June 2005 | VOL. 6 | www.McGowan.pitt.edu

July marks the fourth anniversary of the McGowan Institute. I would like to take this opportunity to extend my thanks and appreciation for the sincere and substantial contributions of our Team that has resulted in the sustained growth and maturation of the Institute. The visibility of Regenerative Medicine is rapidly growing, as well as the role that the McGowan Institute is playing to advance the science and clinical translation of new regenerative technologies. Pittsburgh continues to have increased visibility as a destination for regenerative medicine research and we are also working to make Pittsburgh the destination of choice for regenerative therapies.

Our collective commitment remains to advance the sciences related to regenerative therapies, to train scientists and engineers to pursue technologies related to regenerative medicine, and train a generation of clinicians in the implementation of regenerative therapies. The receipt of two new pre-doctoral training grants this year has facilitated our progress in this area.

The Institute has grown to 180 faculty and over 600 students and staff. I look forward to another exciting year of advances in science and progress in the movement of our emerging technologies to clinical/commercial use. As noted above, our progress is directly attributable to all of the McGowan Team; I trust that next year we will collectively be able to look back and say that we have made more progress along this road that we are traveling together.

Thanks and Best Wishes for Continued Success!
Sincerely,

Alan J. Russell, Ph.D.
Director

 

Jean J. Latimer, Ph.D., Department of Obstetrics and Gynecology, has recently received funding for a study to assess a new paradigm for African American breast cancer involving stem cell differentiation in a novel cell culture system.

By way of background, African American women have a higher incidence of breast cancer than white women before age 40, while the opposite is true after age 40. However, African American women are more likely to die from breast cancer at every age. Although socio-economic factors have in the past been attributed to these differences, data exists to support the less widely held hypothesis that there may be intrinsic biological differences in African American breast tissue compared with white breast tissue. Evidence for this hypothesis includes the fact that a higher percentage of ER negative breast tumors are present in African American women.

In prior studies, Dr. Latimer and her colleagues have developed and published the description of a novel culture system for Human Mammary Epithelial Cells (HMEC), both normal and malignant. This system allows for unusually long-term (3 months or longer) establishment of normal primary cultures that begin as three-dimensional "mammospheres," which are structures made up of 40-100 epithelial cells.

The current study is a one year concept award (paradigm shifting study). In this study the Latimer Laboratory will precisely determine the architectural capacity of epithelial cells from African-American patients involving growth and differentiation in their HMEC system, by analyzing up to 30 cultures from women with known demographic factors such as age, parity and socioeconomic status and compare them with similar samples from Caucasian women.

The hypothesis to be assessed is that greater proportions of stem cells may exist in the Black population than in age- and parity-matched White women. If this hypothesis were true, then one might expect a higher mortality in African American women because of the presence of a stem cell population more likely to form an aggressive tumor type. The research team sees higher mortality in African American women, although this is confounded by economic factors involving assess to health care. The unique HMEC system combined with the efforts and intent of the local University Center for Minority Health will enable the Latimer laboratory to address a fundamental issue involving a new paradigm in terms of the etiology of African American breast cancer.

Jean J. Latimer, Ph.D. is an Assistant Professor in the School of Medicine, Department of Obstetrics, Gynecology, and Women’s Health. She is a member of the graduate faculty of the Molecular Genetics and Biochemistry Program and the University of Pittsburgh Cancer Institute. Her research laboratory is located at Magee-Women’s Research Institute. Dr. Latimer's laboratory specializes in DNA repair deficiency as a causative factor in breast carcinogenesis. Contact Information: latimerj@pitt.edu

 

Dr. Michael Chancellor has reported that a pure culture of muscle-derived stem cells (MDC) may not be needed to cure incontinence; these findings were presented at the 2005 annual meeting of the American Urological Association (AUA), and will be published in abstract 929 in the AUA proceedings.

These scientific studies supplement prior work by Dr. Chancellor and Dr. Huard that demonstrated in preclinical trials that muscle derived stems cells hold promise to treat stress-induced urinary incontinence. That technology has been licensed to Cook MyoSite Inc. who is using those research findings to develop a clinical procedure to treat stress-induced urinary incontinence using adult-muscle derived stem cells. Cook is beginning human clinical trials using adult muscle stem cells to further assess the viability of this methodology.

In the current research studies, reported by Dr. Chancellor at the AUA annual meeting, he notes that “when isolating a specific type of cell for transplantation into the body, the common thought is that the cells must be a pure culture. Getting a pure culture of muscle-derived cells, free of contaminating fibroblasts is difficult, but, if the fibroblasts don’t interfere with the muscle-derived cells, using these cells to regenerate muscle just got a great deal easier.”

