Syam P. Nukavarapu, University of Connecticut
Huinan Liu, University of California, Riverside
Rui L. Reis, University of Minho
Arthur J. Coury, Coury Consulting Services
Symposium Support Aldrich Materials Science
Royal Society of Chemistry
Society for Biomaterials
Teleflex Medical OEM
University of Connecticut - Institute for Regenerative Engineering
University of Connecticut - Sackler Center for Biomedical, Biological, Engineering and Physical Sciences
H2: Advanced Composites and Structures: Micro/Nano/Pico-Technology and Applications
Syam P. Nukavarapu
Monday PM, December 02, 2013
Sheraton, 2nd Floor, Back Bay D
2:30 AM - *H2.01
From Nanotechnology to Picotechnology: What is on the Horizon?
Thomas Webster 1
1Northeastern University Boston USAShow Abstract
Inspired from biological systems, nanotechnology is beginning to revolutionize (and in many cases already has) revolutionized medicine including improved prevention, diagnosis, and treatment of numerous diseases. This talk will summarize efforts over the past decade that have synthesized novel nanoparticles, nanotubes, and other nanomaterials to improve medicine. Efforts focused on the use of nanomaterials to minimize immune cell interactions, inhibit infection, and increase tissue growth will be especially emphasized. Tissue systems covered will include the nervous system, orthopedics, bladder, cardiovascular, vascular, and the bladder. Due to complications translating in vitro to in vivo results, only in vivo studies will be emphasized here. Materials to be covered will include ceramics, metals, polymers, and composites thereof. Self-assembled nano-chemistries will also be emphasized. As the FDA has now approved several nanomaterials for medical applications, recent results from FDA trials will also be discussed. Importantly, this talk will also discuss what further advances we can make in medicine by using picotechnology compared to nanotechnology. In summary, this talk will provide the latest information concerning the design and use of numerous nanomaterials in regenerative medicine while highlighting what is necessary for this field to continue to grow through the exploration of picotechnology.
3:00 AM - H2.02
Functional Inorganic Nanoparticles for Stem Cell Tracking and Ischemic Stroke Treatment
Taeho Kim 1 2 Taeghwan Hyeon 1 2
1Center for Nanoparticle Research, Institute for Basic Science (IBS) Seoul Republic of Korea2School of Chemical and Biological Engineering, Seoul National University Seoul Republic of KoreaShow Abstract
During the last decade, various functional nanostructured materials with interesting optical, magnetic, mechanical, and chemical properties have been extensively applied to biomedical areas including imaging, diagnosis, and therapy. In particular, interdisciplinary collaborative research between material science and biomedicine enabled nanomaterials to translate the medical issues and application to clinical trials. Cellular therapies by the administration of therapeutic cells such as stem cells or immune cells benefit greatly from the inclusion of nanomaterials to achieve high-resolution tracking of the cells. Another application of nanomaterials is as intrinsic chemotherapeutic agents. Long-term cell tracking can be realized by producing highly sensitive nanoparticles or by the control of particle-cell interactions. Engineering physical and chemical properties of nanoparticles - such as size, surface, and composition, enables to obtain optimized therapeutic potentials at target tissues with minimal toxicity. Mesoporous silica-coated hollow manganese oxide (HMnO@mSiO2) nanoparticles were fabricated for highly efficient T1 magnetic resonance imaging (MRI) contrast agent for labeling and MRI tracking of stem cells . Computed tomograpy (CT) cell tracking methods with gold nanoparticles were developed . The protective effects of ceria nanoparticles against ischemic stroke were studied, and demonstrated that intravenously administered ceria nanoparticles considerably reduced the brain infarct volume and nerve damage .
 T. Kim,dagger; T. Hyeon,* and Assaf A. Gilad,* "Mesoporous Silica-Coated Hollow Manganese Oxide Nanoparticles as Positive T1 Contrast Agents for Labeling and MRI Tracking of Adipose-Derived Mesenchymal Stem Cells" J. Am. Chem. Soc. 2011, 133, 2955-2961.
 T. Kim,dagger; T. Hyeon,* and Jeff W. M. Bulte,* “Micro-CT Imaging of Human Mesenchymal Stem Cells Labeled with Gold Nanoparticles” Manuscript in Preparations.
 C. K. Kim,dagger; T. Kim,dagger; S.-H. Lee,* and T. Hyeon,* "Ceria Nanoparticles that can Protect against Ischemic Stroke" Angew. Chem. Int. Ed. 2012, 51, 11039-11043.
3:15 AM - H2.03
Identifying Iron Oxide Based Materials that Can Either Pass or Not Pass through the Blood-Brain Barrier
Di Shi 1 Linlin Sun 2 Gujie Mi 1 Soumya Bhattacharya 3 Suprabha Nayar 3 Thomas J Webster 4
1Northeastern University Boston USA2Northeastern University Boston USA3CSIR-National Metallurgical Laboratory Burmamines India4Northeastern University Boston USAShow Abstract
Abstract: An in vitro blood-brain barrier (BBB) model was developed using murine brain endothelioma cells (b.End3 cells) and confirmed. Confirmation of the BBB model was completed by examining the permeability of FITC-Dextran at increasing exposure times in serum-free medium and comparing such values with values in the literature. After such confirmation, the permeability of five nanoparticles was determined by this model. Through such experiments, magnetic nanoparticles suitable for MRI use which would not pass through the BBB and magnetic nanoparticles suitable for drug delivery which would pass through the BBB to the were identified.
Materials and Methods: According to a patented process entitled “A biomimetic process for the synthesis of aqueous ferrofluids for biomedical applications”, five ferrofluids were synthesized by incubating a ferrous/ferric salt solution in phosphate-buffered saline supplemented with the additives of interest such as collagen, poly(vinyl) alcohol (PVA) and/or bovine serum albumin (BSA) using ammonium hydroxide under highly alkaline conditions. After synthesis, ferrofluids were centrifuged for a stability test so that the supernatant byproducts could be washed away. Dynamic light scattering and TEM were used to characterize their diameter and zeta potential was used to characterize the charge of those superparamagnetic iron oxide nanoparticles (SPIONs). An in vitro blood-brain barrier model based on b.End 3 cells was then used to test the permeability of the various nanoparticles which are GGB (ferrofluid synthesized using glycine, glutamic acid and BSA), GGC (glycine, glutamic acid and collagen), GGP (glycine, glutamic acid and PVA), BPC (BSA, PEG and collagen) and CPB (collagen, PVA and BSA). For this, nanoparticles were diluted 1:19 with HBSS and then inserts were exposed to them for 2 hours. After 2 hours, a 100 mu;L solution was taken from each well and full spectrum absorbance was used to determine the iron concentration that pass through the model. Each experiment was conducted in triplicate and repeated at least three times.
Results and Discussion: Results showed that the highest permeability was obtained from CPB and the lowest permeability was obtained from GGB. Also ferrofluids synthesized using collagen generally had higher permeability than those synthesized using glycine and glutamic acid. These results suggest that for nanoparticles that need to be delivered through BBB (i.e., for treating nuerological diseases), FF should be coated with collagen while, on the other hand, FF should be coated with glycine and glutamic acid to keep the nanoparticles from penetrating the BBB (i.e., for whole body MRI imaging to decrease brain toxicity).
Conclusions: An in vitro model of BBB was established using b.End3 cells. Results showed that in order to be delivered through BBB, ferrofluids should be coated with collagen and ferrofluids should be coated with glycine and glutamic acid to avoid penetration.
3:30 AM - H2.04
Efficacy of Nanoparticle Drug Delivery System in an Embedded Spheroid Tumor Model
Kristie Mercedes Charoen 1 Brian Fallica 1 Muhammad Zaman 1 Mark Grinstaff 1 2
1Boston University Boston USA2Boston University Boston USAShow Abstract
Despite the wealth of research focused on cancer, the testing ground for new drug delivery methods and novel drug candidates is a standard two-dimensional monolayer of cells. However, this method lacks the three-dimensional nature of a tumor, which has been shown to fundamentally alter gene expression. Furthermore, lack of diffusion within a tumor structure fosters necrosis and quiescence in the core. The three-dimensionality coupled with the tumor structure affords an increased resistance to chemotherapeutics. This project establishes a physiologically relevant in vitro model of a tumor that consists of a multicellular spheroid surrounded by a collagen matrix. Theo model is then used for subsequent testing with an expansile nanoparticle drug delivery system. Spheroids were made with a human breast cancer cell line (MDA-MB-231) and incorporated within a matrix to recapitulate stromal elements, whose interactions with tumors are fundamentally involved in the progression of cell phenotype from normal to malignant. Within in vitro models of cancer, both acellular and cellular stromal elements have afforded greater tolerance against chemotherapeutic agents. Cellular stromal elements are introduced via inclusion of fibroblast cells (murine fibroblast NIH3T3 cells) either as part of a spheroid or diffusely seeded within the collagen gel. In addition to the collagen matrix, Matrigel was included within the spheroid to mimic degradation of basement membrane associated with activated tumor stroma. Model plasticity allows for inclusion of any combination of these elements to analyze complex stroma/tumor interactions to evaluate nanoparticle drug delivery systems. Scanning electron and confocal microscopy indicate a well integrated tumor like structure with a necrotic core capable of ingrowth into the collagen matrix. Various spheroid constructs were treated against a polymeric expansile nanoparticle system releasing Paclitaxel (Pax-eNP) and Pax alone given as a bolus dose. Efficacy of a given treatment was judged by overall spheroid growth into the collagen. Nanoparticle and drug presence within the spheroid were measured by confocal microscopy and fluorescence activated cell sorting. Finally, cytotoxicity of the cells was quantified via the metabolic activity of disaggregated spheroids.
3:45 AM - H2.05
Magnesium Oxide Nanoparticles as Novel Materials for Tendon to Bone Insertion Applications
Daniel J. Hickey 1 Linlin Sun 2 Batur Ercan 1 Thomas J. Webster 1
1Northeastern University Boston USA2Northeastern University Boston USAShow Abstract
There are about 100,000 ACL reconstruction surgeries performed every year in the United States, with a failure rate ranging from 5-25%, depending on the criteria of the study . It is believed that this high rate of failure is a result of insufficient healing at the tendon-to-bone insertion site (TBI). The TBI disperses critical stress concentrations that arise naturally between ligaments and bone by providing a compositional and mechanical transition from ligaments, through a fibrocartilaginous zone, to bone. However, this complex, inhomogeneous, and avascular tissue is incapable of regenerating following surgery. Therefore, there is considerable interest in the development of a nanostructured biomaterial that is capable of directing healthy regeneration of spatially controlled tissue across the TBI.
In this study, magnesium oxide (MgO) nanoparticles were used to mineralize poly(l-lactic acid) (PLLA) and tested for their ability to improve the attachment and growth of TBI-related orthopedic tissue. Magnesium is an essential mineral in bone which is thought to regulate the size and density of hydroxyapatite (HA) crystals, and further, Weng and Webster demonstrated that nano-rough MgO increased bone cell density three-fold compared to bulk MgO . Presently, the ability of materials to promote tissue growth at the TBI was characterized via cell adhesion and proliferation experiments with fibroblasts and osteoblasts. Materials were also tested for their mechanical properties, and further characterization was performed using SEM, TEM, XRD, FTIR, EDS, and contact angle tests.
Results indicated for the first time that MgO nanoparticles in plain PLLA or PLLA/HA composites significantly increased osteoblast and fibroblast adhesion on PLLA. Interestingly, both cell lines followed the same general trend of adhesion on each sample, indicating that variations in only the secondary phase of a scaffold material will not be sufficient to direct the formation and maintenance of spatially controlled tissue heterogeneity at the TBI. However, nano-MgO can be used to mineralize different polymer phases to promote the formation of bone tissue at one end of the scaffold and fibrous tissue at the other end.
Mechanical tensile testing revealed that the addition of a secondary nano-phase to plain PLLA hardened the polymer, reducing the material elongation and increasing its elastic modulus. Moreover, the observed changes in mechanical strength of PLLA seemed to be dictated by the size and shape of its secondary nano-phase, indicating that the mechanical properties of PLLA composites can be tailored to align with the strength of bone or ligament tissue.
 Lui et al., Journal of Orthopaedic Surgery and Research, 5 (2010), 59.
 Weng L, Webster TJ, Nanotechnology. 23 (2012), 485105.
4:30 AM - *H2.06
Merging Micro/Nanoscale Technologies and Advanced Biomaterials for Engineering 3D Tissues
Ali Khademhosseini 1 2 3
1Harvard Medical School Cambridge USA2Massachusetts Institute of Technology Cambridge USA3Harvard University Cambridge USAShow Abstract
Tissue engineering is an interdisciplinary field, aimed at maintaining, restoring and enhancing normal tissue and organ functions by merging of engineering, biological sciences and medicine. To date, tissue engineering has been successfully applied to engineer many types of tissues including bone, cartilage and cardiovascular. One of the central themes in the field of tissue engineering is the development of proper biomaterials with tunable characteristics to mimic the body&’s architectural and geometrical intricacies and induce proper cell-cell and cell-matrix interactions. In the past few years, we have been actively involved in the development of innovative biomaterials for tissue engineering and stem cell bioengineering applications. We have demonstrated that that hydrogels are excellent scaffolding materials mainly due to their 3D microenvironment, homogenous cell distribution, and tunable mechanical and chemical properties. For instance, we have used ‘top-down&’ microfabrication techniques (i.e. micropatterning) to develop cell-laden gelatin methacrylate (GelMA) hydrogels with precise geometrical features for bone and cardiovascular tissue engineering applications. Recently, we demonstrated microfabrication of elastin-based hydrogels (methacrylated torpoelastin, MeTro) with remarkable properties for engineering of various types of tissues. We have shown that the fabricated MeTro hydrogels exhibit excellent mechanical characteristics such as high resilience upon stretching and reversible deformation with low energy loss. In addition, we have demonstrated that the mechanical properties of fabricated MeTro gels can be finely tuned to select desirable stiffness for a specific application (i.e. cardiovascular) depending on the methacrylation degree and concentration of the hydrogels. In this talk, I will present recent findings in our lab covering fabrication of novel biomaterials for engineering 3D tissue constructs.
