2006

December

November

Urethral dysfunction is a common complication of diabetes mellitus, spinal cord injury and pelvic trauma. Stress urinary incontinence (SUI) - the involuntary loss of urine secondary to a damaged urethral sphincter mechanism - is particularly common in women and can result from vaginal childbirth. There are currently several approaches to treat SUI, all of which are limited by ineffectiveness or subsequent complications. Regenerative medicine approaches, including cell therapy and tissue engineering, could potentially address these limitations. We believe that utilization of a functional tissue engineered urethral wrap (TEUW) will allow the native urethra to remain intact, while providing enhanced mechanical stability and functional reinforcement through designed regenerative repair mechanisms.
We will explore the use of progenitor cells as the functional component of a TEUW. Specifically, we will explore the following two hypotheses:

• a living, functional, smooth muscle-populated tubular construct can be fabricated in-vitro by appropriately stimulating bone marrow derived progenitor cells (BMPCs) within a natural biological matrix; and
• a tissue engineered urethral wrap (TEUW) derived from these constructs can be successfully implanted and will reverse the short-term changes in mechanical and functional properties of the urethra found in a rat model of SUI. We propose two specific aims to explore these hypotheses.

AIM 1 is to fabricate a living, functional smooth muscle populated tubular construct from BMPCs and a natural biological matrix that is suitable for implantation as a TEUW. This will be determined by determining the in-vitro stimulation regimen, chosen from a cadre of combinations of mechanical strain and biochemicals that yield optimal histological, functional, biomechanical and immunological properties.

October

Breast cancer is endemic in the United States, with nearly 216,000 new cases expected this year (American Cancer Society statistics). For patients undergoing mastectomy, the loss of one or both breasts can cause significant discomfort and psychosocial distress. The number of breast reconstruction operations exceeds 80,000 cases per year (American Society of Plastic Surgeons statistics). Current surgical options including autologous tissue flaps and implants, have significant problems.

The use of adipose precursor cells, or preadipocytes, may represent a better solution for soft tissue reconstruction for cancer defects. We hypothesize that human preadipocytes can be seeded on microcarrier scaffolds and be injected into an animal model to produce a durable engineered soft tissue replacement. Furthermore, we speculate that the preadipocytes will deifferentiate into both adipocytes and elements of the new vascular system that perfuses the fat tissue.

The specific aims of this study are to:

• Characterize cell surface markers of human preadipocytes using flow cytometry and assess the variability of differentiation of cloned subpopulations. Preadipocytes will be isolated from adipose tissue of breast cancer patients for clinical relevance.
• Evaluate the adherence, proliferation and adipogenic differentiation of human preadipocytes within macroporous collagenous beads and small intestinal submucosa (SIS) microparticles in culture.
• Determine the ability of human preadipocytes to generate mature adipose tissue when seeded in collagen and SIS particles and injected subcutaneously into a nude mouse.

September

Early deaths due to battlefield injuries are secondary to exsanguinations or overwhelming central nervous system trauma, whereas late deaths are secondary to sepsis and multiple organ system dysfunction syndrome. Currently, the primary strategy for treating hemorrhagic shock is to control ongoing bleeding and restore intravascular volume by infusing an asanguineous fluid (e.g., Ringer’s lactate solution) and packed red blood cells. However, conventional approaches toward resuscitation require administration of large volumes of fluids that are intrinsically heavy and bulky (e.g., normal saline solution or various colloidal solutions) and/or difficult to store (packed red blood cells). Interestingly, if intravascular volume expansion successfully restores cardiac output and arterial blood pressure before definitive hemostasis has been achieved, then, paradoxically, resuscitation can promote bleeding and shorten survival. Thus, logistical considerations limit the capacity of first responders to provide adequate conventional resuscitation on the battlefield and even in some cases of civilian trauma. In view of these considerations, an ideal initial resuscitation fluid for the initial management of hemorrhagic shock would require administration of just a small volume to improve tissue perfusion and oxygen utilization without increasing blood pressure to such an extent that endogenous hemostatic mechanisms (soft platelet-fibrin plugs) are disrupted.

