Kyriacos A Athanasiou
University Of California Davis
Project start date: 2010-05-05
Project end date: 2014-02-28
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Kyriacos A Athanasiou
TOWARD TISSUE ENGINEERING OF THE KNEE MENISCUS
Kyriacos A Athanasiou, Professor And Chair
University Of California Davis, Office Of Research - Sponsored Programs, Davis, Ca 95618
Grant 2R01AR047839-06 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases
Abstract: This renewal proposal aims to tissue engineer an anisotropic meniscus construct that also captures the regional variations present in the native tissue. Subsequently, the construct will be implanted in a leporine model, using both allogenic and xenogenic cell sources, to achieve both meniscus repair and replacement. It is hypothesized that 1) regionally variant, anisotropic, meniscus-shaped constructs can be engineered by optimizing cell culture and scaffoldless culture conditions; 2) the synergistic and strategic temporal application of certain anabolic and catabolic stimuli will enhance the functional properties of the maturing meniscus construct; and 3) both allogenic and xenogenic constructs can be successfully implanted in a leporine model. These hypotheses will be tested via the following three specific aims 1) to create an anisotropic meniscus construct with regional variations mimicking native tissue, 2) to enhance functional and organizational properties of constructs via synergistic, temporally coordinated exogenous stimulation, and 3) to develop tissue-construct surgical fixation techniques and implant allogenic and xenogenic constructs in the rabbit. In the previous grant, the native meniscus was found to be highly anisotropic and regionally variant both morphologically and biomechanically, motivating our current tissue engineering approach to mimic these characteristics. Aim 1 will thus use co-cultures, meniscus-specific molds, and novel seeding techniques to accomplish this goal. Also identified in the previous grant were stimuli that had positive effects on a scaffold- based approach, but in parallel studies, it was clearly demonstrated that a scaffoldless approach, driven by the differential adhesion hypothesis, was superior. Aim 2 of this proposal will use such a scaffoldless approach, in conjunction with hydrostatic pressure, tension-compression, well confinement time, TGF-1, hypoxia, and chondroitinase-ABC to create tissue engineered constructs. Furthermore, the previous grant underscored the scarcity of meniscus cells that could be used in an in vivo study. Thus, in this proposal, to avoid the use of primary cells, we will investigate the use of passaged allogenic cells and, for the first time, a xenogenic cell source in the in vivo repair and replacement of the meniscus (Aim 3). By examining both allogenic and xenogenic cell sources, this proposal seeks to obviate the issue of tissue scarcity (either autologous or allogenic meniscus and cartilage) and aims to provide a solution to the complex problem of meniscus regeneration. Establishing the means to tissue engineer and implant meniscus tissue would bode well for over one million Americans that undergo meniscal procedures annually. However, the highly anisotropic mechanical properties and morphological regional variance observed in native tissue render recapitulating these structure/function relationship a complex problem. The current tissue engineering approach seeks to mimic these characteristics to produce anisotropic, inhomogeneous allogenic and/or xenogenic constructs that can restore the functional properties of the knee meniscus. Successful completion of this proposal will establish a framework for future scaffoldless meniscus regeneration attempts using alternate cell sources for the repair and replacement of meniscus tissue
Keywords: Adhesions; Allogenic; American; Assay; Autologous; Autologous Fibrin Tissue Adhesive; BMP-2; BMP-2A; BMP2; Bioassay; Biochemistry; Biologic Assays; Biological Assay; Blood Serum; Body Tissues; Bovine Species; Cartilage; Cartilagenous Tissue; Cattle; Cell Attachment; Cell Culture Techniques; Cell-Extracellular Matrix; Cell-Matrix Adhesions; Cell-Matrix Junction; Cell/Tissue, Immunohistochemistry; Cells; Characteristics; Chemicals; Chemistry, Biological; Chondrocytes; Chondroitin ABC Lyase; Chondroitinase ABC; Clinical; Co-culture; Cocultivation; Coculture; Coculture Techniques; Cold-Insoluble Globulins; Collagen; Complement S-Protein; Complex; Deposit; Deposition; Drug Formulations; ECM; Engineering; Engineerings; Epibolin; Extracellular Matrix; FN1; FNZ; Fibrin Adhesive; Fibrin Glue; Fibrin Sealant; Fibrin Sealant