Gulden Camci-Unal, Harvard Medical School
Brendan Harley, University of Illinois, Urbana-Champaign
Eben Alsberg, Case Western Reserve University
Kazunori Kataoka, The University of Tokyo
Symposium Support Aldrich Materials Science
Society for Biomaterials
W2: Polymeric Biomaterials for Engineering Tissues
Tuesday PM, April 22, 2014
Moscone West, Level 2, Room 2003
2:30 AM - W2.01
Silica / Alginate Biohybrids with Covalent Coupling: GPTMS v APTES
Yuliya Vueva 1 Slila Chayanun 1 Daming Wang 1 Gowsihan Poologasundarampillai 1 Louise Connell 1 Frederik Romer 2 John Hanna 2 Julian Jones 1
1Imperial College London London United Kingdom2University of Warwick Coventry United KingdomShow Abstract
Organic-inorganic hybrid materials composed of interpenetrating molecular networks of polymer and silica are potential candidates for synthetic bone grafts as they can mimic the complex structure of a natural bone. Combining the properties of an elastic polymer and bioactive silica with molecular level interactions, the hybrids are expected to exhibit congruent degradation rate and tailored mechanical properties. Property control is primarily achieved through covalent bonds between the components, which can be achieved by functionalising the polymer with silane coupling agents that will then bond to the sol-gel silica. In this work we present new organic- inorganic hybrids of alginate and silica and compare the use of two different coupling agents: 3-glycidoxypropyl trimethoxysilane (GPTMS) or 3-aminopropyltriethoxysilane (APTES). The reaction between alginate and GPTMS involved nucleophilic opening of the GPTMS epoxy ring and formation of ester bonds with alginate carboxylate groups while the reaction with APTES was performed using carbodiimide chemistry. 1H solution state NMR analysis was used to monitor in situ the reaction between the alginate and the coupling agents. It revealed that within 72 hours of reaction most of GPTMS epoxy rings were unreacted or partially hydrolysed to diols indicating a slow functionalization process. 13C CP solid state NMR of aged gel samples did show formation of ester bonds between GPTMS molecules and alginate, showing that the aging step is necessary for covalent coupling to occur (confirmed by SIMS). The reaction between APTES and alginate readily occurred yielding elastic APTES - alginate hydrogels containing amide bonds, confirmed by FTIR, 1H NMR, 13C CP NMR and 15N CP NMR. Depending on the coupling molecule used in the synthesis the silica-alginate hybrids showed different degradation profiles and mechanical properties. GPTMS based hybrids showed faster dissolution in Tris buffer solution at physiological pH compared with APTES based hybrids, which had controllable dissolution behaviour relevant to their coupling degree. Scaffolds were produced by freeze drying APTES / alginate hybrids (pore size 150 µm) and compression tests showed that the scaffolds had an elastic behaviour suggesting that they would be appropriate for cartilage regeneration while the GPTMS based hybrids had improved compressive strength (150 MPa) and toughness comparable with the cortical bone and thus suitable for bone tissue application.
2:45 AM - W2.02
Mussel-Inspired Adhesive Interfaces for Biomedical Applications
Hakan Ceylan 1 Ayse B Tekinay 1 Mustafa O Guler 1
1Bilkent University Ankara TurkeyShow Abstract
Designing artificial instructive coatings at the cell-biomaterial interface have become an appealing strategy to stimulate tissue regeneration and improve implanted material biocompatibility. Guiding cellular activities, such as adhesion, viability, and differentiation, on the biomaterial surface in a robust and cell-selective manner is often essential for both accelerated healing and the long term clinical success of the surgical intervention. In the biological milieu, however, stability of coatings on the material surface is restricted under the abrasive conditions, such as highly hydrated saline environment accompanied with constant mechanical wearing. Various organisms adapted to living in the seashores, or intertidal zones, suffer from the analogously harsh physical and chemical instabilities. To remain sessile, for example, mussels synthesize a highly complex, spatiotemporally evolving glue containing high amount of 3,4-dihydroxy-L-phenylalanine (DOPA). Herein, we imitate this mechanism by incorporating this chemical residue to self-assembling peptide amphiphile building blocks. Self-assembly of these molecules with complementary peptide amphiphiles carrying biofunctional ligands, such as REDV, DGEA, and KRSR, form nanofibrous networks, which closely mimic both structure and biochemical properties of the native extracellular matrix. Being capable of mediating both non-specific surface adhesion and cell type-specific behavioral guidance, we demonstrate general applicability of this supramolecular adhesive concept. We firstly apply this glue as bioactive cardiovascular stent coating, where endothelial cell adhesion and survival is favored over the smooth muscle cells, which is the main cause of re-closure of the arteries (restenosis) in the long term. We further develop an orthopedic/dental implant coating where mineral-depositing cells are selectively promoted over soft tissue forming fibroblasts. We also develop novel mussel-inspired nanofibers with biofunctional ligands that can synergistically deposit bone-like hydroxyapatite in body fluid mimetic conditions. This paves the way to develop a second generation orthopedic/dental implant, which efficiently mediates differentiation of human mesenchymal stem cells into functional osteoblasts. Altogether, we here concentrate on a mussel-mimetic, hybrid supramolecular adhesive interfaces for biofunctionalization of metal implant surfaces in order to direct the cellular behaviors at the molecular level for long-term success of the regenerating tissue.
3:00 AM - *W2.03
Engineering Biomaterials to Control the Wound Healing Response
Guillermo A. Ameer 1 2
1Northwestern University Evanston USA2Feinberg School of Medicine Chicago USAShow Abstract
Biomaterials play a critical role in the design of medical devices and the implementation of new therapies to improve patient care. Our laboratory pioneered the development of citrate-based polyesters, referred to as polydiolcitrates. Polydiolcitrates can be engineered to have properties that improve the function of medical devices and for applications in regenerative medicine. As an example, aberrant oxidative stress and inflammation play a significant role in the development of neointimal hyperplasia, a problem that eventually leads to the failure of small-diameter vascular grafts and stents. By incorporating antioxidant polydiolcitrates into the design of the device, one can control excess reactive oxygen species (ROS) contributing to oxidative stress and inflammation, reduce neointimal hyperplasia, and potentially increase the lifespan of the device. As for applications in regenerative medicine, when polydiolcitrates are combined with mesenchymal stem cells and hematopoietic stem cells one can create vascularized and innervated bladder tissue that may one day benefit patients with bladder cancer or bladder-related complications from spina bifida. In another example, the efficient healing of diabetic skin ulcers can be a significant challenge due to deacreased vascularization, chronic inflammation, high oxidative stress, and biofilm formation. To address this challenge we are investigating the use of thermoresponsive polydiolcitrate oligomers referred to as nanonets. Nanonets self-assemble into an elastic hydrogel with hierarchical nano- and microscale architecture within seconds upon contact with tissue above 30°C. The hydrogel can effectively entrap and slowly release bioactive compounds, proteins, and transgene products that modulate the wound healing response.
3:30 AM - W2.04
Injectable Micropatterned Polymeric Nanosheets for Local Delivery of an Engineered Epithelial Monolayer
Toshinori Fujie 1 5 Yoshihiro Mori 2 Matsuhiko Nishizawa 2 Nobuhiro Nagai 3 Toshiaki Abe 3 Ali Khademhosseini 4 5 Hirokazu Kaji 2
1School of Advanced Science and Engineering, Waseda University Tokyo Japan2Graduate School of Engineering, Tohoku University Sendai Japan3Tohoku University Graduate School of Medicine Sendai Japan4Harvard-MIT Division of Health Sciences and Technology Boston USA5WPI-Advanced Institute for Materials Research (AIMR), Tohoku University Sendai JapanShow Abstract
Age-related macular degeneration is a major ophthalmic disease that causes visual impairment and blindness. Although transplantation of autologous peripheral cells has been tested by injection of cell suspensions, limited visual improvement resulted due to the low viability of the injected cells in the subretinal tissue. As an alternative approach, there have been several reports on the development of natural and synthetic substrates for local delivery of retinal pigment epithelial (RPE) cells using collagen, poly(ethylene terephthalate) and poly(methyl methacrylate). However, these engineered substrates with micrometers in thickness (6 mu;m at thinnest) and several millimeters in size are not sufficiently flexible to be aspirated and injected through a conventional syringe needle into the narrow subretinal space. Thus, a large incision of the sclera and retinal tissue would be required for the injection of these rigid substrates. Such an incision might result in leakage of vitreous fluid and lead to post-surgical infection. Therefore, miniaturization of the substrates is an important approach to achieve minimally invasive delivery of the engineered tissue. In this study, we developed micropatterned polymeric nanosheets consisted of biodegradable poly(lactic-co-glycolic acid) and magnetic nanoparticles (MNPs, 10 nm in diameter) toward local delivery of the RPE cells. The micropatterned nanosheet encapsulating MNPs was fabricated by microcontact printing techniques. Owing to the magnetic property and flexible structure, the micropatterned nanosheet with 170 nm thick was manipulated remotely and delivered to the subretinal space of a swine eye. The micropatterned nanosheet also directed growth and morphogenesis of the RPE cells, and allowed for the injection of an engineered RPE monolayer through syringe needles flexibly without loss of cell viability. Such an ultra-thin flexible carrier has the promise of a minimally invasive delivery of organized cellular structures into narrow tissue spaces.
4:15 AM - *W2.05
Bioactive Materials for Transitioning Cell Phenotypes within Chronic Scar
Mariah Hahn 1
1Rensselaer Polytechnic Institute Troy USAShow Abstract
Introduction: Success in tissue engineering of the vocal fold lamina propria extracellular matrix (ECM) for treatment of chronic vocal fold scarring demands an understanding of how cells integrate the signals presented from the scar microenvironment, in combination with the signals from the biomaterial scaffold, to alter their response. To date, approaches to biomaterial development for scarred lamina propria treatment have examined the response of “normal” (non-scar) vocal fold fibroblasts (VFFs) to the biomaterial. As a result, they have failed to capture cellular responses, from activated macrophages and from resident scar-tissue VFFs (also known as myofibroblasts), from the in vivo implant environment which critically impact the quality and rate of VFF ECM production. In the present work, we conjugate cytokines, previously identified as anti-fibrotic and/or immunomodulatory, to biocompatible poly(ethylene glycol) [PEGDA] hydrogel formulations shown to have mechanical properties which preserve mucosal wave activity with low average phonation threshold pressures. We demonstrate these biomaterials influence macrophage polarization — shifting activated macrophages from a pro-inflammatory phenotype to a phenotype that is anti-inflammatory and pro-healing — and shift myofibroblasts to a “normal” or anti-fibrotic VFF phenotype.