Polymers with a molecular mass >10 exp 6 Da and a relatively linear structure are drag-reducing polymers (DRPs) that have been shown to reduce resistance to turbulent flow in pipes, thereby increasing flow rate at constant pressure (Toms effect). The flow conditions associated with the Toms effect probably do not occur when blood flows through arteries, arterioles, capillaries, venules and veins. Nevertheless, a number of studies have shown that intravenous administration of DRPs to experimental animals increases blood flow rate and decreases blood pressure and calculated peripheral vascular resistance without affecting blood viscosity or vascular smooth muscle tone. Furthermore, in a preliminary series of experiments, Kameneva et al. reported that survival was markedly improved when rats were resuscitated from hemorrhagic shock with a DRP-containing fluid. Prompted by these observations, we used seed-grant funding from DARPA to carry out a study to test the hypothesis that intravenous administration of very small volumes of an aloe vera-based DRP might improve oxygen consumption and extend survival time in rats subjected to otherwise lethal hemorrhage. The aloe vera-derived DRP used for these studies was a mixture of polysaccharides with an average molecular mass of ~4x10 exp 6 Da. Treatment of the rats with 7 ml/kg of the aloe vera-derived DRP (i.e., ~20% of the shed blood volume) significantly prolonged survival relative to treatment with a similar volume of the normal saline vehicle.

August

This study will focus on the cellular characterization of metastatic colon cancer in the liver and a 3-D perfusion culture instrument that recapitulates hepatic vasculature and microenvironment. Colon cancer is a very common cancer second only to lung cancer. Distant metastases are one of the worst prognostic signs as this places the patient in the most advanced staging category. Colon cancers generally spread through the lymphatics or through the portal venous system to the liver. The liver is the most frequent visceral site of metastatic dissemination and is the initial site of distant spread in one-third of recurring colon cancers, with two-thirds of patients having liver involvement at the time of death.  The median survival after the detection of distant metastases range from 6 to 9 months (with heavy liver involvement) to 24 to 30 months (with initially small liver nodules).

Our hypothesis, based on the current cancer stem cells model, is that metastatic colon cancer of the liver is a clonogenic event initiated by cancer stem cells which are optimally adapted to proliferate under the prevailing conditions in the primary tumor and emerges to form the metastatic cancer. Our goal will be to characterize the cancer cells isolated from metastatic colon cancer in the liver by identifying the cancer-initiating cell leading to the metastatic tumor and determine if this cell have the characteristic of a stem cell.

Finally, we propose to reconstitute in vitro tumor growth environment similar to what is found in patient affected with metastatic colon cancer using artificial liver bioreactors.

The specific aims pertaining to the main goal and its hypotheses are listed below:

Aim 1: Identification of colon cancer stem cells.
Aim 2: Expansion of human metastatic colon cancer stem cell populations using bioreactors.

Our approach will be to integrate the fields of stem cell biology, cancer biology, tissue engineering, and clinical treatment of patients to develop promising new therapies for patients with metastatic colon cancer. The overall goal of this project is to use cancer stem cells to create a diagnostic bioreactor using a novel 3-D culture model, which would allow tumor cells to recapitulate their in vivo geno- and phenotype diversity. This “in vitro” regeneration of the patient tumors in bioreactors would allow a new individualized chemotherapy planning and the discovery of novel approaches to effectively target cancer stem cells.

July

The aim of this proposal is to design solutions for vascular, cardiac, and pulmonary organ failure by building interactive teams of researchers focused on specific aspects of cardiopulmonary organ engineering. Our efforts will address a tissue engineered blood vessel, and a myocardial patch. The assembled research teams will function as cores of expertise that address common tasks associated with each of the projects. Five research cores will be established in the following areas:

1) matrix synthesis and surface modification,
2) precursor cell isolation and characterization,
3) biomechanical testing and conditioning,
4) animal model development, and
5) construct assessment.

For each of the organ projects we have design objectives (Specific Aims) that will be achieved in the five-year period of proposed work:

1) Tissue engineered blood vessel - A biological blood vessel will be developed that achieves long-term potency in the rat model and is subsequently evaluated in the porcine model. The blood vessel will be a "biological equivalent" to autologous arteries from a mechanical and biofunctional perspective. During vessel development in vitro, specific mechanical training protocols that have been optimized to direct appropriate cell differentiation and expression of matrix components will be employed.

May

The PediaFlow™ is a miniature magnetically levitated turbodynamic VAD for pediatric patients ranging from birth weight to 15 kg. Our design is based on extensive computational modeling. CFD analysis was used to optimize the flow field for efficiency and hemocompatibility and reach a design point of 0.5 L/min at a 110 mm Hg pressure head.
The current (1st generation) PediaFlow™ design is 51 mm in length with a diameter of 28 mm. Its total mass is approximately 100 g. Prototypes are currently undergoing in vitro performance tests and flow path visualization and quantification. In vivo evaluation is planned during the current grant year.

