be correlated to the degree of crosslinking of the collagen/alginate/fibrin copolymerised gels. It is also found that collagen/alginate/fibrin copolymerised gels prepared with high initial collagen concentration in the solution would degrade during the rheological tests.
9:00 PM - BM3.6.10
Optimization of a Rapidly-Gelling Chitosan Sponge for Cell Delivery—Influence of Concentration, pH and Cross-Linker Solution
Timothee Baudequin 1 , Laila Benameur 1 , Maryam Tabrizian 1
1 Department of Biomedical Engineering McGill University Montréal Canada
Show AbstractA rapidly in-situ gelling scaffold (less than 1.6 second) based on chitosan crosslinked either Guanosine-5’-diphosphate (GDP) [1] or Adenosine 5'-diphosphate (ADP) has been developed in our laboratory as a minimally invasive injectable system for bone regeneration in critical size defects. Besides their chemical and mechanical properties, scaffolds can improve tissue repair by serving as a carrier for cell delivery. The main goal of this study was to investigate the potential of our chitosan sponge for cell encapsulation. The process was optimized with the encapsulation of the pre-osteoblastic cell line MC3T3-E1. The optimal conditions were chosen after chemical, structural and biological characterizations of the chitosan sponge over a week of culture.
Two chitosan solutions (3 mg/mL and pH 5 (C3PH5) or 6 mg/mL and pH 6 (C6PH6)) and two cross-linkers, GDP or ADP, resulting in 4 different sponges, were studied. Chemical and structural characterizations of the different cases were obtained with MicroCT and FTIR measurements. We especially focused on porosity properties over time (open and closed porosities, pore volume, surface/volume ratio at days 1, 4 and 7). The murine pre-osteoblastic cell line MC3T3-E1 was encapsulated over a week to select the best sponge parameters. Cell metabolic activity, proliferation (Alamar blue, DNA quantification, fluorescence and SEM microscopy) and differentiation (ALP staining) were assessed.
The pre-osteoblastic cells were able to survive over the week after encapsulation in the chitosan sponge. The optimal behaviour was found with GDP solution as the cross-linker according to metabolic activity (p<0.05), SEM observations of the core of the sponge and actin filaments staining. We investigated correlations between porosity properties and the evolution of activity over time. Chitosan concentration and pH did not lead to significant differences but C3PH5 solution offered technical advantages (especially regarding solution stability and filtration). The GDP C3PH5 sponge was thus validated as the optimal chitosan sponge for cell encapsulation, and a promising method for cell delivery in critical size defects.
[1] M. Mekhail, J. Daoud, G. Almazan, and M. Tabrizian, “Rapid, guanosine 5’-diphosphate-induced, gelation of chitosan sponges as novel injectable scaffolds for soft tissue engineering and drug delivery applications,” Adv Heal. Mater, vol. 2, no. 8, pp. 1126–1130, 2013.
9:00 PM - BM3.6.11
Multi Material 3D Scaffold Printing with Maskless Photolithography
Ozlem Yasar 1 , Joyce Tam 2
1 Mechanical Engineering Technology City University of New York Brooklyn United States, 2 Mechanical Engineering Technology New York City College of Technology Brooklyn United States
Show AbstractIn today’s technology, organ transplantation is found very challenging as it is not easy to find the right donor organ in a short period of time. In the last several decades, Tissue Engineering was rapidly developed to be used as an alternative approach to the organ transplantation. Tissue Engineering aims to regenerate the tissues and also organs to help patients who waits for the organ transplantation. Recent research showed that in order to regenerate the tissues, cells must be seeded onto the 3D artificial laboratory fabricated matrices called scaffolds. If cells show health growth within the scaffolds, they can be implanted to the injured tissue to do the regeneration. One of the biggest limitation that reduces the success rate of tissue regeneration is the fabrication of accurate thick 3D scaffolds. In this research “maskless photolithography” was used to fabricate the scaffolds. Experiment setup consist of digital micro-mirror devices (DMD), optical lens sets, UV light sources and PEGDA which is a liquid form photo-curable solution. In this method, scaffolds are fabricated in layer-by-layer fashion to control the interior architecture of the scaffolds. Working principles of the maskless photolithography is, first layer shape is designed with AutoCAD and the designed image is uploaded to the DMD as a bitmap file. DMD consists of hundreds of tiny micro-mirrors. When the UV light is turned on and hit to the DMD, depending on the micro-mirrors’ angles, UV light is selectively reflected to the low percentage Polyethylene (glycol) Diacrylate (PEGDA) photo-curable solution. When UV light penetrates into the PEGDA, only the illuminated part is solidified and non-illuminated area still remains in the liquid phase. In this research, scaffolds were fabricated in three layers. First layer and the last layer are solid layers and y-shape open structure was sandwiched between these layers. After the first layer is fabricated with DMD, a “y-shape” structure was fabricated with the 3D printer by using the dissolvable filament. Then, it was placed onto the first solid layer and covered with fresh high percentage PEGDA solution. UV light was reflected to the PEGDA solution and solidified to make the second and third layers. After the scaffold was fabricated, it is dipped into the limonene solution to dissolve the y-shape away. Our results show that thick scaffolds can be fabricated in layer-by-layer fashion with “maskless photolithography”. Since the UV light is stable and does not move onto the PEGDA, entire scaffold can be fabricated in one single UV shot which makes the process faster than the current fabrication techniques.
9:00 PM - BM3.6.12
Microfluidics as a Tool to Generate Simple and Multiple Emulsion for Templating Scaffolds for Tissue Engineering
Piotr Garstecki 2 , Marco Costantini 2 , Cristina Colosi 2 , Jakub Jaroszewicz 3 , Alessia Tosato 2 , Wojciech Swieszkowski 3 , Mariella Dentini 2 , Jan Guzowski 1 , Slawomir Jakiela 1 , Piotr Korczyk 1 , Andrea Barbetta 2
2 Department of Chemistry Sapienza University of Rome Rome Italy, 3 Faculty of Materials Science and Engineering Warsaw University of Technology Warsaw Poland, 1 Institute of Physical Chemistry Warsaw Poland
Show AbstractMultiphase microfluidic systems can be engineered to generate highly ordered templates that can later be used to produce porous matrices from dextran-methacrylate (DEX-MA). We use a flow focusing devices and active on-demand microfluidic systems allow to generate emulsions of tightly controlled droplet size or droplet size distribution and to for multiple droplets. The use of an aqueous solution of DEX-MA and surfactant to break the flow of an organic solvent (cyclohexane) allowed us to generate high volume fraction (above 74% v/v) ordered high internal phase emulsion (HIPE). We collect the crystalline HIPE structure and freeze it by gelling. The resulting polyHIPEs are characterized by an interconnected and ordered morphology. The size of pores and interconnects ranges between hundreds and tens of micrometers, respectively. The technique that we describe allows for precise tuning of all the structural parameters of the matrices, including their porosity, the size of the pores and the lumen of interconnects between the pores.