System; Fibrin Tissue Adhesive; Fibrinogen Adhesive; Fibronectin 1; Fibronectins; Fixation; Formulation; Formulations, Drug; Fungi, Filamentous; Future; GAG; GAG Gene; Generalized Growth; Goals; Grant; Growth; Histology; Hydrostatic Pressure; Hypoxia; Hypoxic; IGF-1; IGF-I; IGF-I-SmC; IGF1; IHC; Immunohistochemistry; Immunohistochemistry Staining Method; Implant; Insulin-Like Growth Factor 1; Insulin-Like Growth Factor I; Insulin-Like Somatomedin Peptide I; Knee; LETS Proteins; Large External Transformation-Sensitive Protein; MR Imaging; MR Tomography; MRI; Magnetic Resonance Imaging; Magnetic Resonance Imaging Scan; Mammals, Rabbits; Mechanical Stimulation; Mechanics; Medial; Medical Imaging, Magnetic Resonance / Nuclear Magnetic Resonance; Meniscus; Meniscus structure of joint; Methods; Methods and Techniques; Methods, Other; Microscopy, Electron, Scanning; Modeling; Molds; NMR Imaging; NMR Tomography; Natural regeneration; Nuclear Magnetic Resonance Imaging; Operation; Operative Procedures; Operative Surgical Procedures; Opsonic Glycoprotein; Opsonic alpha(2)SB Glycoprotein; Oryctolagus cuniculus; Oxygen Deficiency; Performance; Phase; Procedures; Property; Property, LOINC Axis 2; Protocols, Treatment; RGM; Rabbit, Domestic; Rabbits; Regeneration; Regimen; Relaxation; Serum; Serum Spreading Factor; Shapes; Silk; Solid; Solutions; Somatomedin C; Source; Stimulus; Structure-Activity Relationship; Surgical; Surgical Interventions; Surgical Models; Surgical Procedure; Surgical sutures; Sutures; Techniques; Technology; Testing; Time; Tissel; Tissue Engineering; Tissue Growth; Tissues; Treatment Protocols; Treatment Regimen; Treatment Schedule; VTN; Variant; Variation; Vitronectin; Zeugmatography; alpha 2-Surface Binding Glycoprotein; base; bone morphogenetic protein 2; bovid; bovine; chemical structure function; combinatorial; cow; design; designing; engineered tissue; fetal bovine serum; in vitro testing; in vivo; isopropylidene; mimetics; monolayer; novel; ontogeny; propene; propylene; public health relevance; regenerate; repair; repaired; sample fixation; scaffold; scaffolding; structure function relationship; success; surgery; tisseel
Relevance: Establishing the means to tissue engineer and implant meniscus tissue would bode well for over one million Americans that undergo meniscal procedures annually. However, the highly anisotropic mechanical properties and morphological regional variance observed in native tissue render recapitulating these structure/function relationship a complex problem. The current tissue engineering approach seeks to mimic these characteristics to produce anisotropic, inhomogeneous allogenic and/or xenogenic constructs that can restore the functional properties of the knee meniscus. Successful completion of this proposal will establish a framework for future scaffoldless meniscus regeneration attempts using alternate cell sources for the repair and replacement of meniscus tissue
Project start date: 2009-09-18
Project end date: 2011-08-31
Budget start date: 18-SEP-2009
Budget end date: 31-AUG-2010
PFA/PA: PA-07-070
2R01AR047839-06 (2009): $377921
Kyriacos A Athanasiou, Karl F. Hasselmann Professor
Rice University 6100 S Main Houston, Tx 770051892
Grant 5R01DE015038-03 from National Institute Of Dental And Craniofacial Research IRG: ZRG1
Abstract: The application s long-term objective is to use a comprehensive tissue reconstruction approach to successfully address regeneration of the temporomandibular joint (TMJ) disc. There is a clear need to regenerate the TMJ disc to alleviate the need for discectomy in severe cases of disc displacement. Seventy percent of patients with temporomandibular disorders suffer from disc displacement, which can result in disc degeneration and/or perforation and can be manifested in jaw clicking, locking and severe pain. The chief hypothesis of our study is that we can regenerate a TMJ disc construct comparable to the native disc by the use of a peptide-modified, biodegradable scaffold with the appropriate combination of growth factors, cells and mechanical stimuli with enhanced diffusion. To test this hypothesis, we propose the following specific aims 1) To characterize the TMJ disc at the tissue level, 2) to describe the TMJ disc at the cellular level and 3) to engineer the TMJ disc in vitro. This comprehensive study will lay the groundwork for future in vivo studies to replace damaged TMJ discs where implants have failed. Furthermore, since the TMJ disc is a poorly understood tissue, collectively the proposed studies will provide broad and detailed knowledge of structure-function properties of the normal TMJ disc. This