1. School of Biomedical Engineering, Science & Health Systems WWW.BIOMED.DREXEL.EDU/ResearchPortfolio/ V 1.0 SD [020409] BIOMATERIALS RESEARCH Faculty/Contact: Michele Marcolongo, Ph.D., Drexel University. E-mail: marcolms@drexel.edu Tissue Engineered Vascular Graft PROGRAM OVERVIEWPROGRAM OVERVIEW As people age, many accrue a condition known as atherosclerosis in which plaque builds up from cholesterol residues. The plaque build up thickens and hardens the arterial wall creating arterial stenosis, which generates adverse conditions for blood flow through the vessel. When the stenosis occurs within the coronary arteries, the restricted blood flow increases the load on the heart muscle while at the same time decreasing the amount of nutrients and oxygen circulated back to the heart. This begins the cycle to coronary heart disease. Without the necessary oxygen and nutrition, the strength of the heart muscle decreases as cells begin to die from lack of fuel. The decreased strength produces yet more cell necrosis in a cycle that ends in heart failure if left unattended. While the causes of lower back pain remain unclear, it is believed that 75% of the cases are associated with degenerative disc disease, where the intervertebral disc of the spine suffers reduced mechanical functionality as a result of dehydration of the nucleus pulposus. Current treatment options range from conservative bed rest to highly invasive surgical interventions, such as spinal fusion and discectomy, which is aimed at reducing pain, but not at restoring disc function. This project proposes a replacement of the nucleus pulposus with a novel, biocompatible hydrogel of polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP). Nucleus Pulposis Replacement Using Novel Associating Hydrogels Cellular & Protein Attachment to Orthopaedic Biomaterials Present work involves the cellular attachment to orthopaedic biomaterials, specifically titanium. In this study, surface charge and surface are varied in order to help understand their interaction with attachment in vitro. SAOS-2 cells are used in the experiment under influence of an externally applied static electric field. The polarity can varied to induce either a net negative or a net positive surface charge. Implant Retrieval The Implant Retrieval Lab is a collection of explanted hip and knee implants, for use in studies centered on the effects of actual use in the body. Typically, these implants consist of metal components (alloys of titanium-aluminum or cobalt-chromium), which are attached to the body, and ultra high molecular weight polyethelene (UHMWPE), which acts as a wear material and cushion between the metal components. Fibronectin Adsorption to Charged Surfaces For a biomaterial to be successful in vivo, we must study the cellular with the material. Cell adhesion proteins such as fibronectin (Fn) are crucial the biocompatibility and cell adhesive properties of biomaterials used implants. In our lab, we have established a fluorescence technique for measurement of Fn adsorption to biomaterials.

2. School of Biomedical Engineering, Science & Health Systems WWW.BIOMED.DREXEL.EDU/ResearchPortfolio/ V 1.0 SD [020409] The project objective is to design a vascular graft that will suit the replacement of the coronary artery using the principles of tissue engineering to construct a scaffold that mimics the natural structure of the artery and encourages the integration of tissue with the graft concurrent with the degradation rate of the graft. The end product will consist of a neo-artery produced by the body itself. As people age, many accrue a condition known as atherosclerosis in which plaque builds up from cholesterol residues. The plaque build up thickens and hardens the arterial wall creating arterial stenosis, which generates adverse conditions for blood flow through the vessel. When the stenosis occurs within the coronary arteries, the restricted blood flow increases the load on the heart muscle while at the same time decreasing the amount of nutrients and oxygen circulated back to the heart. This begins the cycle to coronary heart disease. Without the necessary oxygen and nutrition, the strength of the heart muscle decreases as cells begin to die from lack of fuel. The decreased strength produces yet more cell necrosis in a cycle that ends in heart failure if left unattended. Current medical practices employ both non-invasive and invasive procedures to retard or stop the cycle. Doctors must often resort to coronary bypass surgery, a method that requires blood flow to be cut off from diseased tissue and rerouted through an autologous or synthetic graft. Unfortunately, differences in the grafts mechanical properties with those of the coronary artery create disturbances in blood flow that result in neo-intimal hyperplasia, a condition in which smooth muscle cells proliferate uncontrollably and occlude the vessel if left the mechanical and structural properties of a natural researchers. PROJECTONEPAGERPROJECTONEPAGER Faculty/Contact: Michele Marcolongo, Ph.D., Drexel University. E-mail: marcolms@drexel.edu Collaborating Researchers: Ericka Prechtel and Ken Young Funding: Laboratories: Laboratory for Biomaterials and Biosurfaces in Tissue Engineering; Implant Research Center. TISSUE ENGINEERED VASCULAR GRAFT The picture above illustrates the structures of the artery and vein. Note the orientation of the fibers, which are of special interest to this project. The middle section or media consists of elastin and smooth muscle cells, which are responsible for the elastic properties of the artery. The goal of a tissue engineered graft is to provide a scaffold so similar to this structure that natural tissue will align itself with the scaffold and begin constructing its own vessel. Typical compliance curves of tissue engineered graft samples those of an ideal vessel. unhampered.The challenge of producing a graft that matches artery while maintaining patency continues to motivate

