ULTRASOUND USED FOR A NON-INVASIVE DIAGNOSTIC SYSTEMInterdisciplinary Biomedical Research Program - IBRP
2001 IBRP Participants: Craig Berman • Kwadwo Bonsu • Lucas Carey • Jason Liang • Cherry Lim • Daniel Medina • Brooke Orosz • Maria Squire • Andrew Sturm • Robert Wlodarczyk
2002 IBRP Participants: Christopher Astefanous• Evren Azeloglu • Craig Berman • Anita Cheruvanky • Edna Choi • Audra Criscione • Daniel Han • Hao-Yu Lin • Evan Spiegel • Lei Wang
2003 IBRP Participants: Ben Adler • Azeema Ameerally • Anita Cheruvanky • Edna Choi• Sara Goldgraben• Anubhav Jain • Erin McCusker • Sravenesh Muralidhar • Gavin Olender • Amit Salkar • Laura Shih • Evan Spiegel • Victor Sutan• Pui Yee Tong • Man Tsz Yau • Hui Jing Yu • Matthew Zafran
2004 IBRP Participants: Ben Adler • Sailaja Akella • Ted Feldman • Sara Goldgraben • Amanda Gorecki • Lamya Karim • Karen Law • Kerry Lanigan • Diana Leung • Isaac Pflaum • Anna Ruvinskaya • An Sung • Christopher Trinh • Hui Jing Yu • Guivion Zumbado
Craig Berman (Yale University); Yi-Xian Qin; Wei Lin; Clinton Rubin, Biomedical Engineering, Stony Brook University
The current research focuses on developing, testing and improving a prototype ultrasound diagnostic system designed for determining bone quality. The ultrasound diagnostic system relies on previously documented properties of ultrasound propagated through bone. Further improvement of the wave analysis algorithms provide for phase velocity and group velocity distinctions which can help limit error in measuring ultrasound velocity through the bone. To test the improved prototype, 38 right feet and femoral heads were obtained from human cadavers under strict protocol. The width of the calcaneous were measured and each calcaneous was scanned at high resolution (0.5 mm) with the prototype ultrasound device. Broadband Ultrasound Attenuation (BUA), ultrasound velocity, and attenuation were recorded. DEXA (dual energy x-ray absorptiometry) data was also collected for both the calcaneous and femoral head. There was a high correlation between ultrasound results and age of measured bones (R > 0.8). Preliminary data demonstrated similar image patterns between ultrasound scanned bone and DEXA determined bone mineral density. These promising results are leading to further study. The data shows that the ultrasound based diagnostic system has promise for analyzing systemic bone quality in a clinical application. This work was supported by a training grant from the National Institutes of General Medical Sciences (R25-GM62492).
BIOABSORBABLE MEMBRANE FOR BONE CELL DELIVERY
Kwadwo Bonsu (Stony Brook University), Kwangsok Kim, Benjamin Hsiao, Benjamin Chu, Nadera Osmani, Meiki Yu, Biomaterials and Chemistry Departments, Stony Brook University
The objective of this project was to investigate the growth of osteoblasts released from a sandwich structured polymer scaffold consisting of nano-structured non-woven membrane. The membrane was a poly (D, L-lactide) (PLA) based blend, and 40 wt% of polymer solution was prepared for electrospinning under DMF as a solvent. The solution was electrospun into a polymer film, which was used for the bottom layer. Bone cell from mouse calvaria was then cast into the bottom layer of the membrane. A thin film was then electrospun for the top layer. The sandwich-structured scaffold was placed in a petri dish with 5ml of medium and placed in an incubator at 37°C to observe the cell release and membrane degradation behaviors. The results showed about 15% of cells released from the scaffold after 2 days. After release, the cells began a gradual growth on the petri dish. In 7 days, 2.0 × 105 cells had grown on the plate. Eventually, we hope to apply this technique towards tissue engineering. The membranes can be introduced into human tissue like bone, into the joints of people such as osteoporosis patients to regenerate the bone through gradual degradation of the membrane and growth of the bone cells. More applications of the membrane are towards more effective and targeted drug delivery procedures. The drugs will be electrospun into membranes and introduced at target sites in the body after surgical procedures and other invasive procedures. These membranes will be made in such a way so as to degrade and release the drugs directly to the affected site at a rate known to the physician. This work was supported by the Center for Biotechnology at Stony Brook and Stonybrook Technology and Applied Research through through a training grant from the National Institutes of General Medical Sciences (R25-GM62492) and a New York State grant.
ESTABLISHING A GENE EXPRESSION DATA BASE FOR FRUIT FLY DEVELOPMENT
Lucas Carey (Stony Brook University) and John Reinitz, Applied Mathematics and Statistics, Stony Brook University
(Abstract not available)
CHANGES IN TRABECULAR BONE ARCHITECTURE INDUCED BY DISUSE AND LOW-LEVEL MECHANICAL STIMULATION
Jason Liang (Massachusetts Institute of Technology); and Clinton Rubin, Biomedical Engineering, Stony Brook University
Three groups of mice were tested under different levels of mechanical demand to determine if these different levels altered trabecular bone quality. Female 16-week-old Balb/C mice were tested control, stimulated, and disuse groups (n=10). The mice femurs were harvested after 3 weeks and examined using Microcomputed Tomography ( µcT). The fan-beam, Scanco Ltd. cT machine was used to generate a three dimensional anaylsis of the trabecular bone properties of the mice. Connectivity, trabecular thickness, SMI were among the properties examined. It was determined that low level mechanical stimulation increased trabecluar thickness by 10% when compared to the control species. Connectivity was increased by 12%. Trabecluar thickness among the disuse group when compared to the control group decreased by 17%. Connectivity decreased by 36%. The results showed that altered mechanical demand can impact trabecular bone quality in Balb/C mice. This work was supported by a training grant from the National Institutes of General Medical Sciences (R25-GM62492).
APPLICATION OF MICROARRAY FILTERING ROUTINES TO ISOLATE GENES INVOLVED IN MECHANICALLY STIULATED BONE FORMATION.
Cherry Jane Lim (Stony Brook University); Michael Hadjiargyrou and Stefan Judex, Biomedical Engineering, Stony Brook University
During the summer of 2001, I worked closely with Dr. Stefan Judex and Bioengineering student, Russell Garman. It was a truly rewarding and learning experience. I was exposed to a gene analysis software called Gene Pix Pro, carried out research, and had opportunity to conduct data analysis. My internship consisted of analyzing gene expression in rats exposed to three different conditions. Rats' legs were either mechanically stimulated or disused. The controlled rats were also added. The stimulated mice were set on a vibrating plate for 10 minutes for 30 days. This mechanically stimulated the tibias of mice. The disused mice were perched by their hind legs so to not use their tibias. After this was accomplished, the rats' mRNAs were extracted and then captured cDNAs from it. Using the newly innovative technique, microarray analysis, cDNAs were analyzed and data was collected from this. These microarray data would eventually show which genes were highly expressed or not expressed in each of the conditions. After analyzing the microarray data on the Gene Pix Pro, I filtered, sorted, and prearranged microarray data on Microsoft Excel. Microsoft Excel is a convenient way of organizing and sorting data especially in large amounts. After Professor Judex and I manipulated our data, we were able to draw conclusions from them. I announced the specific genes that were highly expressed or not expressed in each of the conditions. Russell Garman then performed lab tests and researched these "special" genes. Following this, I made a Power Point presentation on my work and had other professors and graduate students critique my techniques. Another professor, Michael Hadjiargyrou assisted me in ways I could improve my data analysis and also confirmed my conclusions. This work was supported by a training grant from the National Institutes of General Medical Sciences (R25-GM62492).