April

• Susan Braunhut, Ph.D., professor of biological sciences at the University of Massachusetts at Lowell
• Lorraine Gudas, Ph.D., chairman of the pharmacology department and Revlon Pharmaceutical Professor of Pharmacology and Toxicology, Weill Medical College of Cornell University, New York City
• Ellen Heber-Katz, Ph.D., professor, molecular and cellular oncogenesis program, The Wistar Institute in Philadelphia
• Shannon Odelberg, Ph.D., assistant professor, departments of internal medicine and neurobiology and anatomy, University of Utah, Salt Lake City
• Hans-Georg Simon, Ph.D., a developmental biologist and assistant professor of pediatrics, Children’s Memorial Research Center and Northwestern University in Chicago

The regenerative ability of adult human tissues, organs, and appendages is typically very limited.  The default mechanism of wound repair in humans and most other mammals is characterized by scar tissue formation.  However, there is evidence for some site-specific regeneration-like processes during mammalian embryologic development and during the early postnatal period.  In addition, there is lifelong self-renewal capability for selected cell populations such as hematopoietic cells, intestinal epithelium, and hepatocytes.

In contrast, urodele amphibians possess extraordinary abilities to regenerate lost structures, such as the limbs and tail, throughout their lifetime.   These regenerative processes are dependent upon the formation of a blastema at the site of injury.  This regeneration blastema is comprised of a self-organizing pool of proliferating progenitor cells genetically programmed to develop into a phenocopy of the lost structure. 

The blastema carries its own extracellular matrix and its own gene expression signature.  The work described in this project will attempt to unlock the regenerative potential in humans by determining the genetic signature of the developing blastema and attempting to recreate portions of the fetal development process in humans.

The research will involve several milestones including identification of cells that participate in the formation of a blastema-like structure in mammals, the spatiotemporal location of such cells during the remodeling process and the identification of bioactive molecules that induce, maintain, and complete such a process. 

The culmination of this work would eventually be the application of these identified mechanisms and events to the injured mammal in a mouse model. 

A highly interdisciplinary research team has been developed with expertise in developmental biology, molecular biology, matrix biology, pharmacology, immunology, and with training in medicine, veterinary medicine, physics, and computational methods of data mining.  Significant preliminary data has been generated to support the fundamental approach.  Well defined milestones have been identified and a management scheme has been established that assures close collaboration among the principal investigators and their respective laboratories at six different institutions.

March

Breast cancer is endemic in the United States, with nearly 216,000 new cases expected this year (American Cancer Society statistics). For patients undergoing mastectomy, the loss of one or both breasts can cause significant discomfort and psychosocial distress.

The number of breast reconstruction operations exceeds 80,000 cases per year (American Society of Plastic Surgeons statistics). Current surgical options, including autologous tissue flaps and implants, have significant problems. The use of adipose precursor cells, or preadipocytes, may represent a better solution for soft tissue reconstruction for cancer defects.

The study will test the hypothesis that human preadipocytes can be seeded on microcarrier scaffolds and be injected into an animal model to produce a durable engineered soft tissue replacement.

Furthermore, the research team speculates that the preadipocytes will differentiate into both adipocytes and elements of the new vascular system that perfuses the fat tissue.

The specific aims of this study are to:

• Characterize cell surface markers of human preadipocytes using flow cytometry and assess the variability of differentiation of cloned subpopulations. Preadipocytes will be isolated from adipose tissue of breast cancer patients for clinical relevance.
• Evaluate the adherence, proliferation and adipogenic differentiation of human preadipocytes within macroporous collagenous beads and small intestinal submucosa (SIS) microparticles in culture.
• Determine the ability of human preadipocytes to generate mature adipose tissue when seeded in collagen and SIS particles and injected subcutaneously into a nude mouse.

February

Embryonic stem (ES) cell research and scale-up for development of possible clinical therapies is limited by the existing 2D dish culture methods. Our proposed studies present a new approach, in which ES cells are expanded under 3D medium perfusion conditions within four-compartment hollow fiber-based bioreactors. The design of the bioreactors allows integral oxygenation and efficient transfer of nutrients and waste products to and from the cells, cultured at high density involving minimal solute gradients within the cell compartment. Additionally, the interwoven fibers provide a scaffold allowing the cells to form 3D structures where the size of cellular aggregates is limited by the spacing between the hollow fibers. We propose that the well-controlled and versatile culture environment provided by our bioreactor is ideal for both large-scale expansion of undifferentiated ES cells and directed differentiation of ES cells using numerous strategies, including controlled exposure of the cells to molecular reagents and compartmentalized co-culture with mature cells.