References:
M. Costantini, C. Colosi, J. Jaroszewicz, A. Tosato, W. Swieszkowski, M. Dentini, P. Garstecki, A. Barbetta
Microfluidic Foaming: a Powerful Tool for Tailoring the Morphological and Permeability Properties of Sponge-Like Biopolymeric Scaffolds.
ACS Appl Mater Interfaces, 7, 23660-23671 (2015)
J. Guzowski, P. Garstecki
Droplet Clusters: Exploring the Phase Space of Soft Mesoscale Atoms
Physical Review Letters, 114, 188302 (2015)
Costantini, M, Colosi, C., Guzowski, J., Bartetta, A., Jaroszewicz, J., Swieszkowski W., Dentini, M., Garstecki P
Highly ordered and tunable polyHIPEs by using microfluidics
J. Mater. Chem. B, 2, 2290-2300 (2014)
J. Guzowski, S. Jakiela, P.M. Korczyk, P. Garstecki
Custom tailoring multiple droplets one-by-one
Lab on a Chip, 13, 4308-4311 (2013)
J. Guzowski, P.M. Korczyk, S. Jakiela and P. Garstecki
The structure and stability of multiple micro-droplets
Soft Matter, 8, 7269-7278 (2012)
J. Guzowski, P. M. Korczyk, S. Jakiela and P. Garstecki
Automated high-throughput generation of droplets
Lab Chip 11, 3593-3595 (2011)
9:00 PM - BM3.6.13
Dual Injection Multi-Dimensional Embedding of Bio-Polymers Using Custom Built 3D Bio-Printer
Amer Khamaiseh 1 , Tejesh Marsale 1 , Steven Falzerano 1 , Prabir Patra 1 , Isaac Macwan 1
1 University of Bridgeport Bridgeport United States
Show AbstractThe goal of this paper is to demonstrate the use of robotics in multidimensional dual extrusion biological printing with the assistance of a polymer based hydrogel substrate. Current methods of 3D biological printing (bioprinting) are hindered by the need to create scaffolding to support the intended acellular scaffold which supports embedded cells and cellular growth. Modern techniques using multiple extruders and angling are limited in their direction of application and thus their ability to maintain a precise scaffold structure over time. We have devised a means of overcoming the concerns of constructing and maintaining 3D engineered tissue by means of a unique robotics based 3D printing method.The printer features a 150μm extruding needle and finely pitched lead screws each run by finely tuned stepper motors for precision construction. The main printer can be paused, allowing the rotating plate to be properly angled for the robotic arm to continue embedding, as well as to make repairs and provide the means for maintaining the scaffold from any angle. This new technique uses multiple computer aided sliced stereolithographic files to guide the device in overcoming the necessity of printing a single layer by layer structure at any one angle, allowing for greater control of construction, maintenance and repair. Using a cartesian 3D printer in conjunction with a rotating printing platform and a four axis robotic arm, we have managed to develop a bioprinter capable of overcoming several limitations facing standard 3D printing techniques. In addition to this method, we have produced greater structural support through the use of a viscous biocompatible polymer substrate and two biological inks (bio-ink), one capable of generating blood vessel scaffold and one capable of fabricating bone. The substrate used for the suspension of the bio-ink consists of hydrogel made from carbopol and gelatin. This has the necessary shear elastic modulus and buoyancy to allow for the printers needle to move through unhindered without affecting the previously printed layers of the bio-ink. The blood vessel scaffold, printed in the hydrogel substrate, will consist of PCL and the bone scaffold will consist of PLA. Multidimensional 3D printing, with near limitless degrees of freedom is the first step in continued robotic growth and preservation of all tissue engineering projects. Having the ability to electronically apply any structural changes and growth factors will allow for fully automated and uninterrupted production of tissue. With further research we intend to grow stem cells on both types of scaffolds and differentiate them into endothelial cells for blood vessels as well as osteoblasts for bone. These scaffolds will also be tested for their degradation rate, mechanical strength, and cytoskeletal make-up through electron microscopy. Finally we plan to add graphene on both scaffolds as antimicrobial agent by performing a lab testing for different types of bacteria.
9:00 PM - BM3.6.14
Highly Elastic PVA-Gelatin Hydrogels Formed Using Theta-Gel and Cryo-Gel Fabrication Techniques
Patrick Charron 1 , Rachael Oldinski 1
1 University of Vermont Burlington United States
Show AbstractPoly(vinyl alcohol) (PVA) is a synthetic, biocompatible polymer that has been widely studied for use in bioengineered scaffolds due to its highly attractive properties, such as high strength, creep resistance, and porous structure. These properties can be fine-tuned by controlling the physical, non-covalent crosslinks through various techniques. However, due to the inherent lack of cell adhesion ligands, PVA scaffolds must be modified to allow for cell interactions. In addition, PVA hydrogels are relatively brittle, non-elastic materials. Blending in gelatin, a natural collagen derivative, and poly(ethylene glycol) (PEG) with PVA prior to solidification forms a highly organized, cell-instructive hydrogel with improved stiffness. Theta-gels are formed from the solidification of warm solutions and the phase separation of high molecular weight gelatin and PVA from low molecular PEG. While these PVA-gelatin hydrogels can be synthesized in this manner, the hydrogels continue to exhibit low elasticity. Thus, theta-gels were additionally processed using cryo-gel techniques, which involved freezing theta-gels, lyophilizing and re-hydrating. The result was a stronger, more elastic material. Compressive rheological data suggest significant changes in the elastic modulus of the new PVA-gelatin theta-cryo-gels. We hypothesize a formation of stronger bonds between the PVA and gelatin, which allow for significant resilience and high elasticity. Elasticity and tensile properties will be studied through further rheological testing and dynamic mechanical analysis in tensile and shear regimes. The bond strength and crystallinity will be examined through thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR).