PRINCIPLES GOVERNING DESIGN OF HIGH FIELD MRI CONTRAST REAGENTS BASED ON THE CURIER SPIN MECHANISM
Daniel Medina (University of California, Davis) and Charles Springer, Chemistry Department, Brookhaven National Laboratory
(Abstract not available)
THE FEASIBILITY OF USING TOMOSYNTHESIS TO IMPROVE MAMMOGRAM ACCURACY
Brooke Orosz (Stony Brook University), Gene Gindi, and Parmeshwar Khurd, Medical Image Processing Lab, Departments of Radiology and Electrical Engineering, Stony Brook University
This study investigated the feasibility of using tomosynthesis to produce a 3-D mammogram, which could help detect cancers in women with thick, dense breasts. We took x-ray images using the Lorad steriotatic biopsy machine (manufactured by GE), and performed reconstructions using algorithms developed in Matlab. Our technique involved rotating the phantom in the path of the x-ray beam to produce images of an object from multiple angles, which can be combined mathematically to produce a three-dimensional picture of the object. The experiments are still in an early stage, using simple objects, including a cluster of water-filled test tubes. Although I could not produce a well-focused reconstruction, the images gathered may be useful in further refinement of reconstruction techniques. More importantly, I was able to identify many of the technical difficulties involved in taking x-ray images, which can be used for tomosynthesis. I learned that it is crucial to rotate the object without shifting it at all, and that the axis of rotation must be absolutely perpendicular to the x-ray beam. Even a tiny deviation produced a reconstruction so blurred it was useless. The narrow field of the Lorad machine means that we need a test object less that 4 cm in diameter, or parts of it will not be visible from all angles, once again invalidating the reconstruction. However, once these technical difficulties are surmounted, we intend to demonstrate the usefulness of tomosynthesis as a mammographic technique. Partial funding for this work has been provided by: NIH R25-GM-62492
RELATIVE QUANTIFICATION OF GENE EXPRESSION IN BONE IN RESPONSE TO ALTERED LEVELS OF MECHANICAL STIMULATION USING REAL TIME RT-PCR
Maria E. Squire (University of Scranton); Stefan Judex, Department of Biomedical Engineering, Stony Brook University
The purpose of this project was to use real time, reverse transcription polymerase chain reaction (real time RT-PCR) to quantify changes in gene expression in bone in response to altered levels of mechanical stimuli. We have previously showed that small strain events (<5 µE) induced at high frequencies (30-90 Hz) are anabolic in bone. In this study, the expression of specific genes that play a role during mechanically induced bone formation and resorption was analyzed. Sixteen-week female BALB/cByJ mice were randomly assigned to one of following three experimental groups: age-matched control, disuse by tail suspension, and mechanical stimulation (10 min/day@ 0.25g, 45 Hz). Real time RT-PCR targeting the genes that code for collagen type I and beta-2 microglobulin (beta-2) was run on the Light Cycler Instrument (Roche Molecular Biochemicals) for mice euthanized at day 4 of the experiment (n=2 for each group). The preliminary results for the expression of the gene coding for collagen type I, using relative quantification normalized to the housekeeping gene beta-2, showed a 30% (+/- 4.4%) increase in the expression level in the mechanically stimulated group and a 40% (+/- 4.8%) decrease in the expression level in the disuse group in comparison to the age-matched control group. This shows that the gene coding for collagen type I is sensitive to both mechanical stimulation and disuse. In addition, this result shows that real time RT-PCR is a sensitive tool for the quantification of the changes in the expression level of genes in bone in response to altered levels of mechanical stimulation. This study was kindly supported by NSBRI (National Space Biomedical Research Institute) and by a training grant from the National Institutes of General Medical Sciences (R25-GM62492).
THE SYNTHESIS AND STRUCTURE-ACTIVITY RELATIONSHIPS OF MULTI DRUG RESISTANT REVERSAL AGENTS
Christopher P. Borella, Andrew Sturm (Stony Brook University), and Iwao Ojima, Department of Chemistry, Stony Brook University
Presently, two of the most accepted drugs used to treat cancer are paclitaxel and docetaxel. These active anti-tumor (chemotherapeutic) agents act by stabilizing the formation of microtubules during mitosis and inhibiting their depolymerization. Hence, the metaphase/anaphase transition is blocked in the cell cycle and cell apoptosis is initiated. Although these drugs are very effective in the treatment of cancerous tumors, they also present problems, such as undesirable side effects and more importantly the development of multi-drug resistant (MDR) cells. To circumvent the problem, one can either modify the anti-cancer agents to make them more active against resistant cell lines or synthesize compounds called MDR reversal agents. These compounds, when co-administered with anti-cancer agents, lessen the effects of P-gp and increase the anti-cancer drug's effectiveness against multi-drug resistant malignancies. In Dr.Ojima's research laboratory over the summer I worked on synthesizing a novel MDR agent. My synthetic approach utilized a tetra cyclic precursor called 10-deacetlybaccatin III, which is obtained from the Pacific yew tree. The compound I synthesized contained a C-7 substituted hydrocinnamoyl, C-10 acetyl and a C-13 short chain carboxylic acid moiety. The substituted compound was synthesized in order to study polar C-13 substituent effects on the MDR reversal ability. The structure of the compound was confirmed by the use of NMR and MS. Acceptable purity levels were obtained by HPLC. This compound was sent out for in-vitro biological assays, results are soon expected. This research is funded by grants from the National Institutes on Health (GM417980). We also acknowledge support from National Institutes of General Medical Sciences (R25-GM62492)
A NEW MLC TEST PATTERN FOR AUTOMATED IMAGE BASED QUALITY ASSURANCE
R.A. Wlodarczyk (Stony Brook University), K.T. Welsh and L.E. Reinstein, Department of Radiation Oncology, Stony Brook University
Over the past decade there has been a rapid increase in the use of multi-leaf collimators (MLC) for Radiotherapy. Numerous procedures have been described and test patterns developed which address a variety of MLC specific quality assurance parameters. Most, however, rely on manual interpretation and provide non-quantitative results that are not readily tracked in a database. We have designed a new MLC test pattern for specific use with an automated image based quality assurance system (IBQA), and is capable of providing rapid analysis and database tracking of five different QA parameters. The IBQA/MLC test pattern is used to irradiate a 14in x 17in piece of radiographic film that is placed beneath a special phantom containing precisely embedded fixed and moveable fiducial markers for the purpose of independently establishing the coordinate system and scaling factors. The test pattern consists of two rectangular open regions (200mm x 90mm) and two stepped open regions, both of which are offset from the central axis. These regions enable the software to analyze, evaluate, and track the following parameters: inter-leaf leakage, closed-leaf leakage, leaf positioning, penumbra, and light/radiation field coincidence. The incorporated database allows establishment of separate baselines and tolerances for each parameter and each treatment unit, as well as tracking of the results over time. This work was supported by a training grant from the National Institutes of General Medical Sciences (R25-GM62492).
FEASIBILITY OF THREE-DIMENSIONAL MAMMOGRAPHY
Christopher Astefanous (Stony Brook University) and Gene Gindi, Departments of Radiology and Electrical Engineering, Stony Brook University
It is hoped that Three-Dimensional Mammography will make apparent low contrast lesions in high-density breast tissue. Currently, in a conventional mammogram these types of lesions are sometimes undetectable by radiologists. This study's main purpose is to acquire a data set of two-dimensional digital X ray images obtained from a set of different view angles and then use a reconstruction algorithm to assemble these two dimensional slices into a three dimensional image. It was established from a preliminary reconstruction using simulated images that the three-dimensional image will have a lower resolution than a two-dimensional image but will serve to make visible the aforementioned type of lesions. That is, spatial resolution will be lowered in the 3D version, but contrast resolution will be greater. In studying the feasibility of 3D mammography several problems were encountered. Firstly, it is not plausible to rotate an X ray machine around a phantom in order to acquire the two-dimensional images at different angles. Therefore, a rotation stage needs to be fabricated which can rotate a breast phantom to simulate the rotation of the X ray machine. Secondly, in order for a three-dimensional image to be created from two-dimensional slices, there must be an assurance that the rotation axis of the stage is perpendicular to the x-axis of the detector plane and parallel to the y-axis of the detector. Hence, this stage must be adjustable in the x, y, and z planes. In order to establish if the stage is positioned properly, a method can be created where images taken of a calibration phantom are analyzed and used as feedback to properly adjust the stage. Additionally, it must be established where the perpendicular beam emanating from the X ray focal spot strikes the detector in order for the three-dimensional reconstruction algorithm to be accurate. This last problem can be solved from a second calibration phantom tailored to determine if a perpendicular beam is striking it. These projects are at varying degrees of completion. In a related project, the Modulation Transfer Function (MTF) of the X ray system being used was measured by taking the absolute value of the Fourier Transform of a point spread function (PSF). The point spread function was calculated from an image of a plate of lead with a slit ten microns in width. This calculation was carried out in order to characterize the inherent ability of the X ray system to detect spatial frequencies (i.e. details of a breast). There are several corrections that were performed to more accurately measure the MTF. Among these corrections was exponential fitting, a way to correct for irregularities in the PSF curve. Also, the effects of blurring from the finite slit width were corrected for. Lastly, in the MTF curve the value of the spatial frequency being passed was multiplied by the magnification factor. These corrections provide greater accuracy in understanding which spatial frequencies are attenuated. It is hoped that through this study a contribution can be made to the field of mammography which will further the ability of clnical radiologists to better detect lesions in parients. This work was supported by a training grant tfrom the National Institute of Health (NIH GM-62492).