In animal models of stress urinary incontinence, the researchers injected muscle-derived cells, fibroblasts and a mixture of MDC and fibroblasts into the tissue surrounding the urethra. Prior to injection, the models showed a significant decrease in leak point pressure, the point where the bladder leaks passively. Post-injection, all three groups showed an improved leak point pressure of the surrounding urethral tissue. Four weeks post-injection, MDC, fibroblasts and the combination MDC/fibroblast mixture were still present at their respective injection sites. There were no complications.

Fibroblasts did not interfere with the MDC ability to improve leak point pressure. However, the mechanism by which leak point pressure was improved, whether it be muscle regeneration from the MDC or a bulking effect from the fibroblasts is still being investigated. Prior animal model studies by the University of Pittsburgh group have found that MDC were able to regenerate deficient urethral muscle.

In addition to Dr. Chancellor, Irmute Usiene, Ron Jankowski, Ryan Pruchnic, Dongdeuk Kwon, Johnny Huard and Fernando de Miguel, all from the University of Pittsburgh, contributed to this study. MORE

 

The Australian firm Ventracor Limited named the five American heart transplant centers that will take part in the feasibility stage of the U.S. clinical trial of its heart assist device.

Ventracor Limited Chief Executive Officer, Colin Sutton PhD, said that Professor Eric Rose of Columbia University will be the Principal Investigator of the study and Professor Robert Kormos of the University of Pittsburgh Medical Center would be Chairman of the Steering Committee (pictured at the right).

The sites for the U.S. feasibility study involving 10 patients are Columbia University, University of Maryland, University of Minnesota, the University of Pittsburgh Medical Center, and one other site.

Professor Eric Rose said: "We are very excited to have the chance to be involved in the clinical trial of this promising new device. The VentrAssist offers some important potential advantages over existing therapies, which we hope will be shown during the clinical trial."

Professor Robert Kormos added: "The clinical trial of any new therapy requires careful consideration and analysis, and we believe this trial is designed to be both manageable and scientifically rigorous." Also speaking at the meeting were representatives of Vital Engineering (www.vitalengineering.com) who will provide training and field support for the start of the clinical trial. Vital Engineering has experience in over 300 implants of mechanical circulatory support devices, and is highly regarded in the clinical community.

 

Gene therapy for arthritis and other non-terminal, debilitating conditions and diseases is both feasible and safe, report researchers who conducted the world’s first such test on the approach in patients with advanced rheumatoid arthritis.

The results, published in this week’s online edition of the Proceedings of the National Academy of Sciences (PNAS), indicate that introducing a new gene has the potential to block the destructive inflammation process that takes place within arthritic joints. At a time when setbacks have cast doubt on the future of gene therapy, the results also provide assurance that it can be performed without causing undue harm to patients.

The clinical trial, which was conducted at the University of Pittsburgh School of Medicine between 1996 and 1999, involved nine women who had genetically modified cells injected into their arthritic knuckles and marked the first time a gene was introduced into a human joint. The researchers say their results showing that successful gene transfer can target joint inflammation open the door to the development of improved gene-based therapies for both rheumatoid and the more common osteoarthritis, which together affect about 66 million people in the United States.

“At the outset, our primary objective was proving the safety of gene therapy, especially for a clinical application to a nonlethal disease, such as arthritis. Once we learned of the adverse events in the X-SCID trial, we decided it was best to extend follow-up of our patients to five years so that there’d be little question about long-term safety,” explained Christopher H. Evans, Ph.D., D.Sc., the study’s lead author, now director of the Center for Molecular Orthopaedics at Brigham and Women’s Hospital and Robert Lovett Professor of Orthopaedic Surgery at Harvard Medical School.

“Given that gene therapy was so new and we were dealing with an otherwise healthy patient population, the ex vivo protocol was the most appropriate approach to take at the time,” stated Paul Robbins, Ph.D., professor of molecular genetics and biochemistry and orthopaedic surgery at the University of Pittsburgh School of Medicine, who was the study’s co-principal investigator and then director of the University of Pittsburgh’s Viral Vector Core Facility.

In their animal studies that preceded the clinical trial, the researchers had noted that a small percentage of injected cells could escape to other tissues within one week. But with five years follow-up, the authors have seen no evidence that these genetically modified cells have caused any adverse events in their nine patients. MORE

 

The Institute introduced a new web section to highlight some of the innovations that are emerging from the program. The areas addressed are:

• Excellence in Tissue Engineering
• Excellence in Medical Devices
• Innovative Cell Therapy at UPMC
• Acta Biomaterialia
• Multidisciplinary Education
• Musculoskeletal Research
• Nanocarpets for Regenerative Medicine
• Collaboration and Technology Transfer
• Center for Inflammation and Regenerative Modeling

Please enjoy the updates that are presented.