9:00 PM - BM3.6.15
Manufactured Alginate Drug Delivery Vehicles for Disease Prevention and Tissue Regeneration
Michael Karasinski 1 , Rachael Oldinski 1 2 3 , Jennifer Etter 1
1 Mechanical Engineering University of Vermont Burlington United States, 2 Department of Orthopaedics and Rehabilitation in the College of Medicine University of Vermont Burlington United States, 3 Biological Engineering University of Vermont Burlington United States
Show AbstractSoft lithography methods involving a SU-8 photoresist and a Polydimethylsiloxane (PDMS) mold are used to produce channel features in a microfluidic device. A continuous phase fluid (mineral oil) combines at a junction and pinches off the dispersion phase fluid (sodium alginate solution) forming uniform droplets. Using manufacturing techniques identified above, the channel features and flow rates of the microfluidic flow focusing devices (MFFD) are modified in order to create homogeneous alginate microspheres with hydraulic diameters ranging from 1-200µm. The microsphere diameters in different devices will be characterized using scanning electronic microscopy. Microspheres on the order of 1 µm are small enough to pass through cell membranes which means proteins and pharmaceuticals can be delivered intracellularly. Encapsulation efficiency and drug loading of homogeneous alginate microspheres are quantified by using methods developed in our lab. This is accomplished by letting the microspheres absorb dissolved growth factor and measuring absorbance with a 450nm BioRad microplate reader. Drug release is also to be tested. Microspheres in the range of 200µm are large enough to encapsulate entire cells, even stem cells, for use as injectable scaffolds to regrow tissues such as, bone damaged by injury or disease progression. Each use requires microspheres in different distinct size ranges, which is accomplished by fabricating multiple MFFDs and altering fluid input characteristics. Having microspheres of a homogeneous hydraulic diameter is critical and offers distinct advantages for cell study, by making experiments more replicable.
9:00 PM - BM3.6.16
Quiescent and Flow Induced Crystallization in PLLA-WS2 Nanotubes Nanocomposites for Bioresorbable Vascular Scaffolds
Tiziana Di Luccio 1 2 , Karthik Ramachandran 2 , Loredana Tammaro 3 , Francesca Di Benedetto 3 , Carmela Borriello 1 , Fausta Loffredo 1 , Fulvia Villani 1 , Bo Shen 2 , Julia Kornfield 2
1 ENEA Centro Ricerche Portici Portici Italy, 2 Chemistry and Chemical Engineering California Institute of Technology Pasadena United States, 3 ENEA Centro Ricerche Brindisi Brindisi Italy
Show AbstractCoronary Heart Disease (CHD) is a fatal condition which restricts blood supply to the heart due to the buildup of plaque in its arteries. The current treatment is to restore blood circulation through the occluded artery by deploying a metal stent to open the artery. However, being permanent implants, metal stents have serious side effects such as Chronic Angina and Late Stent Thrombosis (LST). Bioresorbable Vascular Scaffolds (BVS), made from biocompatible polymers, are a promising new treatment for CHD as they are transient entities in the body. These scaffolds completely dissolve in two years, leaving no foreign matter behind, which eliminates LST. The polymer which has enabled the first clinically approved BVS is poly (L-lactic acid) (PLLA). Once implanted, PLLA hydrolyses to form L-lactic acid, a natural metabolic product harmlessly processed by the body. However, current PLLA scaffolds are much thicker than metal stents (150mm vs 50mm, respectively) for the polymer scaffold to have comparable mechanical properties to a metal stent. A thicker profile is undesirable as it is more difficult to implant and is unable to access small vessels. We propose to enable thinner and stronger BVSs using biocompatible PLLA nanocomposites.
What we propose in our work is the use of inorganic nanotubes of tungsten disulfide (WS2), to obtain thinner BVS with a proper balance of robustness and flexibility for the scaffold functionality. Apart from imparting favorable mechanical properties to several polymers, biocompatibility assays on WS2 INTs are very encouraging.
The final structure and performances of PLLA scaffolds critically depend upon the crystallization process imparted by the processing parameters of temperature and deformation.
To have insights into the crystallization mechanism under different conditions, we compared neat PLLA and nanocomposites of PLLA-WS2 films prepared by solvent casting, doctor blade, hot press and Flow Induced Crystallization (FIC). The WS2 nanotube concentration in the PLLA was fixed at 0.05wt%.
The thermal, morphological and crystalline properties of the polymeric films were investigated by Differential Scanning Calorimetry (DSC), Polarized Optical Microscopy (POM) and Wide Angle X-Ray Diffraction (WAXS). The mechanical properties of the PLLA and PLLA-WS2 films were tested by uniaxial tensile tests. POM showed that both the neat polymer and the nanocomposites crystallize in spherulites. The morphological and structural changes of the polymer films obtained by FIC were also monitored in-situ by the scattering of a laser beam, showing birefringence when orientation of the polymer chains occurred.
9:00 PM - BM3.6.17
Array of Perfusable Three-Dimensional Microvasculatures on Active Tilting Stage
Rena Shinohara 1 , Junko Enomoto 1 , Tatsuya Osaki 1 2 , Yuka Kobayashi 1 , Tomomitsu Ozeki 3 , Hirotoshi Nakao 3 , Junji Fukuda 1
1 Faculty of Engineering Yokohama National University Yokohama Japan, 2 Mechanical Engineering Massachusetts institute of technology Boston United States, 3 Research amp; Development ULVAC,Inc Chigasaki Japan
Show Abstract
Angiogenesis plays a central role in various physiological phenomena including tumor development and wound healing. It is widely believed that the inhibition of angiogenesis around tumor is effective to prevent cancer progression and metastasis. In a current drug development, anti-angiogenic drug candidates are examined based on proliferation and migration of endothelial cells in a conventional culture dish or in a hydrogel. However, probably because of misunderstanding of cellular responses to drugs and/or insufficient replication of cellular microenvironments, only a limited number of candidates successfully pass through subsequent pre-clinical and clinical trials, making the drug development costly and inefficient. Therefore, robust, reproducible, and biomimetic endothelial cell culture platform is desired to accelerate discovery of anti-cancer drugs.
In this study, we fabricated in vitro vascular-angiogenesis models where cells cultured under a perfusion condition on a tilting stage.
In vitro vascular angiogenesis models were fabricated in collagen gel using our original electrochemical cell detachment, with which cells can be transferred from a gold surface to a hydrogel [1]. Briefly, gold-coated needles (φ500 µm) were modified with an electrically responsive oligopeptide (CGGGKEKEKEKGRGDSP). Then, human umbilical vein endothelial cells (HUVECs) were seeded on the modified gold needles. After two pairs of needles were fixed in a homemade culture device, collagen gel was poured and gelled in a chamber in the device and HUVECs were transferred from the needles to collagen gel by applying a negative electrical potential. The needles were subsequently extracted from the chamber with a specially designed instrument, resulting in endothelialized microchannels. The microchannels were connected in the device to two culture medium reservoirs at both ends. The device was placed on the tilting system where perfusion of culture medium was generated from one reservoir to the other through the microchannels by rotating the tilting stage at 0.2 degree/min.