DETERMINATION OF THE EFFECTS OF REGIONAL ISCHEMIA ON ISOLATED BEATING RABBIT HEART
Evren U. Azeloglu (Stony Brook University), Glenn R. Gaudette, Irvin B. Krukenkamp, Fu-Pen Chiang, Departments of Mechanical Engineering and Biomedical Engineering, Stony Brook University
Unlike many other engineering designs, the heart shows variation within its chambers and surface in terms of mechanical function. In addition, myocardial dysfunction, such as ischemia or infarction, affects the heart regionally. This necessitates a whole field measurement technique with high spatial resolution to assess the viability of such tissue. Computer aided speckle interferometry (CASI), a nondestructive examination technique, is herein developed for this purpose. New Zealand white rabbits were anesthetized and the hearts were isolated (n = 6). A random speckle pattern was created on the anterior surface of the left ventricle. Images of the beating hearts were acquired with a charge-coupled device (CCD) camera for one second at a rate of 50 frames per second. CASI was used to determine the 2-D displacement vectors over regions of approximately 4 x 5 mm with an average spatial resolution of 140 ?m. In order to understand the tissue mechanics during acute regional ischemia, the first diagonal branch of the left anterior descending artery was occluded and measurements were repeated. Regional area stroke work (the integral of the left ventricular pressure with respect to area), the first invariant of the 2-D strain tensor, and systolic contraction were used to determine the regional function. Significant changes were detected with the induction of regional ischemia in all the above mentioned parameters. Commonly used techniques cannot determine 2-D strain and lack the high spatial resolution of CASI. Determination of the 2-D strain can provide useful data on the functionality of the heart. The funds for this study were provided by NSF BES-9903515, and NIH GM-62492.
CONFIRMING THE USE OF FORMIC ACID FOR SIMULATING OSTEOPOROSIS AND THE ABILITY OF ULTRASOUND TO DETERMINE BONE QUALITY
Craig Berman (Yale University) and Yi-Xian Qin, Department of Biomedical Engineering, Stony Brook University
This study sought to establish formic acid as a means for simulating the effects of osteoporosis on trabecular bone and to correlate the artificially simulated bone mineral density (BMD) with non-invasive ultrasound velocity and attenuation to determine bone strength. Thirty-Four bone cubes (10mm x 10mm x 10mm) were cut from right distal condyles of sheep femurs using a diamond blade saw. Orientation information was marked on the cubes with a pencil. In order to calibrate the -CT for sheep bones it was necessary to determine the ratio of bone volume to total volume of the bone cubes. Each of three cubes was further cut into 4 equal cylinders using a diamond wire saw. Leaving one piece of each cube as a control, the other three were acidified in 5% formic acid for different time periods (5.5 min, 11 min, 16.5 min). After being submerged in acid, the bone was immediately dipped into a saturated aqueous solution of sodium bicarbonate to stop the acid and then rinsed with distilled water. The bone's exposure time was marked with a pencil. Three of the acidified and sliced bone cubes were mounted and scanned in the -CT. They were then embedded in polymethacrylate, sliced on the diamond saw and then the bone volume was calculated using Osteomeasure software. The remaining 31 bone cubes were acidified in stages and at each stage were scanned by -CT and scanned by ultrasound. The data collected from this experiment would be able to explain the effects of formic acid on trabecular bone volume and therefore how closely it simulated osteoporosis. The data would also show the correlations between BMD, ultrasound velocity, broadband ultrasound attenuation (BUA) and mechanical strength. Future work may involve determining the effects of formic acid on true bone material modulus using technology such as nanoindentation. These results may help to identify new models for osteoporosis and to evaluate new modality for non-invasive diagnostic of bone quality. This work was supported by the National Space Biomedical Research Institute (TD00207) the New York Center for Biotechnology and by a training grant from the National Institutes of General Medical Sciences (R25-GM62492).
GENERATION OF A NOVEL GENE/FLUORESCENT FUSION PROTEIN AND ITS EXPRESSION ANALYSIS IN MAMMALIAN CELLS
Anita Cheruvanky (Stony Brook University), David Komatsu, Yen Luu, and Michael Hadjiargyrou, Department of Biomedical Engineering, Stony Brook University
This research was conducted in order to analyze the expression of a novel gene in pre-osteoblastic mammalian cells by developing a fusion protein construct. Previous studies show high mRNA expression levels of this gene during rat femoral fracture healing (e.g. callus). Despite of this knowledge, the cellular localization and function of this gene remains unknown. Thus, we reasoned, that a fusion protein composed of our novel gene and green fluorescent protein (GFP) would aid in identifying the cellular localization of this gene and thus provide a clue as to its function. In order to create this fusion construct, we inserted our gene sequence into vectors that encode for GFP. Two fusion proteins were created using two vectors, pEGFP-N2 vector which encodes for a red fluorescent protein gene and pEGFP-C1 vector encoding for GFP (both have been optimized for higher expression in mammalian cells). The MCS (multiple cloning site) of pEGFP-N2 is found at the N-terminus of the GFP gene where as in pEGFP-C1 the MCS is found at the C-terminus of the GFP gene. Having these two constructs allows us to compare whether the location of our gene plays an important role in its expression and localization. To create this fusion protein PCR (Polymerase Chain Reaction) was first used to amplify the segment of DNA containing the novel gene. Vectors pEGFP-C1, pEGFP-N2 and the novel gene were digested with the same restriction enzymes BamH1 and EcoRI and then ligated with using DNA ligase. These recombinant DNA constructs we then used to transform E. coli. Following antibiotic selection, colonies were selected and analyzed for the presence of the plasmid. Positive colonies were then expanded and the plasmid was isolated using commonly used laboratory procedures. This plasmid was then sequenced in order to ensure that it was in-frame (containing no intervening
stop codons that would prevent its translation). Results verified that the fusion protein that was constructed using vector pEGFP-C1 was in-frame. However the fusion protein using pEGFP-N2 was not in-frame and posed a major problem. We are currently repeating the above procedures in order to obtain the pEGFP-N2/novel gene construct. Since, the fusion protein using C1 vector was found to be in-frame it was then transiently transfected into mammalian cells (pre-osteoblastic mouse cell line). The original vector pEGFP-C1 was transfected as control along with the recombinant novel/GFP construct. Fugene6 Transfection Reagent was used in each case in order to ensure high transfection efficiency. Forty-eight hours following transfection the cells were observed under a fluorescence microscope. As the picture below shows fluorescence was more intense within the nucleus. This is consistent with the fact that our protein contains a classical nuclear import signal sequence (PIKKKRP). We can thus hypothesize that our protein localizes in the nucleus and might function in the nucleus. However these results are only preliminary, this transfection along with the newly made fusion protein using pEGFP-N2 will be repeated and further tests performed in order to further confirm my results. This research study was supported by a grant from NIH GM-62492.