The 51st annual meeting of the American Society for Artificial Internal Organs (ASAIO) was held in Washington D.C. in June 2005. Numerous members of the McGowan Institute and the Bioengineering department contributed to the success of the meeting; there twenty presentations on a wide range of topics. Listed below are the abstracts from these presentations. The names of faculty are italicized, graduate students bolded, and undergraduate students are both bolded and italicized.

Development Of A 2D Microfluidic Oxygenation Device

A Vollmer, T Snyder, M Kameneva, B Monzyk, E Burckle, K Dasse, P Martin, H Borovetz , W Wagner, R Gilbert, T Thorsen (Mr. Snyder is completing his PhD in Dr. Wagner's lab)

A novel microfluidic capillary structure was developed for use with photocatalytic oxygen generating thin films. The microfluidic element was fabricated by multilayer soft lithography, and consisted of stacked sets of arborizing channels capable in the current form of diffusing O2 into the flowing medium across a silicone membrane. Fluid was brought into intimate contact with the diffusing surface, facilitating rapid oxygenation in the laminar flow regime. Convective gas transport was modeled analytically and oxygen concentration measured in real-time by microfabricated luminescent O2 sensors embedded in the capillary channels. The microfluidic capillary array was iteratively designed according to physiological principles with the aim of minimizing shear stress. Oxygen sensors provided reliable and precise measurement of oxygenation over a range of flow rates. Hemolysis, as indicated by plasma free hemoglobin, varied with microchannel design. In capillary designs refined to eliminate adverse flow conditions, thrombus deposition and channel occlusion were greatly reduced. This study demonstrates two milestones in the development of the photolytic artificial lung: 1) Design, fabrication, and refinement of a viable blood-bearing microcapillary structure. 2) Integration of micro-sensors to monitor blood O2 concentration during physiological flow conditions.

 

J Antaki, J Wu, T Snyder, H Borovetz, W Wagner, B Paden, C Diao

An important challenge facing the design of the miniature VADs intended for long term support is the optimization of the flow geometry to maximize hydraulic performance while minimizing shear-stress induced hemolysis and thrombosis. For semi-open centrifugal and mixed-flow pumps, as well as axial flow pumps, the complex flow pattern between the blade tip and casing has a dramatic effect on both efficiency and blood damage. This study employed CFD analyses (Star-CD, Adapco), based on a finite volume method, to simulate the 3-dimensional blood flow within a miniature centrifugal impeller. Turbulence was modeled by the standard k-e turbulence model. A multi-block structured mesh was generated to capture the steep velocity gradient near the blade surface. An implicit method based on multiple reference frames allowed coupling between the rotational impeller and stationary blocks. A periodic boundary condition provided efficient computation within the blade-blade region. A power law function was integrated into the code to estimate hemolysis with the shear history being recorded by the Lagrangian particle tracking method. Simulation results at three different tip clearances of 0.1, 0.05 and 0.02mm revealed the sensitivity upon efficiency, tip leakage flow, secondary flows, and hemolysis. Good agreement with the corresponding experimental results achieved with flow visualization studies was obtained.

 

J Antaki, C Diao, J Wu, H Borovetz, W Wagner, T Snyder

A persistent challenge to quantitative design of rotating blood pumps is the great disparity of spatial scales between the primary flow paths and microscopic clearance regions. Flow gaps within journals and blade tips are often on the order of a few cells, confounding the application of macroscopic continuum models. Yet, precisely in these regions exist the highest shear most likely to cause cellular trauma. This study is to determine the kinematics of cellular deformation and screening within small clearances of a small VAD. A transparent model of a centrifugal pump (46mm dia.) with an adjustable tip clearance (0~200 µm) was developed. Microscopic imaging of the blood cells in the tip and back clearances was achieved with a custom designed visualization system, consisting of inverted microscope fitted with 40x objective, Nd:YAG laser and high resolution CCD camera. The acquisition of the blood cell image in the tip clearance was synchronized to the impeller rotation. The cellular deformation vs. gap dimension was thus determined. Experiments with 6µm fluorescent particles were generally in agreement with the corresponding CFD simulation. However, studies with diluted porcine blood revealed the unique features of the cellular deformation, screening, and trajectory that occur in small clearances. As the gap reduces to zero, virtually all cells are excluded from the gap, suggesting a mechanism by which hydrodynamic film bearings may avoid catastrophic hemolysis.