5:00 AM - H2.07
Nanostructured Ceramic and Ceramic-Polymer Composites as Dual Functional Interface for Bioresorbable Metallic Implants
Huinan Liu 1 2 3
1University of California, Riverside Riverside USA2University of California, Riverside Riverside USA3University of California, Riverside Riverside USAShow Abstract
Millions of medical implants and devices (e.g., screws, plates, and pins) are used each year worldwide in surgery, and traditionally the components have been limited to permanent metals (e.g., stainless steel, titanium alloys) and polyester-based absorbable polymers. Because of clinical problems associated with these traditional materials, a novel class of biodegradable metallic materials, i.e., magnesium-based alloys, attracted great attention and clinical interests. Magnesium (Mg) is particularly attractive for load-bearing orthopedic applications because it has comparable modulus and strength to cortical bone. Controlling the interface of Mg with the biological environment, however, is the key challenge that currently limits this biodegradable metal for broad applications in medical devices and implants. This talk will particularly focus on how to create nanostructured interface between the biodegradable metallic implant and surrounding tissue for the dual purposes of (1) mediating the degradation of the metallic implants and (2) simultaneously enhancing bone tissue regeneration and integration. Nanophase hydroxyapatite (nHA) is an excellent candidate as a coating material due to its osteoconductivity that has been widely reported. Applying nHA coatings or nHA containing composite coatings on Mg alloys is therefore promising in addressing the challenges in commercializing biodegradable metallic implants. The composite of nHA and poly(lactic-co-glycolic acid) (PLGA) as a dual functional interface provides additional benefits for medical implant applications. Specifically, the polymer phase promotes interfacial adhesion between the nHA and Mg, and the degradation products of PLGA and Mg neutralize each other. Our results indicate that nHA and nHA/PLGA coatings slow down Mg degradation rate and enhance adhesion of bone marrow stromal cells, thus promising as the next-generation multifunctional implant materials. Further optimization of the coatings and their interfacial properties are still needed to bring them into clinical applications.
5:15 AM - H2.08
Chitosan/Collagen-Coated Gold Nanoparticles Scaffold
Fernanda Silva Tenorio 1 William Candiotto Lueders 1 Mateus dos Santos Silva 1 Ana Paula Lemes 1 Denise Arruda 2 Luiz Rodolpho Raja Gabaglia Travassos 2 Dayane Tada 1
1Federal University of Samp;#227;o Paulo Samp;#227;o Josamp;#233; dos Campos Brazil2Federal University of Samp;#227;o Paulo Samp;#227;o Paulo BrazilShow Abstract
For the last two decades, the advance of material sciences and biotechnology enabled the development of numerous types of scaffolds for regenerative medicine. These scaffolds aim at tissue regeneration involving minimally invasive surgical procedures.
In this work we report the development of injectable scaffold prepared with chitosan and collagen-coated gold nanoparticles (NPs). Chitosan is a hydrogel known for its porous structure, biocompatibility and biodegradation. However, its low mechanical resistance and the requirement of toxic crosslinkers in its preparation, like glutaraldehyde, have hampered its application in biomedical devices. The incorporation of gold NPs aims at improving cell proliferation through mechano-transduction events due to the nanotopography. Besides, gold NPs provide the in situ delivery of bioactive molecules, as for example, collagen type I, which is a bioactive molecule reported to stimulate chondrocytes growth. Finally, since gold is an antibacterial material, the presence of gold NPs may reduce the risk of infection normally caused by scaffold implants.
Gold NPs were prepared by reduction of gold (III) chloride hydrate using sodium citrate. Collagen was linked to gold NPs by EDC/NHS coupling reaction. NPs size distribution and zeta;-potential were measured by dynamic light scattering. The mean diameter was measured as 52.82nm and the zeta;-potential was of -7.6mV. After NPs coating the mean diameter and zeta;-potential were 300nm and -7,3mV, respectively. NPs were also characterized by UV-Vis spectrometry. The maximum of absorption was at 522nm for bare NPs, which is characteristic of gold NPs of 20nm of diameter. Absorbance spectrum of collagen-coated NPs presented two additional bands at 280 and 220nm. These bands are characteristic of proteins, proving that the collagen was linked to the NPs. The concentration of collagen in NPs suspension was determined by Bradford method as being 161mg.mL-1. The hydrogel was obtained by preparing chitosan solution 2% (w/v) in acetic acid 2%. Following, collagen-coated gold NPs were dispersed into the gel and 20mu;L of glutaraldehyde 25% was added. After 12h the hydrogel was acetylated by treatment with acetic anhydride.
Our results demonstrated that the incorporation of gold NPs into chitosan provided chitosan crosslinking at glutaraldehyde concentration 10 times lower than conventional procedures. Additionally, the chitosan collagen-coated gold NPs scaffold presented better mechanical resistance compared to the scaffold of chitosan. Scaffold morphology was characterized by SEM. Compared to the scaffold composed of chitosan only, chitosan-gold NPs scaffold presented a structure with reduced porosity and higher topographical disorder. Studies of cellular proliferation and bacterial test are being performed.
This work has been sponsored by FAPESP.
5:30 AM - H2.09
From Nanofibrous Hollow Microspheres to Nanofibrous Hollow Discs and Nanofibrous Shells
Zhanpeng Zhang 1 Jiang Hu 2 Peter X Ma 1 2 3
1University of Michigan Ann Arbor USA2University of Michigan Ann Arbor USA3University of Michigan Ann Arbor USAShow Abstract
Background: Biomaterials&’ physical shape and structural feature size on the micro and nano scales are increasingly recognized to play important roles in their function as scaffolds for tissue engineering and as vehicles for controlled or targeted therapeutic delivery. Recently, our laboratory developed injectable polymeric nanofibrous hollow microspheres and demonstrated their advantages over traditional cell carriers for knee cartilage regeneration in rabbits. However, the nanofibrous hollow microspheres could only be self-assembled from a star-shaped poly(L-lactic acid) (PLLA) with unclear mechanisms. Objective: To investigate the scientific mechanism of the nanofibrous hollow microsphere formation, to develop facile techniques to generate nanofibrous hollow objects from a variety of polymers, and to employ these vehicles to carry cells for regeneration. Methods: A non-aqueous emulsion system was developed to emulsify polymer solutions into microspheres. A thermally-induced phase separation technique was integrated in the process to generate nanofibrous structure. In one approach, a mixed solvent system was employed to increase the affinity of a polymer solution (such as a linear PLLA) to the emulsion medium to initiate and stabilize double emulsion formation, leading to hollow object formation. In a different approach, appropriate emulsifiers were incorporated into different emulsion systems (including a PLLA solution emulsified in glycerol, a Nylon6 solution emulsified in olive oil, and a polyacrylonitrile (PAN) solution emulsified in silicone oil), which also led to the formation of nanofibrous hollow microspheres. In addition, nanofibrous hollow microspheres with controllable open hole size, nanofibrous hollow discs and nanofibrous shells could be fabricated through adjusting the emulsification process. We seeded pre-adipocytes inside PLLA nanofibrous hollow microspheres, where the cells differentiated into mature adipocytes with rich lipid droplets formation. Conclusion: We formed a generalized theory of nanofibrous hollow structure formation and developed techniques to fabricate nanofibrous hollow microspheres using linear PLLA, PAN and Nylon 6 for the first time. In addition, we developed techniques to control the open hole size on the nanofibrous hollow microspheres. Furthermore, we developed new and facile fabrication techniques to generate nanofibrous hollow discs and nanofibrous shells for the first time. These nanofibrous hollow objects are promising injectable cell carriers for tissue regeneration.
5:45 AM - H2.10
Nanoporous Silicas as Components in Implants and Tissue Engineering Scaffolds
Peter Behrens 1 Nina Ehlert 1 Sina Williams 1 Anne Christel 1 Natalja Wendt 1
1Leibniz University Hannover Hannover GermanyShow Abstract
Nanoporous silica has been widely investigated as a novel material for biomedical applications. We have shown that coatings of nanoporous silica on implants have a favourable biocompatibility  and offer the possibility to locally deliver bioactive molecules (e.g. growth factors)  and small-molecule drugs . Animal experiments have shown that healing reactions after implantation operations could successfully be improved , as the interaction between the surface (coating) of an implant and the surrounding tissue is a crucial factor for the efficiency of a prosthesis.
Nanoporous silica nanoparticles (NPSNPs) offer similar possibilities with regard to growth factor and drug delivery. Often, these nanoparticles are used as suspensions, which are added to cell culture media or injected into animals for testing their efficacy. However, they can also be integrated into tissue engineering scaffolds. Here, we report on the decoration of nanoporous silica nanoparticles with polysialic acid, a polysaccharide involved in neural development, on the NPSNP-based delivery of BMP2, a growth factor, and of dexamethasone, a small-molecule drug. Both are involved in the differentiation of mesenchymal stem cells. Due to their rich surface chemistry, NPSNPs are highly versatile with regard to different surface modifications which allows to adapt their chemical interactions in a way which facilitates their integration into various scaffold materials. As an example, the integration of functionalized NPSNPs into collagen-based scaffolds will be presented. Periodic mesoporous organosilicas (PMOs) offer an interesting alternative to nanoporous silica with a more hydrophobic character.
 C. Turck, G. Brandes, I.Krueger, P. Behrens, H. Mojallal, T. Lenarz, M. Stieve. Acta Oto-Laryngol. 2007, 127, 801-808.
 N. Ehlert, A. Hoffmann, T. Luessenhop, G. Gross, P.P. Mueller, M. Stieve, T. Lenarz, P. Behrens. Acta Biomater., 2011, 7, 1772-79.
 N. Ehlert, M. Badar, A. Christel, S.J. Lohmeier, T. Luessenhop, M. Stieve, T. Lenarz, P.P. Mueller, P. Behrens. J. Mater. Chem. 2011, 21, 752-60.
 N. Ehlert, P.P. Mueller, M. Stieve, T. Lenarz, P. Behrens. Chem. Soc. Rev. 2013, 42, 3847-3861.
H1: Materials and Strategies for Regenerative Engineering
Monday AM, December 02, 2013
Sheraton, 2nd Floor, Back Bay D
9:00 AM - *H1.01
Advanced Biomaterials and Nanotechnology for Improved Health Care
Nicholas Peppas 1
1The University of Texas at Austin Austin USAShow Abstract
Engineering the molecular design of intelligent biomaterials by controlling recognition and specificity is the first step in coordinating and duplicating complex biological and physiological processes. Recent developments in protein delivery have been directed towards the preparation of targeted formulations for protein delivery to specific sites, use of environmentally-responsive polymers to achieve pH- or temperature-triggered delivery, usually in modulated mode, and improvement of the behavior of their mucoadhesive behavior and cell recognition. We address design and synthesis characteristics of novel crosslinked networks capable of protein release as well as artificial molecular structures capable of specific molecular recognition of biological molecules. Molecular imprinting and microimprinting techniques, which create stereo-specific three-dimensional binding cavities based on a biological compound of interest can lead to preparation of biomimetic materials for intelligent drug delivery, drug targeting, and tissue engineering. We have been successful in synthesizing novel glucose- and protein-binding molecules based on non-covalent directed interactions formed via molecular imprinting techniques within aqueous media.
9:30 AM - *H1.02
Composites and Structures for Regenerative Engineering
Cato T. Laurencin 1 2 3 Roshan James 1 2
1University of Connecticut Health Center Farmington USA2University of Connecticut Health Center Farmington USA3University of Connecticut Health Center Farmington USAShow Abstract
Regenerative engineering was conceptualized by bridging the lessons learned in developmental biology and stem cell science with biomaterial constructs and engineering principles to ultimately generate de novo tissue. We seek to incorporate our understanding of natural tissue development to design tissue-inducing biomaterials, structures and composites than can stimulate the regeneration of complex tissues, organs, and organ systems through location-specific topographies and physico-chemical cues incorporated into a continuous phase. This combination of classical top-down tissue engineering approach with bottom-up strategies used in regenerative biology represents a new multidisciplinary paradigm. Advanced surface topographies and material scales are used to control cell fate and the consequent regenerative capacity.
Musculoskeletal tissues are critical to the normal functioning of an individual and following damage or degeneration they show extremely limited endogenous regenerative capacity. The increasing demand for biologically compatible donor tissue and organ transplants far outstrips the availability leading to an acute shortage. We have developed several biomimetic structures using various biomaterial platforms to combine optimal mechanical properties, porosity, bioactivity, and functionality to effect repair and regeneration of hard tissues such as bone, and soft tissues such as ligament and tendon. Starting with simple structures, we have developed composite and multi-scale systems that very closely mimic the native tissue architecture and material composition. Ultimately, we aim to modulate the regenerative potential, including proliferation, phenotype maturation, matrix production, and apoptosis through cell-scaffold and host-scaffold interactions developing complex tissues and organ systems.