Results and Discussion: Pro-inflammatory macrophages and/or myofibroblasts were encapsulated in PEG-bFGF gels. Following 3 days of culture in activation(pro-inflammatory, pro-fibrotic) media, expression of the fibrotic marker αSMA by myofibroblasts in PEG-bFGF gels was reduced over 7-fold relative to non-bFGF containing gels. Similarly, the expression of genes associated with matrix turnover (e.g. MMP1) were increased in response to bFGF. Activated macrophages exposed to bFGF gels displayed over a 50% reduction in inflammatory makers Nos2 and IL-12β relative to non-bFGF hydrogel controls. This reduction in fibrotic markers in myofibroblasts and inflammatory markers in macrophages in bFGF-containing gels occurred despite the continued presence of activating media and is consistent with anti-fibrotic and anti-inflammatory properties reported for bFGF. These results suggest that PEG-bFGF gels could be used to transition the activated cells present in chronic vocal fold scar to more phenotypes more conducive to healing.
Conclusions: The design of material environments to transition activated cells that are present in wound environments to more “normal” cell phenotypes would be desirable in the treatment of chronic scar and other chronic wounds. The present results represent an important step toward using tissue engineering principles to modulate cell phenotypes within an active wound environment.
4:45 AM - *W2.06
New Strategies for Engineering Human Tissues Based on Natural Origin Material Architectures
Rui L. Reis 1 2
1University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine Taipas - Guimaramp;#227;es, Portugal Portugal2University of Minho Braga/Guimaramp;#227;es PortugalShow Abstract
The selection of a scaffold material is both a critical and difficult choice that will determine the success of failure of any tissue engineering (TE) strategy. We believe that natural origin polymers are the best choice for many approaches. In addition, we have been developing an all range of processing methodologies to produce adequate scaffolds for different TE applications. Furthermore an adequate cell source should be selected. In many cases efficient cell isolation, expansion and differentiation methodologies should be developed and optimized. We have been using different human cell sources namely: mesenchymal stem cells from bone marrow, mesenchymal stem cells from human adipose tissue, human cells from amniotic fluids and membranes and cells obtained from human umbilical cords. The potential of each type of cells, to be used to develop novel useful regeneration therapies will be discussed. Their uses and their interactions with different natural origin degradable scaffolds and distinct nano and micro-carriers, and smart release systems, will be described. A great focus will be given to the different sources of stem cells, the isolation of distinct sub-populations, ways of differentiating them, as well as their interactions with different 3D architectures and materials for culturing them. The use of bioreactors to control cell differentiation, as well as the surface modification of the materials in order to control cell adhesion and proliferation will also be illustrated. Several biomimetic and nanotechnology based strategies to engineer mineralized tissues will be described.
5:15 AM - W2.07
Polymer Adhesives for Medical Applications
Hoyong Chung 1 Michael R. Harrison 2 Robert H. Grubbs 1
1California Institute of Technology Pasadena USA2University of California San Francisco San Francisco USAShow Abstract
Biomedical adhesive is important in clinical medicine and various surgical conditions require new and better polymeric adhesives. The biomedical adhesive should be wet adhesive, nontoxic, physiologically stable, rapidly crosslinkable and physically flexible. A new bio-inspired adhesive for medical applications was synthesized from three monomers, N-methacryloyl-3,4-dihydroxyl-L-phenylalanine, acrylic acid N-hydroxysuccinimide ester and acrylic acid. 3,4-dihydroxy-L-phenylalanine (DOPA) containing polymer segment has a function of strong wet adhesion properties. Poly(acrylic acid) is a water soluble segment and N-hydroxysuccinimide ester rapidly forms covalent bonds with thiols on thiol terminated 3-armed poly(ethylene glycol) cross-linking agents. The adhesive with DOPA incorporated demonstrated increased adhesion strength that is measured by lap shear strength tests on wet porcine skin mimicking human tissues. Crosslinking the polymers significantly enhanced the maximum adhesion strength. The novel adhesive shows viscosity drops at high shear rate, demonstrating its potential to be injected through syringe needles. The enhance moduli of crosslinked adhesive revealed that its robust stability after the adhesive attached on intended sites.
Recent developments in various invasive fetal diagnosis and fetal therapeutic technologies has been contributed to improve fetus&’ health, but the limited healing capability of fetal membrane may cause clinically dangerous Preterm Premature Rupture of Membranes (PPROM) from the medical device punctured site. This problem could be solved by the developed new bio-medical adhesives. The wet adhesive properties of the new terpolymer were evaluated in the context of sealing punctured fetal membranes. In order to perform realistic in vitro test on human fetal membrane, a new test device was designed and manufactured to mimic maternal uterus and amniotic sac. The new adhesive successfully sealed needle punctures on fetal membrane. Also the new terpolymer was directly compared to commercially available medical adhesives and was found to be more efficient in the presealing treatment of fetal membrane.
5:30 AM - W2.08
Novel Broad-Spectrum Antimicrobial Polysaccharide-Based Materials
Peng Li 1
1Xi'an Jiaotong University Xi'an ChinaShow Abstract
Microbial infections endanger public health unremittingly and cause a huge economic burden to our society. Numerous efforts have been made to develop molecules which are able to inhibit pathogens. Novel broad-spectrum antimicrobial materials were developed based on natural polysaccharide. Firstly, a group of antimicrobial materials were synthesized by quaternization and alkylation of chitosan.(1) An argon plasma-ultraviolet (UV) induced coating method for hydrogel surface immobilization was developed, which can be applied on diverse soft biomedical surfaces. A novel mechanism of these hydrogels based on “anion sponge” concept was proposed and proven. The optimized coating formulation and conditions show excellent antimicrobial potency. The in vitro and in vivo studies suggest this antimicrobial coating is biocompatible with mammalian cells. Secondly, a peptidopolysaccharide that mimics the bacterial peptidoglycan structure, which is a feature unique to bacterial membrane but absent in mammalian cells, was designed, synthesized and tested.(2) By the ring-opening polymerization of N-carboxyanhydrides (NCA), a polysaccharide backbone was copolymerized with cationic polylysine, and the resulting optimized peptidopolysacchride shows high selectivity to bacteria over mammalian cell. Preliminary results also suggest that the compound stimulates little or no inflammatory response. These two classes of polysaccharide based materials reveal new directions for the design of antimicrobial materials, and they have a promising prospect in further applications.
1. Peng Li, et al. Nature Materials, 2011, 10(2), 149-156.
2. Peng Li, et al. Advanced Materials 2012, 24(30), 4130-4137.
W3: Poster Session: Biomaterials for Regenerative Medicine I
Tuesday PM, April 22, 2014
Marriott Marquis, Yerba Buena Level, Salons 8-9
9:00 AM - W3.01
Building In-Vitro Dentin Like Matrices Using Micro Porous Membranes
Sharmistha Saha 1 Roselyn Odsinada 1 Jiyoung Chang 1 Cheryl Simpliciano 1 Orapin Horst 1 Tejal Desai 1 Stefan Habelitz 1
1University of California San Francisco USAShow Abstract
Controlling the spatial organization of cells with well-defined architecture is a vital aspect of tissue engineering. Odontoblasts are the cells that synthesize dentin. They are densely packed and line the pulpal wall while anchored into dentin through their processes. Recreating the odontoblast layer and morphology may facilitate dentin development in-vitro. Objective: To craft an in-vitro construct that is able to position cells in a configuration that mimics the highly organized layer of odontoblasts in teeth using micro porous membranes and promote secretion of a collagenous matrix that mineralizes. Method: A two-step lithographic process was used to generate a master-mold on a silicon wafer. Polycaprolactone (PCL) was spin cast onto the mold to create a membrane with 1-3µm pores, and pillars with 10µm heights surrounding each pore. Commercial cell culture inserts with 1 or 3mu;m pores and homemade porous PCL membrane were used to enable high-density, single-layer cell accumulation and formation of cell protrusion. MC3T3 cells were seeded at confluent densities onto membranes and centrifuged at 700 rpm to position. Cells were allowed to mineralize under a chemical gradient for up to 21 days. Results: MC3T3 cells formed a monolayer of cells on membrane after centrifugation and produced collagenous matrix, which appeared continuous over large areas. Chemical gradient created with serum, led to the formation of cell protrusions extending through pores at day 21. However, it was observed that MC3T3 cells lose its 3D organization on the porous membranes within 24hr with the absence of pillar structures. Presence of pillars on PCL membrane may prolong cellular organization for a longer time and enable organized matrix production. Conclusion: A porous membranes engineered with micropatterning technology can be used to confine one cell to a single pore while inducing cell protrusion successfully, providing us the possibility of recreating the odontoblastic morphology in-vitro.
9:00 AM - W3.02
Optical Spatiotemporal Characterization of Collagen Systems
Yu Jer Hwang 1 Xuye Lang 1 Julia Lyubovitsky 1
1UCR Riverside USAShow Abstract
Collagen is a naturally occurring biopolymer and its&’ architecture is important in tissue development and repair. We have explored the nucleation, assembly and the 3-D architecture in collagen hydrogels prepared under the conditions commonly employed in biomedical, tissue engineering and medical device research. Employing in situ multi-photon optical microscopy that combines nonlinear optical phenomena of second harmonic generation (SHG) and two-photon fluorescence (TPF) signals we showed that drastically different microstructures are prepared by changing collagen solid content, incubation temperature or ions and moreover that the non-zero cross-linkers for example genipin significantly alter the originally assembled 37 C architectures. Specifically, genipin induces formation of aggregated fluorescent fibers concomitantly with a 3.5 fold drop in SHG contrast (after 24-hour reaction at 37 C) suggesting that this modification disaggregates initial collagen microstructure within the materials at the expense of forming the new fluorescent features. The 800 nm excitation wavelength generates complex TPF contrast within genipin-modified materials. It is centered within the blue-green (~ 490 nm center) as well as red (~ 615 nm center) spectral regions and spatially distinct. The ratio of the intensity of 615 nm emission band to the intensity of 490 nm emission band is 5.3 (after 24-hour reaction at 37 C). There is a slight bathochromic (red) shift when the excitation wavelength is varied between 760 nm and 900 nm. The findings will be discussed within the context of our ongoing tissue engineering and wound repair research.
