Research

Bone Tissue Engineering

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Dynamic Measurement of Biological Processes

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Measurement and Modeling of Inflammation

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Nanotechnology for Therapeutics and Diagnostics

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Tumor Engineering

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Bone Tissue Engineering

Project 1: Non-destructive Real-time Imaging and Control of Bone Tissue Engineering
Graduate Student Researcher: Bryce Whited

The use of tissue engineered scaffolds in combination with progenitor cells has emerged as a promising strategy to restore or replace tissues damaged by disease or trauma. In addition to being biocompatible and exhibiting appropriate mechanical properties, scaffolds must be designed to sustain cell attachment, proliferation, and differentiation to ultimately produce the desired tissue once implanted in the patient. Conventional techniques used to assess successful scaffold design include cell viability stains, DNA assays, and histological sectioning/staining. While significant information can be gained from using these methodologies, they are destructive to the sample and only provide snapshots of scaffold and cell development at a limited number of time points. Consequently, key temporal and spatial information relating to tissue regeneration in the scaffold is lost utilizing these techniques. Thus, the ability to non-destructively monitor cell viability, proliferation, and differentiation in real-time is of great importance for scaffold design and tissue engineering.

To meet this challenge, we have developed a novel imaging-bioreactor system capable of monitoring the development of tissue engineered scaffolds at the cellular level non-destructively and in real time. Our system is based on the incorporation of miniature hollow-core silica fiber (HCFs) imaging microchannels into the scaffold. We have demonstrated the capability of cellular-level imaging of cells labeled with fluorescent proteins through relatively thick scaffolds (~1 mm) (Figure 1). After some modifications, our imaging system can be extended to monitor scaffold development after in-vivo implantation.

Our group is also interested in designing novel tissue scaffolds for bone regeneration. One such scaffold that we have fabricated and characterized is an apatite coated electrospun Poly lactic acid scaffold that enhances cellular infiltration and promotes osteoblastic differentiation (Whited et al., 2010) (Figure 2).

Funding
Institute for Critical Technologies and Applied Sciences Grant

1R01HL098912-01 BRP (G. Wang) 3/01/2010-3/30/2014 NIH/NHLBI

Optical Molecular Tomography for Regenerative Medicine

Relevant Papers
Whited BM, Whitney JR, Hofmann MC, Xu Y, Rylander MN. Pre-osteoblast infiltration and differentiation in highly porous apatite-coated PLLA electrospun scaffolds. Biomaterials. 2011 Mar; 32(9):2294-304.

Project 2: Stress Preconditioning Strategies for Bone Tissue Regeneration
Graduate Student Researcher: Eunna Chung (graduated)

Bone related disorders associated with cancer, injury, abnormal development, and degenerative conditions dramatically diminish the health and quality of life of millions of people. These disorders can cause significant disability through loss of bone or its functionality, creating a need for bone replacements (over 3 million orthopaedic procedures are performed every year) or effective regenerative strategies. Although tissue engineered bone replacements have enormous potential, development of truly viable and functional tissues is limited by numerous aspects such as cell growth rate and formation of connective tissue surrounding cells, extracellular matrix (ECM). Furthermore, there are no effective strategies for stimulating bone regeneration directly in injured or diseased bone in vivo.

Stress conditioning in the form of thermal, tensile, and shear stress can up-regulate ECM production, cell proliferation, and induction of molecular chaperones known as heat shock proteins (HSPs). A clear link has been shown between up-regulated HSPs and enhanced cell proliferation and collagen biosynthesis. However, tissue engineering approaches have not harnessed the potential of HSPs for creation of more viable bone replacements. Furthermore, delivery of HSPs exogenously to diseased bone regions could have tremendous potential to stimulate bone regeneration. The objective of this study is to investigate a transformative approach for bone replacement and regeneration through heat shock protein-based therapies. This research is utilizing combinatorial stress conditioning strategies involving thermal, tensile, and shear stress and exogenous delivery of HSPs to both cell monolayers and scaffolds.

Dynamic Measurement of Biological Processes

Biological processes such as tissue growth, inflammation, and angiogenesis involve a symphony of complex and dynamic interactions among multiple cell types, signaling molecules, and tissue microenvironments. Current normal tissue or tumor models typically consist of two dimensional cell culture systems or in vivo rodent animal models. The “gold standard” for measuring dynamic biological processes requires highly invasive techniques which involve sacrifice of the tissue at specific time points followed by sectioning of the tissue and histological/biochemical analysis. Therefore, we can only obtain a few isolated “static snapshots” of the complex nature of biological processes and disease progression at discrete time points and spatial positions. As a result there often exists a fundamental discrepancy between our understanding of biological processes and the actual events since key tissue response information is lost due to limited temporal and spatial information. The inability to monitor biological processes in real-time non-invasively has limited understanding of processes such as angiogenesis, wound healing, tissue regeneration, and progression of countless diseases. This, in turn, has thwarted development of effective treatments for restoring quality and length of life to millions of patients.