THE IMPACT OF MECHANICAL VIBRATIONS AT VARIOUS MAGNITUDES AND FREQUENCIES ON TRABECULAR STRAIN IN MICE TIBIA
Edna Choi (Stony Brook University) and Stefan Judex, Department of Biomedical Engineering, Stony Brook University
Cortical and trabecular bone tissue is sensitive to mechanical deformation, also known as mechanical strain. Recent data indicates that extremely low magnitude mechanical vibrations delivered at high frequencies (>20Hz) are highly anabolic to trabecular bone. This adaptive response is differential among individuals due to genetic variations that result in unique bone mass and architecture. However, the mechanism by which these very small vibrations are anabolic is not yet known. Here, we investigated whether bone strain is dependent on the magnitude and frequency of the mechanical vibration. Single-element strain gauges were implanted at the anterior-medical surface of the proximal tibia of eighteen adult female mice at the age of 4-months; 6C57BL/6J, 6 BALB/cByJ, and 6 C3H/HeJ mice. Upon recovery, strain data were recorded during normal gaiting and while the mice were sitting on the vibration plate. Until now, only three of the eighteen mice had been strain-gauged because we had encountered difficulties with associated noise in the strain gauge amplifiers as well sa with the surgical techniques.
Preliminary data from two C3HBL/6J mice revealed that a previously identified anabolic stimulus (45Hz, 0.25G) induced cortical bone strain of approximately 5 ?? in magnitude. Longitudinal strains generated by mechanical vibration at 22.5Hz and approximately 0.2G have a range between 3?? and 8?? in magnitude. Results also indicated that bone strains induced by normal gait (walking) ranged from 100?? to 200 ?? in magnitude. Although more through data and statistical analyses are needed to explain the significance of our preliminary data, our study concludes the feasibility of in vivo cortical strain measurement at the proximal tibia of mice. In the future, we will focus on effects of mechanical stimulation, particularly mechanical vibrations, on trabecular strains extrapolated from recorded cortical strains using the Finite Element Method. In addition, bone strains induced by various magnitudes and frequencies will be compared across the three strains of mice to examine whether induced cortical and trabecular strains are influenced by genetic make-up. This study was funded by a training grant from the National Institutes of Health (NIH Gm-62492).
THE DESIGN AND SYNTHESIS OF NOVEL TAXOIDS AS POTENTIAL TREATMENTS FOR MALARIA, LEISHMANIASIS, AND TRYPANOSOMIASIS
Audra Criscione (Providence College), Andrew Sturm, Deric Geng, and Iwao Ojima, Department of Chemistry, Stony BrookUniversity
The drugs currently used to treat malaria, leishmaniasis, and trypanosomiasis have limited efficacy due to drug resistance. Therefore, the discovery and development of new and effective drugs to treat these diseases are desperately needed. Paclitaxel (Taxol®) and docetaxel (currently the most widely used anti-cancer drugs) have properties that target tubulin/microtubules and inhibit mitosis by stabilizing microtubules. It has been shown that human tubulin has good amino acid sequence homology with plasmodium falciparum tubulin, Leishmania major tubulin, and Trypanosoma cruzi tubulin (tubulin of the parasites that cause malaria, leishmaniasis, and trypanosomiasis, respectively). Accordingly, it is likely that the drugs to target human tubulin can also treat the parasitic diseases. The goal of this project is to synthesize three different paclitaxel congeners (taxoids) which will both destroy parasites and be minimally cytotoxic to human hosts. More than twenty reactions have been completed in decent yields to synthesize the designed novel taxoids. Most reactions were performed under nitrogen using syringe technique. Extraction and column chromatography were used to purify the compounds. The progress of each reaction was monitored by proton nuclear magnetic resonance (1H-NMR) and thin layer chromatography (TLC). The structures of the taxoids and their intermediates were characterized mostly by 1H-NMR and the purity of each taxoid was analyzed by high performance liquid chromatography (HPLC). Assays to determine the efficacy of the synthesized novel taxoids against these tropical diseases will be performed by the Department of Infectious and Tropical Diseases at the London School of Hygene and Tropical Medicine. This work was supported by a training grant from the National Institutes of Health (R25-62492).
ANABOLIC EFFECT OF MECHANICAL STIMULATION IN OVARIECTOMIZED RATS IS NOT SUFFICIENT TO STOP OSTEOPENIA
Daniel Han (Cornell University) and Stefan Judex, Maria Squire, R Garman , Clinton Rubin, Department of Biomedical Engineering, Center of Biotechnology, Stony Brook University
Postmenopausal women are a high risk population for developing osteoporosis. In order to study this particular population, an ovariectomized rat model was used to mimic the skeletal changes observed in osteoporotic postmenopausal women. Previous studies have shown that induced non-invasive mechanical stimulation can have an anabolic effect on bones. This study looked at whether a non-invasive mechanical stimulation could also be anabolic to ovariectomized bone even to the point of normalizing the effects of ovarietomy. Ovariectomized retired breeder rats were subjected for 28 days, 10 minutes/day, 5 days/week, to low amplitude (.25 g, where 1g=9.8m/s^2) and high frequency (45 Hz, n=6 or 90Hz, n=6) mechanical signals. Control groups consisted of long term ovariectomized (n=6) and long term non-ovariectomized (n=30) rats that were allowed to roam freely in their cages. Demeclocycline, calceine, and xylenol orange were administered to all of the rats on day 1, 17, and 25 to label sites of bone formation. Bone histomorphometry was used to examine the metaphyseal region of the proximal tibia. Comparing the 90Hz stimulated group to the control ovariectomized, we found a 160% (p<.05) increase in bone formation rate per bone volume, a 60% increase in mineral apposition rate, and a 50% increase in mineralizing surface per bone surface in the trabecular bone. However, there was no significant difference between the 45Hz stimulated bones, the control ovariectomized bones, and the non-ovariectomized control bones in any of the histomorphometric assays. Micro computed tomography (?CT) was used to examine the 3-dimensional static assays of the metaphyseal region of the distal femur. Upon analysis, there were no significant differences in fractional bone volume, trabecular thickness, number, or separation, connectivity density, or SMI, between the stimulated and non-stimulated ovariectomized rats. Although the histomorphometry data showed increased bone formation rates, the static ?CT data did not show significant differences between the stimulated and non-stimulated ovariectomized rats. Further study in bone resorption in stimulated and non-stimulated ovariectomized rats may shed light on the disparity between the histomorphometry and ?CT data. Within the 4 week time span for the experiment, we can conclude that the anabolic signals induced at 90Hz stimulation alone, are not strong enough to increase the overall bone quantity and improve the morphology associated with osteopenia caused by ovariectomy. This study was supported by grants from NIH GM-62492.
NEURONAL SIGNAL PROCESSING FOR MULTI-ELECTRODE NEURAL SENSOR
Hao-Yu Lin (Stony Brook University); Ridha Kamoua, Department of Electrical and Computer Engineering;and Partap Khalsa, Department of Biomedical Engineering, Stony Brook University
For this project, an electronic circuit has been designed for processing multiple neuronal signals acquired by a silicon based multi-electrode sensor. The sensor can help to understand the responses of neural interfaces, which is a desired development for use with patients who have suffered spinal cord injuries, somatic pain and neuromuscular diseases. As detected by the sensor, the neuronal signal has an amplitude in the microvolt range. This small signal is easily contaminated by noise signal from power supply and other nearby instruments. A sequence of signal processing steps is needed to allow this signal to be observed. In this project, we have focused on two critical steps in the signal processing: amplification and filtering. First a buffer stage was implemented to minimize the input noise and provide high input impedance. Next an instrumentation amplifier (INA103) was used with a voltage gain set at 1000 (60dB) by an external resistor. The filtering circuit consisted of a notch filter and a band-pass filter. Both filters were constructed with Burr-Brown's universal active filter (UAF42). The notch frequency was set to 60 Hz to minimize the noise signal from the power supply. The band-pass filter was designed to have a bandwidth from 100Hz to 5kHz since most of the neuronal signals have frequency components within this range. The final stage of the circuit consisted of a programmable amplifier so that the gain could be changed. The circuit was first implemented and tested on a breadboard for processing of a single channel. The input signal was taken from a signal generator with an external attenuator. From the experiments, the output signal frequency responses agreed with the designed expectation. Signal that had frequency below 100Hz and beyond 5kHz was attenuated. Signal between the bandwidth had amplification of 47dB approximately. The lower 3dB point was at around 100Hz and the higher 3dB point was at around 10.2kHz. Particularly the 60Hz frequency had amplification of around 26dB. The overall gain is lower than our design. The reason is due to the unavailability of certain resistor values. The circuit was tested with actual neuronal signal from the rat skin's peripheral nerve. The background noise was much higher than expected. The excessive noise is most likely due to unshielded wires in the circuit as well as the presence of a ground loop. A ground loop occurs when the ground of the various instruments is not connected to the same voltage. This study was supported by a training grant from the National Institutes of Health (NIH GM-62492).