9:00 AM - W3.03
Comparison of Different Decellularization Methods on Adipose Tissue: The Advantage of Physical Method
Shraddha Shrinivas Shanbhag 1 Biawen Luo 1 Selin Foo 2 Nguan Soon Tan 2 Marcus Thien Chong Wong 3 Cleo Choong 1
1NTU Singapore Singapore2NTU Singapore Singapore3Tan Tock Seng Hospital Singapore SingaporeShow Abstract
In the recent years, there is an alarming increase in obesity worldwide. Obesity associated health concerns as well as stigma has driven affected people for reconstructive surgeries. With this, the amount of adipose tissue discarded as a clinical waste has increased proportionally. Literature suggests different protocols that decellularize adipose tissue. These protocols rely on combinations of physical, chemical and enzymatic agents to remove cells and lipids. The end-product of decellularization yields extracellular matrix (ECM) that supports the cells in an organ; both structurally and functionally. Although the previously designed protocols have been successful in the decellularization, they span a number of days. Our study involves a purely enzymatic, chemical or physical process of decellularization. Further, we distinguish them based on their ability to remove lipid and cells. We then tested the capability of these processes in preserving the essential biomolecules such as collagen, Glucoseaminoglyxcans (GAG), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Additionally, we tested the ability of ECMs generated by the enzymatic (ECM-E), chemical (ECM-C) and physical (ECM-P) to support various cell types. We report that the physical method of decellularization is advantageous due to its shorter time; efficient lipid and cell removal; and collagen, GAG, VEGF and bFGF conservation. Therefore, we recommend the use of ECM-P for scaffolds and coats to apply in tissue engineering.
9:00 AM - W3.04
Chitin Nanofiber Micropatterned Flexible Substrates for Tissue Engineering
Pegah Hassanzadeh 1 Mahshid Kharaziha 2 3 Mehdi Nikkhah 2 3 Su Ryon Shin 2 3 4 Jungho Jin 1 Semeiqi He 1 Wei Sun 1 Chao Zhong 1 Mehmet Dokmeci 2 3 4 Ali Khademhosseini 2 3 4 Marco Rolandi 1
1University of Washington Seattle USA2Brigham and Womenamp;#8217;s Hospital, Harvard Medical School Boston USA3Massachusetts Institute of Technology Cambridge USA4Harvard University Boston USAShow Abstract
Engineered tissues require enhanced organization of cells and the extracellular matrix (ECM) for proper function. To promote cell organization, substrates with controlled micro-and nanopatterns have been developed as supports for cell growth, and to induce cellular elongation and orientation via contact guidance. Micropatterned ultra-thin biodegradable substrates with precisely defined chemistry are desirable for implantation in the host tissue. These substrates, however, need to be mechanically robust to provide substantial support for the generation of new tissues, to be easily retrievable, and to maintain proper handling characteristics. Chitin is an ideal substrate. Chitin is a naturally abundant polysaccharide, which is mechanically stable, biodegradable, nontoxic, and physiologically inert. Here, we introduce ultra-thin (<10 mm), self-assembled chitin nanofiber substrates micropatterned by replica molding for engineering cell sheets. These substrates are biodegradable, mechanically strong, yet flexible, and can be easily manipulated into the desired shape. As a proof-of-concept, fibroblast cell attachment, proliferation, elongation, and alignment were studied on the developed substrates with different pattern dimensions. On the optimized substrates, the majority of the cells aligned (<10 mu;m) along the major axis of micropatterned features. With the ease of fabrication and mechanical robustness, the substrates presented herein can be utilized as a versatile system for the engineering and delivery of ordered tissue in applications such as myocardial repair.
9:00 AM - W3.05
Human Cardiomyocyte Response to Micropatterned Feature Widths
Max R Salick 1 2 3 Brett N Napiwocki 1 2 4 Jin Sha 2 5 Gavin T Knight 2 4 Shahzad A Chindhy 6 Timothy J Kamp 6 7 8 Randolph S Ashton 2 4 Wendy C Crone 1 2 4
1University of Wisconsin - Madison Madison USA2Wisconsin Institutes for Discovery Madison USA3University of Wisconsin - Madison Madison USA4University of Wisconsin - Madison Madison USA5East China University of Science and Technology Shanghai China6University of Wisconsin - Madison Madison USA7WiCell Institute Madison USA8University of Wisconsin - Madison Madison USAShow Abstract
Recent developments in stem cell differentiation methods have enabled the derivation of human heart muscle cells, or cardiomyocytes, from stem cell sources at exceedingly high efficiencies. The resulting cells demonstrate many of the properties of immature human cardiomyocytes, but they do not spontaneously align and form mature, highly-structured myofibrils that are typically observed in vivo. In our studies, we&’ve developed a platform that utilizes soft lithography and micropatterning techniques to control cell geometries in a highly specific manner. The patterned geometries consisted of rectangular features of varying sizes and aspect ratios to investigate how cardiomyocyte aggregates responded to the geometric constraint. Pure populations of immature, human embryonic stem cell-derived cardiomyocytes were seeded onto these fibronectin/matrigel geometries. Myofibril assembly of the cardiomyocytes was assessed by observation of cardiomyocyte sarcomere structure using actin and alpha-actinin stains. Alignment of the cells was assessed using DAPI staining and subsequent analysis of nuclear alignment. Results showed that the human cardiomyocytes aligned much more strongly, and formed much more robust sarcomere structures, on geometries with widths ranging from 20µm to 80µm. Features whose shortest side was more than 80µm failed to produce cardiomyocyte aggregates that properly aligned. We hypothesize that this improved alignment of the cardiomyocytes contributes to the enhanced myofibril formation due to improved cell polarity, as well as a focal adhesion/costamere confinement that forces stress fibers to align more consistently with the cell axis. The cells cultured on optimal geometries demonstrate a much more physiologically-representative size, as well as a mature-like myofibril expression.
9:00 AM - W3.08
Porous Si and Biocompatible Electrospun Fiber Composites for Controlled Drug Release in Ophthalmological Applications
Josef Velten 1 Jhansi Kalluri 1 Jeffery Coffer 1
1Texas Christian University Fort Worth USAShow Abstract
Previous investigations have established the utility of porous silicon in electrospun microfibers of biocompatible polymers such as polycaprolactone (PCL) for possible applications such as orthopedic tissue engineering1 and treatment of ocular disease.2 For the latter, the presence of a polymer such as PCL or poly-L lactic acid (PLLA) facilitates processing and provides opportunities for multistage drug delivery dependent on the dissolution kinetics of the porous Si and polymer components, respectively. In this presentation, we outline a process for the spatial control of particle location of nanostructured porous silicon in the polymer network and ideal control of drug release from this platform. Control of the loading amounts is discussed through processing techniques of the composite and the flexibility of the method to easily manipulated variables. We seek to use the porous silicon as a chemical reservoir for the sustained release of either ophthalmological-relevant drugs or cellular growth factors, but begin with proof-of-concept using a fluorescent dye as an easily-tracked proximate. After a discussion of composite microstructure using scanning electron microscopy (SEM) characterization and an evaluation of the stability of such structures in cell-based media, kinetic-based studies of the release of therapeutics from these composites are presented.
1 Dongmei Fan, Giridhar R. Akkaraju, Ernest F. Couch, Leigh T. Canham, and Jeffery L. Coffer, Nanoscale, 2011, 3,354-361.
2 Soheila Kashanian, Frances Harding, Yazad Irani, Sonja Klebe, Kirsty Marshall, Armando Loni, Leigh Canham, Dongmei Fan, Keryn A Williams, Nicolas H Voelcker and Jeffery L Coffer, Acta Biomaterialia , 2010 , 6, 3566-3572.
9:00 AM - W3.10
Ferroelectric Lithium Niobate as a Novel Platform for Tissue Engineering
Craig Carville 1 2 Michele Manzo 3 Katia Gallo 3 Katey McKayed 2 4 Jeremy C. Simpson 2 4 Brian J. Rodriguez 1 2
1University College Dublin Dublin Ireland2University College Dublin Dublin Ireland3KTH - Royal Institute of Technology Stockholm Sweden4University College Dublin Dublin IrelandShow Abstract
The leading cause of implant failure is poor implant osseointegration. The use of electrically polarized biomaterials provides one possibility to address poor osseointegration. Electrically polarized hydroxyapatite has been shown to improve osteoblast attachment and proliferation; however, the mechanism by which this occurs is not fully understood. During the processing of hydroxyapatite, for example, there can be differences in a number of factors that would also influence osseointegration, i.e., surface roughness, Ca/P ratio, etc. In this work, we demonstrate the use of a ferroelectric substrate (lithium niobate, LN) with a permanent polarization (in the z-cut direction) as a novel platform to study the effect of surface charge for improving osseointegration while minimizing the factors described above. Each of the transparent LN samples used has the same chemistry and approximately the same surface roughness. Thus, any differences in the performance between the positively and negatively charged substrates can be ascribed to the surface charge. The biocompatibility of LN and its ability to promote osseointegration were characterized using cell proliferation and mineralization assays.
Fluorescence-based assays in cultured cells demonstrated for the first time the biocompatibility of LN. MC3T3 osteoblast cells cultured for up to 11 days attached and proliferated. These studies revealed an enhancement of cell attachment on the charged LN surfaces compared to uncharged LN, i.e., x-cut LN and a glass control. By 9 days there was a 30% proliferation enhancement on charged compared to uncharged surfaces, further demonstrating biocompatibility and enhanced cell metabolism (via MTT assay). We also report the ability of the cells to undergo mineralization in calcium-supplemented media. The cells on charged LN produced 26% more mineralized calcium than the unpoled LN surface after 30 days. We conclude that the surface charge allows for enhanced osseointegration and provides a novel platform for understanding the effect of surface charge on cellular processes.
9:00 AM - W3.11
Piezoelectricity in Collagen Type II Fibrils Measured by Scanning Probe Microscopy
Denise Denning 1 2 Stefan Habelitz 3 Andrzej Fertala 4 Brian J Rodriguez 1 2
1University College Dublin Dublin Ireland2University College Dublin Dublin Ireland3University of California San Francisco USA4Thomas Jefferson University Philidelphia USAShow Abstract
Electromechanical coupling in collagen type I has been studied extensively since the discovery of piezoelectricity in several biosystems in the mid-1900s. It is known that induced currents (via piezoelectricity) activate the healing process in tissues under compression or tension. The structural similarities between collagen type I and other fibrillar collagens (type II, III, V, etc.) suggest they are piezoelectric as well. Collagen type II comprises 3 identical alpha-1 polypeptide strands twisted together to form a helix; whereas the collagen type I helix comprises two alpha-1 strands and one alpha-2 strand. In this study, piezoresponse force microscopy (PFM) is used to probe the electromechanical properties of reconstituted collagen type II fibrils at the nanoscale. The fibrils are found to exhibit shear piezoelectricity, showing the same cosine dependence of the signal on the angle between the cantilever and fibril axes as has been observed for collagen type I. The piezoelectric coefficient of collagen type II was found to be one order of magnitude lower than that of type I. This reduction could be due to a higher density of cross links in collagen type II, resulting in a lower degree of freedom of movement along the fibril axis and thus, lower shear piezoelectricity. The investigation of piezoelectricity in collagen type II may be relevant for possible regenerative biomaterial devices in the case of damaged cartilage or as piezoelectrically-active cell scaffolds or coatings.