Even immediately after transfer of HUVEC layers to collagen gel, cell-cell junctions such as gap junction and tight junction were preserved, indicating the extraction instrument is beneficial for non-invasive and straight extraction of needles. After 4 days of perfusion culture, the transferred HUVECs oriented to the flow direction. Furthermore, when carcinoma cells were encapsulated in the collagen gel, HUVECs started to migrate and sprout into collagen gel from the vascular wall. Using this angiogenesis model, we demonstrated that the sprouting was downregulated by anti-angiogenic drugs. This endothelial vascular model holds a great potential to become a new platform for in vitro anti-angiogenesis drugs screening.
[1] Osaki T et al. Acceleration of Vascular Sprouting from Fabricated Perfusable Vascular-Like Structures. PLoS ONE. 2015
9:00 PM - BM3.6.18
Biomimetic Polymer Networks for Synovial Fluid Augmentation in the Treatment of Osteoarthritis
Benjamin Cooper 1 , Mark Grinstaff 1
1 Boston University Boston United States
Show AbstractVarious soft tissues undergo degeneration as a result of aging; the disease osteoarthritis, a progressive degeneration of hyaline cartilage in joints, is associated with changes both in soft and hard tissues, as well as in the fluid environment surrounding those tissues. In a healthy state, the fluid surrounding articulating cartilage surfaces effectively prevents cartilage. As the disease progresses, the fluid experiences a decrease in lubricating ability, and cartilage wear occurs. As greater than 15% of the global population is affected by osteoarthritis, there is a significant need to develop biomaterials that can ameliorate the material deficiencies in diseased joints.
To address this need, we have developed polymeric lubricants derived from poly(2-methacryloyloxyethyl phosphorylcholine), a widely biocompatible polymer that provides low coefficients of friction when grafted to surfaces of contact lenses and polyethylene joint prostheses. Polymer networks of various network concentration, crosslinking density, and crosslinker composition were prepared via radical polymerization. Following purification by dialysis and dissolution in phosphate buffered saline, polymer solutions were characterized by rheometry, observed to correlate with polymer network effective molecular weight, and solution lubricating properties were evaluated with ex vivo bovine cartilage. A simultaneous torsion/creep cartilage-on-cartilage friction test was performed at various sliding speeds to explore the effect of articulation conditions on resulting coefficient of friction. More viscous lubricant solutions were found to provide superior fluid film mode lubrication, while less viscous solutions afforded superior boundary mode lubrication. Infrared spectroscopy of histological samples of lubricant-exposed cartilage did not reveal presence of lubricant polymer inside the tissue, confirming the hypothesized mechanism of action as a transient surface lubricant opposed to a bulk material treatment. Finally, as a step towards potential commercialization of this family of lubricants, one formulation was fluorescently labeled (near infrared wavelength) and injected intraarticularly into the knees of 5 rats in vivo. By serially imaging the animals’ knees, a residence time half-life of 30.5 ± 7.1 days was observed, which yields an effective residence time of approximately 120 days, as friction tests reveal positive therapeutic effect at 6.25% of initial lubricant concentrations. The data presented both provide further understanding of how polymer structure relates to macroscopic biomechanical function, as well as advance the clinical potential of this synthetic tissue lubricant.
9:00 PM - BM3.6.19
Engineering an Antimicrobial and Elastic Hydrogel as a Sprayable Wound Healing Patch
Devyesh Rana 1 , Ehsan Shirzaei Sani 2 , Roberto Lara 2 , Nasim Annabi 2 , Suzanne Mithieux 3 , Anthony Weiss 3
1 Bioengineering Northeastern University Boston United States, 2 Chemical Engineering Northeastern University Boston United States, 3 School of Life and Environmental Sciences University of Sydney Sydney Australia
Show AbstractChronic wounds are estimated to affect 6.5 million patients in the U.S annually. These types of wounds do not always heal efficiently and are at risk of bacterial infection. Current difficulties in chronic wound management and treatment are associated with a multi-step approach to patient care where covering the wound, avoiding excessive blood loss, protection from infections, and healing, are separate steps. In this study, we develop a novel elastic and sprayable hydrogel with antimicrobial properties through photopolymerization via visible light. The engineered hybrid hydrogel is comprised of methacrylated tropoelastin (MeTro) and gelatin methacrylate (GelMA) conjugated with an antibacterial peptide (AMP Tet213). The hydrogel mimics the elasticity of the skin and is shown to adhere to porcine skin to form an antibacterial and regenerative barrier. The tensile modulus of the hydrogel was found to be tunable in the range of 5 – 25 kPa based on varying MeTro/GelMA ratios and final polymer concentrations. A hydrogel formulation consisting of 70/30 MeTro/GelMA with a 15% (w/v) final polymer concentration and 0.01% (w/v) antimicrobial peptide (AMP), attains optimal mechanical and antimicrobial properties for the application of wound treatment and bacteria clearance. The engineered hydrogel was effective at preventing the growth of both gram positive (MRSA – CFU decreased 2.5 fold) and gram negative (E.coli – CFU decreased 6.5 fold) bacteria. In addition, in vitro tests showed more than 90% cell viability at days 1, 3, and 5 post-seeding, which suggests no apparent cytotoxic effects on cells in vitro. Our research represents a single-step “smart” treatment for wound management and care.
9:00 PM - BM3.6.20
Preparation of Type I Collagen Nanoworms with Magnetic Targeting For Cancer Treatment
Le Yu 1 , Mei Wei 1
1 University of Connecticut Storrs Mansfield United States
Show AbstractAbstract: Multidrug resistance is one of major challenges in cancer treatment research. In regards to tumor therapy applications, nanoparticles (NPs) have been widely used. The particle properties, such as size and shape, are important factors. It has been proposed that NPs with an elongated morphology are more advantageous compared with spherical NPs as nanocarriers. Increasing evidences showing that non-spherical particles are relatively hard to be taken up by macrophages, therefore prolonging the blood circulation duration and thus enhancing treatment efficacy of nanocarriers. Furthermore, it is possible to graft multi functional groups in NPs with elongated shapes, which includes more than one binding sites and nucleate zones. At last, anisotropic vehicles exhibit more lateral drift compared with spherical particles, resulting in an increased possibility of accumulating at tumor site thus improving treatment efficacy.
In this study, we proposed to use magnetic collagen nanoworms as an anisotropic carrier for tumor treatment. Type I collagen is a long and flexible filamentous protein consists of three types of amino acid molecules. The periodic 40-nm-long gap zone in d-banding collagen fibril has been implicated as the place where apatite crystals nucleate from an amorphous phase, allowing further mineralization of inorganic regents such as silica or iron oxide for extra functionalization (intrafibrillar mineralization).