 

P Wearden , R Kormos, S Badylak , M Kameneva, T Snyder, W Wagner, H Borovetz, J Marks, J Richardson, K Dasse

PURPOSE: The CentriMag® VAS has been used to support >100 adult cardiogenic shock patients as a bridge to decision for up to 14 days. The purpose of this project is to modify/test the system for its potential in treating pediatric patients for the same indication for use.

METHODS: This MagLev System is comprised of a polycarbonate centrifugal pump without mechanical bearings or seals, a motor, drive console and cannulae. The motor levitates the rotor magnetically so that rotation may be achieved without wear to reduce thrombus formation/minimize hemolysis. The system was evaluated in 4 lambs (12–19 kg): 3 acute (<= 24 hours) and one chronic (5-day) study. Pump hemodynamics, Hct, Hgb, platelet count and PFH values were monitored at baseline, every 4 hours for the 1st post-op day and daily thereafter. Necropsy and pump retrieval examination were performed at study termination.

RESULTS: Flows averaged 0.99+/-0.11 lpm (range 0.75 to 1.20 lpm). PFH averaged ~10 mg %. Platelet count, total protein and fibrinogen levels remained stable. No end organ dysfunction was observed. No organ infarcts or thromboembolism were seen.

CONCLUSIONS: This initial trial supports our hypothesis that the CentriMag® VAS can provide cardiac assistance for pediatric patients. We documented satisfactory pump performance in small animals for the intended duration of use. Hemolysis generation (PFH) was low, changes in hematological indices were unremarkable, and organ function was preserved. (Supported by R43 HL07 1376 from NHLBI, NIH).

 

J Wu, J Antaki, T Snyder, H Borovetz, W Wagner, B Paden, C Diao

The configuration of a centrifugal pump has been widely used as a ventricular assist device (VAD) for adults and recently for infants and small children with failing ventricles. For such a centrifugal pump, the back clearance gap together with the central core and outer annular clearance gap jointly form a washout flow path. However, within the washout path, the flow demonstrates a complex 3-dimensional structure, whcih potentially causes cellular trauma and/or thrombosis and also affects hydrodynamic performance. The objective of this study was to eliminate the reversal flow in the back clearance gaps of a miniature pediatric centrifugal blood pump by CFD analyses and design optimization. A commercial CFD code, Star-CD, was utilized to simulate the 3-D blood flow within a miniature centrifugal pump. An implicit method based on multiple reference frames was used for the coupling between the rotational and stationary elements. A power law function was integrated into the code to evaluate hemolysis with the shear history being recorded by the Lagrangian particle tracking method. By CFD analyses and optimization design, it was found that secondary blades, located along the lower and side surfaces of the rotor, can generate antegrade blood flow in the clearance gaps. Through optimizing the secondary blades number and their geometry by CFD, we were able to achieve net antegrade flow with minimal zones of retrograde flow.

 

P Marascalco, J Marhefka, S Shaulis, C Johnson, M Kameneva (Mr. Marascalco and Ms. Marhefka are BioE PhD candidates in Dr. Kameneva's lab)

BACKGROUND: Diabetes is associated with generalized afflictions of the cardiovascular system. Current therapies can at best delay the onset of these complications. Intravenous injections of blood-soluble drag reducing polymers (DRPs) were shown to increase blood flow and tissue perfusion and reduce vascular resistance without affecting the vessel wall. We hypothesized that DRPs might enhance the microcirculation which is thought to be reduced in diabetes.

METHODS: Diabetes was induced in 7 rats after a single dose of streptozotocin (vehicle in controls, n = 8) and characterized by blood glucose levels of 300–400 vs. 80–90 mg/dl in controls. Insulin (0.5–3 U) was administered to maintain blood glucose levels below 450 mg/dl. After 15 weeks of hyperglycemia, DRPs were injected IV at a final concentration of 1 ppm following recording of base hemodynamic parameters in acute experiments. Blood samples were collected before and after DRP infusion.

RESULTS: Body weight, tissue perfusion (TP), MAP and heart rate were significantly lower and vascular resistance and hematocrit were both significantly higher in diabetic animals. An injection of DRPs increased TP by ~40% in both control and diabetic rats with no significant change in MAP.

CONCLUSIONS: This study demonstrated that DRPs were able to improve impaired TP in diabetic animals and may represent a new rheological method for the treatment of impaired microcirculation in diabetes.

 

J Marhefka, P Marascalco, T Chapman, M Kameneva

Purpose: Blood-soluble drag-reducing polymers (DRPs) were demonstrated to have the ability to significantly increase blood flow and tissue oxygenation with no direct effect on vessel tone when injected at nanomolar concentrations in animals with normal and pathological circulation. Several DRPs, including high molecular weight polyethylene oxides (PEO), polyacrylamides, and plant-derived polysaccharides were applied in previous in vivo studies. However, the search continues for new DRPs that would be more biocompatible and mechanically stable than the synthetic DRPs and better defined and more reproducible than the natural DRPs.