To address this challenge, we have developed a first-of-its-kind multifunctional system capable of simultaneous spatiotemporal molecular imaging of dynamic biologic processes and delivery of biological agents in minimally invasive and non-destructive manner within a tissue. This novel system is referred to as the “holey scaffold”. As illustrated in Fig. 1(a), a holey scaffold is built from a network of microchannels embedded in a tissue scaffold. Material for scaffolds can be selected to reflect tissue architecture and is typically composed of biodegradable synthetic (e.g. poly(glycolic acid)) or biological materials (e.g. collagen, laminin, matrigel) to promote tissue growth. Hollow microchannels can be used to perform controlled delivery of biological agents (e.g. nutrients, growth factors, cells, nanoparticles, drugs). Other microchannels can be utilized to introduce µm-scale optical fibers for non-destructive, in situ, and real-time molecular imaging of biological processes. Holey scaffolds can be viewed conceptually as a miniature microscope incorporated within a biological system, capable of fluorescence imaging.

This system will permit tissues of clinically relevant size to be grown in vitro by integrating the holey scaffold in a bioreactor. Within this framework the tissue microenvironment and signaling cues can be tightly controlled to isolate important responses without the presence of the cacophony of signals that can confound interpretation within an in vivo model. The holey scaffold can also be incorporated into in vivo animal models permitting dynamic measurement of biological phenomena within a living animal with minimal perturbation (Fig. 1b). We currently have funding to utilize the holey scaffold to improve bone and blood vessel regeneration and nanoparticle-mediated laser therapies as described below:

Project 1: Characterization of the Tumor Response to Nanoparticle-Mediated Laser Therapy

The goal of this project is to effectively characterize the spatiotemporal nanoparticle transport and dynamic photothermal/photochemical response to nanoparticle-mediated therapy for varying nanoparticle properties and laser parameters within both in vitro and in vivo tumor systems. This objective will be accomplished by creating and utilizing a novel sensing system known as the “holey scaffold” which is capable of minimally invasive and non-destructive measurement and control of dynamic biological and transport processes. The holey scaffold will measure dynamic nanoparticle mass transport and photothermal (temperature, cell viability, and HSP expression), and photochemical (reactive oxygen species production) response in real-time to a variety of nanoparticle types including carbon nanotubes and novel embodiments of carbon nanotubes and fullerenes, targeting approaches, and delivery methods (see Fig. 2). Measured tumor response will be utilized to create a multi-component treatment planning computational model for nanoparticle-mediated laser therapy. The model will consist of unique sub-models for nanoparticle transport, photothermal response, and photochemical effects based on corresponding experimentally measured data (see Fig. 1). Integration of sub-models into an overall treatment planning model will permit prediction of the tumor response and enable determination of ideal nanoparticle properties and laser parameters for maximum therapeutic effectiveness. The model will also provide additional insight into the spatiotemporal tumor response to nanoparticle-mediated laser therapies and extend our physical understanding of these processes for a wider range of experimental conditions than measured. Objectives with associated experimental measurements and computational models are shown in Fig. 1.

Funding
CBET 0955072 (M.N. Rylander) 5/10/10-5/9/15 National Science Foundation CAREER: Holey Scaffold Sensing System for Characterization of the Spatiotemporal Tumor Response to Nanoparticle-Mediated Photothermal and Photochemical Therapy

Project 2: Velocity and Wall Shear Stress Characterization in an Arterial Flow Bioreactor and Tumor Vascular System Graduate Student
Researcher: Elizabeth Voigt

Endothelial cells, the cells lining the inner surface of blood vessels, respond both mechanically and chemically to applied flow conditions. Quantification of this response will assist in the design and development of drug delivery systems. Our objective is to examine how cells grow and react to the presence of different flow environments by directly quantifying the flow conditions to which the cells are exposed. This will be done by directly measuring the flow parameters within an artificial vessel lined with living endothelial cells. We are designing and testing a system that supports the growth of human microvascular endothelial cells in an artificial artery under physiological pulsatile conditions. Particle Image Velocimetry (PIV) is being used to quantify the flow profile within the artery and assist with the design of drug delivery systems.

Initial results using our system indicate that assumptions made in previous work about the flow profiles within such artificial physiological flow systems are incorrect. Direct measurement of flow is necessary for quantitative conclusions about endothelial response to shear conditions to be made.