The formation of collagen fibrils can be taken as precipitation of the protein from a supersaturated solution in an ordered form under particular conditions. The effect of collagen precipitation condition on collagen fibril morphology was investigated. By precisely controlling the precipitation conditions, in this work, semi-flexible type I collagen nanoworms were successfully synthesized and subsequently mineralized with iron oxide in the gap zone of collagen fibrils to grant the nanocarriers with appropriate magnetic properties suitable for cancer treatment. It was observed that, the length and width of nanoworms are about 1 μm and 100 nm, respectively. Besides its suitability for cancer treatment, the magnetic nanoworms is also a promising candidate as a MRI contrast agent.
Acknowledgements: The authors would like to thank the support from NSF grants (CBET-1133883 and CBET-1347130).
9:00 PM - BM3.6.21
An Efficient 3D Porous Hydroxyapatite and Arabinoxylan Bioactive Scaffold System for Bone Tissue Engineering
Saqlain Shah 1 , Mu Aslam Khan 2 , Mu Hashmi 3 , Saifullah Awan 4 , Muhammad Naeem 5 , Muhammad Arshad 6
1 Physics Forman Christian College Lahore Pakistan, 2 Polymer Engineering Technology University of the Punjab Lahore Pakistan, 3 Applied Sciences Superior University Lahore Pakistan, 4 Physics COMSATS Institute of Information Technology Islamabad Pakistan, 5 Physics Islamic International University Islamabad Pakistan, 6 Nanoscience and Catalysis National Center for Physics Islamabad Pakistan
Show AbstractHydrophilic nature of the hydrogels makes it possible to form a 3-D network in water due to H-Bonding. Biocompatibility of hydrogels makes them significant in the field of medicine, biotechnology and tissue engineering. Many researchers have confirmed the remarkable potential of hydrogels in the field of bone tissue engineering. Ceramic composites from a wide range of biodegradable natural hydrogels have been reported as a viable solution in bioactive scaffold synthesis. Porous scaffolds with good mechanical strength and biodegradability make them exclusive due to providing a micro environment for Osteoconduction. This study is about bioactive scaffolds synthesis carried out through freeze drying by using arabinoxylan, a carbohydrate polysaccharide extracted from plantago ovata, and hydroxyapatite as bone cement for bone-tissue engineering. Results show that it provides promising features like porous structure with appropriate mechanical strength, non-cytotoxicity and biodegradability.
9:00 PM - BM3.6.22
Sustained Release of bFGF from Multilayer Nanofilm to Support Human Stem Cell Cultures without Daily Feeding
Hee Ho Park 1 , Uiyoung Han 3 , Jinkee Hong 4 , Juhyun Park 2
1 School of Chemical and Biological Engineering Seoul National University Seoul Korea (the Republic of), 3 School of Chemical Engineering and Materials Chung-Ang University Seoul Korea (the Republic of), 4 School of Chemical Engineering and Materials Chung-Ang University Seoul Korea (the Republic of), 2 Department of Medical Biomaterials Engineering Kangwon National University Chuncheon-si Korea (the Republic of)
Show AbstractThe successful maintenance of undifferentiated state is an essential aspect of stem cell culture. In the case of human pluripotent stem cells including embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, basic fibroblast growth factor (bFGF) plays a crucial role in promoting the undifferentiated state while minimizing spontaneous differentiation into other cell types. However, bFGF has been known to be very labile under normal culture conditions. Therefore, pluripotent stem cell cultures require a daily replacement of culture medium, making the culture costly and labor intensive. Recently, it has been reported that bFGF levels in human pluripotent stem cell culture significantly fluctuate even with daily feeding. Here, a layer-by-layer (LbL) technique-based nanofilm system consisting of charged polymeric materials was established for the sustained release of bFGF. The release of bFGF was continued for over 10 days. Then, the bFGF-releasing nanofilm was constructed on the membrane of transwell permeable support and human iPS cells were cultured with the transwell. As a result, human iPS cells maintained their undifferentiated morphology and expression of pluripotency markers including SSEA-4, Nanog and Oct4 even with less frequent media changes. Furthermore, it was demonstrated that undifferentiated state of iPS cells was preserved during several passages and the differentiation potential was not altered. The bFGF-releasing nanofilm provides a useful method to maintain the undifferentiated state of human pluripotent stem cells. Especially, it is anticipated that the controlled release of bFGF from the biocompatible nanofilm reduces the frequency of media replacement needed to maintain stem cell cultures.
9:00 PM - BM3.6.23
Electrospun Fatty Acid Modified Chitosan/Gelatin Hybrid Nanofiber—A Biomimetic Scaffold for Skin Tissue Engineering
Sayanti Datta 1 , Santanu Dhara 1
1 School of Science and Technology Indian Institute of Technology, Kharagpur Kharagpur India
Show AbstractElectrospinning is an attractive method of fabricating sub-micron range non-woven fiber for tissue engineering. The nano/micro architecture of the fiber provides high surface area, better mechanical property with high porosity and spatial interconnectivity. Thus, electrospun fibers represent a vital role in tissue engineering application. In this present study, fatty acid modification was performed onto chitosan and electrospun scaffold was fabricated with modified chitosan/gelatin and its characterization was done. The chemical identification of that modified chitosan was identuified by FTIR and NMR study. For electrospinning, the polymer solution was placed in a plastic syringe fitted with a blunt end needle. The syringe was kept horizontally on the syringe pump for controlling the solution flow rate from the needle tip. The solution was electrospun at a flow rate of 1-5 µL/ min with an applied high voltage of 18-20 kV. A distance of 15-18 cm was kept between the needle tip and the horizontal collector covered with aluminum foil. Gelatin/fatty acid modified chitosan nanofibrous mats were prepared via electrospinning by using different ratio of gelatin/fatty acid modified chitosan. FTIR, SEM, swelling, degradation, contact angle, hemo-compatibility cytotoxicity, and DNA quantification assay were performed of that blended nanofibrous scaffold. Cell adhesion and fluorescent imaging was evaluated using primary rat fibroblast cells (pRFC) to observe the attachment and proliferation at day 1, 3 and 5 days. FTIR and NMR results showed that successful grafting of that fatty acid chain into native chitosan. The diameter of the fabricated nanofibers was 500-800 nm as observed under SEM. The constructs were significantly prominent with excellent hemo- and cyto-compatibilities compared to tissue culture plate. At day 5, proliferating as well as migrating pRFCs were visible under SEM. Significant cell proliferation rate was also confirmed by higher DNA content and fluorescence microscopy (Rhodamine-Dapi). Therefore, blended nanofibers are very efficient thus can be used as skin graft in severe wounds such as burn wound or diabetic wound. The hydrophobic fatty acid incorporated hybrid nanofibers can be used for drug delivery for future use.