10:00 AM - H1.03
Effects of Surface Modification of 45S5 Bioactive Glass on Bioactivity, Mechanical, and In Vivo Remodeling Properties of Polymeric Biocomposites
Andrew J Harmata 1 2 Sasidhar Uppuganti 2 Jeffry S Nyman 2 Scott A Guelcher 1 2
1Vanderbilt University Nashville USA2Vanderbilt University Nashville USAShow Abstract
Moldable, settable bone grafts that possess mechanical strength exceeding that of host bone and maintain strength while remodeling could improve the clinical management of numerous orthopaedic conditions. Polyurethane (PUR) composites are an attractive alternative to calcium phosphate cements due to their tough mechanical properties and active remodeling. 45S5 bioactive glass (BG) particles have widely been used for bone regeneration purposes due to its osteoconductivity. Although physiological loads are cyclic, fatigue properties of biomaterials utilized in load-bearing applications are rarely reported. We investigated the quasi-static and dynamic compressive mechanical properties of BG/PUR composites. We hypothesized that a BG/PUR composite comprised of surface-modified BG would improve the mechanical properties compared to one made with cleaned-BG, and that this composite would maintain its strength throughout in vivo remodeling. BG particles were grafted with 3-aminopropyl-trietoxysilane (APTES) and surface-polymerized polycaprolactone (PCL). Composites were made from a lysine triisocyanate- poly(ethylene glycol) prepolymer, polyester triol (70% caprolactone, 20% glycolide, 10% lactide polyol, Mn ~300 g mol-1), triethylene diamine catalyst in dipropyl glycol, and BG (56.7 volume %). Under quasi-static compression, cleaned and modified BG/PUR composites had strengths of 7.9 ± 3.2 and 53.8 ± 6.5 MPa, respectively. Fatigue testing was completed in cyclic sinusoidal (5 Hz) compression, reaching maximum stress levels of 5 to 15 MPa. Under a maximum stress level of 5 MPa, the mean fatigue life of the cleaned- and modified-BG composites was 197 ± 257 and 904,172 ± 130,054 cycles, respectively. The compressive modulus of the modified-BG composite gradually decreased until failure. Its residual strain increased throughout the testing due to plastic deformation. The compressive mechanical properties of the composites were dependent on the interfacial bonding between the surface-polymerized PCL chains and the PUR network, formed in situ. Results related to the relative average length of surface chains, with respect to PUR network mesh size, support the hypothesis that the observed increase mechanical strength and fatigue life is due to chain entanglements and physical crosslinks. For the in vivo study, a 11mm diam X 18mm length defect was created in the diaphysis of sheep femur and BG composites were implanted/cured. After 8 and 16 weeks, the defect site was explanted and quasi-static compression mechanical testing was conducted. The mechanical properties of explanted BG/PUR composite from the sheep femoral plug defect maintained strength above the local native bone control. By comparing these compressive mechanical properties of cleaned- versus modified-BG/PUR composites, we conclude that surface modification significantly extends the BG/PUR composite&’s ability to withstand physiologically relevant dynamic stresses, while actively remodeling in vivo.
10:15 AM - H1.04
Gradient Matrix Design for Osteochondral Tissue Engineering
Deborah L Dorcemus 1 3 Syam Nukavarapu 1 2 4
1University of Connecticut Farmington USA2University of Connecticut Farmington USA3University of Connecticut Storrs USA4University of Connecticut Storrs USAShow Abstract
Osteochondral (OC) tissue is a complex structure comprised of an upper layer of articular cartilage, the subchondral bone and the central cartilage-bone interface. In order to facilitate proper regeneration of the tissue it is essential to devise a matrix that ensures cartilage-bone interface formation along with the regeneration of the individual tissue layers. Although mono-phasic and bi-phasic matrices were previously applied to OC defect repair, they failed to establish the proper osteochondral interface upon regeneration. In this study, we design and develop a gradiently porous matrix with increasing pore volume from one end to other, along the scaffold length. We hypothesize that such a design will enable OC tissue regeneration, including the cartilage-bone interface. For this matrix polylactide-co-glycolide or PLGA 85:15 microspheres were combined with a water-soluble porogen in a layer-by-layer fashion, increasing the porogen content (from bottom to top), and thermally sintered. The resulting matrix was then porogen-leached to form a gradiently porous structure. Micro-CT scanning was performed to establish the gradient pore structure with pore volume continuously increasing from 30-60%. A biodegradable hydrogel was infused into the gradient pore structure to form a unique OC graft where the microsphere and hydrogel phases co-exist with opposing gradients. When the individual phases are loaded with osteogenic and chondrogenic growth factors, the structure would ensure the spatial control of growth factor delivery necessary to regenerate osteochondral tissue structure. The uniqueness of this approach is to design a matrix system where bone- and cartilage-forming phases are unified in a way that supports the complexity of OC tissue regeneration. As determined by immunostaining, the gradient matrix system seeded with human bone marrow stromal cells show osteogenic as well as chondrogenic differentiation. Overall, through this study we designed a gradient matrix system that would support osteochondral tissue engineering while forming a seamless interface between the articular cartilage and the underlying bone matrix.
11:00 AM - *H1.05
Composite Scaffolds for Guiding Stem Cell Differentiation
Helen Lu 1
1Columbia University New York USAShow Abstract
Musculoskeletal joint motion is facilitated by synchronized interactions between multiple tissue types and the seamless integration of bone with soft tissues such as ligaments, tendons or cartilage. Many of these soft tissues transit into bone through a multi-region fibrocartilaginous interface, which serves to minimize the formation of stress concentrations while enabling load transfer between soft and hard tissues. With its functional significance, re-establishment of the soft tissue-to-bone interface is thus critical for promoting the integration of biological as well as synthetic soft tissue grafts. A pressing challenge for interface tissue engineering is how to harness the repair potential of mesenchymal stem cells for the simultaneous regeneration of more than one type of tissue, which is essential for multi-tissue integration. To this end, our approach centers on identifying and optimizing composite scaffold design parameters such as scaffold composition and fiber organization, especially when coupled with mechanical loading in directing stem cell differentiation into the ligament fibroblasts, fibrochondroytes or osteoblasts. This lecture will describe our work with nanofiber-based scaffolds and how controlling scaffold diameter, alignment, composition and/or mechanical loading can direct MSC differentiation into interface-relevant cell populations, without concurrent stimulation with growth factor or inductive media. These insights provide new understandings of stem cell-biomaterials interactions, and will lead to a new generation fixation devices for integrative connective tissue repair and regeneration.
12:00 PM - H1.07
One- and Two-Dimensional Carbon and Inorganic Nanomaterials as Reinforcing Agents for Biodegradable and Biocompatible Polymeric Tissue Engineering Implants
Behzad Farshid 1 2 Gaurav Lalwani 1 Balaji Sitharaman 1
1Stony Brook University Stony Brook USA2SUNY Stony Brook Stony Brook USAShow Abstract
Synthetic biodegradable polymers have been widely investigated as scaffolds for tissue engineering applications. For certain applications (e.g. loading bearing hard tissues), the polymeric scaffolds need to possess sufficient mechanical strength. Reinforcing agents possessing high intrinsic mechanical property allow efficient load transfer, increasing the mechanical properties of the polymeric implants. These reinforcing agents should not only improve the bulk properties of the polymer, but also not elicit any adverse effects on cells and tissues. In this study, we report the efficacy of several organic and inorganic one- and two- dimensional nanomaterials (single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), single-walled graphene oxide nanoribbons (SWGONRs), multi-walled graphene oxide nanoribbons (MWGONRs), graphene oxide nanoplatelets (GONPs), tungsten sulfide nanotubes (WSNTs) and molybdenum sulfide nanoplatelets (MSNPs) as reinforcing agents to enhance mechanical properties of polypropylene fumarate (PPF) for tissue engineering applications. We also report the in vitro biocompatibility of the PPF nanocomposites at nanomaterials loading concentrations that provide maximum mechanical reinforcement. PPF nanocomposites were prepared by dispersing SWCNTs, MWCNTs, SWGONRs, MWGONRs, GONPs, WSNTs and MSNPs in PPF [1,2]. Nanocomposites were used for compression and flexural testing. Subsequently, cytotoxicity of nanocomposites (yielding maximum mechanical reinforcement) against MC3T3 pre-osteoblasts was evaluated. Confocal and scanning electron microscopies (SEM) were performed to study cell attachment.
All PPF nanocomposites showed statistically significant enhancement in the mechanical properties with 0.2 wt% MoS2 nanoplatelets showing the highest Young&’s modulus of asymp; 10GPa; a 100% increase compared to PPF (control). Inorganic nanoparticles showed equivalent or better mechanical reinforcement compared to carbon nanoparticles. Cytotoxicity study showed more than 78% viability for cells in contact with crosslinked samples, which increases to ~100% with media dilution, as expected for a dose dependent cytotoxicity. Confocal and SEM imaging analysis showed about 40-49% cell attachment.
1Dand 2D carbon and inorganic- nanostructures as reinforcing agents significantly improve the mechanical properties of PPF. PPF nanocomposites exhibit low cytotoxicity, good cell attachment and spreading. The results taken together indicate that 1Dand 2D carbon and inorganic- nanostructures are promising materials as reinforcing agents to improve the bulk and functional properties of polymeric implants for tissue engineering applications.
This work was sponsored by National Institutes of Health (grants No. 1DP2OD007394-01).
. Lalwani, G., et al., Biomacromolecules, 2013. 14(3): p. 900-90.
. Lalwani, G., et al., Acta Biomaterialia (In Press)
12:15 PM - H1.08
Can Tough Corneal Scaffolds Be Created?
Khaow Tonsomboon 1 Michelle Oyen 1
1Cambridge University Cambridge United KingdomShow Abstract
Fracture toughness has occasionally been neglected in the development of tissue engineering scaffolds. In fact, almost all recent developments aim to achieve transparent scaffolds with the tensile strength and elastic modulus closely-matched to those of native cornea despite the fact that cornea is normally subjected to below-ultimate-strength cyclic tensile loadings due to intraocular pressure, ocular muscle contractions and eye blink. Similarly to other soft collagenous tissues, toughening mechanisms in cornea are not well understood, but the lamellar structure of orthogonally aligned collagen fibrils in corneal stroma is thought to account for its toughness. To examine this, transparent laminates of gelatin nanofibers in chitosan-alginate gel, mimicking the corneal lamellar structure, were created in a three-step process. First, stacks of orthogonally aligned gelatin nanofibers were created by electrospinning followed by chemical cross-linking. Next, dehydrated cross-linked gelatin fibers were swollen in chitosan-alginate solution, forming fiber-reinforced hydrogel composites. Finally, the resulting structures were subjected to cycles of dehydration and chemical cross-linking to increase their mechanical properties and optical transparency. Fracture toughness and time-independent tensile behaviors of the orthogonally-aligned fiber-reinforced hydrogels were characterized using trouser tearing and uniaxial tensile tests. Their behaviors were compared to those of pure hydrogels and hydrogels reinforced with randomly-oriented or parallel gelatin fibers. Relative orientation of fibers in adjacent layers was found to significantly affect the overall fracture toughness and time-independent tensile behaviors of the fiber-reinforced hydrogels and is therefore a key to achieve tough biomimetic scaffolds for corneal tissue engineering.
12:30 PM - H1.09
Issues in Translation of Advanced Composites from the Bench to the Medical Marketplace
Arthur J. Coury 1
1Coury Consulting Services Boston USAShow Abstract
Advanced composites comprise a growing component of research and development of medical products. Composites range in complexity from heterophase single materials to multi-component constructs of combination medical products. The diversity of requirements leading to regulatory approval and successful marketing of medical products based on the range of composites is enormous. For all regulated medical products, there are criteria which must be met in order to achieve a commercially successful outcome. Technical, regulatory, economic and societal considerations can be subdivided into several essential components each. Careful consideration of each of these criteria should be given before committing substantial resources to product development. A good point at which to make this evaluation is often just following proof of concept. Numerous examples exist of successful and unsuccessful medical product development efforts. The failure to develop a successful medical product may well be determined by even one or a few of the critical criteria not being met.
Syam P. Nukavarapu, University of Connecticut
Huinan Liu, University of California, Riverside
Rui L. Reis, University of Minho
Arthur J. Coury, Coury Consulting Services
Symposium Support Aldrich Materials Science
Royal Society of Chemistry
Society for Biomaterials
Teleflex Medical OEM
University of Connecticut - Institute for Regenerative Engineering
University of Connecticut - Sackler Center for Biomedical, Biological, Engineering and Physical Sciences
H4: Advanced Scaffolds for Tissue Engineering I
Tuesday PM, December 03, 2013
Sheraton, 2nd Floor, Back Bay D
2:30 AM - *H4.01
Advanced Composites and Structures for Tissue Engineering
Nuno M. Neves 1 2
1University of Minho Guimaraes Portugal2University of Minho Braga/Guimaraes PortugalShow Abstract
Natural origin biodegradable polymers have outstanding properties for many biomedical applications including as scaffolds for tissue engineering. The cell recognition sites, the biodegradability and the cytocompatibility of many natural origin biomaterials are particularly appealing in those applications. However, the limitations in its processing and the narrower spectra properties when compared to its synthetic counterparts, frequently hinder its application in more demanding biomedical devices. The composition of those materials with synthetic biodegradable polymers allows conferring them easier processability, which is a key issue for many applications but in particular for tissue engineering scaffolding. The effective reinforcement of biodegradable biomaterials with fibres or with particulates enable tailoring its properties for defined applications and provide enhanced opportunities for using biomimetic approaches in the biomaterial development for advanced therapies.
A critical ingredient of many strategies aiming the development of Advanced Therapies and in particular using Tissue Engineering concepts is the need for scaffolds providing temporary structure to assist the cells in the regeneration of tissue defects. The scaffolds should be specifically designed to create environments that promote tissue development and not merely to support the maintenance of communities of cells. To achieve that goal, highly functional and porous scaffolds may combine specific morphologies and surface chemistry with the local release of bioactive agents.
Many composites were already proposed in scaffolds aiming the regeneration of a wealth of tissues. We have a particular interest in developing systems based in biodegradable polymers for the regeneration of bone and articular cartilage. Those mechanically demanding applications require a combination of mechanical properties, processability, cell-friendly surfaces and tuneable biodegradability that need to be adjusted for the specific application envisioned (the properties of both bone and cartilage are highly dependent of the anatomical site considered). Those biomaterials may be processed by different routes into devices with wide range of morphologies such as fibers and meshes, films or particles with outstanding biological and structural performance for biomedical applications. We will review herein our latest developments in this fascinating research area.