DETERMINING THE SPECTRAL CHARACTERISTICS OF DIAPHRAGMATIC EMG ACTIVITY IN THE C57/B6 WILD TYPE MOUSE
Evan T. Spiegel (Stony Brook University); Ki Chon, Irene C. Solomon,and Marvin H. O'Neal III, Departments of Physiology and Biophysics,and Biomedical Engineering, Stony Brook University
The spectral composition of diaphragmatic electromyogram (EMG) activity was studied in urethane anesthetized C57/B6 wild type mice to determine their respiratory characteristics. The spectral characteristics of respiration in the C57/B6 wild type have never been examined so this study attempts to establish a ruler against which future studies can be measured. EMG data were collected from respiratory-related diaphragmatic contractions while the mouse, which was properly anesthetized, inhaled a hyperoxic gas mixture (60% N2, 40% O2). Spectral analysis of EMG data was performed using both Short-Time Fourier Transform (STFT) and Fast Fourier Transform (FFT) algorithms. FFT analysis was implemented to determine change in spectral composition from minute to minute in a 10 min EMG recording for each mouse, while STFT analysis was used to investigate the spectral composition change within one respiratory cycle because diaphragmatic EMG is a time varying signal. FFT Analysis showed discrete peaks in regards to spectral composition of diaphragmatic EMG in wild type mice. Two distinct peaks were evident in the high frequency oscillation (HFO) range of the wild type mouse around 120Hz and 210Hz. HFO frequency peaks as identified in the C57/B6 mouse have also been observed in similar studies on cats and rabbits. Another peak, less apparent than the others, was present in some of the wild type mice within the medium frequency oscillation (MFO) range, generally around 50Hz. No trend has been established about the change in spectral composition over the period of EMG activity. STFT analysis of wild type mouse EMG activity exhibited similar frequency peaks as the FFT method, but revealed that the period of these specific oscillations with respect to the period of a diaphragmatic contraction is short. Examining the FFT analysis of EMG data of wild type mice suggests that there is a specific frequency composition for the C57/B6 wild type mouse even though the location of these discrete peaks exhibits some variability over time. This study was supported by a training grant from the National Institutes of General Medical Sciences (R25-GM62492), and a grant from the National Heart Lung and Blood Institute (R01-HL63175).
THE ENERGY LANDSCAPE OF PROTEINS: FROM COMPLEX TO SIMPLE
Lei Wang (Stony Brook University) and Ilya Vakser, Department of Applied Mathematics and Statistic, Stony Brook University
One of many steps scientists are now taking to better understand biological systems of organisms is to identify protein structures, their functions, and how they interact with each other. Protein structure is critical for understanding the protein function and is mostly determined by X-ray crystallography, NMR spectroscopy, and computer modeling. A program called GRAMM (Global RAnge Molecular Matching) developed by Dr. Ilya Vakser predicts (docks) possible position of a protein (ligand) with respect to another protein (receptor) at multiple resolutions (high, low, intermediate). For each predicted position, there is energy associated with the location. Because the high-resolution predictions are more dispersed than the low-resolution ones (Fig.1), we hypothesized that high-resolution docking will result in a more complex energy landscape than the low-resolution one. Proteins from 2PTC ( -Trypsin), 2HHB (Hemoglobin), and 1CHO ( -Chymotrypsin) were docked at both high resolution and low-resolution, and 1000 matches for each resolution were generated. The ligand's center of mass shift was used as a parameter to plot energy landscape for all 1000 matches at both resolutions (Fig.2). We found that there is a clear distinction between the high-resolution energy landscape and the low-resolution energy landscape. In high-resolution graphs the data points are dispersed, whereas in low-resolution ones the points are concentrated into columns. Thus there is a clear correlation between complexity of energy landscape and resolution. Visualization tools and quantitative expressions must be developed to reflect the degree of complexity at different resolutions. The bigger-picture of this study is to find the global-minimum at the low-resolution, and then to refine the structure to detect the global-minimum at the high-resolution. Acknowledgment:
This study was supported by a grant NIH GM-62492.We would like to thank Dr. Andrei Tovchigrechko for his technical support.
NANOINDENTATION AND SYNCHROTRON INFRARED MICROSPECTROSCOPY OF BONE: CORRELATING BONE MATERIAL AND CHEMICAL CHARACTERISTICS
Benjamin Adler (Stony Brook University); Bhavin Busa, Lisa Miller, Clinton Rubin, and Stefan Judex, Department of Biomedical Engineering, Stony Brook University and the National Synchrotron Light Source, Brookhaven National Lab
Introduction: Osteoporosis fractures are associated with declines in bone strength, which is affected by bone quantity and quality. Although bone quality is not well understood, it is influenced by mechanical material properties. These material properties have an unclear relationship with bone chemical properties, knowledge of which may be beneficial in understanding the etiology of bone diseases. A method has been developed to combine material and chemical testing of bone in such a way that material-chemical correlations may be determined.
Materials and Methods: Due to the high resolution of the two techniques (data points are as small as 100 µm² for spectroscopy, and for nanoindentation about 0.071µm²) it is important that we have a way to accurately test the same areas. This requirement necessitated the creation of a coordinate system universal to the stages of both devices. It was found that reference points bored into the epoxy resin surrounding the samples, with a sharpened pin, could be used as local origins for our coordinate systems. At the Brookhaven National Lab spectroscopy facility, the microscope is fitted with a stage that can lock slides at specific, determinable and repeatable angles (orientations). The nanoindenter stage however, is merely an open magnetized plate. Taking advantage of the locking microscope stage, magnetic microscope slides were fabricated for sample mounting. In order to align these slides on the nanoindenter stage, a thin aluminum strip was affixed plumb to the edge of the stage. These steps enabled the calculation of a conversion factor between the two stages based on the ratios of the relative distances between reference points. To obtain preliminary micromechanical data femurs from seven adult Sprague-Dawley rats were harvested. Three sections (distal head, diaphysis, and proximal head) were cut (diamond wafer saw) and then dried in a series of alcohol concentrations ranging from 70-100%. Bones were then embedded in epoxy resin under vacuum, and sanded using silicon-carbide abrasive papers of grits 320, 600, 800, and 1200. Polishing cloths impregnated with diamond suspensions of sizes 3, 1, 0.25, and 0.05 µm provided the smooth surfaces necessary for nanoindentation and spectroscopy. Tests included 32 indents made on the distal epiphysis, and 162 indents made on the distal diaphysis, distributed evenly between the medial and lateral regions, of the one set of prepared samples.
Results: A total of 16 indents selected in each condyle produced moduli (mean ± standard deviation) of 23.95 ± 2.47 (GPa) in the medial condyle compared to 27.3 ± 3.55(GPa) in the lateral (p=0.01). In the distal diaphysis the medial aspect measured 32.2 ± 4.6 (GPa), and the lateral aspect measured 34.1 ± 5.1 (GPa) (p=0.037). A convergence study of the 81indents made in the medial aspect revealed 20 indents as a sufficient sample size for a precise mean. Chemical testing of the samples is still under way.
Discussion: A method for the correlation of micromechanical and chemical properties of bone has been developed. Preliminary micromechanical data showed significant and repeatable variations in Young's modulus between the medial and lateral regions of the distal epiphysis and diaphysis. The observed differences between the medial and lateral regions will provide us with targets to test in the future. To this end the hind limbs of six adult (four male, two female) Sprague Dawley rats will be prepared for testing using the above methodology. It is expected that curve fitting of scatter plots between material and chemical properties will demonstrate correlations which may provide insight into the etiology of bone diseases such as osteoporosis. This work was kindly supported by grants from NASA, and by the National Institutes of Health (NIGMS); R25-GM62492.