9:00 AM - W3.12
Collagen Remains Piezoelectric in a Moisture-Rich Environment
Denise Denning 1 2 Michael V Paukshto 3 Stephen Jesse 4 Stefan Habelitz 5 Andrzej Fertala 6 Sergei V Kalinin 4 Brian J Rodriguez 1 2
1University College Dublin Dublin Ireland2University College Dublin Dublin Ireland3Fibralign Coorporation Sunnyvale USA4Oak Ridge National Laboratory Oak Ridge USA5University of California San Francisco USA6Thomas Jefferson University Philidelphia USAShow Abstract
The functional role of piezoelectricity in collagenous materials is a topic which has been under debate since the discovery of piezoelectricity in bone in 1957. Various studies have shown the potential of piezoelectricity in collagen to play a role in bone regeneration. However, in order for collagen piezoelectricity to have biological significance in the case of bone, the property must be observed on the length scale of the piezoelectric component (collagen) in physiological conditions; a topic in the literature with contradictory results. Macroscopically, hydrated bone has been shown to have an insignificant piezoelectric effect compared with dry bone. However, at the nanoscale the piezoelectric effect is observed in both dry and wet bone. Here, using single and multifrequency (band excitation (BE)) piezoresponse force microscopy (PFM), the electromechanical properties of collagen type I have been investigated as a function of humidity, from ambient to 90% relative humidity (RH), which surpasses the hydrated range for collagen in physiological bone (12% moisture content corresponding to ~ 40/50% RH). BE PFM allows for the detection of the cantilever response across a band of frequencies corresponding to the contact resonance at each pixel in an image, enabling contact resonance frequency and quality factor (energy dissipation) mapping in addition to electromechanical property imaging. The role of the water layer between the tip and surface with varying humidity and applied voltage has also been investigated, revealing no change in tip-sample capillary adhesion between on and off voltage states. The results show that collagen remains piezoelectric in a highly humid environment. These results lay to rest the question of whether collagen is piezoelectric in physiologically-relevant moisture-rich environments, and will facilitate future studies investigating the biofunctionality of piezoelectricity in physiological conditions.
9:00 AM - W3.13
DNA Nucleobase Crystals as Functional Biomaterials: Investigation of Unexpected Electromechanical Behavior of Thymine Microcrystals Grown by Slow Evaporation
Sabine Neumayer 1 2 Igor Bdikin 3 Andrei Kholkin 4 Jia Jun Li 1 Brian Vohnsen 1 Brian J. Rodriguez 1 2
1University College Dublin Dublin Ireland2University College Dublin Dublin Ireland3University of Aveiro Aveiro Portugal4University of Aveiro Aveiro PortugalShow Abstract
Piezoelectricity in biomaterials is a ubiquitous phenomenon of high scientific interest, not only because of a possible biological function, but also in terms of potential applications as cell scaffolds and biocompatible sensors and actuators. Deoxyribonucleic acid (DNA) is one of the most important bionanomaterials that shows potential for biopiezoelectric applications. As DNA strands are stable polymers that are easy to replicate, they are ideal candidates for molecular nanodevices. Thymine is one of the nucleobases in DNA that are held by backbones of deoxyribose and phosphate. The coupling of nucleobases through hydrogen bonds and van der Waals forces might play a fundamental role in electric and possibly electromechanical behavior and are therefore interesting to study separately in the form of crystals.
In this work, thymine crystals were grown from aqueous solutions having different thymine concentrations (0.1 - 4.5 mg/ml). Centrosymmetric anhydrate and monohydrate crystals were prepared at room temperature in a covered beaker containing 40 ml of 4.5 mg/ml solution. X-ray diffraction (XRD) measurements show that both forms of these crystals are monoclinic with space group P21/c, in agreement with literature. The centrosymmetric structure of thymine anhydrates was corroborated using second harmonic microscopy (SHM) and local piezoresponse force microscopy (PFM) measurements, which showed an absence of both second harmonic signal and piezoelectric response. However, evidence of possible non-centrosymmetric crystal structure was observed for micro-sized crystals prepared under certain evaporation conditions. These microcrystals exhibited second harmonic signal and piezoelectricity. In plane (IP) and out of plane (OOP) piezoelectric response was measured with local effective piezoelectric coefficients of around 1 pm/V IP several pm/V OOP. SHM measurements confirmed that the piezoresponse is not a surface effect. However, the exact origin of this non-centrosymmetric behavior could not be determined, as microcrystals are unsuitable for conventional XRD.
In some cases, temporal switching of domains was observed. As cell adhesion and proliferation are known to be influenced by charge (e.g., poled hydroxyapatite) this opens up possible applications for nucleobase crystals as functional biomaterials in regenerative tissue engineering.
9:00 AM - W3.16
Fabrication of Conductive Alumina Membranes as a Biomaterial for Neural Tissue Engieering
Sevde Altuntas 1 Buket Altinok 2 Belma Aslim 2 Fatih Buyukserin 3
1Tobb Univ. of Econ. Tech. Ankara Turkey2Gazi University Ankara Turkey3TOBB Univ. of Econ. Tech. Ankara TurkeyShow Abstract
Biomaterials that simultaneously present electrical, chemical and topographic cues are ideal substrates for neural tissue engineering. Significant amount of research has been conducted to utilize combinations of these cues on a rainbow of different substrates for increased cell adhesion, proliferation and alignment as well as enhanced neurite outgrowth. The nature of anodized aluminum oxide (AAO) membranes intrinsically provides fine control over topographic and chemical cues for enhanced cell interaction; hence these biomaterials are widely used for connective tissue applications. Neural tissue engineering research with AAO substrates, however, is still very limited and the reported literature mainly focuses on the influence of topography on neural response. Herein, we present the fabrication and characterization of conductive AAO (CAAO) surfaces for the ultimate goal of integrating electrical cues for improved neural tissue behavior on the nanoporous substrate material. Parafilm was used as a protecting polymer film, for the first time, in order to obtain large area (50 cm2) free-standing AAO membranes. Carbon (C) was then deposited on the AAO surface via sputtering. Morphological characterization of the CAAO surfaces revealed that the pores remain open after the deposition process. The presence of C on the material surface and inside the nanopores was confirmed by XPS and EDX studies. Furthermore, I-V curves of the surface were used to extract surface resistance values and conductive AFM demonstrated that current signals can only be achieved where conductive C layer is present. Finally, novel nanoporous C films with controllable pore diameters and one dimensional (1-D) C nanostructures were obtained by the dissolution of the template AAO substrate. Regarding cellular data, naked AAO surfaces showed cell growth stimulation, negligible cytotoxicity and improved cell adhesion for differentiated PC 12 cells. Cellular adherence, cytotoxicity as well as neurite formation are currently being investigated for CAAO surfaces and the details of these studies will be presented. The combination of chemical, topographic and electrical cues will investigated for enhanced nerve regeneration by comparing growth factor doped and/or electrically stimulated CAAO surfaces having different porous topography.
This work was supported by The Scientific and Technological Research Council of Turkey, Grant No. MAG-111M686
9:00 AM - W3.19
Photo-Responsive Polyethylenimine Hydrogels for Microfabrication of Cell-Active Platforms
M.Gabriella Santonicola 1 2 Antonio Paciello 3 4
1Sapienza University of Rome Rome Italy2MESA+ Institute for Nanotechnology / University of Twente Enschede Netherlands3Istituto Italiano di Tecnologia Naples Italy4University of Naples Federico II Naples ItalyShow Abstract
Supramolecular photo-responsive hydrogels are prepared from partial methacrylation of branched polyethylenimine (PEI). The properties of the PEI-based hydrogels in terms of swelling and porosity can be controlled during synthesis by the amount of functional methacrylate groups on the polymer backbone. Different reaction conditions were used, and the synthesis was optimized to give superabsorbent hydrogel materials with water retaining properties higher than 95%. The hydrogel microstructure was characterized using several techniques including small-angle x-ray scattering and fluorescence microscopy to visualize the gel porous network. The PEI-based hydrogels are activated by multi-photon laser irradiation and can be patterned on the micron scale when molecular probes with free carboxylic acid or hydroxyl groups are present in solution. This approach offers the possibility for precise immobilization of bioactive signals into three-dimensional matrices without the need of a photoinitiator. Direct patterning of the hydrogel matrix in solutions with several types of biomolecules was demonstrated in multi-photon confocal microscopy experiments. In combination with bioactive molecules, these hydrogels represent a novel cell-instructive platform that can be selectively encoded with active signals, with relevant applications in biotechnology and medicine.
9:00 AM - W3.22
Design Stretchable, Tough, Yet Stiff Hydrogel via Multi-Scale Strengthening
Shaoting Lin 1 Xuanhe Zhao 1
1Duke Univeristy Durham USAShow Abstract
Hydrogel application is limited by its poor mechanical property for structurally demanding tissue engineering. Here, we designed highly stretchable, tough, yet stiff hydrogels based on fiber reinforced hydrogel composites. We used 3D printing technology to fabricate three dimensional patterned fibrous structures. Impregnating the fiber mesh with highly stretchable and tough PAAM-alginate hydrogel constructs the fiber reinforced hydrogel. Synthetic gels can reach fracture energies of around 9,000 J m-2. But modulus of these tough hydrogels is only about 100 kPa. Here, we designed fiber reinforced hydrogels, which can reach fracture energy of about 20,000 J m-2 and modulus of around 3MPa. Stretrability of these fiber reinforced composites can still reach about 10, which is even larger than many pure gels. The enhancement of toughness is due to multi-scale energy dissipation mechanism which spans over multiple length scales ranging from nanometers to millimeters. This design of fiber reinforced hydrogel composites can serve as a model to expand the application of hydrogels for biomedical devices and soft machines.