Funding
This work is supported in part by a Clare Boothe Luce Graduate Fellowship, AEThER, and the Nanotherapeutics and Bioheat Transfer Lab. We would like to thank Wake Forest University for providing a sample bioreactor for our redesign.

Project 3: Non-destructive real-time imaging techniques to asses cellular events and cell/biomaterials interactions
Graduate Student Researcher: Bryce Whited

The use of tissue engineered scaffolds in combination with progenitor cells has emerged as a promising strategy to restore or replace tissues damaged by disease or trauma. In addition to being biocompatible and exhibiting appropriate mechanical properties, scaffolds must be designed to sustain cell attachment, proliferation, and differentiation to ultimately produce the desired tissue once implanted in the patient. Conventional techniques used to assess successful scaffold design include cell viability stains, DNA assays, and histological sectioning/staining. While significant information can be gained from using these methodologies, they are destructive to the sample and only provide snapshots of scaffold and cell development at a limited number of time points. Consequently, key temporal and spatial information relating to tissue regeneration in the scaffold is lost utilizing these techniques. Thus, the ability to non-destructively monitor cell viability, proliferation, and differentiation in real-time is of great importance for scaffold design and tissue engineering.

To meet this challenge, we have developed a novel imaging-bioreactor system capable of monitoring the development of tissue engineered scaffolds at the cellular level non-destructively and in real time. Our system is based on the incorporation of miniature hollow-core silica fiber (HCFs) imaging microchannels into the scaffold. We have demonstrated the capability of cellular-level imaging of cells labeled with fluorescent proteins through relatively thick scaffolds (~1 mm) (Figure 5). After some modifications, our imaging system can be extended to monitor scaffold development after in-vivo implantation.

Funding:
Institute for Critical Technologies and Applied Sciences Grant 1R01HL098912-01 BRP (G. Wang) 3/01/2010-3/30/2014 NIH/NHLBI Optical Molecular Tomography for Regenerative Medicine

Relevant Papers
Whited BM, Whitney JR, Hofmann MC, Xu Y, Rylander MN. Pre-osteoblast infiltration and differentiation in highly porous apatite-coated PLLA electrospun scaffolds. Biomaterials. 2011 Mar; 32(9):2294-304.

Measurement and Modeling of Inflammation

Measurement and Modeling of Inflammation
Graduate Student Researcher: Samuel Shimp

We are making significant advances in defining mechanisms of cell signaling networks associated with inflammation through extensive in vitro laboratory work as well as in vivo mouse studies. Our mechanistic, computational models are enhancing our understanding of autoimmune pathogenesis and cancer thermal therapies, leading to the design of novel therapeutic strategies. Some of our most significant results show modulation of the immune response through control of heat shock protein chaperone activity.

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disorder that can affect nearly every organ in the body. One of our areas of expertise in the heat transfer and nanotherapeutics lab is that of imaging and measuring heat shock protein activity. Heat Shock Protein 90 (Hsp90) is known to chaperone numerous signaling client proteins including those involved with inflammation and anti-apoptotic pathways. I am exploring the link between Hsp90 and chronic autoimmune disease pathogenesis through the use of Hsp90 inhibitors to regulate Hsp90 activity. One inhibitor employed is an anti-cancer drug in phase 3 clinical trials known as Alvespimycin (17-DMAG). 17-DMAG inhibits Hsp90 function by binding to the carboxyl (C-) terminal domain resulting in deactivation, destabilization, and degradation of Hsp90 inflammatory signaling clients, such as Akt, for decreased inflammation. Our long-term goal is to elucidate and mathematically model the cellular mechanisms of the Hsp90 modulated inflammatory pathways in autoimmune diseases such as SLE.

My research uses J774 mouse macrophage cells and mouse kidney (mesangial) cells for in vitro studies. I also perform studies using New Zealand Black x White (NZB/W) and MRL/lpr mouse models of lupus nephritis for in vivo studies.