Methods: High molecular weight poly(n-vinylformamide) (PNVF) was synthesized using an inverse emulsion technique and characterized using chromatography, rheology, and hydrodynamic methods. Then, the PNVF was tested in vivo to determine its ability to enhance blood circulation after intravenous injections.

Results: The synthesis yielded PNVF with a molecular weight of 4.5x106 Da and molecular and viscoelastic properties similar to well-known DRPs. PNVF solutions demonstrated the ability to significantly reduce flow resistance in both in vitro and in vivo models. Studies of the polymer mechanical degradation caused by high stresses showed that the PNVF molecules degraded at a slower rate than the most commonly used DRP, PEO. Since PNVF is known to have no toxicity, our results warrant further investigations of this polymer for potential clinical use.

 

S Vandenberghe, P Segers, J Antaki, R Shihab, P Verdonck

The Deltastream (Medos Medizintechnik GmbH) is a diagonal pump intended for use in miniaturized CPB or ECMO circuits. It can also be used for short-term left ventricular (LV) assist. This in vitro study aimed to gain insight in the heart-device interaction during pulsatile modulation. The Deltastream was inserted with apical-to-ascending aorta cannulation in the UGhent cardiovascular simulator consisting of a pneumatically driven silicon heart and a lumped mock circulation. Experiments were performed for 3 heart rates (50 - 100 - 150 BPM) and 3 heart failure conditions (none - mild - severe). The Deltastream was first run in continuous mode and the rpm was incrementally increased (0 - 5000 rpm). Next, a sine-modulated rpm was enforced in pulsatile mode. The effect of (de)synchronization between heart and Deltastream was evaluated with several combinations of heart and pump rates and triggering. Hemodynamic and energetic parameters were assessed and analyzed. Pump failure (0 rpm) results in backflow and aortic pressure drops up to 46 mmHg. The pump flow below baseline in continuous mode was found to be compensated by diminished aortic flow, thus yielding constant total flow. High continuous speed (5000 rpm) resulted in acute hypertension (MAP up to 178 mmHg). With pulsatile assist, unmatched heart and pulsatile pump rates yielded complex unphysiologic pressure and flow patterns. LV unloading was very dependent on synchronization and optimal unloading was achieved when the minimum rpm occurs at end-systole. Pulsatile support can be beneficial only if triggering is accurately controlled.

 

L Santelices, M Druzdzel, R Schaub, B Uber, R Kormos, J Antaki (Ms. Santelices is completing her BioE MS in Dr. Antaki's lab; Ms. Uber is a BioE PhD candidate in Dr. Antaki’s lab)

While VADs have demonstrated their therapeutic role for cardiac rehabilitation, a consensus on weaning protocol and markers of cardiac recovery has yet to be reached. The expertise for managing these devices is constantly evolving, multi-disciplinary, and limited to few centers. This has motivated the development of a Clinical Decision Support System (CDSS) to assess a patient’s readiness for weaning. A decision model was developed using statistical analysis of patient data and organized expert input. Data from all VAD weaning candidates at UPMC between 1996 and 2003 (n=29) were subjected to a longitudinal and cross-sectional analysis. Data Mining and Natural Language Processing were used to spot trends and isolate data that differed significantly between patients who were successfully weaned and those who remained VAD-dependent. Experts were consulted regarding weaning protocol and treatment preferences. The resulting model consisted of 2 main phases: a multi-parameter health status screening followed by a 3-tier evaluation of cardiac capacity. The decision structure and associated uncertainties were translated into an influence diagram using decision modeling software developed at U. Pittsburgh (GeNIe 2.0). The final model was tested on a random subset of retrospective patient cases and successfully predicted their outcomes. For accessibility, the model has been implemented on a hand-held computer featuring an efficient GUI. In addition to aiding the standardization of weaning protocol, we aim for this CDSS to promote evidence-based decision-making in healthcare teams with varying ranges of clinical experience.