9:00 AM - W3.23
Multi-Layered Fibrin Gel Polymerization of Laser Etched Poly(epsi;-caprolactone) Dual- Scale Electrospun Scaffolds for Co-Cultured Cartilage Tissue Engineering
Danielle Traphagen 1 2 Eun Jung Kim 1 Annie Ouyang 1 Ellen Liebenberg 1 Benjamin Lee 3 Xinyan Tang 1 Jeffrey Lotz 1 Shuvo Roy 1
1University of California, San Francisco San Francisco USA2California Institute of Regenerative Medicine San Francisco USA3University of California, Berkeley Berkeley USAShow Abstract
Damaged articular cartilage (AC) tissue shows poor regenerative capacity due to its avascularity. Current (AC) treatment modalities lack longevity or are primarily palliative in nature. The authors aim to respond to the clinical challenges presented by cartilage cell culture in electrospun scaffolds by exploring the optimal delivery environment or Bilaminar Cell Pellets (BCPs). BCPs are comprised of mesenchymal stem cells and nucleus pulposus cells that undergo chondrogenesis without hypertrophy.^1 BCPs were applied to biodegradable Poly(ε-caprolactone) (PCL) scaffolds. These multi-layer scaffolds were electrospun to provide 3D architecture for defect applications, while FDA-approved fibrin was selected for its adhesive and wound healing properties. The scaffolds provide superior cellular infiltration due to their dual-scale and laser-etched architecture. BCPs were seeded into laser-etched and non-etched scaffolds with fibrin gel. Single layer scaffolds were encased in fibrin gel for immunofluorescence to visualize type 2 collagen and aggrecan. The scaffolds were cultured for 21 days for biochemical and histological analysis, which is currently pending. Cellular attachment and infiltration was assessed using Scanning Electron Microscopy (SEM), Glycol-Methacrylate and Paraffin histology. In addition, quantitative Real Time Polymerase Chain Reaction was performed to quantitatively assess the gene expression of chondrogenic markers. The PCL nanofiber diameter was analyzed by SEM to be approximately 200-600 nm, while the PCL microfiber diameter was around 2-3 um. The laser-etched pore diameter was approximately 467 nm. SEM images indicated that the nanofiber and microfiber scaffold structure was retained after laser etching. This is a novel study showing that dual-scale electrospun meshes can be modified by commercial laser etchers to increase porosity, allow for the application of pelleted BCPs, and can be held together with fibrin gel to provide desired thickness for cartilage tissue engineering techniques.
1. M.E. Cooke, A.A. Allon, T. Cheng, A.C. Kuo, H.T. Kim, T.P. Vail, R.S. Marcucio, R.A. Schneider, J.C. Lotz, T. Alliston. Structured three-dimensional co-culture of mesenchymal stem cells with chondrocytes promotes chondrogenic differentiation without hypertrophy. Osteoarthritis and Cartilage. 2011;10:1210-1218
9:00 AM - W3.24
Aligned Electrospun Hyaluronic Acid-Based Scaffolds Containing Multivalent Peptide Conjugates
Nikhil A. Rode 1 Natalie C. Marks 2 Min Ju Lee 1 Kevin E. Healy 1 2
1UC Berkeley Berkeley USA2UC Berkeley Berkeley USAShow Abstract
The natural extracellular matrix (ECM) is made up of protein nanofibers, which provide mechanical support as well as topographical and chemical signals to the embedded cells. Bioinspired materials designed as scaffolds for regenerative therapies attempt to replicate this environment by taking into account the chemical, biological and physical cues to the ECM. Electrospinning has emerged as a versatile, inexpensive, and scalable method to create materials that that readily recapitulate the physical environment of the ECM. In order to fully realize the potential of electrospun materials as ECM scaffolds, it is necessary to devise a method of tuning the biological signaling component independently of the electrospinning process. To accomplish this, we have created a scaffold combining the use of electrospinning to control the morphology of the material with multivalent peptide conjugates embedded in the fibers to control their biochemical environment. The material is based on 800 kDa hyaluronic acid that has been conjugated with the cell-binding peptide bspRGD(15) using carbodiimide and maleimide-based chemistry. The final valency of the conjugate averaged 23 peptides bound to each hyaluronic acid molecule as measured by in-line SEC, multi-angle light scattering, differential refractive index and UV absorbance measurements. This conjugate was electrospun from a water-based solution with a high molecular weight 1 MDa poly(ethylene glycol) carrier and 200 Da poly(ethylene glycol) diacrylate, which was crosslinked to render the structure water-stable. The fibers produced had an average diameter of 538 ± 262 nm. Alignment was achieved by using a 76 mm drum rotating at 6000 rpm as the electrospinning target, resulting in 86.4% of the fibers being aligned to within 10 degrees of the main axis. Induced pluripotent stem cell-derived cardiomyocytes were seeded onto the electrospun fibers and attached and maintained their cardiac phenotype. The cells formed beating structures that aligned with the underlying fibers. Long range beating capable of deforming the scaffold was observed after five days and continued for over two months. This study has demonstrated we can independently vary the macroscale mechanical properties, nanoscale topography and alignment of the electrospun fibers, and the chemical and biological signals presented to cells. Possible applications of this material include use as a cardiac patch to reinforce the weakened myocardial wall post infarction and as a wound dressing .
9:00 AM - W3.25
Advanced Microstructural and Compositional Characterization of Hydroxyapatite-Boron Nitride Nanocomposites
Feray Bakan 1 Meltem Sezen 1 Yapincak Goncu 2 Nuran Ay 3
1Sabanci University Istanbul Turkey2BORTEK Boron Technologies amp;Mechatronics Inc. Eskisehir Turkey3Anadolu University Eskisehir TurkeyShow Abstract
As the formation of hard tissues in human consists of nano-sized hydroxyapatite (nHAp), research on the probable bio implant applications of nHAp has been increasingly gaining importance. In spite of having sufficient biocompatibility, HAp cannot perform the expected mechanical properties of a hard tissue. It is considered that, the production of composites by adding a variety of materials to nHAp would improve mechanical properties. Accordingly, nano-sized hexagonal boron nitride (hBN) having different compositional percentages was added to the nHAp to form novel composites with expected properties. For the advanced characterization of the materials, structural and morphological analysis methods were used. For the structural characterization, XRD, Raman Spectroscopy and EDS were used, and for the identification of morphology and surface features, TEM, SEM and FIB and AFM techniques were utilized. The comprehensive analytical study was helpful to reveal the microstructural and compositional changes information in detail.
Acknowledgements: The authors would like to thank TUBITAK 112M592 and 112M591 projects for financial support, also the support of Prof. Dr. Mehmet Ali Gulgun for TEM investigations is gratefully acknowledged.
9:00 AM - W3.28
Dental and Orthopedic Biomaterials - Surface Modification of Dental Implants
Fernando Luzia Franca 1 Eduardo Luzia Franca 1 Lucas Botelho Franca 1
1CEFETMG Araxamp;#225; BrazilShow Abstract
The interaction between cells and implant materials is influenced by the composition of the surface structure and / or the surface of the material, subject studied and proven by several authors.
The process of osseointegration is benefited by changes in the surfaces of dental implants which are responsible for the acceleration of adhesion, migration and cell proliferation.
An ideal surface roughness is desired to occur matrix deposition and growth of bone tissue in close contact with the bone . The surface treatments are performed with the aim of increasing the mechanical and chemical bond between the implant and bone.
Dental and orthopedic biomaterials should promote osteoblast adhesion optimizing the process of integration between surgical implants placed and biological tissues.
The results show that the chemical treatment and the roughness of the surface appear to act in shear forces , especially evaluating the implant removal torque . Thus , the authors observed that the changes in the surface of the implants can optimize osseointegration and allow loading and earlier use in areas with lower density or auxiliary application in bone regenerated recentemente.
Surface modification of implants can result in morphological phenomena and physicochemical significant bone response . Another possible explanation for the good results is that changes in the chemical structure of the surface may be more favorable for binding marrow.
Thus, based on histomorphometry and biomechanical data , implants with changes in structure showing an anchorage higher than the unmodified implants . These implants allow greater integration with the bone implants unmodified after a short time of healing.
Key-words: Treatment of surface, Dental implantation.
9:00 AM - W3.29
The Absorption of Organic Dyes by Hollow Activated Carbon Fibers Fabricated from Kapok Fibers
Dickon H.L. Ng 1 Caihong Zhang 1 Jia Li 2
1The Chinese University of Hong Kong Shatin Hong Kong2The University of Jinan Shandong ChinaShow Abstract
Hollow activated carbon fiber (ACF) had been fabricated by using kapok through carbonization and activation processes. In the sample preparation, pieces of kapok were washed by HCl to remove surface wax before they were annealed at 600oC in argon for an hour. The samples were further activated for 10 to 35 min at 850oC under constant flow of CO2. During activation, the unstable carbon atoms in the fibers were selectively removed and more pores were created and increasing the surface area. The SEM analysis confirmed that the ACF samples were with morphology similar to that of the original Kapok. The average diameter of the fibers was 15mu;m and the thickness of the wall of the hollow fibers was 100nm. The Raman scattering patterns of the ACF samples showed two peaks at 1339cm-1 and 1596cm-1 corresponding to the disorder and graphitic carbon, respectively. This implied that the samples contained both crystalline and amorphous carbon.
The nitrogen adsorption and desorption isothermals were obtained from the AFC samples which were activated in CO2 for (a) 10, (b) 15, (c) 25, and (d) 35 min. Isotherm (a) showed a sharp increase in the adsorbate upon a slight increase of gas pressure before it achieved a plateau. This indicated that there was a limited pore size range and a monolayer adsorption. Thus, majority of the pores in this ACF sample were micropores. The isotherm (b) for sample after 15 min of CO2 treatment, a gentle shoulder appeared first, indicating that it was a mixed mono- and multi-layer adsorption. In isotherms (c) and (d) of samples after >15 min of CO2 treatment, their shoulders diminished suggesting the presence of larger pores for multi-layer adsorption and a mesoporous structure. All the isotherms exhibited hysteresis which was related to the filling and emptying of mesopores by pore condensation or by capillary, respectively, during the adsorption/desorption processes. Based on these results, we confirmed that these ACF demonstrated a microporous structure after short activation, but developed a mesoporous structure after prolong activation. We also determined that the maximum BET surface area of these ACF samples was 1517m2/g.
The methylene blue (MB) dye adsorption test by using our AFC samples was performed and the concentration of MB in water was monitored at different time. To study the kinetic mechanism of the adsorption, the pseudo first order and second order models were used to interpret our measured results. We found that the behavior of MB adsorption by ACF was consistent with the pseudo-second-order rate model having the correlation coefficients higher than 0.99. The equilibrium absorbance of the dye was as high as 526 mg/g in using the ACF sample treated in CO2 for 25 min. In summary, the kapok fibers are promising precursors for producing ACF. Its superior absorption ability for absorbing organic dye MB had been confirmed in this work.