Relevant Publications & Conferences:
C. Chafin, S. Muse, R. Hontecillas, J. Bassaganya-Riera, D. Caudell, S. Shimp, M. N. Rylander, J. Zhang, L. Li, and C. Reilly, 2010, "Deletion of PPAR-gamma in Immune Cells Enhances Susceptibility to Anti-GBM disease," Journal of Inflammation Research, 3: 127-134.
Shimp III, S. K., Reilly, C. M., Rylander, M. N., "Computational Modeling of Hsp90 inhibition illustrates a potential therapy to inhibit immune mediated inflammation in systemic lupus erythematosus", American Association of Immunologists, Annual Meeting May 7-12, 2010, Baltimore, MD.
Shimp, S. K., Reilly, C. M., Rylander, M. N., "Computational modeling of Hsp90 as a therapeutic target to inhibit immune-mediated inflammation in systemic lupus erythematosus", College of William & Mary 9th Annual Graduate Research Symposium, March 26-27, 2010, College of William & Mary, Williamsburg, VA.
A. Peairs, R. Dai, L. Gan, S. Shimp*, M. N. Rylander, L. Li, C.M. Reilly. 2010, "Epigallocatechin-3-gallate (EGCG) attenuates inflammation in MRL/lpr mouse mesangial cells," Cellular Molecular Immunolology, 7(2):123-32.
Shimp, S. K., Peairs, A., Reilly, C. M., Li, L., Rylander, M. N., 2008, "Cytokines and hepatoneogenesis through an indirect mechanism involving heat shock protein 90", Poster Presentation at 6th Annual Via Research Recognition Day 16 Oct. 2008, Virginia College of Osteopathic Medicine, Blacksburg, VA.
Bradley, A. T., Evans, W. C., Reed, J. L., Shimp, S. K., Fitzpatrick, F. D., 2008, "TEM Cell Testing of Cable Noise Reduction Techniques from 2 MHz to 200 MHz – Part 1" - Asia-Pacific EMC Week And Technical Exhibition Singapore, May 19-23, 2008
Bradley, A. T., Evans, W. C., Reed, J. L., Shimp, S. K., Fitzpatrick, F. D., 2008, "TEM Cell Testing of Cable Noise Reduction Techniques from 2 MHz to 200 MHz – Part 2" - Asia-Pacific EMC Week And Technical Exhibition Singapore, May 19-23, 2008
Shimp, S. K., Southward, S. C., Ahmadian, M., 2007, "Detecting Crew Alertness With Processed Speech" ASME/IEEE Joint Rail Conference and Internal Combustion Engine Spring Technical Conference, March 13-16, 2007, Pueblo, CO, USA

©2011 Virginia Tech - Wake Forest School of Biomedical Engineering / Institute for Critical Technology and Applied Science/ Virginia Tech

Nanotechnology for therapeutics and diagnostics

Project 1: Photothermal and Photochemical Response to Laser Therapy with Nanoparticles
Graduate Student Researchers: Jon Whitney and Saugata Sarkar (graduated)

Laser therapy based on photothermal (heat generation) and photochemical (reactive oxygen species production (ROS)) can provide minimally-invasive and potentially more effective alternatives to conventional resection, chemotherapy, and radiation, reducing complication rates and increasing patient recovery and quality of life. Although laser therapies have enormous promise, their effectiveness is limited due to nonspecific excitation or heating of target tissue, which can lead to healthy tissue injury. When laser therapies are utilized as cancer treatments their therapeutic efficacy can be compromised due to induction of molecular chaperones known as heat shock proteins (HSP) in regions of the tumor where non-lethal temperature elevation occurs. Thermal induction of HSP expression by tumor cells can increase tumor recurrence by enhancing tumor cell viability and diminish effectiveness of chemotherapy and radiation treatments generally employed in conjunction with thermal therapy.

Nanoparticles utilized in combination with laser therapy can improve treatment effectiveness by enhancing photothermal and photochemical mechanisms and increasing treatment selectivity when nanoparticles are targeted to tumor cells. When incorporated in cancer therapy, the selective photothermal/photochemical capability afforded by nanoparticles increases tumor cell death and diminishes HSP expression in the tumor through targeted heat generation and production of reactive oxygen species. We are measuring the response of breast, brain, prostate, and kidney cancer cells and tumors to a variety of nanoparticles such as carbon nanotubes (CNTs) and novel embodiments of carbon nanotubes including single walled carbon nanohorns and carbon nanotube peapods. Figure 1 shows the varying types of nanoparticles we are investigating. CNTs are graphene sheets of carbon atoms rolled into a tube and capped by fullerene hemispheres. The two major types of CNTs are single walled carbon nanotubes (SWNTs) which have one seamless tube and multi-walled nanotubes (MWNTs) which possess two or more concentric tubes. The diameter and length of SWNTs are 1.5-3.0 nm and 20-1000 nm respectively whereas the corresponding dimensions are 5.0-100 nm and 1-50 µm for MWNTs. Another unique embodiment of SWNTs are single-walled carbon nanohorns (SWNHs) which are composed of an aggregate of SWNTs with overall diameters of 50-100 nm.