3:00 AM - H4.02
Honeycomb-Shaped Scaffolds for Control Cellular Adhesion, Proliferation, and Differentiation by Altering Mechanical and Topological Properties
Hiroshi Yabu 1 2 Hiroki Satoh 1 Yuta Saito 1 Takahito Kawano 1 Masatsugu Shimomura 1 2
1Tohoku University Sendai Japan2JST Sendai JapanShow Abstract
Cell culture scaffolds act as a template for tissue regeneration, and encourages cells to form healthy and functional tissues. Cell behaviors, including cellular adhesion, proliferation, migration, and differentiation, are regulated by the interactions between cells and the microenvironment of the cells; therefore, the chemical, topological, and mechanical properties of scaffold surfaces are significant for regulating the cell behavior. We have reported that honeycomb-patterned porous polymer films can be prepared by casting a polymer solution under humid conditions and using condensed water droplets as templates of pores. In this report, we show the preparation of honeycomb scaffolds for cell culturing by using above-mentioned “breath figure” method, and we found that their mechanical and topographical properties strongly affect the adhesion of fibroblasts. By photo-crosslinking of the poly(1,2-butadiene), the hardness of the honeycomb scaffold can be successfully controlled without any surface chemical changes, and detail modulus values of scaffolds were measured by atomic force microscopy. We found that only small numbers of the cells adhered on the softer honeycomb scaffolds, which has even higher modulus value than conventional gels, comparing with flat films and a hard honeycomb scaffold. These results indicate that the elastomeric honeycomb substrates are useful for evaluating the effect of the mechanical signal-derived geometry on the transduction system of cells.
We also found that honeycomb scaffolds led human mesenchymal stem cells (hMSCs) to osteospecific and myospecific differentiations depending on the size of pores without any hazardous chemicals and supplements. Polystyrene honeycomb scaffolds with different pore sizes were successfully fabricated by casting a polymer solution under humid conditions in order to investigate the effect of porous microtopography on hMSC differentiation. We have used honeycomb scaffolds to achieve the microtopography-induced differentiation of hMSCs without any hazardous chemicals. Honeycomb scaffolds led hMSCs to osteospecific and myospecific differentiations depending on the size of pores, which identified by immunofluorescent microscopy. This selective differentiation suggested that surface microtopography may be effective for using hMSCs in regenerative medicine and tissue engineering.
3:15 AM - H4.03
Advanced Scaffold Design for Cartilage Mediated Bone Tissue Engineering
Paiyz E. Mikael 1 2 Syam Nukavarapu 1 2 3
1University of Connecticut Storrs USA2University of Connecticut Farmington USA3University of Connecticut Farmington USAShow Abstract
Treating long-bone defects still to date is a major challenge in orthopedic surgery. Current treatment modalities such as autografts and allografts have many limitations and often fail. Tissue Engineering has emerged as a promising and more fitting alternative. Despite the tremendous efforts by researchers, the traditional scaffold designs support intramembranous ossification through the direct differentiation of mesenchymal stem cells into bone forming cells (osteoblasts). Bone formation via this route does not involve vascularization process and therefore relies solely on diffusion for nutrient and waste transport; this results in extremely hypoxic conditions in the central regions of scaffolds and leads to limited amount of bone regeneration. On the other hand, long-bones development, naturally, occurs through cartilage mediated ossification (endochondral); where bone formation occurs through cartilage matrix formation, matrix vascularization and finally ossification . As such, in order to address the unique challenges of long-bone regeneration, an advanced polymer-hydrogel scaffold is proposed. Our system is composed of a poly(85 lactide-co-15 glycolide) acid microsphere scaffold as a load-bearing phase. Thermal sintering and porogen leaching technique was used to develop PLGA microsphere scaffolds with tunable pore volume .The second component of our system is a hydrogel matrix that will closely mimic the natural three dimensional extra cellular matrix (ECM) network required for stem cells differentiation into chondrocytes and their subsequent maturation into hypertrophic chondrocyte. In addition, the hydrogel will serve to encapsulate cells rapidly and efficiently, as well as providing a venue for drug and agent delivery. Through this study, we successfully developed a hydrogel-polymer scaffold system where the hydrogel phase supports cartilage-mediated bone formation, while the stable polymer phase acts as a load-bearing graft essential for long-bone repair. Together, the polymer-hydrogel matrix design is proposed as a novel matrix system for bone regeneration via endochondral ossification.
 Amini A R, Laurencin C T and Nukavarapu S P 2012 Bone tissue engineering: recent advances and challenges Critical reviews in biomedical engineering 40 363-408
 Amini A R, Adams D J, Laurencin C T and Nukavarapu S P 2012 Optimally porous and biomechanically compatible scaffolds for large-area bone regeneration Tissue engineering. Part A 18 1376-88
3:30 AM - H4.04
Nanoclays with Biomineralized Hydroxyaptite for Design of Biodegradable Polymeric Scaffolds for Bone Regeneration
Avinash H Ambre 1 Dinesh R Katti 1 Kalpana S Katti 1
1North Dakota State University Fargo USAShow Abstract
Existing strategies for bone regeneration based on tissue engineering principles are driven by challenge of developing scaffolds capable of eliciting cell response favorable for tissue formation and providing mechanical support until the regenerating tissue has enough strength to support itself or restore function at defective sites. The effect of mechanical stimuli on cellular processes has broadened the scope of mechanical properties with the need to consider the mechanical properties at nanoscale in addition to the macroscale. Biodegradable composites prepared by incorporating inorganic fillers in biodegradable polymers have great potential for fabricating scaffolds that can meet the challenges of bone tissue engineering. Biomimetic nanoclay-hydroxyapatite (HAP) hybrid was prepared using sodium montmorillonite (Na-MMT) clay modified with an unnatural amino acid and for preparing biodegradable polymer (both natural and synthetic) composite films and scaffolds in our studies. The choice of MMT clay was based on insights gained through “altered phase theory” for polymer-clay nanocomposites (PCNs), reinforcing capability of MMT clay, medicinal properties of MMT clay and its reported use for pharmaceutical applications. A biomimetic approach was used to mineralize HAP in modified MMT clay galleries to obtain the nanoclay-HAP hybrid and it was found through infrared spectroscopy studies that carboxylic groups of unnatural amino acid in modifed clay were involved in HAP mineralization. Nanoindentation tests performed on biodegradable polymer (synthetic) composite films containing nanoclay-HAP hybrid showed significant improvement in nanomechanical properties thus emphasizing the reinforcing effect of nanoclay-HAP hybrid at nanoscale. Biocompatibility and osteoconductive potential of the biodegradable polymer composite films and scaffolds was studied through appropriate assays involving human mesenchymal stem cells (hMSCs). In addition to studies related to differentiation of hMSCs on these composite scaffolds and films, hMSC behavior was studied using phase contrast microscopy while scanning electron microscopy (SEM) was used to study scaffold microstructure, cell morphology in scaffold microenvironment and cell infiltration in scaffolds. This work also demonstrates a unique “two-stage cell seeding” experiment involving hMSCs as an approach to increase tissue formation under in vitro conditions. Our experiments indicate that nanoclays mediate hMSCs to differentiate and proliferate inside scaffold three dimensional structures. This new nanoclay-HAP-Polymer system represents a viable new material system in tissue engineering.
3:45 AM - H4.05
Biphasic Scaffolds for Bone Repair
Clarke Nelson 1 Yusuf Khan 1 2 3 Cato T. Laurencin 1 2 3
1University of Connecticut Health Center Farmington USA2University of Connecticut Health Center Farmington USA3University of Connecticut Storrs USAShow Abstract
Statement of Purpose: Sintered composite microsphere matrices have shown potential toward fulfilling the goal of autograft replacement, but studies have suggested that 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 can be synthesized within the pore structure and potentially increase cell migration and residence throughout the scaffold.
Methods: Sintered, composite microsphere matrices were fabricated according to reported procedure and then submerged in three separate concentrations of PLLA in DMF and cooled to allow thermally induced phase separation (TIPS) to occur. Mechanical integrity of hybrid scaffolds was measured using an Instron Uniaxial Testing Machine. Viability was assessed with confocal microscopy. MC3T3 cells were cultured for 21 days and assayed for DNA content. For fluorescence microscopy, bone marrow stromal cells were harvested from fluorescent reporter mice and seeded on scaffolds. The scaffolds were imaged with fluorescent microscopy, and at 28 days, scaffolds were cryosectioned, and cells distribution data was determined through image analysis (FIJI).
Results: Mechanical data showed that the TIPS process did not appear to affect the mechanical integrity of the scaffolds. Confocal data showed cell residence throughout the pore space. Fluorescent microscopy data showed that the hybrid scaffolds appear to promote osteogenic differentiation at further distances from the periphery, but this increased differentiation may be at the cost of total cell population. The DNA assay exhibits no significant differences at any time point. Results for the fluorescent imagery show evidence for increased osteogenic differentiation for the 1% TIPS-permeated scaffolds compared to controls. The cell distribution data from day 28 shows lower cell population but increased osteogenic differentiation farther from the periphery of the scaffold in hybrid scaffolds compared to control scaffolds.
Conclusions: Results indicate that an ECM-mimetic fibrous network could be created in the pore spaces of a sintered, composite microsphere scaffold. Importantly, TIPS fibrous networks share a similar size to natural ECM networks on the order to 50-500 mu;m and have pore spaces large enough to facilitate human osteoblast migration. Taken together, these studies conclude that an ECM-mimetic network can increase the population of cells committed to the osteogenic lineage at an increased distance from the periphery of the scaffold.
4:30 AM - *H4.06
Fabrication of Bioactive Glass Scaffolds with Tailored Biodegradability
Himanshu Jain 1 Mona K Marei 2 Matthias M Falk 3
1Lehigh University Bethlehem USA2Alexandria University Alexandria Egypt3Lehigh University Bethlehem USAShow Abstract
An ideal bioscaffold for tissue development must meet several requirements. For instance, it should be biocompatible, preferably bioactive for rapid tissue growth; must have interconnected macro (~200mu;m) porosity for cell migration and proliferation; resorb at ~ the growth rate of new tissue; should possess suitable mechanical properties for the duration of tissue growth; and can be fabricated in irregular shapes & sizes at moderate cost. We have pursued these goals by developing novel fabrication methods that produce a special class of bioactive tailored amorphous multiporous (TAMP) structures with superimposed nano and macro porosities. The interconnected pores larger than ~100mu;m allow infiltration by cells, blood vessels, collagen fibers, etc. At the same time, nanoscale porosity is exploited to match the degradation rate of the scaffold with the rate of tissue growth. Fortunately, nanoporosity is also shown to enhance cell response under in vivo and vitro conditions. Thus nano-macro porous bioactive glass appears to be an ideal candidate for use as scaffold for bone regeneration. Recently, certain glass compositions are shown to be also useful for soft tissue regeneration and wound healing.
The creation of pores differing in size by several orders of magnitude is a challenging problem of material processing. We have pursued its novel solutions by exploiting multi-scale phase separation, followed by the selective removal of one or more phases by leaching or evaporation. Two independent approaches have been pursued utilizing the classic melt-quench and sol-gel methods of glass preparation, respectively. Glass compositions with varying functionalities and corresponding processing conditions are identified, which lead to spinodal decomposition that is required for the interconnectivity of pores. Typically, bimodal or multi-modal porosity is obtained with pores ranging from ~10 nm to ~100 mu;m, and it is possible to control the nano and macro porosities independently for tailoring to the needs of a specific patient or application. Additional fabrication processes are introduced if macropores significantly larger than ~100 mu;m are desired. This presentation will review and compare the pros and cons of the currently available methods for fabricating nano-macro porous bioactive glass structures. Results of in vitro tests with MC3T3 pre-osteoblast and in vivo tests with animal models, which demonstrate the superior performance due to the presence of nanoscale porosity, will be also reviewed.
5:00 AM - H4.07
Biodegradable Nanocomposite Based Spiral Structures for Bone Tissue Engineering
Aja Aravamudhan 1 2 Roshan James 1 2 Xiaojun Yu 4 Sangamesh Kumbar 1 2 3
1University of Connecticut Health Center Farmington USA2University of Connecticut Health Center Farmington USA3University of Connecticut Storrs USA4Stevens Institute of Technology Hoboken USAShow Abstract
Introduction: In scaffold based tissue engineering, successful bone regeneration depends on material composition, topography and pore properties. Specifically polymer-ceramic nano-composites have shown great promise as scaffolds for bone regeneration. Cylindrical porous scaffolds offer superior mechanical properties but fail to improve transport features such as metabolic waste removal and nutrient supply. These limitations translate into limited in vitro cell infiltration, and poor tissue ingrowth following implantation. To overcome these limitations, we have previously demonstrated the benefits of an open spiral scaffold structure and its ability to promote cell infiltration. A uniform distribution of nano-hydroxyapatite (nHA) within the scaffold will achieve desirable scaffold performance in terms of its physical properties and ability to support cellular events. For instance, non-uniform nHA deposition restricts favorable cellular responses to regions having optimal nHA deposition. To overcome these shortcomings, we hypothesize that an open scaffold design composed of sintered microsphere spiral structures incorporating nanofibers and osteoinductive nHA will combine mechanical strength, material transport and ECM mimicry. Electrospinning and layer-by-layer (LbL) deposition technique was used for the uniform deposition of nanofibers and nHA throughout the thickness of the sintered scaffold.
Results: Controlled surface deposition of nHA on sintered microsphere scaffolds was achieved by LbL technique. By applying multiple coatings each of thickness 100-200nm an nHA concentration of ~60mu;M/mm2 was achieved. Optimal level of nHA for maximum osteoinduction has been reported to be 45-70mu;M/mm2. These structured composite scaffolds have a compressive modulus of 52MPa, comparable to trabecular bone. When bone marrow stromal cells (BMSC) were seeded on these spiral scaffolds and on cylindrical microsphere/n (control) scaffolds, it was observed that the multilayered spiral scaffolds supported greater cell proliferation and osteogenic progression than conventional cylindrical scaffolds.
Conclusion: LbL polyelectrolyte based nHA coating approach provided uniform nHA deposition. Further, the spiral structured composite scaffold promoted osteoblastic progression of seeded BMSCs.