vital information will allow the definition of design and validation criteria to engineer disc constructs. To characterize the disc at the tissue level, native porcine TMJ discs will be examined to determine ultrastructure, biomechanical properties under tension and compression, and biochemical content and organization. To describe the disc at the cellular level, cellular topography will be elucidated, subpopulations will be identified, and cells will be cultured on two dimensional poly(propylene fumarate-co-ethylene glycol)-GRGD surfaces where proliferation and biosynthesis will be measured with varied growth factors present. Once the native disc is characterized, an engineered disc will be created using the optimal growth factors incorporated within the scaffold in the shape of a native disc, using a combination of a rotating bioreactor, intermittent hydrostatic pressure and direct compression/tension. At various time points, properties of the engineered constructs will be compared to the determined native disc properties. The proposed research represents a novel approach to regenerate the TMJ disc in that we intend to first perform the necessary characterization studies and then use cell-seeded scaffolds, bioactive factors, mechanical signals and enhanced diffusion to facilitate disc regeneration.
Keywords: biodegradable product, growth factor, peptide, regeneration, temporomandibular joint, tissue engineering, tissue support frame, biochemistry, biomechanics, biosynthesis, cell proliferation, mechanical stress, animal tissue, artificial membrane, bioreactor, biotechnology, hydrostatic pressure, immunocytochemistry, in situ hybridization, three dimensional imaging /topography, tissue /cell culture, transmission electron microscopy
Project start date: 2004-07-01
Project end date: 2009-06-30
5R01DE015038-03 (2006): $283608
5R01DE015038-02 (2005): $290433
1R01DE015038-01A2 (2004): $290433
Self-Assembling Process In Tissue Engineering Of Articular Cartilage
Kyriacos A Athanasiou, Karl F. Hasselmann Professor
Rice University 6100 S Main Houston, Tx 770051892
Grant 1R01AR053286-01A2 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: MTE
Abstract: The application s long-term objective is to use a comprehensive bioengineering approach to successfully address articular cartilage regeneration. To this end, a self-assembling process was recently developed to tissue engineer articular cartilage using high density seeding of native chondrocytes in molds made of agarose. The study s main hypothesis is that the self-assembling process can be enhanced synergistically by the addition of growth factors and mechanical stimulation. To test this hypothesis, we propose the following specific aims 1) To determine optimal growth factor conditions for the self-assembling process for in vitro articular cartilage regeneration. 2) To enhance this process using mechanical stimuli. 3) To engineer articular cartilage combining optimal growth factor and mechanical conditions for the self-assembling process. Application of four growth factors (TGF-(31, TGF-(33, BMP-2, and IGF-I) will be implemented in the self-assembling process at varying concentrations and dosage frequencies. The most favorable concentrations and dosage frequencies of each growth factor will be determined and combinations of these will be examined to select the optimal growth factor conditions. The effects of hydrostatic pressure and direct compression will also be examined at varying magnitudes, frequencies, and application times. The best conditions for each mechanical stimulus and their combinations will be selected from the multiple treatments. Finally, the effects of interactions between the optimized growth factor conditions combined with the optimal mechanical stimulation conditions on the self-assembling process will be assessed. At various time points, morphological, biochemical, and biomechanical properties will be quantified and compared to native tissue values, enabling the selection of optimal conditions. More specific hypotheses include 1) The combination of growth factors will exhibit a synergistic effect by improving the self-assembled constructs functional properties approaching two-thirds of native tissue. 2) Interactions between hydrostatic pressure and direct compression will enhance the regeneration process when compared to unloaded controls or constructs under a single mechanical stimulus. 3) The synergistic effects of combined growth factor and mechanical stimulation conditions will yield articular cartilage constructs with combined functional properties approaching those of native articular cartilage.