 

G Pantalos, C Ionan, S Koenig, J Speakman, C Lucci, M Gartner

The ability of the 4“ and 3” pediatric cardiopulmonary assist system (pCAS) pumps to restore acceptable hemodynamics while providing left ventricular assistance (LVA), cardiopulmonary support (CPS), and veno-venous ECMO (VVE) was evaluated using an instrumented infant mock circulation that creates clinically relevant normal ventricular function (NVF) and left or right ventricular failure (LVF, RVF). The left atrium was cannulated with a 20 Fr. Baxter pediatric cannula (LVA), and the right atrium with an 8 Fr. Medtronic Biomedicus uptake cannula (CPS) and a 14 Fr. OriGen double lumen cannula (VVE). Aortic return was via an 18 Fr. Cannula. Data were collected while providing (1) LVA during LVF, (2) CPS during RVF and NVF, and (3) VVE during NVF with the pCAS operating in continuous and pulsatile modes using water (1 cP viscosity) and 40% glycerin/water (3.5 cp) as test fluids. For continuous flow operation, 4“ pCAS was able to restore acceptable hemodynamics by generating flows of 1.0 l/min at 1200 RPM for LVA, 1800 RPM for CPS, and 3300 for VVE with a zero flow RPM of 530 for LVA and 700 for CPS. Pulsatile and continous mode performance was comparable. Equivalent pCAS performance resulted using 40% glycerin/water. Initial 3” pCAS performance for LVA had a similar trajectory as the 4“ pCAS, but shifted by an additional 250 RPM with a zero flow RPM of 780. These data demonstrate acceptable pumping performance of the pCAS for infant circulatory support. [NHLBI Contract No. HHSN268200449189C]

 

A Polk, D McKeel, W Federspiel , W Wagner (Ms. Polk is a BioE PhD candidate in Dr. Wagner's lab)

A bioreactor was developed as a prototype biohybrid lung to facilitate study of endothelial cell (EC) response to varying shear stress and oxygenation over a 7-day period. The bioreactor was based on an exchangeable, endothelialized microporous hollow fiber (MHF) module that rotates (1–1500 RPM) while also permitting gas transport. Bioreactor component materials were tested individually and together for degradation and cytotoxicity in the presence of culture medium and ECs. MHF surface modification with radio frequency glow discharge and adhesion proteins was investigated to augment EC adhesion and growth. Emphasis in reactor design was placed on minimizing culture medium use and providing easy access for medium assay. EC confluence was achieved on modified MHF modules prior to bioreactor placement. Preliminary trials demonstrated EC survival for several days on MHFs at shear stresses up to 21 dyn/cm 2 ( Fig.). Gas transfer capacity was demonstrated in water; future studies will include ECs on the modules. This bioreactor provides a first step towards a mixing oxygenator with endothelialized surfaces that might be compatible with extended patient support periods.

 

K Burgess (Henchir), Q Yang, W Wagner, W Federspiel (Ms. Henchir is a BioE PhD candidate in Dr. Federspiel's lab)

We are developing artificial alveolar-capillary (AAC) modules for use in next generation biohybrid artificial lungs as an improvement over existing hollow fiber bundle technology. Our approach uses microfabrication and soft-lithography techniques applied to polydimethylsiloxane (PDMS) to create intimate arrays of blood and gas microchannels that approach the microvascular scale of the gas exchange units found in the natural lung. Our AAC modules will be designed to 1) increase the surface area to blood volume ratio and decrease diffusion distances compared to current artificial lungs and 2) allow endothelialization of the blood microchannels to improve biocompatibility and reduce thrombosis. In this study we examined endothelial cell seeding and viability in 3-D prototypes containing blood microchannels. Prototypes were fabricated by molding PDMS around an array of 100 wires (100µm in diameter) or by molding PDMS on microfabricated channels and stacking the PDMS layers to form a module. The microfabricated prototypes contained 700 channels (20 µm high and 100µm wide). The modules were incorporated into a perfusion loop and human umbilical vein endothelial cells were seeded statically and dynamically into fibronectin-modified modules. We found that static seeding was initially more efficient than dynamic seeding (31% vs. 13% reduced using alamar blue assay), but that cell viability decreased over ten days to less than 5% reduced. Future work will focus on additional cell culture studies, incorporating gas pathways, and optimizing the gas exchange characteristics of the AAC modules.

 

N Hagglund, B Frankowski, B Hattler, W Federspiel

Our lab is pursuing the development of a more compact intravenous respiratory assist catheter. In this study we assessed the potential to increase gas exchange efficiency and hence reduce device size by using a rotating hollow fiber bundle in our respiratory catheter. A bench prototype was constructed of a respiratory catheter with a small fiber bundle (25 French, 0.1 m 2 membrane area) that could be steadily rotated up to 10,000 RPM. The performance of the rotating bundle catheter was characterized in a mock vena cava loop using water as the test fluid. The CO 2 and O 2exchange rates increased with increasing rate of bundle rotation, but plateaued above 6000 – 7000 RPM. The maximum gas exchange rates were 529 ± 4.1 and 263 ± 2.3 ml/min/m 2 for CO 2 and O 2, respectively, which were over 2-fold greater than achieved in the same test using control respiratory catheters based on our pulsating balloon design. Bench tests using bovine blood indicated that the rotation of the fiber bundle per se does not appear to cause significant red cell hemolysis. On-going work is aimed at 1) reducing the degrees of plateau in gas exchange with increasing rotation rate; and 2) designing an implantable version of the catheter with improved seals and bearings and a mechanism to prevent the fiber bundle from damaging the vena cava.