9:00 PM - BM3.6.24
Synthetic Modifications of Silk Fibroin for Application as Adhesive Biomaterials
Danielle Heichel 1 , Kelly Burke 1 2
1 Polymer Program University of Connecticut Storrs United States, 2 Chemical and Biomolecular Engineering University of Connecticut Storrs United States
Show AbstractSilk fibroin, isolated from the cocoons of Bombyx mori (B. mori) silkworms, is a high molecular weight protein that contains high concentrations of glycine, alanine, serine, and tyrosine amino acid residues. Silk fibroin is comprised of both hydrophilic regions and hydrophobic regions, where amino acids in the hydrophilic segments can be modified using aqueous chemistries. Hydrogen bonding along the protein’s backbone in the hydrophobic regions results in the formation of beta sheet secondary structures that physically crosslink the protein. Tuning beta sheet content has been shown to afford control over the mechanical properties and degradation rate of silk-based materials. This work seeks to extend the applicability of silk as an aqueous-based adhesive, with particular emphasis on adhesion to negatively-charged surfaces. Primary amines were conjugated to carboxylated side chains via carbodiimide coupling. These amines were hypothesized to enhance the material’s bond to negatively charged surfaces at neutral pH. Successful conjugation was confirmed using nuclear magnetic resonance (NMR) spectroscopy and chromogenic assays. Crosslinkers of varying molecular weights and hydrophilicity were used to tailor the mechanical properties and swelling of the networks. The effect of protein functionalization on beta sheet secondary structure formation was quantified using Fourier Transform Infrared (FTIR) spectroscopy. The silk-based materials prepared in this work are envisioned to find utility as aqueous adhesives tailored for different surfaces.
9:00 PM - BM3.6.25
Preparation and Characterization of Exceptionally Small Molecule Hydrogelator with Potential Applications in Tissue Engineering
Belete Legesse 1 , Arthur Gonzales III 1 , Hicham Fenniri 1
1 Chemical Engineering Northeastern University Boston United States
Show AbstractBecause of the remarkable sensitivity to external stimuli incorporated with biocompatible property, supramolecular hydrogel based on low molecular weight gelator has gained immense interests. As a result, the design and preparation of low molecular weight organic gelators as well as understanding of the mechanism of the gelation process have attracted great attention recently and become the key point for supramolecular hydrogel preparation. Here we present the rational design of a very low molecular weight (170 Da) nucleobase derivative hydrogelator 2,4,6-trihydroxypyrimidine-5-carboxamide (THPCA) and the characterization of its aggregation properties in various aqueous environments. The organic hydrogel scaffold contains nitrogen heterocyclic ring and amide groups that can boost its self-assembly in aqueous medium into nanofibers/microfibers through hydrogen bonding interactions. The characterization of the hydrogel was performed by rheology, infrared spectroscopy, scanning electron microscopy and transmission electron microscopy studies. The results obtained indicated that high value of storage modulus, G’ (∼104 Pa) can be obtained in phosphate buffer and in the presence of various salts. Our findings demonstrated that the hydrogel is remarkably strong and can be a suitable scaffold for tissue engineering.
9:00 PM - BM3.6.26
Composition of Gold Nanoparticle and RGD Peptide for Enhancing Electrochemical Signals of Human Embryonic Stem Cells
Ho-Chang Jeong 2 , Sung-Sik Choo 1 , Keun-Tae Kim 2 , Ki-Sung Hong 3 , Sung-Hwan Moon 3 , Hyuk-Jin Cha 2 , Tae-Hyung Kim 1
2 Sogang University Seoul Korea (the Republic of), 1 Chung-Ang University Seoul Korea (the Republic of), 3 Konkuk University Seoul Korea (the Republic of)
Show AbstractThe precise monitoring of remaining undifferentiated human pluripotent stem cells (hPSCs) and their proper eliminations are highly important for safe teratoma-free stem cell therapies. Previously, we have reported a powerful in situ label-free cell chip platform, which enabled sensitive detection of as few as 72,000 cells of human embryonic stem cells (hESCs), based on hPSCs-specific electrochemical signals. However, to fullly eliminate the risk of teratoma formation from hESCs using cell-based sensing system, it is certain that the sensitivity of electrochemical sensing system needs to be further improved. Herein, we report a new strategy that could significantly enhance the sensitivity of hPSCs chip, by incorporating gold nano particles (GNPs) and branched arginyl-glycyl-aspartic acid (RGD) peptides in matrigel layer as electrical signal-enhancing materials and cell adhesion promoters, respectively. The developed cell chip platform containing hybrid matrigel layer was found to be capable of detecting the electrical signal of as few as 25,000 cells (hESCs) with a linear range from 25,000 to 890,000 cells, which was 2.8 times more sensitive than the previous hPSCs chip . Hence, it can be concluded that the newly developed material, the hybrid matrigel layer containing GNPs and branched RGD peptides, would be highly beneficial to develop cell-based sensing system whose electrochemical performance is sensitive enough to assess the risk of teratoma formation prior to clinical application of hPSCs-based products.
9:00 PM - BM3.6.27
Characterization of Aluminum Substituted Hydroxyapatite for Biological Studies
Joo Ho Kim 1 , Jennie Kunitake 1 , Lara Estroff 1
1 Cornell University Ithaca United States
Show AbstractHydroxyapatite (HA) is highly susceptible to ionic substitution, with biogenic HA never being found in its pure, stoichiometric form (Ca10(PO4)6(OH)2). Various types of substituted synthetic HAs have been investigated as prosthetic and scaffold materials, such as strontium-substituted HA for augmenting osteogenesis. Many types of divalent cations have been studied as ionic substitutions in HA but few studies have been done on trivalent cations such as aluminum. From this body of work, several reports on aluminum substituted HA (Al-HA) suggest Al-HA as a viable candidate for biomaterials. Conversely, there are reports of aluminum detection in pathological calcifications of exostoses and ductal carcinoma in-situ. These conflicting reports suggest a better understanding on Al-HA and its biological properties is necessary. The current study is focused on characterization of Al-HA synthesized via aqueous precipitation to obtain biologically-relevant particle sizes and crystallinities. Powder X-ray diffraction (pXRD) confirmed the synthesis of HA and Al-HA, while also indicating a decrease in crystallinity of Al-HA with higher aluminum concentrations. Infrared and Raman spectroscopy further corroborated the decrease in crystallinity and implied the inclusion of water in the less crystalline structures of Al-HA. Through electron microscopy (SEM, TEM), morphological changes such as elongation and thinning of HA rods were observed with increasing aluminum concentration. Further studies are ongoing to deeper characterize and assess the effect of Al-HA on cancer related cells compared to HA.