Keywords: articular cartilage, conditioning, growth factor, tissue engineering, Fungi, bioengineering /biomedical engineering, cell, chondrocyte, collagen, compression, culture, density, dosage, experimental design, extracellular matrix, histology, hyaline substance, hydrostatic pressure, immunocytochemistry, indexing, model, morphology, mucopolysaccharide, regeneration, relaxation, respiratory reflex, scanning electron microscopy, seed, stress, tissue
Project start date: 2007-04-01
Project end date: 2012-02-29
1R01AR053286-01A2 (2007): $277518
Toward Tissue Engineering Of The Knee Meniscus
Kyriacos A Athanasiou, Karl F. Hasselmann Professor
Rice University 6100 S Main Houston, Tx 770051892
Grant 1R01AR047839-01A2 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: ZRG1
Abstract: The application s long-term objective is to use a cogent and comprehensive tissue engineering approach to successfully address meniscus regeneration, which remains one of the most elusive problems in musculoskeletal medicine. The study s main hypothesis is that the meniscus can be regenerated by a series of steps that involve the use of a scaffold with bioactive agents, cells, a bioreactor, mechanical stimuli, and an animal model. To test this hypothesis, we propose the following specific aims 1) To design, fabricate, and characterize meniscus-specific biodegradable scaffolds. 2) To engineer the meniscus. 3) To test the meniscus in an animal model. The methodology involves an analysis and a synthesis phase In the analysis phase, topographical and spatial properties of the meniscus will be identified using biomechanics, biochemistry, ultrastructural methods, and cell culture. The objective is to define gold standard properties against which the properties of the regenerated meniscus will be compared. In the synthesis phase, fibrochondrocytes will be seeded onto meniscus-specific scaffolds and exposed to mechanical forces. The scaffolds, made of poly(propylene fumarate-co-ethylene glycol), are designed to be bioabsorbable, biocompatible, have mechanical integrity, allow for directed attachment of cells through the use of the GRGD peptide, and provide biosynthetic signals through the use of a growth factor. A hydrodynamic focusing bioreactor, operating in a low-shear environment, will then be used to enhance nutrient transport and to modulate mechanical signals. Furthermore, the effects of hydrostatic pressure and direct compression/tension will also be examined using custom-made instruments. At various time points, the properties of the engineered constructs will be quantified and compared to native tissue properties. The tissue engineered constructs will then be implanted in rabbits to evaluate the in vivo functional characteristics of the new meniscus. The clinical significance of the approach described in this proposal is enormous, since meniscal problems continue to be some of the most vexing in orthopaedics.
Keywords: knee, orthopedics, tissue engineering, biochemistry, biomechanics, bioreactor, biotransformation, chondrocyte, joint prosthesis, mechanical pressure, skeletal regeneration, tissue support frame, laboratory rabbit, tissue /cell culture
Project start date: 2002-12-10
Project end date: 2007-11-30
1R01AR047839-01A2 (2003): $273561
Kyriacos A Athanasiou, Professor And Chair
Rice University, 6100 Main, Houston, Tx 77005-1892
Grant 5R01DE015038-05 from National Institute Of Dental & Craniofacial Research
Abstract: The application´s long-term objective is to use a comprehensive tissue reconstruction approach to successfully address regeneration of the temporomandibular joint (TMJ) disc. There is a clear need to regenerate the TMJ disc to alleviate the need for discectomy in severe cases of disc displacement. Seventy percent of patients with temporomandibular disorders suffer from disc displacement, which can result in disc degeneration and/or perforation and can be manifested in jaw clicking, locking and severe pain. The chief hypothesis of our study is that we can regenerate a TMJ disc construct comparable to the native disc by the use of a peptide-modified, biodegradable scaffold with the appropriate combination of growth factors, cells and mechanical stimuli with enhanced diffusion. To test this hypothesis, we propose the following specific aims 1) To characterize the TMJ disc at the tissue level, 2) to describe the TMJ disc at the cellular level and 3) to engineer the TMJ disc in vitro. This comprehensive