The objective of this 2-year project is to take the first step toward applying our bioreactor technology to ES cell research, by expanding and maintaining undifferentiated mouse embryonic stem (mES) cells in laboratory-scale versions of our bioreactor. We hypothesize that undifferentiated mES cells can be expanded and maintained in the perfused 3D environment provided by our bioreactor, and that within this 3D culture model mES cell pluripotency can be maintained by culturing mES cells and fibroblast feeder cells in two separate bioreactors perfused within one circuit (compartmentalized co-culture).

The specific aim of the project is to:
1. Develop a 3D culture model for bioreactor expansion and maintenance of undifferentiated mES cells, incorporating compartmentalized co-culture of mES cells with fibroblast feeder cells and allowing for enzymatic mES cell harvesting.

The research plan consists of the following tasks:

1.1 Develop the 3D culture model incorporating direct co-culture of mES cells and feeder cells;

1.2 Develop the culture model incorporating compartmentalized co-culture of mES cells and feeder cells; and

1.3 Develop a protocol for enzymatic mES cell harvesting from intact bioreactors.

January

John A. Kellum, MD

McGowan Institute Investigators: Gilles Clermont, MD; William Federspiel, PhD; Yoram Vodovotz, PhD; William Wagner, PhD

The NIH has announced the funding of a $7 million grant entitled "Systems Engineering of a Pheresis Intervention for Sepsis (SEPsIS)" to design and test an extracorporeal device for the treatment of severe sepsis, based on the principle of hemoadsorption. John A. Kellum, MD , Department of Critical Care Medicine, will lead a multi-disciplinary team comprised of basic science and clinical researchers, bioengineering and biomaterials experts, and experts in complex systems modeling.

Severe sepsis (acute onset organ failure in the setting of infection) is a major health problem that kills nearly 250,000 Americans each year and costs billions of dollars. Available therapies for sepsis, including those recently approved, are suboptimal and new therapies are urgently needed. However, the complexities of the inflammatory response network and the high cost of clinical trials, particularly in the critically ill, renders the traditional drug/device development paradigm obsolete.

The research team has previously developed and tested an extracorporeal blood purification device for treatment in chronic renal disease and has adapted this device for the treatment of acute inflammatory diseases. They have also developed and partially calibrated—in both rodents and humans—a mathematical model of sepsis. SEPsIS will integrate these two achievements and, through an iterative design process, develop a device that can be used to treat severe sepsis.

The goal of the SEPsIS grant is to design an extracorporeal blood purification device for the treatment of severe sepsis. The project brings together investigators from departments of Bioengineering, Chemical Engineering, Critical Care Medicine, Surgery, Medicine, and Mathematics. Investigators will also call on the expertise of two companies, one specializing in adsorbent polymer technology (MedaSorb Technologies, LLC) and the other in complex systems modeling (Immunetrics, Inc.).

McGowan Institute faculty and their roles in the SEPsIS Grant are:

• Dr. Gilles Clermont is the Co-Director of the Simulation Core and will focus on simulation and the development of a mathematical model of blood purification. As the project matures, his efforts will also include the design of the clinical pilot study. In years 4 and 5 he takes on the additional responsibility of managing the clinical pilot study and contributing to data analysis and manuscript preparation.

• Dr. William Federspiel's expertise is in biotransport phenomena, especially as related to artificial lungs, blood transport and cardiovascular devices. He is overseeing all aspects of the research on the hemoadsorption device and related models of the hemoadsorption process. Dr. Federspiel will also be responsible for the University component of the design and manufacturing work aimed at developing early investigational prototypes of the hemoadsorption device. He will design, supervise, and coordinate the associated experimental studies, analyze results of experimental and theoretical investigations, and help prepare reports.

• Dr. Yoram Vodovotz's laboratory will be responsible for the large number of cytokine assays required in this project. These cytokine measurements will be used to calibrate the mathematical model of inflammation and to determine the efficacy of hemoadsorption by the hemoadsorption device. His lab has the capability for multiple assays from a single sample making multiple analyses feasible in a small animal model.

Dr. William Wagner is an expert in the area of cardiovascular device biocompatibility, having worked extensively with circulatory and pulmonary support devices. His lab performs evaluations from in vitro surface analyses to clinical trials focusing on thrombosis, thromboembolism, and inflammation. In this partnership, he is responsible for directing the work in the biocompatibility core.