9:00 PM - BM3.6.29
Study of Vegetal Based Polyurethanes/Hydroxyapatite and Curcumin Nanocomposites for Tissue Engineering
Llilian Coimbra 2 , Thais Moraes Arantes 2 , Dayane Tada 3 , Fernando Cristovan 3 , Tatiane Arantes 1
2 Chemistry Instituto Federal Goiano Ipora Brazil, 3 Federal University of São Paulo Sao Jose dos Campos Brazil, 1 Federal University of Goiás Jatai Brazil
Show AbstractThe search for materials with properties of biocompatibility with the human body constitutes a major challenge for researchers in the field of new materials. Biodegradable polyurethane (PU) is one of the most biocompatible materials used as temporary extracellular matrices in bone tissue engineering scaffolds. On the other hands, the curcumin has attracted interest, because properties antioxidant, antimicrobial properties and improve the adhesion, flexibilities and resistance. Allied to the polymers, a promising alternative in the field of biomaterials is the preparation of polymer nanocomposites, associating the properties of polymers with bioactive ceramics, such as hydroxyapatite nanoparticles, which in addition provides the best interaction between human tissue and biomaterial. Thus, in this work, it was prepared nanocomposite of polyurethane with hydroxyapatite (HA) nanoparticles modified curcumin seeking to obtain biocompatible materials for use as scaffolds in medical/odontological applications. The nanoparticles will be embedded in the polymer matrix by in situ polymerization of the polyurethane. The best conditions for the synthesis of nanocomposites as well as the synergistic effect of the incorporation of nanoparticles were studied. The HA nanoparticles were synthesized by hydrothermal processing and were characterized by X-ray diffraction (XRD), Raman spectroscopy and transmission electron microscopy (TEM). The XRD and Raman spectra showed crystalline hydroxyapatite colloidal nanoparticles were obtained in the hexagonal phase. TEM images showed HA nanoparticles presented a well-defined nanorod shapes and narrow size distributions with dimensions (width and length) around of 5 nm and 10 nm. The nanocomposites were prepared by in situ polymerization. The PU nanocomposites were obtained by two-shot technique, which was initially obtained from a polyol from Ricinus communis oil and then this polyol was cured with toluene diisocyanate (TDI) in the presence of HA/curcumin nanoparticles at 60 ° C. The complete polymerization of the monomer could be proved by typical spectra showed by 13C, 1H nuclear magnetic resonance, infrared and Raman spectroscopies. Results of antibacterial tests indicate that the HA/curcumin nanoparticles have antibacterial properties against both Staphylococcus aureus and Escherichia coli. The cell viability of the PU and PU/HA/curcumin was analyzed by reduction of the tetrazolium salt. Embryonic mouse fibroblast cells were grown in the presence of nanocomposites for a total period of 96 hours. Analyses were made in 24h, 48h, 72h and 96h. The suspensions at the end of each period were analyzed in spectrophotometer. The 24h experiments were the most conclusive, with the HA/curcumin presence in the polyurethane, there is an increased in cellular proliferation. The results demonstrated that the PU/HA/curcumin nanocomposites have potential use as biomaterials in medical/odontological applications.
9:00 PM - BM3.6.30
Rupture Force of Cell Adhesion Ligand Tethers Modulates Biological Activities of a Cell-Laden Hydrogel
Min Kyung Lee 1 , Jooyeon Park 1 , Xuefeng Wang 1 , Mehdi Roein-Peikar 1 , Eunkyung Ko 1 , Ellen Qin 1 , Jonghwi Lee 2 , Taekjip Ha 1 , Hyunjoon Kong 1
1 University of Illinois at Urbana-Champaign Urbana United States, 2 Chungang University Seoul Korea (the Republic of)
Show AbstractThe development of synthetic extracellular matrix with different types and spatial organization of cell adhesion ligands has drawn much attention for cell culture, engineering, and therapies. Recently, it has been suggested that ligand-matrix bond strength can regulate cell adhesion and activities. Therefore, in this study, we demonstrated the effects of ligand-matrix bond strength on cell adhesion, differentiation, and secretion activities using a hydrogel coupled by integrin-binding deoxyribonucleic acid (DNA) tethers with pre-defined rupture forces.
Integrin-binding DNA tethers were prepared by annealing two single-stranded DNA (ssDNA) chains. One ssDNA chain was modified with thiol group at 3’ end and Cy5 at 5’ end, while the complementary DNA strand is modified with biotin at different sites. Then, these two ssDNAs were annealed together. The DNA tether coupled with RGD peptides, termed RGD-DNA tether, was immobilized to alginate conjugated with biotin, termed alginate-g-biotin. The hydrogel was formed by cross-linking alginate-g-biotin molecules with adipic acid dihydrazide (AAD) in aqueous media using NHS/EDC chemistry. The molar ratio of AAD was tuned to control the elastic modulus of the gel. Mesenchymal stem cells were cultured on RGD-DNA tether-conjugated gels, and cell adhesion, differentiation, and function were examined.
As a result, we were able to regulate the rupture force of DNA tethers by the spatial arrangement of matrix-binding biotin groups. Avidin was used to conjugate biotin-tagged DNA tethers and hydrogel of alginate grafted with biotin. DNA tethers with higher rupture forces enhanced the adhesion, neural differentiation, and paracrine secretion of mesenchymal stem cells. Therefore, we propose that this study can provide a guideline in developing hydrogels for cell implantation, which should be structurally stable and provide an appropriate environment for cell survival and activities, by allowing orthogonal regulating of bulk gel properties and cell-gel interface. In sum, this study presents a new perspective of material design for cell culture and transplantation by engineering cell-hydrogel interface mechanics.
9:00 PM - BM3.6.31
High Modulus Biodegradable Bone Fixation Devices
Bryant Heimbach 2 , Montgomery Shaw 3 , James Olson 1 , Mei Wei 2 3 4
2 Department of Biomedical Engineering University of Connecticut Storrs United States, 3 Institute of Material Science University of Connecticut Storrs United States, 1 Teleflex Medical Coventry United States, 4 Department of Materials Science and Engineering University of Connecticut Storrs United States
Show AbstractEvery year there are over 3 million bone fractures in the United States, and over 30% of these require some kind of internal mechanical fixation to help heal the bone. The current standard for such a device is the use of metal plates, typically stainless steel or titanium, because they have great mechanical properties for stabilizing bone and are relatively inert in the body. However, due to the high stiffness of metals relative to bone, metals impart stress shielding, which transfers the loads from the bone into the fixation plate and causes degradation of the surrounding bone. Also, overtime metal fixation plates tend to leach metal ions into the body. For these reasons, there is often a need for a second surgery to remove the implant, sacrificing patient comfort.