W1: Biomaterials for Tissue Regeneration
Tuesday AM, April 22, 2014
Moscone West, Level 2, Room 2003
9:30 AM - W1.01
Nanoscale Crater Interfaces Guide Cell Migration and Patterning
Wilie Mae Reese 1 Sangmo Koo 2 Hojeong Jeon 2 3 Costas P. Grigoropoulos 2 Kevin E Healy 1 4
1University of California at Berkeley Berkeley USA2University of California at Berkeley Berkeley USA3Korea Institute of Science and Technology Seoul Republic of Korea4University of California at Berkeley Berkeley USAShow Abstract
Although cell adhesion to nanostructured interfaces has been extensively studied, few studies have focused on tuning nanotopographical surfaces to direct cell migration for cell patterning. Using multi-photon ablation lithography, we fabricated arrays of nanoscale craters in quartz substrates with a variety of geometries and spacing (i.e. pitch). Changing the nanocrater diameter (600-1000 nm), depth (110-350 nm), and/or pitch (1-10 um) alters the planar surface area available for cells to establish stable focal adhesions (FAs) and induces migration away from regions of high nanocrater density. This persistent migration can be used to dictate cell patterning (e.g., lines, circles) according to the nanocrater parameters. To further investigate interactions of these surfaces and the cell&’s adhesion mechanisms, we probed the effects of extracellular ligand presentation and intracellular contractile protein (e.g., Talin) activation on patterning. Nanocraters patterned in a gradient array, and presenting a RGD peptide ligand for cell adhesion, promoted cell migration in a ligand density dependent manner that encouraged cell patterning determined by the nanocrater pitch. Additionally, we found that cells that overexpress the N-terminus of Talin-1, which is one of the major proteins responsible for stable focal adhesion formation, lack the necessary balance of integrin activation to quickly spread and migrate from low pitch to high pitch regions. These nanoscale surfaces serve as tools for mechanobiology studies and understanding the attributes of surfaces necessary to physically pattern cells.
9:45 AM - W1.02
Sustained BMP-2 Release from Densified Titanium for Orthopedic Application
Hyun-Do Jung 1 Hyoun-Ee Kim 1 Young-Hag Koh 2 Yuri Estrin 3 4
1Material Science and Engineering, Seoul National Univ., Seoul Republic of Korea2Korea University Seoul Republic of Korea3Department of Materials Engineering, Monash University Clayton Australia4NITU MISiS Moscow Russian FederationShow Abstract
Orthopedic implant surfaces are often coated with biologically active agent to improve the surface biocompatibility of materials . Bone morphogenetic protein-2 (BMP-2) has been widely used to accelerate the healing of bone defects and is also used in medical and dental implants. Therefore, many studies have incorporated BMP-2 in implants to stimulate and augment bone formation . However, negative side effects such as heterotopic bone formation, retrograde ejaculation and osteoclast activation are often found when supraphysiologic doses of BMP-2 were delivered over relatively short periods. Various materials have been proposed and investigated for use as BMP carriers . However, in many cases, BMP-2 adsorbed superficially on the metal surfaces is released with an initial burst and diffuses away from the implantation site too quickly. Therefore, a more efficient growth factor delivery strategy is needed to promote better orthopedic implant healing.
It is expected that titanium implants containing and continuously releasing BMP-2 will enhance bone formation at the implant-bone interface and promote faster bone regeneration at the injured site. We proposed this novel drug delivery system in which we utilize a porous metal scaffold as the drug carrier, coat the inner pore walls of scaffold with growth-factors, and compact the scaffold, so that the growth factors get trapped within the interconnected pores of the scaffold. When the scaffold is implanted in the body, the growth factors will be released from the surface initially, and the ones in the inner pores will be released slowly over time.
In this study, we fabricated BMP-2-embedded Ti implants using a novel technique and demonstrated effective embedment and long-term release of growth factors in metal scaffolds. For loading the growth factor, porous Ti specimens were soaked in BMP-2 solution in vacuum, and then air-dried. After drying, the porous Ti discs coated with BMP-2 were pressed uniaxially. We report the results of the first in vitro BMP-2 release studies for a prolonged period as well as those of an accompanying in vivo study. Fabricated titanium implant released growth factors for extended periods of time of up to 5 months. The BMP released from Ti specimens that had been immersed in a PBS solution for several months remained biologically active. In comparison with the conventional growth factor release systems, this approach surmounts the limitations associated with rapid denaturalization and diffusion of growth factors, which potentially reduces the required growth factor dose.
 Bae SE et al. The Journal of cell biology 1991;113:681-7.
 Jun S-H et al. Journal of Materials Science: Materials in Medicine 2013:1-10.
 Kitajima T et al. Journal of Controlled Release 2012;160:676-84.
10:00 AM - *W1.03
Exploring and Engineering the Cell Material Interface for Regenerative Medicine
Molly M Stevens 1
1Imperial College London London United KingdomShow Abstract
This talk will give an overview of our research into the development of new materials and materials-based characterisation approaches for regenerative medicine [1-4]. The ability to control topography and chemistry at the nanoscale offers exciting possibilities for stimulating growth of new tissue through the development of scaffolds that mimic the nanostructure of the tissues in the body and advances here will be presented. By applying state of the art materials-based approaches we can better engineer the cell-material interface and can also elucidate disease processes within tissues. Recent examples from our group utilising state of the art analysis to investigate the structure of engineered tissue will be presented.
 E. T. Pashuck, M. M. Stevens "Designing Regenerative Biomaterial Therapies for the Clinic."
Science Translational Medicine.2013. 4 (160) 160sr4.
 S. Bertazzo, E. Gentleman, K. Cloyd, A. Chester, M. Yacoub, M.M.Stevens
“Nano-analytical electron microscopy reveals fundamental insights into human cardiovascular tissue calcification.”
Nature Materials. 2013. 12(6):576-83.
 E. Gentleman, R. Swain, N. Evans , S. Boorungsiman , G. Jell , M.Ball , T. Shean , M. Oyen , A. Porter, M. M. Stevens "Comparative materials differences revealed in engineered bone as a function of cell-specific differentiation."
Nature Materials. 2009. 8(9): 763-770.
 M. D. Mager, V. LaPointe, M. M. Stevens “Exploring and exploiting chemistry at the cell surface.”
Nature Chemistry. 2011. 3(8): 582-589.
10:30 AM - W1.04
Modifying Minimalist Self-Assembled Peptide Systems for Regenerative Medicine Applications
Rui Lui 1 Alexandra Rodriguez 2 Colin Barrow 1 David Russell Nisbet 2 Richard James Williams 1
1Deakin University Geelong Australia2Australian National University Canberra AustraliaShow Abstract
Producing functional biomaterial scaffolds for Tissue engineering applications involves the design and fabrication of three-dimensional (3D) environments that are reminiscent of the native extracellular matrix (ECM): a morphologically, mechanically and chemically rich environment whose biological function is to support and provide a dialogue between the structure and the attendant cells, controlling their function and behaviour (1). Nanofibrous materials yielded by the self-assembly of peptides are rich in potential; particularly for the formation of scaffolds that mimic the landscape of the host environment of the cell. Such scaffolds should be endowed with the capacity to promote endogenous or exogenous cell survival and integration within an injury site for tissue repair. Here, we report on a range of novel approaches for the formation of supramolecular structures presenting desirable amino acid sequences and biological macromolecules via the co-assembly of multicomponent systems.In the first example, we demonstrate an enzyme mediated scaffold formed from short peptides and ECM components (2). In the second approach we present a novel methodology to include bioactive epitopes in rationally designed minimalist peptides that cannot otherwise yield the desired scaffold structures under biologically relevant conditions (3). Through the combination of these approaches, we show that we can rationally design self-assembled structures for the facile fabrication of biochemical and mechanical environments which can directly influence cell behavior.
1)Nisbet, D.R., and Williams, R.J.(2012) Self-assembled peptides: Characterisation and in vivo response, Biointerphases. 7, 1-4.
2) Williams, R.J. et al (2011) The in vivo performance of an enzyme-assisted self-assembled peptide/protein hydrogel, Biomaterials. 32, 22, 5304-5310
3)Rodriguez, A., Parish, C.L, Nisbet, D.R., and Williams, R.J. (2013) Tuning the amino acid sequence of minimalist peptides to present biological signals via charge neutralised self assembly. Soft Matter. 9, 3915-3919
11:15 AM - W1.05
Scaffolds in Tissue Engineering: Some Selected Maxillofacial Applications
Erhan Piskin 1
1Hacettepe Univeristy Ankara TurkeyShow Abstract
Tissue engineering is one of the recent theraputic approaches for both soft and hard tissue repair. Healthy cells taken from the host own tissues or from other sources are used together with scaffolds. Target specific (eg., osteoblasts, chodrocytes, etc.) or preferentially stem cells are isolated, differentiated (in the case of stem cells), or even genetically modified (for instance to express growth factors, eg., BMPs) and are used in two diffetrent approaches. In the first approach they are just loaded into the scaffolds (as seeds) and apply as biohybrid implants. Alternatively, cells are propagated within the pores of scaffolds within bioreactors (in vitro) to form tissue-like structures and then they are implanted for tissue replacement. Scaffolds have large and interconnected pores which allows 3D-cell ingrowth are used. They have to be degradable in vivo, means that they should degrade such a rate that the new forming tissues to replace them properly. Of course both they and their degradation products must be biocompatible. They are produced several techniques, such as moulding/salt extraction, electrospinning, cryogelation, etc. They are made of several natural polymers (e.g., collagen and its denaturated form gelatin) and synthetic polymers (e.g., lactides, glycolide and ε-caprolactone). Several bioactive agents (e.g., growth factors, etc.) may be also incorporated (usually as controlled release formulations) to trigger the regeneration rate and proper new tissue formation. After careful in vitro biocompatibility test, tissue engineering scaffolds (loaded with cells) or biohybrid implants are applied in vivo in proper animal models. Critical size defects (means that the defects do not recover by themselves) are created in animals. In the maxillofacial applications, cranium, cleft palate, zgyoma, mandibula, etc. models have been used for bone tissue engineering. Ear defects are created to study cartilage repair. Several macro-, histological, molecular techniques are used to investigate tissue regeneration. This talk briefly reviews the topics mentioned above by using the experience of the author&’s group in this field.