study will lay the groundwork for future in vivo studies to replace damaged TMJ discs where implants have failed. Furthermore, since the TMJ disc is a poorly understood tissue, collectively the proposed studies will provide broad and detailed knowledge of structure-function properties of the normal TMJ disc. This vital information will allow the definition of design and validation criteria to engineer disc constructs. To characterize the disc at the tissue level, native porcine TMJ discs will be examined to determine ultrastructure, biomechanical properties under tension and compression, and biochemical content and organization. To describe the disc at the cellular level, cellular topography will be elucidated, subpopulations will be identified, and cells will be cultured on two dimensional poly(propylene fumarate-co-ethylene glycol)-GRGD surfaces where proliferation and biosynthesis will be measured with varied growth factors present. Once the native disc is characterized, an engineered disc will be created using the optimal growth factors incorporated within the scaffold in the shape of a native disc, using a combination of a rotating bioreactor, intermittent hydrostatic pressure and direct compression/tension. At various time points, properties of the engineered constructs will be compared to the determined native disc properties. The proposed research represents a novel approach to regenerate the TMJ disc in that we intend to first perform the necessary characterization studies and then use cell-seeded scaffolds, bioactive factors, mechanical signals and enhanced diffusion to facilitate disc regeneration
Keywords: 2-dimensional; Address; Adhesions; Aminoacetic Acid; Anabolism; Animal Model; Animal Models and Related Studies; Arginine; Arginine, L-Isomer; Articular Disk, Temporomandibular; Aspartic Acid; Au element; Biochemical; Biocompatible Materials; Biomaterials; Biomechanics; Bioreactors; Body Tissues; Cell Attachment; Cell Communication and Signaling; Cell Signaling; Cell-Matrix Adhesions; Cell-Matrix Junction; Cells; Chondrocyte-like Cell; Chondroitin Sulfates; Chondroitin, hydrogen sulfate; Collagen; Collagen Fiber; Coupled; Data; Development; Diffusion; Dimensions; Elastin Fiber; Engineering; Engineerings; Exhibits; FGF-2; FGF2; FLR; Face; Failure (biologic function); Family suidae; Fibroblast Growth Factor 2; Fibroblast Growth Factor, Basic; Fibroblasts; Future; GFAC; Gene Expression; Genetics, in situ Hybridization; Glycine; Glycosaminoglycans; Gold; Growth Agents; Growth Factor; Growth Factors, Proteins; Growth Substances; HBGF-2; Heparin-Binding Growth Factor 2; Heparin-Binding Growth Factor Class II; Hydrogels; Hydrostatic Pressure; Implant; In Situ Hybridization; In Vitro; Inferior; Intracellular Communication and Signaling; Jaw; Jaw Joint; Knowledge; L-Arginine; L-Aspartic Acid; Literature; Mandibular joint; Measures; Mechanics; Medial; Microscopy, Electron, Scanning; Monitor; Morbidity; Morbidity - disease rate; Mucopolysaccharides; Musculoskeletal; Natural regeneration; Nutrient; PDGF; Pain; Painful; Patients; Peptides; Perforation; Phase; Pigs; Platelet-Derived Growth Factor; Population; Property; Property, LOINC Axis 2; Prostate Epithelial Cell Growth Factor; Prostatropin; Protocols, Treatment; RGM; Regeneration; Regimen; Research; Research Specimen; S Period; S Phase; S phase (cell cycle); Shapes; Signal Transduction; Signal Transduction Systems; Signaling; Site; Specimen; Standards; Standards of Weights and Measures; Stimulus; Structure; Structure of articular disc of temporomandibular joint; Structure-Activity Relationship; Suidae; Surface; Swine; Synthesis Period; Synthesis Phase; TEM; TMJ; TMJ Disc; TMJ Diseases; TMJ Disorders; TMJD; Temporo-mandibular joint disorder; Temporomandibular Disorders; Temporomandibular Joint; Temporomandibular Joint Diseases; Temporomandibular Joint Disk; Temporomandibular Joint Disorders; Temporomandibular joint disorder; Testing; Time; Tissues; Transmission Electron Microscopy; Treatment Protocols; Treatment Regimen; Treatment Schedule; Validation; bFGF; base; biological signal transduction; biosynthesis; chemical structure function; design; designing; facial; failure; improved; in situ Hybridization Staining Method; in vivo; model organism; new approaches; novel approaches; novel strategies; novel strategy; p(PF-co-EG); poly(propylene fumarate-co-ethylene glycol); porcine; reconstruction; regenerate; response; scaffold; scaffolding; size; structure function relationship; success; suid; two-dimensional; wasting