We are also exploring the capability of carbon CNT/SWNH peapods for dual imaging and therapy (Figure 2). These structures are comprised of a CNT or SWNH (pod) encapsulated with endohedral metallofullerenes (EMFs) (a.k.a. buckyballs), The EMFs are spherical fullerenes containing gadolinium. This embodiment leverages CNTs/SWNHs as photoabsorbers and buckyballs containing gadolinium as imaging agents providing a nanoparticle capable of photothermal treatment and diagnostic imaging. Our lab is investigating the optimal nanoparticle properties and laser parameters to achieve selective and effective tumor treatment using both in vitro and in vivo experiments and computational modeling. We are measuring the impact inclusion of these nanoparticles has on optical and thermal tissue properties. We employ infrared imaging to determine the spatial temperature distribution in vitro and magnetic resonance thermometry to determine the temperature distribution in vivo in response to nanoparticle enhanced therapies. We have recently developed a novel cell viability algorithm to determine the temporal and spatial tumor response to therapies involving nanoparticles and laser therapy. Figure 3 shows a representative fluorescence image with live cells fluorescing green due to calcein staining (A), brightfield image showing dead cells stained wit trypan blue (B), and corresponding quantified viability distribution showing live (red) and dead (blue) cells (C). There is close correspondence between measured and quantified cell viability distributions. There is a high degree of tumor death in the center ring which is encompassed by the laser beam, however in the dish periphery where lower temperature elevations occurs, cell viability remains high. Radial viability measurements were computed and the radial viability percentages in 1000 pixel increments and are shown in Figure 3D.

Funding
CBET 0955072 (M.N. Rylander) 5/10/10-5/9/15 
National Science Foundation 
CAREER: Holey Scaffold Sensing System for Characterization of the Spatiotemporal Tumor Response to Nanoparticle-Mediated Photothermal and Photochemical Therapy

1R21CA156078-01 (C.G. Rylander) 8/10/2010-8/09/2012 
NIH/NCI 
Fiberoptic Microneedle Device for Nanoparticle-Enhanced Photothermal Therapy of Aggressive, Infiltrative Bladder Cancer

CBET 1034026 (J. Freeman) 8/10/10-8/9/13 
National Science Foundation 
A Novel Treatment for Connective Tissue in Ehlers-Danlos Patients and Strained and Sprained Ligaments Investigating Carbon Nanostructure Enhanced Prolotherapy

ZRG1 BCMB-S (50) (S. Torti) 4/01/2008 – 3/31/2013 NIH/NCI 
Nanotubes in Tumor Imaging and Therapy

CBET 0933571 (C.G. Rylander) 8/16/2009 – 8/15/2012 
National Science Foundation 
Fiberoptic Microneedle Device for Combined Light and Nanomedicine Delivery: Mimicking Nature’s Design of a Mosquito

CBET 0731108 (M.N. Rylander) 8/1/2007 – 7/31/2011
National Science Foundation 
Characterization and Model Development for the Cellular Response to Nanotube-Mediated Laser Therapy

1R21CA135230-01 (M.N. Rylander) 7/1/2008 – 6/31/2011 NIH/NCI 
Trimetallic Nitride Templated Endohedral Metallofullerenes as Dual Imaging and Multimodal Therapeutic Agents for Laser Cancer Therapy

1 R41 EB008907-01 (Naha) NIH/ ZRG1 SSMI-Q (10)7/1/2008 – 6/31/2010 
A Novel Nanomaterials Approach for Cancer Imaging and Therapeutic Treatment

Related Papers
Photothermal or Hyperthermia Therapy alone or in combination with Nanoparticles (most recent to earliest)