3. School of Biomedical Engineering, Science & Health Systems WWW.BIOMED.DREXEL.EDU/ResearchPortfolio/ V 1.0 SD [020409] Faculty/Contact: Michele Marcolongo, Ph.D., Drexel University. E-mail: marcolms@drexel.edu Collaborating Researchers: Jonathon Thomas, Graduate Student, Drexel University. Funding: NSF Laboratories: Laboratory for Biomaterials and Biosurfaces in Tissue Engineering; Implant Research Center. NUCLEUS PULPOSIS REPLACEMENT USING NOVEL ASSOCIATING HYDROGELS Chronic lower back pain is the number one cause of lost work days in the United States, making it one of the most expensive health care issues today. While the causes of lower back pain remain unclear, it is believed that 75% of the cases are associated with degenerative disc disease, where the intervertebral disc of the spine suffers reduced mechanical functionality as a result of dehydration of the nucleus pulposus. Current treatment options range from conservative bed rest to highly invasive surgical interventions, such as spinal fusion and discectomy, which is aimed at reducing pain, but not at restoring disc function. This project proposes a replacement of the nucleus pulposus with a novel, biocompatible hydrogel of polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP). These polymer gels are stable in physiological fluid as a result of physical crosslinks consisting of intramolecular hydrogen bonds within PVA crystallites and intermolecular hydrogen bonds between PVA and PVP. In addition, these implants can be inserted in a minimally invasive fashion, using current arthroscopic techniques. We are currently examining the potential applicability of PVA / PVP hydrogels of varying compositions through stability and mechanical property analysis. Polymer fractional mass loss over time in vitro shows that gels containing 1% PVP maintained the greatest percentage of their initial mass (nearly 97%). The addition of PVP in small concentrations stabilizes the polymer network with intermolecular hydrogen bonds between PVA and PVP. A combination of superior stiffness and mass retention in vitro is seen for blends prepared with 1% PVP. The dominant mechanism responsible for mechanical property reduction with increasing PVP compositions is the degree of dissolution. The following data represents the Youngs Modulus and fractional mass loss of PVA (Mw=143K) / PVP (Mw=40K) blends with immersion in vitro. Youngs Modulus of hydrogel blends after 56 days immersion in vitro. Mass Loss after 120 days immersion in vitro. PVA/PVP associating hydrogels may be a viable candidate for nucleus pulposus replacement. The polymer gel blend is a swollen network that can be dehydrated and rehydrated, thus allowing this nucleus replacement to be inserted arthroscopically, thereby negating expensive, time consuming surgery. PROJECTONEPAGERPROJECTONEPAGER

4. School of Biomedical Engineering, Science & Health Systems WWW.BIOMED.DREXEL.EDU/ResearchPortfolio/ V 1.0 SD [020409] PROJECTONEPAGERPROJECTONEPAGER Faculty/Contact: Michele Marcolongo, Ph.D., Drexel University. E-mail: marcolms@drexel.edu Collaborating Researchers: Paul Heipp, Graduate Student, Drexel University; Thomas Jefferson University; Cornell University (sample preparation). Funding: NIH/NIAMS Laboratories: Laboratory for Biomaterials and Biosurfaces in Tissue Engineering; Implant Research Center. CELLULAR & PROTEIN ATTACHMENT TO ORTHOPAEDIC BIOMATERIALS Present work involves the cellular attachment to orthopaedic biomaterials, specifically titanium. In this study, surface charge and surface roughness are varied to help understand their interaction with cellular attachment in vitro. SAOS-2 cells are used in the experiment under the influence of an externally applied static electric field. The polarity can be varied to induce either a net negative or a net positive surface charge. Initially, surface roughness is kept to a minimum (approximately 2-5 nm RMS) to reduce the effects of this parameter on cellular attachment. Results show an increase in cellular attachment with a negative surface charge in the absence of serum proteins, and a decrease in cellular attachment with both negative/positive surface charges in the presence of serum proteins. Work is also being pursued with an increase in surface roughness and the effects of serum/serum free media under the same experimental conditions stated above. Electrostatic optimization for osseointegration