DETERMINING MATERIAL PROPERTIES OF THE HUMAN LUMBAR FACET JOINT CAPSULE: USING OPTICAL IMAGE CORRELATION TECHNIQUES
Azeema Ameerally (Stony Brook University); Jesse Little, Partap Khalsa, Department of Biomedical Engineering, Stony Brook University
Purpose: This study was performed to determine tensile material properties of the lumbar spine facet joint capsule (FJC) to further develop a more accurate computational model. A computational model facilitates simulations of spinal manipulations that cannot be performed in vivo, paving the way for a greater understanding of the causes and eventual treatment of low back pain (LBP). Methods: Intact facet joints (n = 7) were harvested from cadaveric human lumbar spines. The FJC (bone-capsule-bone, BCB) were trimmed into a rectangular geometry and the thickness, width, & lengths were measured using a digital micrometer. A specimen was uniaxially loaded (Tytron 250, MTS, Inc.) and preconditioned before testing. Silicone carbide particles were sprinkled onto the ligamentous region of the BCB and were illuminated to create light intensity patterns recorded by a CCD camera at 7.5, 15, & 30 Hz. The region of interest was divided into subimages. By using an image correlation function in Matlab (v.6.1, The MathWorks, Inc.), a fourier transform on the light intensity pattern of the sub image was formed which tracked the u,v displacement vectors and allowed for Eulerian plane strains calculations. BCB specimens were loaded (10-50% strain) parallel to the collagen fibers, at loading rates of 1, 5, 10 and 15% strain/sec. Results: Plane strains were non-uniform and were averaged to obtain a mean value for each loading for each specimen. The instantaneous viscoelastic modulus of elasticity was significantly different (p<0.001) at increasing strain magnitudes (10-50%), though it was unaffected by loading rate (p = 0.59, 2-way repeated measures ANOVA). Despite the controlled uniaxial loading, significant shear strains (?xy) occurred and corresponded to a substantial mean principal angle (17.88º + 13.61º) of the principal strains. Hence, the first principal strain was used to calculate material properties. The mean uniaxial, viscoelastic and elastic tensile moduli were of an exponential form , where a & b were 0.83 & 11.7 for the viscoelastic and 0.41 & 12.9 for the elastic responses, (r2 = 0.97 and 0.96, respectively). The mean Poisson's ratio was 0.22 + 0.20. Conclusions: The FJC exhibits viscoelastic properties and displayed its greatest anisotropic properties with increasing strain magnitudes. Further analysis of the elastic and viscoelastic response of additional specimens are necessary for a more validated computational model. Currently there are estimates of material properties of the FJC, however this study is unique, in that, it uses optical image correlation techniques to calculate the material properties. This study was supported in part by NIGMS R25-GM62492 and by R03 AR46865 (Khalsa).
CLONING AND EXPRESSION ANALYSIS OF PAIRED MESODERM HOMEOBOX PROTEIN GENE 1 AND T-BOX TRANSCRIPTION FACTOR 15 DURING FRACTURE REPAIR
Anita Cheruvanky, David Komatsu and Michael Hadjiargyrou, Department of Biomedical Engineering, Stony Brook University
The identification and expression analysis of transcription factors involved in bone regeneration is crucial in determining whether they may be used for gene based therapies for fracture repair. Suppressive subtractive hybridization performed with RNA isolated from intact rat femurs and from post-fracture (PF) days 3, 5, 7, 10, 14 and 21 calluses showed the up-regulation of transcription factors Paired Mesoderm Homeobox Protein gene 1 (PRX-1) and T-Box Transcription Factor 15 (T-Box 15). These data were correlated with previous research showing their involvement in limb development and to the central physiological processes occurring during the fracture healing process. It was thus hypothesized that PRX-1 and T-Box 15 play a vital role in the processes of chondrogenesis and ossification in the maturation of the fracture callus. To begin to address this hypothesis, the temporal expression pattern of these genes was determined using Northern Blot analysis. A gene specific cDNA probe was synthesized using Reverse Transcription Polymerase Chain Reaction (RT-PCR) where RNA extracted from gestational day 18 rat embryos was used as a template along with pairs of degenerate oligonucleotide primers designed specifically to amplify a 320 base pair and 376 base pair sequence of PRX-1 and T-Box 15, respectively. Each amplified band was purified and ligated into the linearized PCR-Trap vector using T4 DNA ligase. These recombinant constructs were used to transform E.coli and screened for the presence of PRX-1 and T-Box-15. However, no bacterial colonies were seen on the plate corresponding to T-Box 15. On the other hand, many colonies were present on the plate corresponding to PRX-1 and few were selected for further analyses. The plasmid containing PRX-1 was extracted from the positive colonies, sequenced for verification and used as a probe for Northern analysis. Total RNA extracted and pooled from intact rat femurs and fracture calluses at PF day 3, 5, 7, 10, 14 and 21 were separated by gel electrophoresis and transferred to a filter which was hybridized using the P-32 labeled PRX-1 cDNA probe. Figure 1 shows the transcriptional activity of PRX-1 in intact femurs and at PF days 3, 5, 7, 10, 14 and 21. The transcriptional activity of PRX-1 is high at PF days 3, 5 and higher still at PF days 14 and 21 compared to intact. Normalizing this data to the 18S rRNA band and comparing it to the gene's transcriptional activity in intact femurs produced similar results. High transcriptional activity of this gene at PF days 3 and 5 (put numbers) suggests that the gene may play a role in the intramembranous ossification of a fracture callus. In addition the high transcriptional activity of this gene at PF days 14 and 21 also suggests that PRX-1 may play a role in chondrogenesis and possibly bone remodeling. Further research on this project would involve obtaining a Northern Blot analysis on T-Box 15. In addition, the spatial expression of PRX-1 could be determined using in situ hybridization. This research study was supported by a grant from National Institutes of Health (NIGMS) R25-GM62492.
THE IDENTIFICATION OF TARTRATE-RESISTANT ACID PHOSPHATASE AS A QUANTITATIVE ASSESSMENT OF BONE RESORPTION
Edna Choi (Stony Brook University) and Stefan Judex, Department of Biomedical Engineering, Stony Brook University
Introduction
Bone quantity is heavily influenced by bone remodeling, an interactive and coordinated process between bone formation and resorption. Yet, most studies at the tissue level have focused on the quantification of bone formation in response to mechanical stimuli and/or disuse, hence, limiting our understanding of bone's catabolic response to its mechanical environment. In order to more fully assess bone remodeling, it is essential to evaluate the anti-catabolic effects of a mechanical stimulus as well. In this project, we were interested in quantifying bone resorption via a histological approach, known as tartrate-resistant acid phosphatase (TRAP) staining. TRAP acts as a marker enzyme for bone resorption because it is released by osteoclasts, bone resorbing cells, to resorb bone matrix. Therefore, the amount of TRAP released would reflect the net rate of bone resorption. Methods
Bone samples were stored in -80 C immediately after harvesting. Each femur sample was separated into four regions, as shown in the figure to the right; proximal epiphysis-metaphysic (EM1), proximal diaphysis (MD1), distal diaphysis (MD2), and distal epiphysis-metaphysis (EM2). Each region was fixed in formalin, followed demineralization in a cocktail of formic acid and formaldehyde. Before embedding in glycol methacrylate (GMA), samples were infiltrated in a GMA infiltration solution until they appeared transparent. Resulting GMA blocks were sectioned at 3 to 5 m with a microtome (Microtome: R-Jung, Germany. Blade: Delaware Diamond Knives, Inc., USA). Sections were adhered to gelatin-coated slides and pre-incubated in 0.2 M Tris Buffer, pH 9.0. Sections were stained for 30 minutes at 37 C in a mixture of NaNO2, acetate buffer (pH 5.8), Na-Tartrate, napthol-ASTR-phoshate, and n,n-dimethylformamide. Sections were counterstained with methyl-green for 15 sections.