5:15 AM - H4.08
Fabrication of Pure Cellulosic and Composite Scaffolds by Unidirectional Freeze Drying Method for Cartilage Tissue Engineering
Avinav Nandgaonkar 1 Lucian Lucia 2 Wendy Krause 1
1North Carolina State University Raleigh USA2North Carolina State University Raleigh USAShow Abstract
Osteoarthritis is a degenerative disease of articular cartilage which is second most leading cause of disability after cardiovascular diseases. Various method and materials had been used in order to mimic the structure and regenerate the cartilage. In order to mimic the directionality of collagen fibrils in native cartilage, we intend to work on a novel material known as bacterial cellulose (BC) which is secreted by Glucanactobacter xylinus because of its added advantages of nanofibrous structure which can mimic the native extra cellular matrix (ECM) and high wet tensile properties. The Glucanactobacter xylinus had been synthesized by using two carbon source, glucose and mannitol. The chemical structure and functional groups were found to be same for these products by Fourier Transform Infrared (FTIR). However, the crystallinity of mannitol sample was low compared to glucose sample, confirmed by X-ray diffraction (XRD) which is useful for degradation of scaffold. The average fiber diameter of the fibrils was found to be 50-100 nm analyzed by scanning electron microscopy (SEM). Pure cellulosic and composite scaffolds were prepared by unidirectional freeze drying method. The composite scaffold properties were carefully tailored by adjusting the chitosan (Ch) concentration from 1, 1.5, and 2 wt%. SEM images confirmed the presences of well inter-connected porous network having uni-directionality of fibrils with the porosity varying from BC (75±2%), BC-Ch-1wt% (86±1%), BC-Ch-1.5wt% (79±2%), and BC-Ch-2wt% (74±1%). The surface areas by Brunauer-Emmett-Teller (BET) were found to be 201 m2g and 29.5 m2g for pure BC and BC-Ch scaffolds, respectively, which confirmed the reduction in porosity and pore size by the incorporation of chitosan at 2wt%. Thus, the unidirectional porous structure with low crystallinity index would be suitable for its potential application in cartilage tissue engineering.
5:30 AM - H4.09
Multiphasic Eosin Y and UV Crosslinked Hyaluronic Acid Based Hydrogels for Integrated Osteochondral Tissue Regeneration
Spencer L Fenn 1 Meredith Koch 1 Rachael A. Oldinski 1 2
1University of Vermont Burlington USA2University of Vermont Burlington USAShow Abstract
Statement of Purpose: Osteoarthritis (OA) is the predominant form of arthritis in our aging population. With individuals living increasingly longer lives, we face new challenges relating to the degradation of tissues within the body. Regenerative tissue engineering utilizing bio-polymer scaffolds allow treatment to begin at the first sign of OA, alleviating pain as well as returning them to a pre-osteoarthritic condition. As the osteochondral tissues display a gradient of properties and structure, an integrated multi-phasic scaffold made of extracellular matrix (ECM) components was designed to mimic natural tissue. Our goal is to fabricate an integrated multi-phasic scaffold in the presence of drug-encapsulated microspheres to direct osteochondral tissue regeneration. Methods: Methacrylation of hyaluronan (HA) was performed through a novel method in dimethyl sulfoxide by ion exchange with an ammonium salt. The solution was reacted with methacrylic anhydride for 24 hours with dimethylaminopyridine as the catalyst, hydrolyzed, lyophilized, and characterized by 1H-NMR. Cytocompatibility of the HA-MA was verified with primary human mesenchymal stromal cells (hMSCs). Hydrogels of 2 and 4% (w/v) 1.5 MDa HA-MA were crosslinked using either UV or visible green light in the presence of photoinitiators Irgacure 2959 and eosin Y/triethanolamine/1-vinyl-pyrrolidinone, respectively. Unconfined compression tests were performed and swell ratios were determined. Results and Discussion: Varying degrees of modification were achieved ranging from 10-90%. HA-MA (87% methacrylation) was formed into hydrogels and tested in unconfined compression, yielding moduli ranging from 156-597 Pa for UV-crosslinked hydrogels and 160-730 Pa for eosin Y (EY) crosslinked hydrogels. Swelling in PBS was reduced in EY crosslinked samples at 875-959% wt. increase versus 1154-1566% for UV crosslinking; the increased stiffness resisted swelling. Integrated multi-phasic scaffolds can be formed by successive layering and polymerization of varied polymer blends in a custom mold using UV and EY crosslinking techniques individually or in combination. In addition, scaffolds were formed in the presence of novel PEG-modified alginate (AA-g-PEG) microspheres for controlled drug release. Conclusions: The HA-MA-based scaffolds demonstrate efficacy of incorporating AA-g-PEG microspheres into the hydrogel network. Additional mechanical testing will be performed on varying degrees of modification and polymer solution concentrations to optimize properties of each scaffold layer. Differentiation and proliferation of hMSCs within our scaffold will be investigated to determine the optimal concentration of ECM components and drug release profiles within each layer of the scaffold.
5:45 AM - H4.10
Biopolymer/Graphene Nanocomposites for Bio-Applications
Ananta Raj Adhikari 1 Wei-Kan Chu 1 2 Ardebili Haleh 3 Fernanda Laezza 4
1University of Houston Houston USA2University of Houston Houston USA3University of Houston Houston USA4University of Texas Medical Branch Galveston USAShow Abstract
This study is on the fabrication of biopolymer nanocomposites based on Poly(lactic-co-glycolic) acid (PLGA) with Graphene platelets (GNPs). Various PLGA/GNP nanocomposites (1 and 5 wt% of GNPs) were prepared using a solution based technique. Transmission electron microscopy (TEM), X-ray diffraction (XRD), Differential scanning calorimeter (DSC), and Thermogravimetric analyzer (TGA) were used to analyze the morphology and the thermal properties. We demonstrated that GNPs in PLGA provide a favorable platform resulting in enhanced polymer crystallization. Functionalized GNPs decrease the thermal stability with the expense of crystallization. PLGA/GNP nanocomposites were also studied their cytocompatibility and found that 1wt% PLGA/GNP showed dramatic increase of cells irrespective of the chemical modification of GNPs. These results provide a strong evidence for a new class of materials that could be important for biomedical applications.
H5: Poster Session
Syam P. Nukavarapu
Arthur J. Coury
Tuesday PM, December 03, 2013
Hynes, Level 1, Hall B
9:00 AM - H5.01
Cells Culture and Control of Growth Orientation via Wet Processed Opportunely Tailored Nanostructures
Ennio Capria 1 Stefania Corvaglia 1 Loredana Casalis 1 Alessandro Fraleoni-Morgera 1
1Elettra - Sincrotrone Trieste Basovizza ItalyShow Abstract
Wet processing methods are in principle the best options to obtain large area depositions of nanosized patterns, They permit the best performances in terms of low costs, rapidity, and simplicity. Auxiliary Solvent-Based Sublimation-Aided NanoStructuring (ASB-SANS) is a novel, fast, low-cost and versatile wet-processing method to create large area, ordered arrays of filamentary nanostructures. The technique exploits the unique properties of a sublimating crystal able to sublimate (SS) as a template for the material to be structured/patterned (Target Material, TM). An appropriate auxiliary solvent (AS) is added to SS and TM substances to obtain a ternary mixture able to be wet processed at room temperature and ambient pressure, over large areas. A satisfactorily control on the pattern shapes can be achieved over a wide range of structures: aligned fibers, nets, mesoporous, dendrimers, and bulky 3D structures. In particular the potential of this technique has been demonstrated by fabricating controllable and reproducible patterns out of different polymers [i.e. Polyvynilpyrrolidone, PVP; Poly(lactic acid), PLA; Polyisoprene; Poly(ethyleneglycol), PEG; Poly(methylmethacrylate), PMMA] and with carbon nanotubes.
A consequent test of cell cultures on ASB-SANS generated patterns has been carried out with HeLa cells on different polymers. Substrates showed good compatibility with cells. Moreover, where polymer patterns where present, a preferential orientation to cell growth and a cells alignment along the nanostructures has been deeply investigated.
9:00 AM - H5.02
Biological Characterization of TAMP Scaffolds for Hard Tissue Regeneration
Tia J. Kowal 1 Shaojie Wang 2 Jutta Y. Marzillier 1 Mona Marei 3 Himanshu Jain 2 Matthias M. Falk 1
1Lehigh University Bethlehem USA2Lehigh University Bethlehem USA3Alexandria University Alexandria EgyptShow Abstract
The medical community is moving forward rapidly from tissue replacement to tissue regeneration with the advent of tissue engineering. The ideal tissue engineering material should be bioactive (cells of the body can interact with it), contain pores to allow for nutrient exchange and cell migration, mimic the structure/morphology of the tissue to be regenerated, and be a temporary scaffold during the regeneration process. Based on these requirements, bioactive glass scaffolds, termed TAMP (Tailored Amorphous Multi Porous), have been developed in our laboratory using a novel sol-gel technique. These TAMP scaffolds are manufactured on site allowing for the control of many material parameters independently including chemical composition, surface roughness, porosity, pore distribution, and surface area.
The 70 mol% SiO2-30 mol%CaO model composition has shown excellent biocompatibility via the rapid formation of hydroxyapatite in simulated body fluid, as well as in tests with bone forming cells. This composition meets all criteria described above: (1) it has large macro-pores (100µm-200µm) allowing for cell migration and colonization of the interior of the scaffolds and nano-pores (2.5nm-50nm) that allow for enhanced cell adhesion, (2) a morphology similar to trabecular bone, and (3) in vitro experiments analyzing degradation indicate that TAMP scaffolds have a half-life of 15.4 days. To examine the capacity of the TAMP scaffolds to support hard tissue regeneration, an extensive biological characterization of cellular response to the TAMP scaffolds including analysis of cell adhesion, proliferation and differentiation was performed using techniques that evaluated these processes on the mRNA, protein, and enzymatic levels. Adhesion and proliferation analyses were performed using immunofluorescence and Western blots, and differentiation of MC3T3-E1 pre-osteoblasts into mature bone forming osteoblasts was evaluated by measuring Alkaline Phosphatase (ALP) enzyme activity and by observing bone specific proteins (transcription factors and secreted extracellular matrix proteins) by immunofluorescence and Western blot analyses. The results of these in vitro observations are supported by in vivo experiments in animal models. Results confirm that TAMP scaffolds support adhesion, proliferation and differentiation of osteoblast precursor cells indicating the superiority and usefulness of the material.
Acknowledgements: Research in the Falk lab is supported by NIH-NIGMS (GM55725). Research in the Jain lab is supported by the NSF-IMI (DMR-0844014) and Materials World Network (DMR-0602975) grants.
9:00 AM - H5.03
Poly (3-Thiophene Hexylacetate)/Poly(Hydroxybutyrate-co-Valerate) Blends for Use in Tissue Engineering
Mariana Silva Recco 1 Mateus Sousa Franco 1 Adjaci Uchoa Fernandes 2 Tatiane Moraes Arantes 3 Fernando Henrique Cristovan 1
1Universidade Federal de Samp;#227;o Paulo Samp;#227;o Josamp;#233; dos Campos Brazil2Universidade de Sao Paulo Sao Paulo Brazil3National Institute for Space Research Sao Jose dos Campos BrazilShow Abstract
Conducting polymers discovered in 1980s promoted a revolution in the construction of electronic devices. Today these materials have been investigated for replacing traditional materials in the construction of these devices. Furthermore, it was observed that such materials stimulated adhesion and proliferation of various cell types to be biocompatible materials for use in scaffolds for tissue engineering. [1, 2]. Although, the poor mechanic properties and biodegradability limit their applications. Blends of polythiophenes (PT) and polythiophenes derivatives with the biodegradable thermoplastic polymer poly(hidroxybutyrate-co-valerate) (PHBV) can improve the mechanical performance of this material . PT/PHBV blends are candidates for biological application such scaffold for tissue engineering applications. In this work, the monomer 3-hexyl-thiophene acetate (TAcHex) was synthesized by Steglich esterification using DMAP and DCC as catalysts in dry dichloromethane solution and molecular sieve. The polymerization of the monomer was made by FeCl3 chemical oxidation, as suggest by Sugimoto. PTAcHex/PHBV blends were prepare by solution method. Films of PHBV: PTAcHex blends were prepared in four different weight ratios 100:0, 98:2, 92:8 and 88:12. The materials were characterized by 1H-MNR, FTIR, UV-vis, x-ray diffraction, differential scanning calorimetry (DSC) and thermogravimetry analysis (TGA). The yield for the esterification and polymerization were 54% and 32%, respectively. The final product of polymerization can be confirmed by the FT-IR spectra. The disappearance of the band around 736 cm-1 , visible in the monomer&’s spectrum, and the emergence of another band around 830 cm-1 in the polymer&’s spectrum occur due to the formation 2.5 bonds in the thiophene ring. The UV-Vis analysis of the films showed the increase of the absorbance due concentration ascendance of PTAcHex in the blends. PHBV matrix do not contributes with the absorbance at this region. In the thermal analysis, a curve of pure PHBV showed two melting peaks (159.1°C and 172.3°C). With the increase of PTAcHex amount in the PHBV matrix, both of the melting peaks became wider and shifted to lower temperatures. The decrease trend of first and second melting points with increase of PTAcHex amount, suggests a reduction in the crystallinity of the blends. This behavior was confirmed by WAXS.
Acknowledgments: This work was supported by CNPq, CAPES and FAPESP (2011/17475-6).
 a) R.D. McCullough, Adv. Mater., 1998, 10, 2. b) M. M. P. Madrigal, M. I. Giannotti, G. Oncins, L. Franco, E. Armelin, J. Puiggalí, L. J. del Valle, C. Alemán, Polym. Chem., 2013, 4, 568.
 Mattioli-Belmonte, M.; Giavaresi, G.; Biagini, G. Int. J. Artif. Organs. 26, 2003, 1077.
9:00 AM - H5.04
Effects of Cellulose Nanocrystals on the Properties of Chitosan Hydrogel for Medical Applications
Mateus Santos Silva 1 Thais L.A. Montanheiro 1 Joao Paulo Machado 2 Nelson Duran 3 4 Dayane Batista Tada 1 Ana Paula Paula Lemes 1 3
1Universidade Federal de Samp;#227;o Paulo Samp;#227;o Josamp;#233; dos Campos Brazil2Instituto Nacional de Pesquisas Espaciais Samp;#227;o Josamp;#233; dos Campos Brazil3Universidade Estadual de Campinas Campinas Brazil4Universidade Federal do ABC Santo Andramp;#233; BrazilShow Abstract
Although chitosan hydrogels has attracted significant interest for production of biomaterials due its biocompatibility and antibacterial properties, the low mechanical properties of chitosan hampers its application. The improvement of many polymeric matrixes has been achieved by the introduction of cellulose nanocrystals (CNC). They are interesting not only as reinforcing agent in biopolymers, but also as providers of new properties.