 

R Svitek, B Frankowski, W Federspiel

We are developing a compact paracorporeal respiratory assist lung (PRAL) that uses a rotating annular fiber membrane bundle to both enhance gas exchange and pump blood. Our ultimate goal is to achieve significant CO 2 removal (100–120 ml/min) at relatively low veno-venous blood flowrates (500–1000 ml/min) without the need for a separate pump. This study examined the effect of fiber bundle porosity on the gas exchange and pumping performance of the PRAL. We fabricated two PRAL devices with bundle porosities of 0.43 and 0.80, but otherwise similar with membrane areas of 0.42 m 2 and 0.50 m 2 respectively. The devices were tested for gas exchange in a flow loop using water as the test fluid at 3 L/min. The PRAL with the higher bundle porosity achieved CO 2 removal at 1500 RPM of 153 ml/min/m 2 compared to only 116 ml/min/m 2 for the PRAL with lower bundle porosity. In bovine blood, the PRAL with the higher bundle porosity at 1500 RPM achieved a CO 2 removal rate of 182 ml/min/m 2 at a blood flowrate of only 750 ml/min. In a separate pump test in water, the fiber bundle with higher porosity generated 67 mmHg compared to only 52 mmHg for the fiber bundle with the lower porosity at 0.75 L/min flow at 1500 RPM with water as the test fluid. The fiber bundle with increased porosity is within 10% of our gas exchange target, and the pumping ability is consistent with generating 750 ml/min blood flow through percutaneous cannula less than 20 Fr.

 

H Eash, S Budilarto, B Hattler, W Federspiel

Our lab is exploring further improvements to our respiratory support catheter while its current version (the Hattler Catheter ®) is undergoing commercialization and readiness for human clinical trials. Our current respiratory support catheter uses a pulsating balloon to enhance O 2 and CO 2 exchange, with pulsation at a constant beat rate and balloon volume. In this study, we tested the hypothesis that using random balloon pulsation rates and volumes could disrupt fluid entrainment within the fiber bundle, improve overall mixing, and increase gas exchange. We compared random pulsation rates and volumes with standard constant pulsation at the average rates and balloon volumes used in random pulsation. Gas exchange of our catheter with 100% O 2 sweep gas was measured in a 3 L/min water flow loop at 37 °C. For constant balloon volume, CO 2 gas exchange with random pulsation from 200–400 beats per minute (BPM) was not significantly different than for constant pulsation using the average beat rate (286 BPM) of the random pulsations: 302.2 ± 1.4 versus 299.5 ± 0.9 ml/min/m 2. For a constant pulsation rate of 300 BPM, CO 2 gas exchange using random versus constant balloon volume was statistically significant but negligible: 294.3 ± 0.6 versus 301.1 ± 1.7 ml/min/m 2. Random versus constant rate and volume pulsation also showed a negligible change for O 2 exchange. We conclude that random balloon pulsation of either rates or volumes does not impact overall mixing sufficiently to improve gas exchange in our respiratory catheter.

 

R Miller, S Safta, A Hallab, J Patzer II (Mr. Safta is a BioE PhD candidate in Dr. Patzer's lab)

Detoxification support for liver failure patients by dialysis against albumin-containing dialysate (bound solute dialysis or BSD) has shown clinical potential. Theoretical and experimental analysis of BSD indicates that it can be effective at as little as 0.2% human serum albumin (HSA), making it possible to practice BSD with readily available dialysis equipment in single pass by adding commercially available 25% HSA to the dialysate. Since optimal dialysis performance requires well-mixed dialysate, two factors mitigate against directly metering HSA into the dialysate: laminar flow profile in the dialysate, and density segregation between the HSA concentrate and the dialysate solution. A static mixer was designed to promote mixing of HSA into dialysate. Photography was used to highlight flow patterns in both the mixer and dialysis cartridge as a function of the number of static mixer elements and Reynold’s number. We found that a Kenix® type mixer with 18 segments was sufficient to well-mix a stream of dialysate with HSA. The pressure drop across a prototype device remained below 10mmHg for flow rates common to dialysis. We have designed the device so that it can be inserted into the dialysate flow upstream of the dialyzer. The static mixer accomplishes several goals: (1) it provides a well-mixed dialysate/HSA solution; (2) it provides a port for metering HSA into the dialysate; (3) it has minimal pressure drop so that it can be used with available dialysis equipment.