Results
Although our sections successfully demonstrated TRAP activities, severe non-target background staining was observed. Such background staining lowered the contrast between cell nuclei and enzyme activities, hence, the identification of TRAP was made uneasy (Fig3). When sections were counterstained with Mayer's Hematoxylin rather than Methyl Green, it was found that TRAP activities were masked due to the similarity in pigmentations of enzymes and nuclei (Fig4). Discussion
Bone resorption was evaluated with TRAP staining. Staining results were highly dependent on tissue preparation, which is the earliest part of the protocol. Therefore, if problems arose during tissue preparation, we would not be able to recognize them until the end of the protocol. In addition, we encountered enormous amount of irreversible background staining and our sections were floating off the slides during incubation and staining. Such severe background staining may have resulted from uneven sectioning. During sectioning, the securing clamp on the microtome deformed GMA block (approx. 1in. X 1in.) by pushing the center of the block upward. The block also deformed while microtome blade was cutting through, resulting in uneven and thick sections. Although microtome was set to section at 3 to 5 m, resulted sections appeared to have approximately 20 m in thickness due to block deformation. Extended staining time might also be accounted for the severe background staining. With paraffin blocks, background staining can be minimized by deplasticizing the block. However, GMA cannot be deplasticized; therefore, the background staining could only be alleviated by 1). creating thin sections with even thickness, 2). shortening length of staining incubation (max of 20 minutes rather than 30 minutes), and 3). employing Poly-L-Lysine as an alternative section adhesives (known to cause less background staining and to provide stronger adhesion than gelatin). We have not been able to collected quantitative data on bone resorption due to the technical difficulties encountered. In the future with better staining sections, bone resorption will be quantified by means of image analysis. We would then expect to observe an increase in TRAP activity in mechanically stimulated bones and decreased TRAP activity in bones of mechanical disuse.
Acknowledgements
This research study was kindly funded by grant from the National Institutes of Health (GM62492).
REGIONAL MECHANICAL CHARACTERIZATION OF CHRONIC ISCHEMIA
Sara Goldgraben (Stony Brook University), Evren U. Azeloglu, Paul Kochupura, Glenn R. Gaudette, Department of Biomedical Engineering, Stony Brook University
Coronary artery disease is a leading cause of death in America. This disease leads to a region of inadequate blood flow (ischemia). The creation of new blood vessels may re-supply the ischemic tissue with blood. The purpose of this study was to analyze a small region of the heart that was made chronically ischemic. A constrictor was placed on a branch of the circumflex coronary artery, of the New Zealand white rabbit, which slowly narrowed the vessel over 2-3 weeks and created an ischemic region. Six weeks later a second surgery was performed where the heart was isolated and placed on a modified Langendorff apparatus. Two cameras were used to take images of the heart. Data were collected for an ischemic region and perfused region. The data were saved on a computer for later analysis. The tissue was then stained for infarct size. Using high density mapper (HDM), the displacement along the x and y-axis was determined. From this, regional area stroke work and systolic contraction was calculated. The ischemic region of interest that was analyzed was 6.3 x 5.2mm, and it was divided into sixteen smaller elements sized 2.1mm2 each. The perfused region of interest that was analyzed was 8.3x8.2 mm and it was divided into sixteen smaller elements sized 4.24 mm2 each. Four beats were analyzed for each region and were consistent with each other. The average maximum pressure was 107 mmHg and the average minimum pressure was 11mmHg. The average systolic contraction was -3.2% and the regional area stroke work was -.06mmHg, for the ischemic region. The perfused region had a systolic contraction of 7.5% and a regional area stroke work of 4.8mmHg. These results show that perfused tissue ejects more blood. In conclusion, our results suggest that tissue subject to ischemia cannot perform work as well as perfused tissue, and that this dysfunction can be determined in very small regions. In future studies, the determination of the amount of work for a specific region will help us decide the best location to inject growth factors so that functional vessels will grow and supply the ischemic tissue with blood. This work was supported by grants from NIH #GM62492 and New York State Office of Science Technology and Academic Research #C020088.
MEASUREMENT OF R2* CHANGES IN A PHANTOM INDUCED BY Z-ROTATIONS
Anubhav Jain (Cornell University); Thomas Ernst, Medical Department, Brookhaven National Lab, Upton New York; Elisabeth Caparelli, Medical Department, Brookhaven National Lab, Upton New York
Functional Magnetic Resonance Imaging (fMRI) is a relatively new, safe imaging technique that provides in vivo spatial and temporal mapping of brain activity. Typically fMRI studies exploit the Blood Oxygenation Level Dependent (BOLD) effect, which reveals that the magnetic susceptibility of blood depends on its level of oxygenation. Consequently, areas near functioning areas, which receive oxygenated blood through the hemodynamic response, have a lower R2* and can be detected on an fMRI scan. However, previous studies have shown that rotations of a head phantom about axes perpendicular to the MRI magnetic field (x- or y-axes) also yield significant changes in R2*, which may compromise fMRI studies relying on the BOLD effect. Since z-rotations do not change the magnetic permeability distribution relative to the magnetic field (along z-direction), we hypothesized that z-rotations should not induce changes in magnetic field or R2*. This study tested whether R2* changes occur in a head phantom due to z-rotations. The phantom consisted of a plastic skull filled with gelatin (plus 50 mM NaCL and .25 mM azide). Three sets of images, each consisting of one GRE scan (TE/TR=10/700 ms, flip-angle=30 , FOV=20×20 cm, 128 × 128 matrix, 4mm slices, 1-mm gap, 22 slices) and one EPI scan (TR= 7.5 s, 100 TE values from 20 ms to 305 ms, 3 ms increment, FOV=20 × 20 cm, 64 × 64 matrix, 4mm slices, 1 mm gap, 22 slices), were acquired at 0 , 8 and 12.5 rotations. For 0 rotation, an additional EPI image was acquired immediately following the first. The phantom was rotated manually, and the images were taken axially on a 4T Varian/Siemens scanner. For each voxel, R2* was calculated by fitting a mono-exponential function to the signal-decay in the multi-echo EPI scans. R2* maps were created for all rotations by setting the intensity of each voxel proportional to its R2*. Coregistration was used to align the R2* maps to the first 0 R2* map (reference map), using the high-resolution GRE scans to accurately determine rotation angles. R2* maps were calculated by subtracting these aligned maps from the reference. We found small changes in R2* for no (0 ) rotation, and larger, more significant changes for the 8 and 12.5 areas. The changes for 0 rotation were concentrated on the edge of the phantom and a small area in the center. These edge effects may be due to poor signal data on the edge due to large susceptibility changes. A foreign substance in the phantom (e.g. bacteria) may be responsible for the other changes. Much more pronounced differences were caused by the 8 and 12.5 rotations, although these differences were smaller than those measured previously for x and y rotations. A large portion of these differences may be caused by EPI distortions, which occur in different parts of the image for different rotations, making perfect coregistration impossible. Future work will focus on correcting for EPI distortions, investigating the phantom for any foreign materials that may compromise results, and scanning at more rotation angles. This study was supported by grants from R25-GM62492.Reference: Caparelli, E.C., Tomasi D., Ernst, T.: "Measurement of R2* changes in a head phantom induced by small rotations". Proc. Intl. Soc. Mag. Reson. Med. 11 (2003)p.1765
CHRONIC EFFECTS OF MICROSPHERE-DELIVERED VEGF DNA ON ARTERIOLAR RESPONSES IN THE HAMSTER CHEEK POUCH
Erin McCusker (FW Olin College of Engineering, MA); William Chen, Patrick Valane, and Mary D Frame, Department of Biomedical Engineering, Stony Brook University
Objective: Our purpose was to determine the impact of chronic exposure to the angiogenic cytokine, VEGF (vascular endothelial cell growth factor) on normal microvascular responses. Methods: We made hyaluronan (HA) microspheres containing either VEGF (vascular endothelial growth factor) or salmon (control) DNA, or (control) PEG-g-chitosan, poly-lactide-co-glycolide (PLGA) microspheres containing albumin (BSA). Microspheres were infiltrated into the cheek pouch of male hamsters (N = 6, n = 9) up to 9 days prior to acute microvascular testing. On Day 7 or Day 9 post-injection, a spectrum of microvascular responses was evaluated in the acute hamster cheek pouch model. The arteriolar diameter was measured using intravital microscopy in the cheek pouch of the anesthetized (70mg/kg, pentobarbitol) hamsters; all agents were micropipette applied. Results: Responses in controls did not differ from historical laboratory responses in untreated animals. In comparison to the historical controls, the receptor-mediated constriction to endothelin (10-8 M) was enhanced (26% increased response), suggesting an upregulation of the ET1A receptor. Both local dilation to nitroprusside (SNPl, 10-4 M, 50% decrease in response) and remote dilation to LM609 (10ug/ml, 35% decrease), a response dependent on both cGMP and Cx43, were attenuated. This suggests an interference with the cGMP metabolism. An enhanced arteriolar network preconditioning response to L-arginine (LA2, 27% increase) supports that connexin 43 (Cx43) activity may be increased. Local constriction to potassium chloride (10-2 M) and dilation to adenosine (10-4 M), and remote dilation to nitroprusside (SNPr) were unchanged from control. Conclusions: Thus, chronic treatment with VEGF DNA via microsphere delivery alters specific vasoactive responses. It may upregulate the ET1A receptor and potentially interferes with cGMP pathways. Thanks to NIH HL55492, AHA EI0040197N and NIGMS GM62492.