11:30 AM - *W1.06
Understanding the Foreign Body Reaction in Tissue Engineering
Stephanie J Bryant 1 Mark D Swartzlander 1 Luke D Amer 1 Anna K Blakney 4 Themis R Kyriakides 2 Kurt D Hankenson 3
1University of Colorado Boulder USA2Yale University New Haven USA3University of Pennsylvania Philadelphia USA4University of Washington Seattle USAShow Abstract
Synthetic hydrogels are promising in situ cell carriers for tissue engineering due to their ease of formation and tunablity. However, nearly all implanted non-biological materials elicit a foreign body reaction (FBR), characterized by an acute inflammatory reaction followed by the formation of a dense, avascular fibrous capsule surrounding the implant. With the promise of synthetic-based hydrogels for in vivo applications, questions arise as to the role the FBR plays in tissue engineering, in particular when cells are present inside a hydrogel. Our group has investigated the FBR to poly(ethylene glycol) (PEG)-based hydrogels and demonstrated that although PEG is considered bioinert and largely non-fouling, a FBR ensues evidenced by the presence of macrophages at the hydrogel-host interface and the formation of a fibrous capsule. We have further shown that creating a softer and more biomimetic PEG hydrogel (e.g., YRGDS incorporation) attenuates the severity of macrophage activation and the FBR, although does not abrogate it. Our recent work has focused on understanding (a) the mechanisms by which macrophages sense and respond to PEG-based hydrogels and (b) how macrophages and the FBR affect and are affected by encapsulated cells. Towards addressing the former, we have investigated the FBR to PEG hydrogels and PEG hydrogels with YRGDS or YRDGS. Our results from in vivo studies indicate that proteins adsorb loosely to all three hydrogels with similar signatures. The fibrous capsule thickness, however, was significantly lower for YRGDS, when compared to YRDGS (p<0.01) and PEG (p=0.057), suggesting that macrophages recognize the underlying peptide and that integrin-mediated events in part modulate the FBR. Towards addressing the latter, we have investigated the FBR to cell-laden hydrogels when the encapsulated cells are dermal fibroblasts, mesenchymal stem cells (MSCs) or osteogenically differentiating MSCs, all derived from C57BL/6 mice, and implanted subcutaneously into C57BL/6 mice. Our findings demonstrate that MSCs are able to attenuate the FBR, while differentiating MSCs have a reduced ability, and differentiated fibroblasts led to a more severe FBR. The latter is attributed to a negative affect of the macrophages on the encapsulated fibroblasts, which in turn contributed to a more severe FBR. Taken together, our findings suggest that the chemical and mechanical nature of the hydrogel dictates the severity of the FBR and when cells are encapsulated in the hydrogels they are affected and can affect the severity of the FBR. Interestingly, MSCs are able to reduce significantly the FBR and our preliminary studies point towards PGE2 as a key immunomodulatory molecule secreted by MSCs. In conclusion, our studies indicate that the FBR will indeed play an important role in tissue engineering.
12:00 PM - W1.07
Graphene-Hydroxyapatite Biocompatible Nnanomateriales
Hector Eduardo Cid 1 Claramaria Rodriguez Gonzalez 1 Pedro Salas 1 Luz M. Lopez 1 Victor M. Castano 1
1Universidad Nacional Autonoma de Mexico Santiago de Queretaro MexicoShow Abstract
Tissue engineering is a branch of science that deals with the design and synthesis of biomaterials with properties such that promote tissue regeneration by cell proliferation and differentiation. Biocompatible polymers have been widely investigated for the development of biomaterials due to their low cytotoxicity, together with its ability to form polymeric scaffolds with morphology suitable for mechanical support of the tissue. Also, various techniques have been developed which allow functionalization of polymeric scaffolds in order to improve such mechanical and biological properties, thus yielding, high quality hybrid materials with application in the area of tissue engineering. In this study, graphene oxide sheets (GrO) were functionalized with hydroxyapatite nanoparticles (nHAp) through a simple and effective hydrothermal treatment and a new physicochemical process. Microstructure and crystallinity of the new hybrid nanomaterial GrO/nHap were investigated by Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray diffraction (XRD) and thermo-gravimetric analysis (TGA). Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were performed to characterize the morphology of the functionalized material. To analyze biological properties, the composite material was functionalized using three different GrO:nHap ratios. In order to evaluate the cytotoxicity and cell proliferation the obtained materials were subjected to a MTT assay using the NIH-3T3 cell line. Polymeric scaffolds based in chitosan-polyvinyl alcohol (PVA-Ch) co-polymer were fabricated, integrating the hybrid material GrO/nHap at different concentrations (1-5 wt %), using a physical technique. The morphological characteristics of the resulting biomaterials were observed by SEM. The technique parameters were modified to increase the surface area and pore size of the biopolymer scaffolds. Confocal electron microscopy and SEM were performed to observe cell adhesion on the composite. Cell viability of the polymeric scaffolds reinforced with the hybrid materials was measured by MTT assay, using the same cell line described before. The mechanical and physical properties will be characterized using thermal analysis (TGA and DSC) and mechanical tester. The resulting novel materials combine the biocompatibility of the nHap with the strength and physical properties of the graphene improving the properties of the polymer matrices, thereby obtaining a biomaterial with potential applications in tissue engineering.
12:15 PM - W1.08
Nanoscale Piezoelectricity in Fmoc-Diphenylalanine Hydrogels and Their Potential for Application as Multi-Functional Scaffolds
Kate P Ryan 1 2 Jason Beirne 3 Gareth Redmond 3 Andrei Kholkin 4 Brian J Rodriguez 1 2
1University College Dublin Dublin 4 Ireland2University College Dublin Dublin 4 Ireland3University College Dublin Dublin 4 Ireland4University of Aveiro Averio PortugalShow Abstract
Fluorenylmethyloxycarbonyl diphenylalanine (Fmoc-FF) hydrogels have biocompatibility and viscoelasticity comparable to extracellular matrices and commonly used biopolymers. As such, they are proposed as a promising new material for regenerative medicine applications. Fmoc-FF hydrogels self-assemble through molecular stacking of molecules to form three-dimensional networks of ordered fibril structures, ideal for use as tissue engineering scaffolds or biomedical device coatings. Additionally, the self-assembly process is a simple, cost effective method for manufacturing these nanomaterials on a large scale.
The existence of piezoelectric properties could facilitate the further application of Fmoc-FF fibrous networks to applications where electrical or mechanical stimuli can be used to promote tissue regeneration. For example, bone and nerve regeneration have both been identified as being sensitive to piezoelectric properties. The direct piezoelectric effect has been linked with the ability of bone to remodel in response to an applied stress. Piezoelectricity has also been shown to promote in-vitro axonal regeneration following nerve injury.
Here, we report local shear piezoelectricity in self-assembled peptide hydrogels composed of Fmoc-FF nanofibrils, measured by piezoresponse force microscopy (~1-2 pm/V - comparable to collagen ~1-2 pm/V). The nanofibrillar nature of the gel is further confirmed by scanning electron microscopy, transmission electron microscopy, and helium ion microscopy. Also, comparisons of fluorescence emission spectra measured for Fmoc-FF in solvent and in the gel phase suggest that pi-stacking interactions between Fmoc moieties facilitate nanofibril formation. Structural analyses (circular dichroism and attenuated total reflectance-Fourier transform infrared spectroscopy) confirm the Fmoc-FF molecules within the fibrous network are predominantly in a β-sheet conformation, similar to the dominant structure observed in diphenylalanine nanotubes. Therefore, the non-centrosymmetric nature of the β-sheet is likely to be responsible for the observed piezoelectricity in the Fmoc-FF hydrogels as well.
Gulden Camci-Unal, Harvard Medical School
Brendan Harley, University of Illinois, Urbana-Champaign
Eben Alsberg, Case Western Reserve University
Kazunori Kataoka, The University of Tokyo
Symposium Support Aldrich Materials Science
Society for Biomaterials
W5: Biomaterials for Regenerative Therapies
Wednesday PM, April 23, 2014
Moscone West, Level 2, Room 2003
2:30 AM - W5.01
Synthetic Hydrogel Scaffolds with Vascular Network for Tissue Engineering
Alessandro Tocchio 1 2 Margherita Tamplenizza 1 Federico Martello 1 Irini Gerges 1 Eleonora Rossi 1 2 Paolo Milani 1 3 Cristina Lenardi 1 3
1Fondazione Filarete Milano Italy2European School of Molecular Medicine, Campus IFOM-IEO Milano Italy3Universitamp;#224; degli Studi di Milano Milano ItalyShow Abstract
One of the major limitations in tissue engineering is the lack of proper vascularization . Nowadays skin and cartilage grafts are successfully used in-vivo mainly thanks to their low requirement for nutrients and oxygen that can be met by the host vascularization. However this approach fails when applied to complex and massive tissues. The formation of new blood vessels is indeed a slow phenomenon and the deficiency of oxygen and nutrients supply rapidly cause widespread cell death in the graft core. With the aim to overcome this hindrance we developed an innovative technique based on sacrificial elements together with a library of novel synthetic hydrogels . In this approach fluidics channels are deeply embedded within the hydrogel porous scaffold in order to favor biomimetic synthetic vasculature generation.
This approach was demonstrated to be suitable for the creation of densely populated vital tissue constructs with perfusable branching endothelialized channels. In vitro perfusion was efficiently performed using a customized bioreactor, specifically developed for the perfusion of soft hydrogel scaffolds. One key advantage of this technology is that the perfusable scaffold is formed in a one step process simply by filling the empty volume around the template, crosslinking the matrix and then dissolving the template. This rapid method prevents the formation of a necrotic core in thick cellular construct, indicating that this technology could provide a solution to scale current engineered tissue to clinically relevant dimensions.
This technique allows the development of scaffolds with vascular network for treatment of big defects in tissue regeneration based on innovative and complementary technologies which combine material properties, network architecture, rapid prototyping, 3-D stem cells culture and scaffold bio-functionalization.
 A. Khademhosseini, J.P. Vacanti, R. Langer, Progress in tissue engineering, Sci Am. 2009, 300(5), 64-71
 F. Martello, A. Tocchio, M. Tamplenizza, I. Gerges, V. Pistis, R. Recenti, M. Bortolin, M. Del Fabbro, S. Argentiere, P. Milani, C. Lenardi, Poly(amidoamine)-based Hydrogels with Tailored Mechanical Properties and Degradation Rates for Tissue Engineering, Acta Biomaterialia, in press.
2:45 AM - W5.02
An Injectable Piezoelectric Drug Delivery System to Target Tissue Regeneration in Tissue Engineering Applications
Daniela P. Pacheco 1 2 Rui L. Reis 1 2 Vitor M. Correlo 1 2 Alexandra P. Marques 1 2
13B's Research GroupGroup - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine AvePark, 4806-909 Caldas das Taipas, Guimaramp;#227;es Portugal2ICVS/3Bamp;#8217;s - PT Government Associate Laboratory Braga/Guimaramp;#227;es PortugalShow Abstract
Improved design of efficient drug delivery systems (DDS) capable of responding to biological stimuli within an extended time window are a constant pursue in the field of tissue engineering and regenerative medicine (TERM). Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a natural and biodegradable polymer with piezoelectric properties, i.e. capable of suffering electric polarization due to mechanical stress, and vice-versa. Naturally occurring electric currents are an intrinsic property of human skin tissues, likely to act as an integrator of cells organization, development as well tissue regeneration. Under this context, this work reports an injectable piezoelectric DDS system incorporating hydrophilic and hydrophobic bioactive molecules aiming at modulating defined biological functions and tackle tissue regeneration.