 

S Safta, R Miller, J Patzer II

Accumulation of albumin-bound toxins, which have a range of detrimental effects on the liver, other organs, and patient physiology, is common in liver failure. Due to extremely low solubility in physiologic solutions, these toxins cannot be removed by conventional dialysis. Bound solute dialysis with slow continuous ultrafiltration (BSD-SCUF) has the potential of removing albumin-bound toxins.

Methods: An in-vitro system consisting of a reservoir containing surrogate blood (dialysate with 40 g/L human serum albumin (HSA) saturated with unconjugated bilirubin), the Gambro Prisma System, and a Baxter CT-110G dialyzer was employed to measure reservoir bilirubin clearance as a function of time over a 3-h experiment. BSD-SCUF variables included blood and dialysate flow rates, ultrafiltration rate, and dialysate HSA concentration in clinically relevant ranges.

Results: Clearance of bilirubin was 5 to 10% without HSA in the dialysate stream. Clearance increased to 20–25% when HSA was added to the dialysate stream, irrespective of blood and dialysate flow rates. Notably, bilirubin clearance was not significantly different throughout the experimented ranges of HSA concentration (0.4 to 1.6 g HSA/L). Ultrafiltration rate did not affect clearance of bilirubin.

Conclusion: In accord with the BSD model, ultrafiltration, which removes solute at free solute concentration, does not affect bound solute clearance. Adding HSA to the dialysate stream increases bilirubin removal, independently of the blood or dialysate flow rates. The results show that toxin removal is independent of the HSA concentration in the dialysate.

 

S Budilarto, H Eash, B Hattler, W Federspiel

The Hattler Catheter® is an intravenous respiratory assist device that uses a centrally located pulsatile balloon within a hollow fiber bundle to enhance gas exchange by active blood mixing. Flow visualization techniques were used to investigate flow patterns induced by balloon pulsation in this respiratory catheter. We tested the hypothesis that the nonsymmetric inflation and deflation of the balloon leads to nonuniform balloon-generated perfusion of the fiber bundle. The respiratory catheter was placed in a 1“ ID rigid test section of an in-vitro test loop (3 L/min DI water). Particle image velocimetry (PIV) was used to map the velocity vector field arising from balloon pulsation at 120 beats/min. in lateral cross-sections of the test vessel. Velocity measurement showed that radial velocity varied between 5 cm/s in the region adjacent to the top and bottom quarters of the fiber bundle and 0 cm/s in the region adjacent to the right and left quarter of fiber bundle. We hypothesized that the nonuniform radial flow caused by nonsymmetrical balloon collapse would lead to different gas exchange in the affected fiber regions. An in-vitro study of local gas exchange was performed by selectively perfusing sweep gas flow (100% helium) to four quarter regions of the fiber bundle. Preliminary results showed 15–45% variation in CO 2 exchange among these regions. The variation was not only attributed to nonuniform radial velocities, but also visual observations of nonuniform fiber motion around the bundle. Quantification of fiber motion, via PIV, will be pursued to analyze the relative velocity.

 

J Wu, J Antaki, G Bearnson, J R Boston, C Diao, J Gardiner, J A Hawkins, G Jacobs,
M Kameneva, B Keller, P Khanwilkar, J Kirk, R Kormos, V Morell, J Long, C Li, S Miles, E Prem, B Paden, D Paden, R E Shaddy, M Simaan, T Snyder, H Tsukui, S Vandenberghe, W Wagner, S Webber, H S Borovetz

Engineering analysis towards the development of a miniature circulatory assist pump has lead to a 2 nd generation design, hybridizing features from HeartQuest™ and Streamliner™ maglev VAD systems. The current embodiment, the PediaFlow™, arises from a rigorous analysis of engineering objectives, coupled with numerical optimization of hemodynamics, electromagnetics, and heat transfer. A multidisciplinary strategy was used to balance key criteria related to (1) anatomic compatibility, (2) performance, (3) biocompatibility, (4) suspension robustness, and (5) manufacturability. The current specifications are 38 mm length, 21 mm diameter, 48 gram weight, 0.73 cc priming volume, nominal flow point of 0.5 lpm against 100 mmHg, and shock load rejection of 6g. External temperature rise, constrained to 2° C, proved to be critical determent of size of the assembly. Ongoing efforts of our combined university-industry consortium addresses challenges of biocompatibility, cannulation, control, and human factors design. In vivo tests on lambs are scheduled for Q3–2005.

Advances in Quantitative Biomedical Models
James Antaki

Molecular Biocompatibility Issues
William Wagner

For more Information see the ASAIO Journal’s Website: www.asaiojournal.com

 

McGowan@pitt.edu