5. School of Biomedical Engineering, Science & Health Systems WWW.BIOMED.DREXEL.EDU/ResearchPortfolio/ V 1.0 SD [020409] Faculty/Contact: Michele Marcolongo, Ph.D., Drexel University. E-mail: marcolms@drexel.edu Collaborating Researchers: Tom Coulter, Graduate Student, Drexel University; Exponent Inc, Stryker Howmedica Osteonics Corporation, and The Rothman Institute at Thomas Jefferson University. Funding: Laboratories: Laboratory for Biomaterials and Biosurfaces in Tissue Engineering; Implant Research Center. IMPLANT RETRIEVAL UHMWPE Hip Component The Implant Retrieval Lab is a collection of explanted hip and knee implants, for use in studies centered on the effects of actual use in the body. Typically, these implants consist of metal components (alloys of titanium-aluminum or cobalt-chromium), which are attached to the body, and ultra high molecular weight polyethelene (UHMWPE), which acts as a wear material and cushion between the metal components. The current study involves developing a technique for using Atomic Force Microscopy (AFM) to determine the material surface properties of the UHMWPE component of the implant at both loaded and unloaded locations on the sample. The image above shows the original surface of an unloaded portion of the sample. The vertical lines are a result of the original machining of the part. The image to the right was under load. Nanometer structure of UHMWPE sample as shown using AFM These results can then be compared with in vitro wear test data. This study is being pursued in order to provide information for the Implant Research Center, a collaboration with Exponent Inc, Stryker Howmedica Osteonics Corporation, and The Rothman Institute at Thomas Jefferson University. PROJECTONEPAGERPROJECTONEPAGER

6. School of Biomedical Engineering, Science & Health Systems WWW.BIOMED.DREXEL.EDU/ResearchPortfolio/ V 1.0 SD [020409] Faculty/Contact: Michele Marcolongo, Ph.D., Drexel University. E-mail: marcolms@drexel.edu Collaborating Researchers: Hanako Yananaka, Graduate Student, Drexel University. Funding: Laboratories: Laboratory for Biomaterials and Biosurfaces in Tissue Engineering; Implant Research Center. FIBRONECTIN ADSORPTION TO CHARGED SURFACES For a biomaterial to be successful in vivo, we must study the cellular interaction with the material. Cell adhesion proteins such as fibronectin (Fn) are crucial to the biocompatibility and cell adhesive properties of biomaterials used for implants. Many researchers have been studying biomaterial properties and their effects on protein adsorption to the biomaterial surfaces. In our lab, we have established a fluorescence spectroscopy technique for measurement of Fn adsorption to biomaterials. Current techniques used in studying Fn include radiolabeling and immunoassays. Although immunoassays allow protein site-specific labeling, the amount of protein adsorbed is commonly expressed as a relative quantity, and conversion of this quantity into surface density units is difficult. Our technique is intended to provide quantitative information with the high sensitivity of radiolabeling, while eliminating the use of radioactive isotopes. We add to the development of fluorescent measurement of protein adsorption through analysis of the sensitivity, accuracy, and reproducibility of the technique. The technique involves labeling Fn solution with a fluorescent dye Oregon Green 488 (Molecular Probes, Eugene, OR) at a specific molar ratio of dye to Fn. Dye unattached to Fn is removed by dialysis. Absorbance at 280 nm (OD280) and 496 nm (OD496) of the labeled Fn solution are measured with a spectrophotometer (DU 640 Spectrophotometer, Beckman). Fn concentration and the number of molecules of dye attached to each Fn molecule are calculated from the values of OD280 and OD496. Figure 1. Comparison of fluorescence spectroscopy technique to radiolabeling and sensitivity of technique in measurement of Fn adsorption to TCP and BSA-preadsorbed TCP. Comparison of Figures 1 and 2 shows variability in Fn adsorption with experiments performed on different days. Variability is also seen in comparison of data from different authors using radiolabeling. For Fn on TCP, radiolabeling has shown differences ranging from 83-500%, with reduced sensitivity at lower plating densities. Fluorescence spectroscopy provided similar consistency and sensitivity to that obtained by radiolabeling. With the standard, the technique measures the surface density of Fn adsorbed to materials allowing comparison to data obtained by other methods, and making it favorable over immunoassays [HY1]. The technique may be a convenient alternative to current methods of quantitative analysis of protein adsorption to biomaterials. Figure 2. Consistency of Fn surface density measurements with fluorescence spectroscopy technique. PROJECTONEPAGERPROJECTONEPAGER