Microparticles of PHBV incorporating a model protein, Bovine Serum Albumin (BSA), and glucocorticoid, Dexamethasone (Dex), were produced by a double emulsification-solvent evaporation method with modifications. Variations of the composition of the organic phase during processing allowed tuning surface topography, size distribution and core porosity of the PHBV microparticles. Likewise, the entrapment efficiency of Dex, but not BSA was modulated by varying the processing method. However, the in vitro release profile studies confirmed an initial burst effect which was followed by a sustained pattern, typical of a first order release kinetics independently of the conditions and the incorporated molecules.
An innovative approach that tackle the reduced residence time of microparticles at the injection site, and takes advantage of its piezoelectric character to release the loaded bioactive molecules was designed. Injectable formulations of Gellan Gum hydrogel, already proposed by us for diverse tissue engineering applications, were considered as carriers. Combined and well integrated systems of PHBV microparticles within GG hydrogel, responsive to electrical stimulation, were successfully achieved. By varying the properties of the hydrogel and the intensity of the provided signal, we were able to design systems with different release profiles, which can then be tuned according to tissue and pathology/injury specific requirements.
In this sense, the development of an injectable piezoelectric drug delivery system, which guarantees a localized delivery and allows the release of biochemical cues with different physicochemical features, as well as its localized deliver, was achieved representing a versatile tool to prepare instructive cell microenvironments towards tissue regeneration in TERM applications.
3:00 AM - *W5.03
Injectable Regenerative Biomaterials for Treating Cardiovascular Disease
Karen L. Christman 1
1UC San Diego La Jolla USAShow Abstract
Cardiovascular disease remains the leading cause of death in the western world. Two major types of cardiovascular disease, myocardial infarction and peripheral artery disease, have few available treatments and therefore numerous patients continue to decline towards heart failure for the former and amputation for the latter. Current clinical trials have focused on cell therapies. It is however largely acknowledged that these cells act via paracrine mechanisms to recruit endogenous cells to help repair and regenerate the tissue. In animal models, it has been established that cellular recruitment to the damaged tissue can also occur via implantation of biomaterial scaffolds. In particular, injectable hydrogels derived from decellularized extracellular matrix (ECM) can provide tissue specific cues to promote endogenous regeneration. Rather than surgical implantation, these materials can be delivered minimally invasively, including via catheter-based injection into the heart. Hydrogels derived from porcine ventricular ECM and skeletal muscle ECM have shown significant promise for treating myocardial infarction and peripheral artery disease, respectively. Recent developments and translational progress with these materials will be discussed.
4:00 AM - *W5.04
Fibrin Gels as Cell-Instructive Substrates for Regenerative Medicine
Kent Leach 1 2
1Univ. of California, Davis Davis USA2UC Davis School of Medicine Sacramento USAShow Abstract
Fibrin is a provisional matrix during tissue repair and is an attractive biomaterial for cell delivery. Fibrin hydrogel properties can be tuned using a variety of techniques including altering protein concentrations, pH, calcium content, and overall ionic strength. As an alternative to increasing the concentration of clotting proteins, we demonstrated that tailoring the NaCl content in the pre-gel solution alters gel stiffness, pore size, fiber diameter, and permeability. The supplementation of fibrin gels with increasing NaCl concentrations slowed gelation time from 4 minutes to 10 minutes, retaining gelation time within in a clinically acceptable period without risking osmotic shock. We hypothesized that fibrin gels with increased bulk stiffness would provide substrate-mediated cues to entrapped cells, and these cues would instruct cell function. We examined this hypothesis in two distinct applications: neurogenesis and osteogenesis.
Neurite extension from dorsal root ganglia (DRGs) entrapped in fibrin gels correlated with substrate stiffness. We observed significant decreases in neurite length as NaCl content increased from 0.8% (w/v) to 3.5% (w/v). Furthermore, we observed significant reductions in degradation area surrounding the entrapped DRG with increasing salt concentration. We determined that neurite extension within fibrin gels is dependent on fibrinolysis and is mediated by the secretion of serine proteases and MMPs by entrapped DRGs, as confirmed by culturing cells in the presence of inhibitors against these enzymes and real-time-polymerase chain reaction.
We also explored the contribution of fibrin gels with varying NaCl content toward the dual potential of mesenchymal stromal cells (MSC) in bone repair: osteogenic differentiation and trophic factor secretion. Gel volume was better preserved in fibrin hydrogels containing greater NaCl concentrations (which also exhibited higher compressive moduli), while human MSC entrapped in fibrin gels with lower NaCl content rapidly contracted the material. Early MSC proliferation was markedly accelerated (over 200% increase in DNA) in gels containing less NaCl compared to gels with the highest NaCl content. Alkaline phosphatase activity and calcium deposition, early and late indicators of osteogenic differentiation, respectively, also were highest in fibrin gels with more NaCl, yet secretion of vascular endothelial growth factor (VEGF) was lowest. Fibrin gel osteoconductivity was dramatically improved by adding polymeric substrata coated with bone-like mineral, exhibiting near linear increases in both calcium and phosphate entrapment over 21 days.
Collectively, these data demonstrate that the biophysical properties of fibrin gels can be tailored by the simple addition of NaCl. Salt-mediated control of fibrin gel properties is sufficient to significantly direct cell function as it relates to tissue engineering and regenerative medicine.
4:30 AM - *W5.05
Bioactive Hydrogels and Nanomaterials for Vascular Regeneration
Hyunjoon Kong 1
1University of Illinois at Urbana-Champaign Urbana USAShow Abstract
Human bodies are highly vascularized to transport gaseous molecules, nutrients, and cell metabolites to and from cells residing in tissues and organs. As such, cardiovascular disease resulting from vascular occlusion, leakage, and rupture is the world&’s leading cause of death. Extensive efforts were made to treat these cardiovascular diseases by recreating vascular networks sing regenerative medicine including growth factors and cells. These medicines are often integrated with biomaterial systems that can enhance their therapeutic efficacy. To contribute these efforts, we have been developing various implantable devices that can elevate the quality of vascular regeneration. In this talk, I will introduce a few advanced biomaterial systems including (1) a hydrogel-based “Living” microvascular stamp that can control spatial organization of blood vessels during regeneration and (2) a self-folding hydrogel that can modulate vascular drug release with its shape change. I will also introduce nanomaterials used to engineer stem cell surface for cell delivery ischemic tissue. These material systems will greatly serve to take quality of revascularization therapies to the next level.
5:00 AM - W5.06
Skin Printer: Microfluidic Approach for Skin Regeneration and Wound Dressings
Lian Leng 1 Phoenix Qing Ba 1 Saeid Amini-Nik 1 2 Axel Guenther 1 Marc Jeschke 1 2
1University of Toronto Toronto Canada2Sunnybrook Health Sciences Centre Toronto CanadaShow Abstract
Skin is an important organ that forms a protective barrier against the external environment. Once broken, the process of wound healing is immediately set in motion. However, in conditions associated with severe skin loss, normal wound healing cannot reconstitute the barrier, leading to high mortality. To mitigate this, different wound dressings are routinely employed in surgical practice. However (a)more cost-effective dressings that offer shorter recovery times and (b)dressings that better resemble important physiological features of skin with minimal morbidities are needed.
A number of microfluidic approaches have been suggested for drug screening in microscale organ-mimetic platforms, as well as for the continuous formation, assembly and in vivo application of cell populated microfibers. Function of skin cells and wound-healing behavior have been studied using microfluidic platforms, and bioprinting efforts are beginning to make their way towards the repair of burn wounds and cartilages.
Here, we present to our knowledge the first approach for the in-flow formation and in vivo application of cell-populated wound dressings that accurately reproduce key features of human skin using biomaterials. The skin printer consists of a microfabricated cartridge that enables the continuous formation of wound dressings from mosaic hydrogel sheets, with the controlled incorporation of viable human fibroblasts, and the ability to define multilayered and vascularized(i.e.perfusable) hydrogel sheets.
In order to print cell-populated sheets with materials promoting both cell viability and proliferation while easy to handle, two materials are used:(1)providing stiffness to the sheet, (2)for cell printing. Precise control over the combination of these two materials is critical, as the printed patterns size and pitch will directly impact on the sheet stiffness. Different patterns were produced by varying the valve actuation time and inlet gas pressure. Such pattern was repeated on a bilayer system to illustrate the ability to create a structure that would enable the co-localization of keratinocytes and fibroblasts. These patterns can either be hollow to promote vascularization while providing relevant thicknesses, or cell-populated with local control over cell seeding density. As a case study, we printed biopolymer sheets seeded with human fibroblasts and showed their proliferation and attachment in vitro.
By performing excisional skin biopsy on immunodeficient mice, we replaced excised skin with these biopolymer sheets. Preliminary data suggests that our printed biopolymer sheets led to an improved skin regeneration as compared to our control.
We believe the skin printer provides a scalable approach(up to 35mm wide sheets were produced at rates of up to 10mm/s) and uniquely addresses the need to provide skin substitutes at affordable prices that are easy to handle and apply, while potentially reducing wound recovery times along with improving clinical outcomes.
5:15 AM - W5.07
Evaluation of Nanofiber-Permeated Scaffolds for Bone Repair in a Transgenic Mouse Model
Clarke Nelson 1 Yusuf Khan 1 2 3 David W Rowe 4 Cato T Laurencin 1 2 3
1University of Connecticut Health Center Farmington USA2University of Connecticut Health Center Farmington USA3University of Connecticut Storrs USA4University of Connecticut Health Center Farmington USAShow Abstract
Statement of Purpose: Sintered composite microsphere matrices have shown potential as autograft replacements, but cellular migration is often limited to the periphery in static culture. Fibrous networks of the physical scale of collagen ECM in bone may increase cellular retention and migration leading to improved bone repair. While a fibrous structure alone would have limited clinical utility due to no load-bearing potential, we propose to increase cell migration throughout a mechanically stable microsphere matrix using a secondary, nanofibrous phase within its pore structure. We hypothesize that a nanofiber mesh within the pore structure may increase cell migration, residence, and differentiation throughout the scaffold.
Methods: Sintered, composite microsphere matrices were fabricated according to reported procedure, submerged in three separate concentrations (0.25%, 1%, and 2%) of PLLA in DMF, and cooled to allow thermally induced phase separation to occur. For in vitro studies, bone marrow stromal cells were harvested from bone restricted GFP reporter mice, seeded on three different nanofiber-infused scaffolds, and assayed for DNA content and alkaline phosphatase (ALP) activity at 1, 3, 7, 14, and 21 days. Scaffolds were then implanted for 6 weeks in calvarial defects of transgenic mice carrying the same GFP reporter and the animals received a single injection of alizarin complexone 1