This work evaluated the effect of CNC on the properties of chitosan hydrogels crosslinked with glutaraldeyde. The CNC were produced by acid hydrolysis of cellulose microcrystals with H2SO4 (64% w/w), and their average dimensions were obtained by atomic force microscopy (AFM). The mean length was of 140.7 nm and the mean diameter was of 24.5 nm. The glutaraldehyde was used in molar proportion of 1: 2 (glutaraldehyde: amine groups of chitosan) to promote the polymer crosslinking, which was confirmed by infrared spectroscopy. The FTIR spectra showed the increased intensity of the band related to C=N at 1660 cm-1 and the decreasing of intensity of bands related to C-N of amine groups of chitosan between 1050 and 1200 cm-1, indicating the crosslinking of chitosan chains by glutaraldehyde. The chitosan/CNC hydrogels with 0, 2, 4 and 6% (w/w) of CNC were characterized by swelling tests and scanning electronic microscopy (SEM). A high swelling degree was observed for pure chitosan hydrogel (~ 9000 %) and for chitosan/CNC hydrogels, but the values showed that the addition of CNC decrease the swelling degree of chitosan hydrogels. The swelling degree of chitosan hydrogels containing 2, 4 and 6% (w/w) of CNC were 9.2%, 13.0% and 22.8% lower than pure chitosan. The hydrogels morphology changed with the increasing of CNC as observed in the cryo-fractured surface of samples analyzed by SEM. The chitosan/CNC hydrogels with 0 and 2% (w/w) of CNC showed a fragile structure without defined pores; however, the chitosan/CNC hydrogels with 4 and 6% (w/w) of CNC were more resistant to the fracture process and showed a homogeneous surface with defined pores. Studies of cellular proliferation and bacterial test are being performed.
9:00 AM - H5.05
Formulation of Ag/BSA Nanoparticles Adsorbed Collagen Grafted PHBV Scaffold for Potential Use in Joint Arthroplasty
Rotimi Bakare 1 Chandra Bhan 1 John Stubbs 2 Dharmaraj Raghavan 1
1Howard University Washington USA2Howard University College of Medicine Washington USAShow Abstract
Infections with bacteria have become a serious concern in joint arthroplasty. The primary objective of this study is to formulate silver nanoparticles loaded cytocompatible poly(3-hydroxylbutyrate-co-3-hydroxylvalerate) (PHBV) matrix so that the released nanoparticles can maintain an antimicrobial environment while promoting the growth of bone cells. Cytocompatibility of PHBV was enhanced by grafting collagen on microporous PHBV film. To this end, the salt leached film was subjected to thermal radical copolymerization with hydroxyl ethyl methacrylate (HEMA), activated with carbonyldiimidazole (CDI), and coupled with collagen. A sleuth of spectroscopic (FTIR, NMR, XPS), physical (SEM) and thermal (TGA) analytical techniques were used to characterize the functionalized porous PHBV films. The amount of collagen present on PHBV film was quantified by Bradford assay. The antimicrobial agent, BSA stabilized silver nanoparticles (Ag/BSA nanoparticles), was then adsorbed on the collagen immobilized PHBV functionalized film at 4oC and pH 7.4. The amount of nanoparticles adsorbed was determined by Atomic Absorption Spectroscopy and was found to be 0.26 microgram/cm2. Future studies will investigate the antimicrobial efficacy of Ag/BSA nanoparticles loaded collagen grafted PHBV film against E. coli.
Acknowledgments : NSF-DMR
9:00 AM - H5.06
Changes in Conformational Structure and Crystalline Morphology of Poly(3-Hydroxybutyrate-co-3-Hydorxyhexanoate)s During Electrospinning Investigated by FT-IR and Raman Spectroscopy
Liang Gong 1 D. Bruce Chase 1 Isao Noda 1 John Rabolt 1
1University of Delaware Newark USAShow Abstract
Bacterially synthesized polyhydroxyalkanoates (PHAs) are a class of naturally occurring biodegradable and non-cytotoxic thermoplastic polyesters. Recent research has found that among all the species of PHAs, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHB-HHx), possesses the unique property of preserving the phenotypes of chondrocytes and osteoblasts when used as a tissue engineering construct. PHB-HHx also promotes chondrocyte regeneration[3,4] and initiates the chondrogenesis of mesenchymal stem cells (MSCs). In addition, it has been found that the cell-substrate interaction with PHB-HHx electrospun mesh is enhanced compared to that of the corresponding cast films and the cells show a specific orientation along the fiber axis on the surface of the mesh[6,7], but, thus far, there is no explanation for this observation.
In our present research, random and aligned nanofibers of poly(3-hydroxybutyrate-co-3-hydorxyhexanoate)s (PHB-HHx) with different HHx content (3.9, 7.6, 13mol%) were fabricated via electrospinning and investigated by Raman and FT-IR spectroscopy to identify changes in conformational structure and crystalline morphology due to processing. There are obvious differences in the Raman and FTIR spectrum compared to that of the corresponding spun-coated thin films and this may be indicative of a change in the chain conformation, which may influence the interaction between cells and the polymer nanofibrous mesh at the molecular level. In addition, we continue to explore the real time dynamics of crystallization during fiber formation by planar array infrared (PA-IR) spectroscopy, which has a time resolution of < 1 millsec and allows us to follow chain organization and order on that time scale.
The observed results from FT-IR, Raman and PA-IR will be correlated with thermal analysis and wide angle x-ray diffraction in order to provide an insight into how the nanofibrous structure can influence its properties and thus its interaction with cells.
9:00 AM - H5.07
Low Temperature Fabrication of Hydroxyapatite-Titanium Nanocomposites for Bone Replacements
Isabella Grabski 1 Deok-Yang Kim 1
1Bergen County Academies Hackensack USAShow Abstract
Titanium alloys are frequently used as bone replacements due to their high mechanical strength, but titanium-based metals do not closely match the physical properties of bone, causing stress shielding and bone resorption in the long run. In this study, fabrication of hydroxyapatite-titanium nanocomposites at low temperature was attempted as a simple model system to understand the behavior of nucleation/crystallization of hydroxyapatite nanocomposites. Our preliminary results reveal that the organic-inorganic hybrid sol-gel process resulted in inconsistent Ca/P ratio confirmed by elemental analysis, but that the precipitation method from Ca-rich solutions provided a facile synthetic route for crystalline hydroxyapatites at 20 °C. Transmission electron imaging confirmed regular rod-shaped nanocrystals about 20 nanometers in length. Effects of various concentrations of titanium nanoparticles as seeds for heterogeneous nucleation of rod-shaped hydroxyapatites at low temperature will be discussed. Understanding the formation of crystalline hydroxyapatites at metal interface may facilitate formulations of different hydroxyapatite nanocomposites, especially for the future usage of emerging technology like 3D printing to mimic the extracellular matrix found in bone tissue.
9:00 AM - H5.09
Engineered Mineralization Using Protein-Derived Peptides for Dental Tissue Repair
Mustafa Gungormus 1 Hanson Fong 1 Sefa Dag 1 Greg Huang 1 Candan Tamerler 2 Mehmet Sarikaya 1
1University of Washington Seattle USA2University of Kansas Lawrence USAShow Abstract
Distinguishing the enzymatic versus nucleation or morphogenesis effects of protein-derived peptides (PDPs) in biomineralization of hydroxyapatite is intriguing in the use of this new class of newly discovered peptides in dental and bone therapeutics. Amelogenin is the main protein instrumental in the developing enamel; its effect on enamel formation and its utility as a therapeutic agent have been a subject of intense study for the last 3 decades. As demonstrated recently, using combinatorial mutagenesis and bioinformatics similarity approaches, this collaborative interdisciplinary group has identified specific, 10-25 AA-long sequences along amelogenin with specific mineral binding and/or mineralization capabilities. Furthermore, these ADPs have been demonstrated to have fast and slow mineralization capabilities in the remineralization of human teeth with great potential as versatile therapeutic agents, such as hypersensitivity, incipient caries, and white spot lesions. Herein, we study the mechanism of hydroxyapatite mineralization by specifically using two peptides ADP5 and ADP7 which affects mineral formation and its kinetics within homogeneous solutions and on tooth surfaces. Two approaches, experimental and computational, are undertaken using mutagenic peptides in which the charged moieties are knocked out or knocked-in, or otherwise their location are modified to influence enzymatic versus growth effects. Experimental approaches involve the use of recently developed biomineralization assay in solution of mutant peptides to quantify kinetics and allotropic and morphological forms of the mineral(s) formed. The computational approach, based on molecular dynamics and mechanics, analyze folding pattern of the WT and mutant peptides in the presence of Ca2+ and (PO4)2- ions and early stage mineral nucleation to bring about a detailed hierarchical structural developments. The combination of the experimental and modeling studies, while shedding more light to the capacity of these multifunctional peptides and form the fundamental mechanisms with high potential in the development of remineralization strategies in the eventual clinical dental care.
9:00 AM - H5.10
3D Scaffolds with Inverted Colloidal Crystal Geometry from Expandable Cationic Poly(DMAA-co-AMTAC) Hydrogel for Well-Plate Format
Joong Hwan Bahng 1 Yi Chun Wang 1 Nicholas Kotov 1 2 3
1University of Michigan Ann Arbor USA2University of Michigan Ann Arbor USA3University of Michigan Ann Arbor USAShow Abstract
Importance of cell studies in 3D culture evidenced in academics as well as in industries lead to significant growth in the development of 3D tissue culture scaffolds. Amongst the varieties, hydrogel have been one of the most popular choices as scaffolding material due to its chemical versatility as well as its mechanical similarity to living tissues. In this study, we introduce a positively charged polyelectrolyte hydrogel scaffold for tissue culture. The hydrogel is made by free radical copolymerization of N,N-Dimethylacrylamide (DMAA) and (3-Acrylamidopropyl)-trimethylammonium chloride (AMTAC) chemically cross-linked by N,N&’-Methylenebisacrylamide (NMBA) for the manufacture of scaffolds with inverted colloidal crystal (ICC) geometry. The poly(DMAA-co-AMTAC) was found biocompatible through cell studies and its chemical properties demonstrated following features: (1) cationic nature promoted cell-adhesive interface between various tissue types, (2) high transparency allowed for optical-based evaluation protocols, (3) homogenous gel formation avoided lot-to-lot variability . As a 3D tissue support scaffold, the ICC poly(DMAA-co-AMTAC) demonstrated numerous advantages due to its unique topology: unique manufacturing process rendered (i) simplicity in its manufacture, (ii) highly-ordered array structure (iii) high porosity allowing efficient nutrient delivery and metabolite diffusion, (iv) facile cell inoculation of adherent cell types, (v) structural resemblance of several in vivo organs. Combination of benefits and consistencies in material properties and geometrical features enabled standardization to our 3D ICC scaffold, which is critical in the experimental reproducibility. Moreover, the poly(DMAA-co-AMTAC) hydrogel was found to undergo volume transition where the degree of the swelling was dependent on the contents of the cross-linking agent, charged monomer, and ionic strength of the external environment. This property was utilized to control the physical dimensions of ICC geometry (cavity size and interconnecting channels) significant for many tasks of ex-vivo tissue engineering and for tight fitting the ICC scaffolds into a well-plate. Along with the standardized feature, easy integration into current infrastructure deployed in the current pharmaceutical industries makes ICC poly(DMAA-co-AMTAC) hydrogel scaffold a practical 3D tissue culture and in vitro early stage assay platform for the future drug discovery and development.
9:00 AM - H5.11
Fabrication and Characterization of a Porous Hydroxyapatite Scaffold for Bone Tissue Engineering
Garima Tripathi 1 Ashutosh Sharma 2
1Indian Institute of Technology Kanpur India2Indian Institute of Technology, Kanpur Kanpur IndiaShow Abstract
Bone tissue has dazzling ability of smart adaptation and retort to ever changing mechanical stimuli, which allows self-repairing and remodeling upon limited damages. This is due to its structure that, particularly in long bones, is characterized by a hierarchically organized porosity conferring high strength, elasticity and ability to disperse biomechanical loads down to the smallest trabeculae. The need of structurally complex scaffolds becomes crucial in case of regeneration of long bones, as they have to withstand complex biomechanical loads. Presently, serious diseases involving long segmental bones can be treated only by repeated and painful surgery with the aid of metallic components. However this solution does not allow patients to recover the functionality of their original bones, thus penalizing the quality of life. The present study refers to the preparation and characterization of porous hydroxyapatite scaffolds to be used as matrices for bone regeneration or as specific release vehicles. Ceramics are widely used for bone tissue engineering purposes and in this study, hydroxyapatite porous scaffolds were produced using the polymer replication method. Polyurethane sponges were used as templates and impregnated with ceramic slurry at different ratios, and sintered following a specific thermal cycle. The characteristics of the hydroxyapatite porous scaffolds and respective powder used as starting material, were investigated by using scanning electron microscopy, particle size distribution, X-ray diffraction, Fourier transformed infrared spectroscopy and compressive mechanical testing techniques. It was possible to produce highly porous hydroxyapatite scaffolds presenting micro and macropores and pore interconnectivity. The obtained preliminary results are very promising for further development of scaffolds for segmental bone regeneration.
9:00 AM - H5.13
Fabrication of Biphasic Calcium Phosphate Ceramics/Biodegradable Polymers as Artificial Bone Materials
Yusuke Yanagi 1 Masahiro Yoshizawa-Fujita 1 Yuko Takeoka 1 Mamoru Aizawa 2 Masahiro Rikukawa 1
1Sophia University Tokyo Japan2Meiji University Kanagawa JapanShow Abstract
There has been considerable interest in the use of calcium phosphates, which are principal inorganic constituents of natural bone, as bone substituents and scaffold materials for bone tissue engineering. Recently, composites of biodegradable polymers and calcium phosphate ceramics have been applied for artificially-grafting materials. These composites are expected to improve the poor mechanical properties of calcium phosphate ceramics by mixing flexible biodegradable polymers, and also encourage bone re-mineralization with biodegradation of polymers. In this study, we tried to fabricate biphasic calcium phosphate (BCP) ceramics containing hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP) which induces osteoblast cells. In addition, the composites of BCP ceramics and poly(L-lactic acid) (PLLA) which is most promising biodegradable polymer for artificial bone materials were fabricated.