S. Sarkar, Abhijit A. Gurjarpadhye, C. Rylander, M. N. Rylander, 2011, “Optical Properties of Breast Tumor Phantoms Containing Carbon Nanotubes and Nanohorns,” Journal of Biomedical Optics, Accepted and in press
S. Sarkar, K. Zimmermann, W. Leng, P. Vikesland, J. Zhang, H. Dorn, T. Diller, C. Rylander, and M. N. Rylander, 2011, “Measurement of the Thermal Conductivity of Carbon Nanotube-Tissue Phantom Composites with the Hot Wire Probe Method,” Ann Biomed Eng. [Epub ahead of print].
J. Whitney, S. Sarkar, J. Zhang, T. Do, T. Young, M. K. Manson, T.A. Campbell, A. A. Puretzky, C. M. Rouleau, K. L. More, D. B. Geohegan, C. G. Rylander, H. C. Dorn, M. N. Rylander, 2011, “Carbon Nanohorns as Photothermal Agents for Cancer Therapy,” Lasers in Surgery and Medicine, 43(1):43-51.
J. W. Fisher, C. F. Buchanan, C. Szot, S. Sarkar, C. Rylander, and M. N. Rylander, 2010, “Photothermal Response of Human and Murine Cancer Cells to Multiwalled Carbon Nanotubes and Laser Irradiation,” Cancer Research, 70 (23): 1-10.
M. N. Rylander, Y. Feng, K. Zimmermann, and K. R. Diller, 2010, “Measurement and Mathematical Modeling of Thermally Induced Injury and Heat Shock Protein Expression Kinetics in Normal and Cancerous Prostate Cells,“ International Journal of Hyperthermia Special Issue on Prostate Cancer Therapy, 26(8): 748-764.
J. Zhang, J. Ge, M. Shultz, E. Chung, G. Singh, C. Shu, P. Fatouros, S. Henderson, F. Corwin, D. Geohegan, A. Puretzky, C. Rouleau, K. More, C. Rylander, M. N. Rylander, H. Gibson, H. Dorn, 2010, “In Vitro and In Vivo Studies of Single-Walled Carbon Nanohorns with Encapsulated Metallofullerenes and Exohedrally Functionalized Quantum Dots,” Nano Letters, 10(8): 2843-2848.
S. Sarkar, C. G. Rylander, and M. N. Rylander, 2010, Photothermal Response of Tissue Phantoms Containing Multi-walled Carbon Nanotubes: Modified Optical and thermal properties,” Journal of Biomechanical Engineering, 132: 044505-1-044505-5.
M. Kosoglu, R. Hood, Y. Chen, Y. Xu, M. N. Rylander, and C. Rylander, 2010, “Fiberoptic Microneedles for Transdermal Light Delivery: Ex Vivo Porcine Skin Experiments”, Journal of Biomechanical Engineering, 132(9): 091014-1-091014-7.
C. Shu, J. Ge, J. Zhang, J. Hyun Sim, B. G. Burke, K. A. Williams, M. N. Rylander, T. Campbell, D. Geohegan, A. R. Esker, H. W. Gibson, and H. C. Dorn, 2010,"A Facile High-Speed Vibration Milling Method to Water-Disperse Single-Walled Carbon Nanohorns,” Chemistry of Materials, 22, 347–351.
A. Burke, X. Ding, Ravi Singh, R. A. Kraft, M. N. Rylander, C. Szot, C. Buchanan, J. Whitney, J. Fisher, N. Levi-Polyachenko, H. C. Hatcher, R. D’Agostino Jr., N. Kock, P. M. Ajayan, D. L. Carroll, F. M. Torti, S. V. Torti, 2009, “Long-term survival following a single treatment of kidney tumors with multiwalled carbon nanotubes and near-infrared radiation,” PNAS, 4, pp. 12897-12902.
Computational Models for Cancer Laser Therapy alone or with Nanoparticles (most recent to earliest)

Y. Feng, D. Fuentes, A. Hawkins, J. Bass and M. N. Rylander, 2009, "Optimization and Real-Time Control for Laser Treatment of Heterogeneous Soft Tissues," Computer Methods in Applied Mechanics and Engineering, 198, Issues 21-26, 1742-1750.
Y. Feng, D. Fuentes, A. Hawkins, J. Bass, M. N. Rylander, A. Elliott, A. Shetty, R. J. Stafford, and J. T. Oden, 2009, "Nanoshell-Mediated Laser Surgery Simulation for Prostate Cancer Treatment," Special issue on Computational Biomedical Engineering for Journal of Engineering with Computers, 25, 3-13.
Y. Feng, J. T. Oden, and M. N. Rylander, 2008, " A Two-State Cell Damage Model Under Hyperthermic Conditions: Theory and In Vitro Experiments," Journal of Biomechanical Engineering, 130: 0410161-10.
J. Fisher and M. N. Rylander, 2008, “Effective Cancer Laser Therapy Design Through the Integration of Nanotechnology and Computational Treatment Planning Models,” Proc. SPIE, 6869, 68690D1-11.
J. T. Oden, K. R. Diller, C. Bajaj, J. C. Browne, J. Hazle, I. Babuška, J. Bass, L. Biduat, L. Demkowicz, A. Elliott, Y. Feng, D. Fuentes, S. Prudhomme, M. N. Rylander, R. J. Stafford, Y. Zhang, 2007, “Dynamic Data-driven Finite Element Models for Laser Treatment of Cancer,” Numerical Methods For Partial Differential Equations, 23, 904-922.
M. N. Rylander, Y. Feng, J. Bass, and K. R. Diller, 2007, “HSP Expression and Damage Optimization Algorithm for Prostate Cancer Therapy Design,” Lasers in Surgery and Medicine, 39, 731-746.
J. T. Oden, K. R. Diller, C. Bajaj, J. C. Browne, J. Hazle, I. Babuska, J. Bass, L. Demkowicz, Y. Feng, D. Fuentes, S. Prudhomme, M. N. Rylander, R. J. Stafford, and Y. Zhang, 2006, “Development of a Computational Paradigm for Laser Treatment of Cancer,” Lecture Notes in Computer Science, 3993: 530-537.
Y. Feng, M. N. Rylander, J. Bass, J.T. Oden, and K. R. Diller, 2005, “Optimal Design of Laser Surgery for Cancer Treatment Through Nanoparticle-Mediated Hyperthermia Therapy,” NSTI-Nanotech. 1, 39-42.
M. N. Rylander, Y. Feng, Y. Zhang, J. Bass, R. J. Stafford, J. Hazle, and K. R. Diller, 2005, “Optimizing HSP Expression in Prostate Cancer Laser Therapy Through Predictive Computational Models,’ Journal of Biomedical Optics, 11, 0411131-16.
Project 2: Measurement of Nanoparticle Transport at the Cellular and Tissue Level
Graduate Student Researcher: Kristen Zimmermann