For these reasons, there has been interest in creating a biodegradable fixation plate that would disappear in the body within a year or two as well as have mechanical properties matching those of natural bone. Natural bone has a flexural modulus and strength ranging from 7 to 25 GPa and 100 to 130 MPa, respectively, so ideally a fixation plate would fall in that range. However, attempts to make a biodegradable option has resulted in materials with subpar mechanical properties, particularly in the balance between modulus, strength, and toughness.
The present research uses the combination of pultrustion and compression molding methods to make a composite with both long fiber and particle reinforcement to achieve the desired mechanical properties for bone fixation. The current design relies on either poly-L-lactic acid (PLLA) or silk fibroin (SF) fibers for long fiber reinforcement and the bioceramic hydroxyapatite (HA), which is the mineral phase in natural bone, for the particle reinforcement. These components are bound with the matrix polymer polycaprolactone (PCL) to create a dense composite bar suitable for use as an internal fixation device. To start, PLLA or SF fibers are coated with a suspension of PCL and HA, run through a pultrusion die, and consolidated on a metal frame where the coated fibers are then hot pressed into the final composite bar. When using PLLA as the long fiber reinforcement, a flexural modulus and strength of 10.5 GPa and 200 MPa, respectively, have been achieved. When SF is used as the long fiber reinforcement, a flexural modulus and strength of 16.5 GPa and 265 MPa have been achieved. Perhaps the most important aspect of the composite is the toughness shown. By taking advantage of the fiber pullout and crack deflection benefits in composites, samples made with PLLA long fibers have shown remarkable toughness, with no catastrophic failure during mechanical testing. In addition to the mechanical properties of the composite being suitable for internal fixation, all of the materials are already FDA approved and should degrade within the one to two year period, making this a very promising material for use as a biodegradable bone fixation device.
9:00 PM - BM3.6.32
Engineered Nano-Clay Based 3D Bone Tissue Engineering Scaffolds Duplicate Genetic, Mechanical and Biochemical Microenvironments for Prostate Cancer Tumor Model
MD Shahjahan Molla 1 , Dinesh Katti 1 , Kalpana Katti 1
1 North Dakota State University Fargo United States
Show AbstractThe focus of this study is to use a regenerative medicine approach to create a bone-mimetic humanoid environment for evaluation of migration of prostate cancer to bone. Prostate cancer (PCa) is the sixth leading cause of cancer originated deaths among men worldwide and second most frequently diagnosed form of cancers in the United States. A complex metastatic cascade is involved to the dissemination of cancer cells to the distant organs. It includes the sequence of phases: local invasion, intravasation, circulation, arrest and extravasation, proliferation and angiogenesis. Initially single cells or clumps of cancer cells leave the primary tumor to invade the local stroma and ECM (extracellular matrix). On the second step of metastatic cascade, cancer cells intravasate either into blood vessels or lymphatic system. They translocate through the bloodstream to the microvessels of a distant site, extravasate from the bloodstream, and the survived cells are arrested at a distant tissue and acclimate to the foreign microenvironment resulting in metastatic colonization. Traditional prostate cancer bone metastasis models contain many limitations about mimicking human bone microenvironment. The complex nature of cancer metastasis leads to the development of a cancer model based on specific metastatic stage. Here we report mesenchymal to epithelial transition and colonization of disseminated prostate cancer cells on nano-clay based in vitro osteotropic tumor model. A novel cell culture system termed as ‘sequential culture’ has been applied to create a more bone-mimetic niche for colonization of prostate cancer cells. Human mesenchymal stem cells were seeded on nano-clay based bone scaffolds, where they differentiated into bone cells without osteogenic media. Then prostate cancer cells were seeded on the differentiated bone cells. Multicellular tumoroids with distinct tight cellular junctions and hypoxic core regions were observed on these scaffolds with sequentially cultured cells. Extensive qRT-PCR experiments were performed to evaluate the expressions of key genes related to osteoblastic bone metastasis of prostate cancer cells. Gene expression analysis data suggest mimicking of mesenchymal to epithelial transition (MET) stage of cancer metastasis on our in vitro cancer model. Our gene expression results also indicate not only 2D vs 3D cell culture method but also coculture vs sequential culture has significant effects on expression of key metastasis-related genes. These studies demonstrate the use of regenerative medicine to study prostate cancer through duplication of the humanoid 3D metastatic environment.
9:00 PM - BM3.6.33
Effect of Nanohydroxyapatite and Nanodiamonds Coated Electrospun Cellulose Acetate Scaffolds on Cell Morphology
Jaime Santillan 1 , Samir Bello 2 , Yaiel Rodríguez 2 , Eduardo Nicolau 3
1 Physics University of Puerto Rico San Juan United States, 2 Biology University of Puerto Rico San Juan United States, 3 Chemistry University of Puerto Rico San Juan United States
Show AbstractBone grafting is a clinical procedure typically employed to repair and remodel bone structures that have suffered of diseases such as cancer or infection and to reconstruct bone after a traumatic injury. One of the mayor drawbacks associated with bone grafting is the risk of disease transmission and rejection given that this requires invasive surgery. Bone tissue engineering and the development of novel nanobiomaterials may enhance the current scaffold materials whose purpose is to mimic the function of the extracellular matrix structure. In this study we prepared cellulose acetate (CA) scaffolds using the electrospinning technique and then tested the interaction between cells and the obtained biomaterial using inmunohistochemical markers. Cell morphology was identified by immunostaining and cell proliferation was measured using a BrdU assay. The non-woven membrane was achieved using 17% CA solution containing Acetone/Dimethylacetamide (2:1) as the solvent. Electrospraying of hydroxyapatite (nHAP) nanoparticles and nanodiamonds onto the surface of CA membranes was also done in order to provide a novel substrate topography. This surface roughness is suitable to promote a better cell-membrane interaction and initiate the osteogenic differentiation. CA membranes were characterized using Scanning electron microscopy (SEM), Atomic force microscopy (AFM), Fourier transform Infrared spectroscopy (FTIR) and X-Ray Diffraction (XRD). Cell morphology was characterize using Fluorescence and Confocal microscopy. These results confirmed the production of uniform nanofibers and shows the potential of nHAP and nanodiamonds coated CA nanofibers as bone scaffold and suitable biomaterial for regenerative medicine.