BCP ceramics containing HAp and β-TCP were fabricated by sintering mixtures of fibrous HAp (f-HAp) and DNA. The porosity and HAp/β-TCP ratios of the BCP ceramics were controllable by the ratios of f-HAp and DNA, and increased with increase of additive amount of DNA. Scanning electron microscopy measurements showed that the BCP ceramics had interconnected macro and micro porous. The pore size of macro and micro pores of BCPs were about 20-100 µm and 1 µm, respectively. The bending strengths and elastic moduli of HAp and BCP ceramics were in the range of 7.4-59 MPa and 0.71-22 GPa, respectively. PLLA was synthesized in HAp or BCP ceramics by bulk polymerization to fabricate PLLA/HAp or PLLA/BCP composites without using any additional catalyst. When the polymerization reactions were carried out for 7 days at 130 °C, the weight-average molecular weights (Mw) of PLLA obtained in HAp and BCP ceramics were about 6,700-8,500 g mol-1 with the monomer conversion rate of about 100%. The introduction ratios of PLLA in the composites were in the range of 16-30 wt%. The crystallinity of PLLA was about 10%. The mechanical strength of the composites was increased by the hybridization with PLLA, and the bending strengths and elastic moduli of PLLA/HAp and PLLA/BCP composites were 32-66 MPa and 6.4-18 GPa, respectively. Biocompatibility of BCP ceramics were also evaluated by using osteoblast like MC3T3-E1 cell. BCP and TCP ceramics showed better cell proliferation than porous HAp ceramics.
9:00 AM - H5.14
Preparation of Biodegradable Polymer Nanofibers and Their Applications for Biomaterials
Yuri Shiomura 1 Masahiro Yoshizawa-Fujita 1 Yuko Takeoka 1 Masahiro Rikukawa 1
1Sophia University Tokyo JapanShow Abstract
Recently, many approaches have been taken effort to regenerate tissues by proliferating stem cells in artificial scaffolds instead of damaged extra cell matrix in regenerative medical fields. Biocompatibility, biodegradability, mechanical properties, and porous structures are required for the scaffold materials. To prepare the scaffolds having these properties, two approaches using poly(L-lactic acid) (PLLA) were examined in this study.
First, in order to create high-porosity scaffolds for bone tissue engineering, PLLA/nanoparticle hydroxyapatite (PLLA/HAp) composite fibers were prepared by the electrospinning technique. PLLA (Mw=300,000) was dissolved in trifluoroethanol to obtain viscous solution, and n-HAp (200 nm diameter) was added to the solution with the PLLA/HAp composite weight ratio of 1:1. The high viscous solution obtained was electrospun at a high input voltage of 30 kV to create nanofibers. In the same way, PLLA fibers were prepared. The scanning electron microscopy (SEM) results showed that the average fiber diameter of the PLLA/HAp composites and the PLLA fibers were 673 nm and 1037 nm, respectively. The porosity of both the fiber mats was about 82%. Tensile tests showed the mechanical strength and elongation of the PLLA/HAp composite fibers and the PLLA fibers were 3.7 MPa, 87%, and 5.2 MPa, 163%, respectively. To evaluate biomineralization propereties, both of the fiber mats were soaked in simulated body fluid (SBF). After soaked in SBF, the deposition of calcium phosphate was observed for PLLA/HAp composite fiber mats, although PLLA fiber mats did not show biomineralization. Cell culture tests were examined with osteoblast-like MC3T3-E1 cells. PLLA/HAp and PLLA fiber mats showed suitable cell adhesion and proliferation, independent of the addition of HAp.
As the second approach, synthesis of PLLA based diblock copolymers including catechol units was examined to improve cell adhesion. The chain end of PLLA obtained by ring-opening polymerization of L-lactide with Tin (II) octoate was modified with a RAFT agent, followed by the RAFT polymerization of N-(3,4-dihydroxyphenethyl) methacrylamide (DOPMA) having catechol groups. The cell culture tests using osteoblast-like MC3T3-E1 cells showed that initial adhesion rates on the diblock copolymers were higher than that on PLLA. Phase separation of these polymers was also investigated.
9:00 AM - H5.15
In Vitro Examination of Poly(glycerol sebacate) Degradation Kinetic: Effects of Porosity and Cure Temperature
Nadia Krook 1 Courtney LeBlon 2 Sabrina Jedlicka 1 3 4
1Lehigh University Bethlehem USA2Lehigh University Bethlehem USA3Lehigh University Bethlehem USA4Lehigh University Bethlehem USAShow Abstract
Poly(glycerol sebacate) (PGS) is a biodegradable and biocompatible elastomer that has been used in a wide range of biomedical applications, including drug delivery, microfluidic devices, and tissue engineering scaffolds. The material possesses similar mechanical properties to those of soft body tissues and is mechanically tunable by altering cure temperature. An increased cure temperature correlates to an increased amount of cross-linking, resulting in a greater elastic modulus. While a porous format is common for scaffolds, to allow cell ingrowth, PGS degradation has been primarily studied in a nonporous format. The purpose of this research was to investigate the degradation of porous PGS at three frequently used cure temperatures: 120°C, 140°C, and 165°C. The thermal, chemical, mechanical, and morphological changes were examined using thermogravimetric analysis, differential scanning calorimetry, Fourier transform infrared spectroscopy, compression testing, and scanning electron microscopy. Over the course of the 16-week degradation study, the samples&’ pores collapsed. The specimens cured at 120°C demonstrated the most degradation and became gel-like after 16 weeks. Thermal changes were most evident in the 120°C and 140°C cure PGS specimens, as shifts in the melting and recrystallization temperatures occurred. Porous samples cured at all three temperatures displayed a decrease in compressive modulus after 16 weeks. This in vitro study helped to elucidate the effects of porosity and cure temperature on the biodegradation of PGS and will be valuable for the design of future PGS scaffolds.
9:00 AM - H5.16
Synergistic Action of Pancreatic Iselts and Human Adipose-Derived Stem Cells in Fibrin Gel to Cure Type 1 Diabetes Mellitus
Yong Hwa Hwang 1 Suk Ho Bhang 2 Min Jun Kim 1 Yong-kyu Lee 3 Byung-Soo Kim 2 Dong Yun Lee 1
1Hanyang University Seoul Republic of Korea2Seoul National University Seoul Republic of Korea3Korea National University of Transportation Chungju Republic of KoreaShow Abstract
While subcutaneous tissue has been proposed as a potential site for pancreatic islet transplantation, concern remains that the microvasculature of subcutaneous tissue is too poor to support transplanted islets. In an effort to overcome this limitation, we evaluated whether fibrin gel with human adipose-derived stem cells (hADSCs) and rat pancreatic islets could cure diabetes mellitus when transplanted into the subcutaneous space of diabetic mice. Subcutaneously co-transplanted islets and hADSCs showed normalization of the diabetic recipient&’s blood glucose levels. The result was enhanced by co-treatment of fibroblast growth factor-2 (FGF2) in the fibrin gel. The hADSCs enhanced islet viability after transplantation by secreting various growth factors that can protect islets from hypoxic damage. Afterward, hADSCs could maintain islet viability by recruiting new microvasculature nearby the transplanted islets via overexpression of vascular endothelial growth factor (VEGF). The hADSCs did not directly differentiate into endothelial cells (no detection of biomarkers of human endothelial cells), but showed evidence of differentiation toward insulin-secreting cells (detection of human insulin). Mice receiving islet transplantation alone did not become normoglycemic. Collectively, co-transplantation of fibrin gel with islets and hADSCs will expand the indications for islet transplant therapy and the potential clinical application of cell-based therapy.
9:00 AM - H5.17
Preparation and Characterization of Electrospun Biodegradable Polyurethane Scaffold for Cardiac Repair
Tzu-Hsiang Shih 1 Wei-Fang Su 1 2
1National Taiwan University Taipei Taiwan2National Taiwan University Taipei TaiwanShow Abstract
Heart failure is a major cardiovascular disease with high mortality. Via the application of cardiac tissue engineering, damaged myocardium can be replaced by cardiac scaffold. The cardiac scaffold can not only enhance cardiac function but also improve cardiac remodeling. In cardiac tissue engineering, scaffolds must be porous, resilient, biodegradable, biocompatible and similar mechanical properties matching with native tissue. Polyurethane have been considered good candidates with its elasticity and toughness for utilizing in cardiac tissue engineering. In this study, we have synthesized biodegradable polyurethane from polycaprolactone diols (PCL), isophorone diisocyanate (IPDI) and 1,4-diaminobutane (DAB) by reacting PCL with IPDI first then with DAB. The chemical structures of synthesized polyurethanes were confirmed by NMR and their molecular weight was determined by GPC. Their thermal, mechanical, and physical properties were also investigated to exhibit high elasticity and strength. Polyurethanes were further fabricated into porous scaffolds by electrospinning using dimethylformamide(DMF) as a solvent. The MTT cytotoxicity and cell growth of scaffolds are currently under investigation. This type of electrospun biodegradable polyurethane scaffold has potential for the repair of damaged heart tissue.
9:00 AM - H5.18
Novel Absorbable Polyurethane Biomaterials and Scaffolds for Tissue Engineering
Alarbi M. Emmakah 1 Andrew J. Luzzi 2 Rao S. Bezwada 3 Syam Nukavarapu 1 2 4
1University of Connecticut Storrs USA2University of Connecticut Farmington USA3Bezwada Biochemical, LLC Hillsborough USA4University of Connecticut Farmington USAShow Abstract
The quest for new biomaterial design is warranted to meet scaffolding needs in tissue engineering. Polyurethanes (PUs) are an important class of biomaterials developed in the past primarily for biostable biomedical devices, and currently in the form of biodegradable scaffolds for tissue engineering and regenerative medicine applications. The main drawback of PU polymers is the lack of complete degradation. The aim of this study is to introduce a new class of polyurethanes that show hydrolytic degradation by not only the soft segments, but also the hard segments of these block co-polymers. Aromatic isocynates are commonly used in biodegradable polyurethane synthesis, however; PUs synthesized using methylene diphenyl diisocynate (MDI) only yielded partially bio-absorbable polymers. We designed a new family of polyurethanes comprising of aromatic diisocynate similar to MDI, but the non-degradable methylene linkage in MDI is replaced with units derived from absorbable, safe, and biocompatible glycolic acid, lactic acid, p-dioxazones, caprolactone monomers. In addition, the mechanical strength and degradation rate for the new generation of polyurethanes are tunable based on the degradable linkage between the two aromatic rings of the isocynate. For this study, a polymer was synthesized from absorbable isocynate and polycaprolactone (MW 2000) via melt polymerization. The polymer was solvent casted and bored into polymer films for biocompatibility evaluation. Polymer films seeded with human bone marrow derived stromal cells establish cell compatibility of the newly designed polyurethane. In order to develop a three-dimensional and porous structure, a novel scaffold fabrication method, Dynamic Solvent Sintering, was applied for the first time to polyurethanes. In this method, the polymer was processed to obtain microparticles, and then those particles were brought together into three-dimensional and porous structures using tetrahydrofuran and n-Hexane as solvent/non-solvent, respectively. Microstructural studies reveal particle-particle bridging, and the effect of solvent, non-solvent composition on the degree of sintering. In conclusion, this study establishes a new polyurethane biomaterial that is fully absorbable for tissue engineering applications. The study further establishes a polyurethane scaffold with potential applications in bone and osteochondral tissue engineering.
9:00 AM - H5.19
In-Situ Forming, Thermogelling Chitosan-Hyaluronic Acid Based Injectable Hydrogels Prepared via Ionic/Covalent Co-Crosslinking for the Encapsulation of Live Cells
Ghulam Jalani 1 Lisbet Haglund 2 Fiorenzo Vetrone 3 Marta Cerruti 1
1McGill University Montreal Canada2McGill University Montreal Canada3Universitamp;#233; du Quamp;#233;bec Varennes CanadaShow Abstract
In-situ forming, injectable hydrogels are important class of biomaterials widely used for biomedical applications. The ideal hydrogel system would contain bioactive, nontoxic and biodegradable materials crosslinked with nontoxic covalent crosslinkers with fast gelling ability. Furthermore, it would maintain physiological pH and remain injectable solution at room temperature to encapsulate live cells while convert to gel at body temperature within short time. To combine all these characters we hereby report a facile strategy to produce injectable, in-situ forming hydrogels from chitosan and hyaluronic acid interpenetrating networks, crosslinked via ionic and covalent co-crosslinking with considerably short gelation time. Chitosan solution prepared in hydrochloric acid and Dulbecco's Modified Eagle Medium (DMEM) in a volume ratio of 1:1, was neutralized with β-glycerophosphate followed by mixing with hyaluronic acid solution prepared in DMEM. Genipin solution prepared in ethanol was then added to the above solution. The mixed solutions were incubated at 37 degree C to prepare the gels. This scheme overcomes the problem of precipitation of chitosan at neutral pH. Interestingly hyaluronic acid can be mixed easily with chitosan solution neutralized previously with β-glycerophosphate without the precipitation of chitosan or hyaluronic acid. The gelation time of these hydrogels is quite short as compared to the gels crosslinked with only genipin. This results in highly elastic, non-toxic, covalently crosslinked, thermogelling and injectable hydrogels at physiological conditions. This scheme does not need the modification of chitosan or hyaluronic acid, contains no photoinitiator or toxic crosslinker but still produces tough, elastic and biocompatible hydrogels with short gelation times. Nucleolus populous (NP) bovine cells could be dispersed homogenously inside the gels. The cells remained alive and metabolically active in the gels containing different combinations of chitosan and hyaluronic acid co-crosslinked with β-glycerophosphate and genipin. The prominent properties like biactivity, covalent crosslinking, better elasticity, short gelation time and ability to encapsulate the live cells at physiological conditions make these hydrogels a desirable choice for tissue engineering applications.