Carbon nanomaterials have been used for a variety of biomedical applications, from biosensing to drug delivery. Over the past decade, groups in Japan and China have begun investigating a new type of carbon nanomaterial, called single-walled carbon nanohorns (SWNHs). This material is unique in structure, yet exhibits the excellent mechanical, chemical, and thermal properties characteristic of carbon nanotubes (CNTs). The unique structure of SWNHs provides advantages over CNTs, such as larger surface areas both externally and internally to allow for greater surface modification or drug loading. Additionally, SWNHs can be synthesized without the use of a metal catalyst, reducing potential toxicity. Due to such advantages, SWNHs have been studied previously as drug delivery systems for chemotherapeutic agents and have also been investigated for nanoparticle-enhanced laser therapies for cancer malignancies; however, studies have been limited by lack imaging techniques. Therefore, our group is developing a highly fluorescent SWNH that can be used to study the transport of these particles in vitro and in vivo at both the cellular and tissue levels. The fluorescently tagged SWNHs will serve to image these particles optically, study their transport mechanisms and distribution both in vitro and in vivo, and to detect tumor margins in vivo once targeted with a receptor-specific ligand.



Funding
Kristen is funded by a National Science Foundation Graduate Research Fellowship. 
CBET 0955072 (M.N. Rylander)5/10/10-5/9/15 
National Science Foundation 
CAREER: Holey Scaffold Sensing System for Characterization of the Spatiotemporal Tumor Response to Nanoparticle-Mediated Photothermal and Photochemical Therapy

Project 3: Nanoparticles and Electroporation

Graduate Student Researchers: Chris Arena and Jose Umanzor-Alvarez

This research is focused on utilizing carbonaceous nanomaterials to enhance the ablation of tumors through irreversible electroporation (IRE). IRE employs high-voltage, ultra-short electric pulses to destabilize cell membranes. Nanoparticles afford us the ability to localize the pulsed electric field based therapy to targeted cancer cells.

Additionally, we are evaluating the heat shock response to IRE. While IRE is inherently non-thermal, it is hypothesized that the stress caused by the permeabilization of membranes could lead to the induction of heat shock proteins (HSP), which are known to play a role in tumor recurrence.

Relevant Papers:
C. B Arena, J. L Caldwell, M. N. Rylander, R. V. Davalos, 2009, “Theoretical study for the treatment of pancreatic cancer using electric pulses.” Conf Proc IEEE Eng Med Biol Soc., 2009:5997-6000.

C. B Arena, J. L Caldwell, M. N. Rylander, R. V. Davalos, 2010, “Theoretical Considerations of Tissue Electroporation with High Frequency Bipolar Pulses,” IEEE Trans Biomed Eng., Published online. Funding Information: CBET 0933335 (R. Davalos) 8/1/2009 – 7/31/2012 National Science Foundation Combinatorial Brain Cancer Therapy through Irreversible Electroporation and Carbon Nanotubes

Tumor Engineering

Project 1: Shear stress-mediated cross talk between endothelial cells and breast cancer cells and its effect on the angiogenic response and metastatic potential of breast tumors
Graduate Student Researcher: Cara Buchanan

The proximity of endothelial cells to cancer cells within the tumor microenvironment suggests reciprocal growth factor exchange and cross-talk could directly stimulate tumor growth and metastasis, however, the role of the tumor vascular niche in regulating expression of pro-angiogenic growth factors is not well understood. It is hypothesized that endothelial cells directly influence the angiogenic response and metastatic potential of breast cancer cells through regulation and stimulation of tumorigenic growth factors. The expression profiles of these factors, such as vascular endothelial growth factor, the angiopoietins and matrix metalloproteinases are a critical determinant of the angiogenic response and metastatic potential, and should be well understood to overcome tumor resistance to anti-angiogenic therapies.