Project start date: 2004-07-01
Project end date: 2010-06-30
Budget start date: 1-JUL-2008
Budget end date: 30-JUN-2010
5R01DE015038-05 (2008): $0
5R01DE015038-04 (2007): $275383
Self-Assembling Process In Tissue Engineering Of Articular Cartilage
Kyriacos A Athanasiou, Karl F. Hasselmann Professor
Bioengineeringrice University
Grant 5R01AR053286-03 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: MTE
Abstract: The application´s long-term objective is to use a comprehensive bioengineering approach to successfully address articular cartilage regeneration. To this end, a self-assembling process was recently developed to tissue engineer articular cartilage using high density seeding of native chondrocytes in molds made of agarose. The study´s main hypothesis is that the self-assembling process can be enhanced synergistically by the addition of growth factors and mechanical stimulation. To test this hypothesis, we propose the following specific aims 1) To determine optimal growth factor conditions for the self-assembling process for in vitro articular cartilage regeneration. 2) To enhance this process using mechanical stimuli. 3) To engineer articular cartilage combining optimal growth factor and mechanical conditions for the self-assembling process. Application of four growth factors (TGF-(31, TGF-(33, BMP-2, and IGF-I) will be implemented in the self-assembling process at varying concentrations and dosage frequencies. The most favorable concentrations and dosage frequencies of each growth factor will be determined and combinations of these will be examined to select the optimal growth factor conditions. The effects of hydrostatic pressure and direct compression will also be examined at varying magnitudes, frequencies, and application times. The best conditions for each mechanical stimulus and their combinations will be selected from the multiple treatments. Finally, the effects of interactions between the optimized growth factor conditions combined with the optimal mechanical stimulation conditions on the self-assembling process will be assessed. At various time points, morphological, biochemical, and biomechanical properties will be quantified and compared to native tissue values, enabling the selection of optimal conditions. More specific hypotheses include 1) The combination of growth factors will exhibit a synergistic effect by improving the self-assembled constructs´ functional properties approaching two-thirds of native tissue. 2) Interactions between hydrostatic pressure and direct compression will enhance the regeneration process when compared to unloaded controls or constructs under a single mechanical stimulus. 3) The synergistic effects of combined growth factor and mechanical stimulation conditions will yield articular cartilage constructs with combined functional properties approaching those of native articular cartilage
Keywords: articular cartilage, conditioning, growth factor, tissue engineering Fungi, bioengineering /biomedical engineering, cell, chondrocyte, collagen, compression, culture, density, dosage, experimental design, extracellular matrix, histology, hyaline substance, hydrostatic pressure, immunocytochemistry, indexing, model, morphology, mucopolysaccharide, regeneration, relaxation, respiratory reflex, scanning electron microscopy, seed, stress, tissue
Project start date: 2007-04-01
Project end date: 2012-02-29
7R01AR053286-04 (2009): $177711
Sponsored Links Excellgen http://Excellgen.com
Kyriacos A Athanasiou
University Of California Davis
Project start date: 2003-07-01
Project end date: 2017-02-28
Toward Tissue Engineering Of The Knee Meniscus
Kyriacos A Athanasiou, Karl F. Hasselmann Professor
Bioengineeringrice University
6100 S Main
houston, Tx 770051892
Grant 5R01AR047839-05 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: ZRG1
Keywords: cell, knee, tissue engineering biochemistry, biomechanics, bioreactor, biosynthesis, cell type, collagen, compression, dicarboxylate, environment, ethylene glycol, gold, growth factor, histology, hydrostatic pressure, immunocytochemistry, implant, medicine, model, mucopolysaccharide, orthopedics, peptide, regeneration, seed, synovial membrane, tissue, tissue /cell culture
Project start date: 2002-12-10
Project end date: 2008-11-30
5R01AR047839-05 (2007): $237124
5R01AR047839-04 (2006): $244206
5R01AR047839-03 (2005): $272680
5R01AR047839-02 (2004): $273111