9:00 AM - H5.20
The Influence of Thickness on Differentiation of Dental Pulp Stem Cells Cultured on Polyisoprene and Gutta Percha Films
Liudi Zhang 1 Christopher Joubert 2 Alexa Aseel-Fine 4 Chung-Chueh Chang 3 Yingjie Yu 1 Marcia Simon 2 Miriam Rafailovich 1
1Stony Brook University Stony Brook USA2Stony Brook University Stony Brook USA3Stony Brook University Stony Brook USA4Jericho High School Jericho USAShow Abstract
Dental pulp stem cells (DPSC) have been used in many studies for biomedical application. Chemical differentiation into specific tissue requires special media i.e. high glucose or high oxygen (20%) (Blaine T. Mischen, M.D. Plast. Reconstr. Surg. 122: 725, 2008.). These conditions are nearly impossible to reproduce in vivo. Therefore, we should find a trigger which induces differentiation without globally changing the chemical or metabolic environment. Our previous studied indicated that polybutadiene substrates of moduli higher than 2.5MPa induced upregulation of dental scialoprotein (DSP) gene expression and deposition of crystalline hydroxy apatite deposits without other soluble induction agents, such as dexamethasone or BMP-2. Here we present an investigation of another class of elastomers, polyisoprene (PI) and ZnO filled PI, commonly known as Gutta Percha, which a material currently used in endodontic practice can also be potentially used in regeneration of the pulp, rather than obduration of the canal.
To study the effect of substrate mechanics on DPSC function and differentiation, we dissolved PI in toluene and spun cast into films with different thickness (from 15 to 200nm), on hydrofluoric acid etched Si wafers. These substrates were then annealed at 170°C to remove residual solvent and stress in a high vacuum oven. Shear force atomic microscope (SMFM) was used to determine the influence of thickness on relative modulus of PI. Substrates were also produced by spin casting films approximately 150nm thick of three commercial brands of Gutta Percha: Calamus, Protaper, and Lexicon. DPSCs were then seeded on these substrates at an initial density of 5,000/cm2 and assayed for proliferation up to 14 days and differentiation at 28 days. Results from Samples, where 10 nM dexamethasone (DEX) was added to induce differentiation were compared to those without induction factors. The results indicated that DPSC proliferated on all “hard” films; thin PI films, and following an initial three day lag period, on all the Gutta Percha films doubling times comparable to the control samples on TCP. Much longer doubling times were observed on the soft PI films. The mechanics of DPSC on different thickness PI were tested by SMFM at day 4 and day7, and in the absence of DEX, were found to follow substrate mechanics. At day 28, HA biomineralized deposits, as well as upregulation of osteocalcin and ALP were observed only on ‘Hard, G>2.5 MPa” films. Further testing is planned to confirm whether these results are also applicable in vivo.
9:00 AM - H5.21
Control of Cell Adhesion and Detachment on Temperature-Responsive Block Copolymer Langmuir Films
Morito Sakuma 1 2 Yoshikazu Kumashiro 2 Masamichi Nakayama 2 Nobuyuki Tanaka 2 Kazuo Umemura 1 Masayuki Yamato 2 Teruo Okano 2
1Tokyo University of Science Tokyo Japan2Tokyo Women's Medical University Tokyo JapanShow Abstract
Our laboratory has investigated temperature-responsive poly(N-isopropylacrylamide) (PIPAAm)-grafted surfaces for controlling cell adhesion and detachment, which was able to be modulated by the densities, molecular weight, and molecular composition of the polymer. This study developed a Langmuir-Schaefer (LS) method for fabricating PIPAAm surfaces of which the properties were accurately designed. LS surfaces of polystyrene-block-PIPAAm (PSt-PIPAAm), which was synthesized by a reversible addition-fragmentation chain transfer polymerization, were fabricated at an air-water interface, and their surface properties were characterized. In addition, cell adhesion and detachment on the surface in response to temperature was also evaluated. PSt-PIPAAm Langmuir film was produced at an air-water interface using a Langmuir-trough apparatus. A PSt-PIPAAm chloroform solution was dropped on an air-water interface, and the surface pressure of the interface was measured by a Wilhelmy plate during squeezing the Langmuir film with barrier plates. The phase of Langmuir films was estimated by plotting their surface pressure-area (πminus;A) isotherms, and the density of the film was regulated by the position of barrier plates. After being compressed and horizontally transferred on a hydrophobically-modified glass substrate, PSt-PIPAAm Langmuir film was obtained and the surfaces were analyzed by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), attenuated total reflectance Fourier transfer infrared spectroscopy (ATR/FT-IR), and ellipsometric measurement. In the πminus;A isotherms of PSt-PIPAAm Langmuir films at an air-water interface, at the initial compression, Langmuir film having larger PIPAAm ratio in PSt-PIPAAm was reached to a liquid-to-solid phase with a larger occupied area, indicating that the chain entanglements among PSt-PIPAAm molecules with a larger PIPAAm ratio were stronger than those of other PSt-PIPAAms. After the slope of πminus;A isotherms after approximately 30 mN/m, the second phase transition from liquid phase to solid phase was observed. In particular, Langmuir film having smaller PIPAAm ratio showed the transition at a large area. Since PSt is a water-insoluble and higher rigidity polymer than PIPAAm, the transition of a solid phase should be occurred at a large occupied area in Langmuir film having the higher composition ratio of PSt. Cell adhesion was observed at 24 h after incubation at 37 °C on the PSt-PIPAAm LS surfaces as well as on a conventional tissue culture polystyrene surface. On the other hand, the rate of cell detachment was found to be enhanced on the PSt-PIPAAm with increasing PIPAAm ratio by reducing temperature to 20 °C. The mechanism underlying cell adhesion and detachment on the LS surfaces will be discussed in the symposium.
9:00 AM - H5.22
Biofunctional Thermo-Responsive Polymeric Surface with Micropatterns for Label Free Cell Separation
Yoshikazu Kumashiro 1 Jun Ishihara 1 2 Terumasa Umemoto 1 Kazuyoshi Itoga 1 Jun Kobayashi 1 Masayuki Yamato 1 Teruo Okano 1
1Tokyo Women's Medical University Tokyo Japan2University of Tokyo Tokyo JapanShow Abstract
Thermo-responsive polymeric surfaces can allow adhered cells and cell sheet to be spontaneously recovered by lowering temperature from normal condition (37 degrees) without proteolytic enzymes and this technology allows us to transplant cell sheets to host tissues without using biodegradable scaffolds. In this study, striped-polyacrylamide (PAAm) patterns were immobilized a thermo-responsive surface for controlling cell adhesion and proliferation as a next generation of biomaterials. A thermo-responsive surface with spin-coated a positive photo-resist was irradiated through 3 mu;m/3 mu;m black and white striped photomask, followed by the development using a developer solvent. Then, the radical polymerization of hydrophilic AAm was performed on the photoirradiated area, followed by the removal of a residual photo-resist. The stripe-patterned surface was clearly observed by a stain using Alexa-488 bovine serum albumin at 37 degrees in 1M phosphate buffered saline and a phase contrast image of an atomic force microscope in an air condition. Prepared stripe-patterned surface as cell separating intelligent interfaces were characterized by observing the adhesion and detachment of three types of cells: HeLa cells (HeLas), human umbilical vein endothelial cells (HUVECs), and NIH-3T3 cells (3T3s). Although cell adhesion and detachment in response to temperature were observed for all cells on a PIPAAm surface without patterns, PIPAAm surface with 3 mu;m striped-patterns exhibited different cell adhesion properties. HeLas hardly adhered on the stripe-patterned surface even at 37 degrees. On the other hand, HUVECs adhered on the stripe-patterned surface until 12 h incubation at 37 degrees, but the adhered HUVECs detached themselves through the next 12 h incubation at 37 degrees. And 3T3s adhered on the stripe-patterned surface and adhered 3T3s detached themselves after reducing temperature to 20 degrees as well as on a PIPAAm surface without patterns. Utilizing the different cell adhesion properties, a mixture of HeLa, HUVEC and 3T3 was separated determined by a flow-cytometry. After being allowed to adhere on the stripe-patterned surfaces at 37 degrees for 24 h, the adhered cells on the surfaces were incubated at 20 degrees. In the initial period of incubation at 37 degrees for 12 h, repelled HeLas were recovered from the stripe-patterned surface. After the additional incubation at 37 degrees for 12 h, HUVECs were released and recovered. Finally, 3T3s were released and recovered after reducing temperature to 20 degrees. These results indicated that a PIPAAm surface with striped-patterns functioned as a cell-separating interface by utilizing the intrinsic cell adhesion properties of individual cells. In conclusion, the stripe-patterned surface has a potential for breakthrough in the next generation of biomaterials.
9:00 AM - H5.24
Preliminary Investigation of a Sacrificial Process for Fabrication of Polymer Membranes with Sub-Micron Thickness
Luke Armitage Beardlsee 1 Dimitrius A. Khaladj 1 Magnus Bergkvist 1
1University at Albany (SUNY) Albany USAShow Abstract
Fabrication of sub-micron thin films of biodegradable plastics having well-defined through-holes for use as tissue engineering scaffolds or cell culture substrates represent a challenge. Many commonly used biomaterials are not directly photopatternable so soft-lithography molding is commonly used; however the demolding approach can cause thin polymer structures to tear, which limits the minimum thickness of the materials (typically 5-15 µm).
Here we present a single mask, sacrificial mold process that allows ultrathin 2-dimensional membranes to be fabricated using biocompatible polymeric materials. For initial investigations polycaprolactone (PCL) was chosen as a model material. Here we demonstrate that the process is capable of creating 250-500 nm thin, through-hole PCL membranes with various pore-sizes and spatial features down to 2.5 micrometers using contact photolithography.
The technique uses a mold created from two layers of lift-off resists. The upper layer is patterned, while the lower layer acts as a sacrificial release layer for the polymer membrane. Photoresist on top of the layers of lift-off resist is patterned using conventional photolithography. During development the mask pattern is transferred onto the first sacrificial layer and the photoresist is removed using acetone, leaving behind a thin mold. The mold is filled with a solution of the desired polymer. Subsequently, both the patterned and lower LOR layers are dissolved by immersion in a basic aqueous solution. This process gently releases the patterned thin polymer membrane with no strenuous forces applied to the structure. The membrane can be recovered post-release or mounted onto support structures pre-release to facilitate handling.
In the iteration of the process presented here, SC1813 photoresist is used along with either LOR 3A or LOR 10A baked at 150 C as the top-most layer. The lower sacrificial layer is PMGI SF9 photoresist (Microchem, Newton MA). The PMGI is baked at 200 C, and dissolves approximately 5 times slower than LOR. A spin coating step is used to fill the mold with polymer. For these experiments a 0.025 g/ml PCL in toluene solution was coated at rates varying between 1000-2000 rpm. The PCL was then reflowed at 95 C to ensure that the mold is filled evenly. The polymer membranes are then released in the time frame of 30 min by placing the molds in alkaline solution (i.e., photoresist developer). We demonstrate fabrication of various pattern designs and show that the membranes can be mounted to a fixture, with either PCL or SU8 as an adhesive, and then released. Endothelial cell culture on 300 nm thin, through-hole membranes is also demonstrated.
9:00 AM - H5.25
Prevention of the Growth of Pseudomonas Aeruginosa through the Use of Ceria Nanoparticles
Qi Wang 1 Thomas Jay Webster 2 J. Manuel Perez 3
1Northeastern University Boston USA2Northeastern University Boston USA3University of Central Florida Orlando USAShow Abstract
Although ceria nanoparticles have been studied for numerous traditional science and engineering applications, there have been almost no studies regarding their potential use in biomedical applications until recently when researchers demonstrated that ceria nanoparticles possess antioxidant properties at physiological pH values. The ability of these nanoparticles to act as an antioxidant lies in their ability to reversibly switch from Ce3+ to Ce4+. Moreover, there have been almost no studies focusing on their possible antibacterial properties despite the fact that such nanoparticles may alter the presence of reactive oxygen species to kill bacteria. In this study, we coated ceria nanoparticles with dextran or polyacrylic acid to minimize its potential toxicity and clearance by the immune system. The coated ceria nanoparticles were then tested in bacterial assays involving Pseudomonas aeruginosa, one of the most significant bacteria responsible for infecting numerous medical devices. The results showed that ceria nanoparticles with either coating significantly inhibited the growth of Pseudomonas aeruginosa by up to 55.14% after 24 hours compared with controls (no particles). Inhibition of bacterial growth was concentration dependent. In summary, this study revealed for the first time that the ceria nanoparticles could be a potential novel material for numerous antibacterial applications.
9:00 AM - H5.27
Fabrication and Morphological Investigation of Multi-Walled Electrospun Polymeric Nanofibers
Jamal Seyyed Monfared Zanjani 1 Burcu Saner Okan 2 Mehmet Yildiz 1 Yusuf Menceloglu 1
1Sabanci University Istanbul Turkey2Sabanci University Istanbul TurkeyShow Abstract
Outstanding properties of nanofibers with layered design make them promising materials for different area of applications ranging from self-healing materials, drug delivery systems, biosensors, and many other state-of-the-art technologies. This research has investigated fabrication of tri-axial structured electrospun polymeric nanofibers. The process is based on a nozzle that allows for tri-axial extrusion of three different fluids, which consists of concentric inner, middle and outer tubes. Two spinnable polymers for middle and outer walls of nanofibers, poly (methyl methacrylate) and polyacrylamide, respectively, and low molecular weight polyethylene glycol (Mw=300), with low spinnability properties for the inner part were chosen. Hansen Solubility parameters were used to systematic optimization of solvent selecting process for each layer. Degree of miscibility of each solution, polymer solution concentration, polymer molecular weight, dielectric constant, solution vapor pressure, interfacial tension between each layer, applied voltage, electrospinning distance, and flow rate ratio effects on morphology of resultant nanofibers and Taylor cone formation were studied. Characterization was done using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), thermo gravimetric analysis (TGA) and differential scanning calorimetry (DSC).