By integrating tissue engineering strategies with cancer biology, micro-scale fluid mechanics, and optical flow diagnostics, we will create a novel 3D in vitro tumor vascular model in which the interaction between tumor and endothelial cells under physiologically relevant fluid shear conditions can be monitored (Fig. 1). We are currently developing and validating a first-of-its kind in vitro breast tumor vascular model in which the co-culture of breast cancer cells and endothelial cells can be conducted under physiologically accurate conditions. Utilizing a tissue engineering approach of novel scaffolds within a bioreactor, this model will serve as a tool to investigate bidirectional cross-talk between breast cancer cells and endothelial cells, as well as the cellular response to dynamic conditions representative of the physical tumor microenvironment at various stages of development. Our ultimate goal is to understand shear stress-mediated cross talk between endothelial cells and breast cancer cells and its effect on the angiogenic response and metastatic potential of breast tumors. Understanding the role of the vascular niche in supporting the growth and metastasis of tumors may provide insight for the design of new therapeutic strategies to eradiate angiogenesis-dependent tumors. This research strategy can also be used to understand how region-specificity for tumor metastasis is related to different shear stress/flow patterns.

Related Papers
C. S. Szot, C. F. Buchanan, P. Gatenholm, M. N. Rylander, J. W. Freeman, 2011, “Investigation of Cancer Cell Behavior on Nanofibrous Scaffolds,” Materials Science and Engineering C, 31: 37-42.

Funding
Cara is funded through a National Science Foundation Graduate Research Fellowship.

Project 2: Development of a 3D in vitro Vascularized Breast Cancer Model
Graduate Student Researcher: Chris Szot

Despite rising success rates when cancer is diagnosed and treated in its early stages, a tumor that has developed neovascularization and metastasized poses a significantly greater risk of mortality. Current therapeutic options, some of which were developed over a quarter century ago, include surgery, radiation therapy, chemotherapy, hormone therapy, and immunotherapy. Problems with current treatments include recurrent tumors, non-specific targeting, targeting that is too specific, damage to vital organs, aggravation of existing conditions, and infertility. There is a need for innovative cancer therapeutics that are not prone to these problems and which can effectively inhibit tumor development.

The progression of cancer research, and subsequent discovery of permanent treatment options, is limited by the experimental systems available for studying its complex mechanisms. A majority of cancer research is conducted using small animal models; intricate, living systems that can be variable and contain many uncontrollable factors. Current in vitro models of tumorigenesis are restricted by the use of static, 2D cell culture monolayers that lack the structural architecture necessary for proper cell-cell interaction and an in vivo phenotype and 3D cell culture systems that restore the cellular morphology and phenotypes observed in vivo but are too simplistic for studying the pathological mechanisms of angiogenesis, invasion, and metastasis. Tissue engineering, specifically the unique cell culturing techniques provided by the use of 3D scaffolds and bioreactor technology, offers an exciting approach for studying cancer development in vitro.

Similar to normal tissue progression, an emerging tumor requires oxygen and nutrients supplied by the vasculature to maintain cell function, growth, and survival. Tumors experience a transition from an avascular to a vascular state by responding to changes in the tumor microenvironment and initiating an angiogenic response from the host vasculature. Typical stages of tumor development and important markers associated with hypoxia and angiogenesis are shown in the figure below.

Our group is investigating the ability to induce an angiogenic shift in human breast cancer cells in vitro through employing a tissue engineering approach in which cancer cells are subjected to culturing conditions that mimic specific tumor microenvironmental cues. The goal of this project is to construct an environment in which we can adjust each aspect of the tumor microenvironment to synergistically control the angiogenic shift and direct it towards the development of a 3D in vitro vascularized breast cancer model that can be used to better understand the pathological mechanisms of tumorigenesis, angiogenesis, and metastasis. The Figure to the right shows formation of a neovessel in response to culturing of endothelial and cancer cells together in a hydrogel.

Related Papers
C. S. Szot, C. F. Buchanan, P. Gatenholm, M. N. Rylander, J. W. Freeman, 2011, “Investigation of Cancer Cell Behavior on Nanofibrous Scaffolds,” Materials Science and Engineering C, 31: 37-42.