VISCOELASTIC PROPERTIES OF FIBROBLASTS SEEDED ON A HYALURONAN (HA)/RECOMBINANT FIBRONECTIN (RFN) COMPOSITE BEHAVE AS A FUNCTION OF CROSS-LINKING DENSITY AND RFN FUNCTIONAL DOMAINS
Sravanesh Muralidhar (Stony Brook University), Kaustabh Ghosh, Xiao Zheng Shu, Shouren Ge, Xiaohua Fang, Miriam Rafailovich, Glenn D. Prestwich, and Richard A.F. Clark, Department of Biomedical Engineering, State University of New York at Stony Brook
Extracellular matrix (ECM) molecules have a profound effect on cell growth, migration, and differentiation. Cell receptor binding to these molecules alters the cell's cystoskeletal arrangement giving an easily monitored display of cell phenotype. In engineering tissue, one can take advantage of known cell-ECM interactions to design scaffolds that have appropriate molecular clues for the desired cell function. Hydrogels are an ideal scaffold for engineering molecular specificity since their hydrophilicity eliminates non-specific protein binding. HA, a natural occurring hydrogel, can be thiol-derivitized so that specific molecules can be covalently linked to the HA during polymerization. HA hydrogels were prepared at different cross-linking densities of cross-linker, polyethylene glycol divinylsulfone (PEGDVS), varying the gel's stiffness, and linked to varying concentrations of the rFN functional domains, varying the adhesiveness of the cells to the HA matrix. We hypothesized varying cross-linker and rFN concentration would modulate the stiffness of the cell's surface (the surface modulus) and influence the cell's cytoskeleton. Fibroblasts were seeded on the HA matrix and their response, quantified by the surface moduli, to the underlying substrate was measured using the DI-3000 Scanning Force Microscope (SFM). Images of fibroblasts stained with alexa fluor-488 phalloidin for the actin cytoskelton were taken using the Leica confocal microscope showed semi-quantified cell spreading. The cell surface moduli were found to be a function of cross-linking density. The cells also showed a direct relationship between spreading on the substrate and their surface moduli. This indicates cells respond to both the viscoelastic properties and the adhesiveness of the engineered HA/rFN composite. The ability to easily monitor direct relationships between hydrogel physical properties and cell phenotype will greatly enhance our potential to design hydrogel composites for specific tissue engineering purposes. This study was supported by a training grant from the National Institute of General Medical Sciences (R25-GM62492).
CHARACTERIZATION OF TRABECULAR BONE ELASTIC MODULUS AND HARDNESS USING NANOINDENTATION
Olender, G D (Rensselaer Polytechnic Institute); +E. Mittra; Yi-Xian Qin, Stony Brook University
INTRODUCTION:
Osteoporosis is a debilitating skeletal disease that is causing the annual expense of hospitalization for fractures to reach $47 billion. Trabecular tissue is the main constituent afflicted by a loss of bone density and degradation of material strength. In the study to treat this degenerative muscoskeletal disease, research is focusing on smaller areas, specifically the nano-scale to determine the intrinsic elastic modulus and hardness of bone microstructures. Understanding the relation of trabecular structural orientation, mineral composition and material property will provide a better model to evaluate osteoporosis for therapeutic and preventative measures. The object of this study is to perform mechanical testing on embedded trabecular bone samples harvested from human calcanai to determine elastic modulus and hardness at local and regional locations. Nanoindentation equipment will be used to make pinpoint depressions to obtain material properties [Turner et al 1999.]METHODS:
The bone specimens were collected from both men and women from either their left or right heel. Of the 19 samples harvested; 9 were from women, 7 from men (2 sex undetermined) and 12 came from the right foot, 7 from the left foot. To prepare for nano- indentation, the follow steps had to be taken. A plug of trabecular bone was bored from the donor heel and cut along the sagittal plane using a diamond saw. Debris and tissue artifact were removed from the sample using a water jet. The samples were then dehydrated in a series of ethanol concentrations. Specimens were embedded in a hardening epoxy resin and had the surface sanded down to reveal bone using decreasing grit size (600, 800 and 1200 grit). Diamond suspension concentrations (3µ, 1µ, ¼ µ and 1/20µ) were used to complete the metallographic polishing. Finally, 15mm magnetic disks were glued opposite of the polished end so the sample could be placed in the indenter [Rho, Tsui, and Pharr 1997].
The nanoindenting equipment determines the elasticity of the material substrate by monitoring loads and displacement produced by a diamond Berkovich tip. The area and depth of the depression are calculated by the indenting software while sensors record the elastic material response to draw a stress strain diagram for the plastic and elastic region. This data reveals the modulus and hardness of the material from the slope of the unloading curve [Rho, Tsui, and Pharr 1997.] No relevant data is gathered from the plastic deformation of the bone sample. Making a permanent mark can not be avoided when determining the elastic properties of the material
The samples are arranged in the indenter according to height closest to the tip and oriented with respect to the superior and anterior planes. Over the cross-section of the bone, 27 indents are made to determine an average modulus and hardness per sample. The Berkovich tip makes a single depression mark at a constant rate of 100 µN/s until a maximal force of 600µN is reached. The tip is held at that position, unloads and then moves to the next point. 9 regions are mapped in a grid over each sample where 3 localized indents (20 um apart) are made over single trabeculae. The overall modulus is calculated from this data as well as comparative results between regions.
RESULTS:
Out of the 6 samples tested on the nanoindenter significant difference was found between LB v RP, RT v LD, LD v RP. The Tukey T test was performed to evaluate a confidence interval of 95%. The range from these values would corroborate a significant difference if zero is not be included in the interval. When comparing local to regional differences, it was found that the elastic modulus did not vary greater than ?2 GPa when indents were made close together. The standard deviation for regional points was 3.74, averaged for all samples.
DISCUSSION:
This data suggests that individual trabeculae which differ in shape and structure would also possess different material properties. There was a wide variance in elastic modulus recorded from the samples between regions, but the deviations were not as great when examined from local indents. The small deviation at local points gives strong evidence for consistency of elastic modulus over the trabeculae. The anisotropic behavior of trabecular bone supports the research that the bone has a heterogeneous material property over a cross section. Certain areas of trabecular tissue can have a predetermination for fracture which can be shown by their lower than average modulus. By combining the characterization of trabecular bone with improved imaging techniques, diseased or weak tissue could be targeted to prevent stress related fractures. In the future, the quality of human bone could be assessed using noninvasive imaging to determine the bone's modulus, mineral composition and microarchitecture to better diagnose and treat osteoporosis.REFERENCES:
Turner C, Rho J, Takano Y, Tsui T, Pharr G. Elastic properties of trabecular and cortical tissue are similar: results from two microscopic measurement techniques. Journal of Biomechanics 32 (1999) 437-441Rho J, Tsui T, Pharr G. Elastic properties of human cortical and trabecular lamellar bone measured by nanoindentation. Biomaterials 1997 vol. 18 ACKNOWLEDGEMENT:
This work is kindly supported by the National Institutes of Health (NIGMS); R25-GM62492
THE EFFECTS OF HIGH FREQUENCY, LOW MAGNITUDE MECHANICAL VIBRATION ON HUMAN BONE AND MUSCLE
Amit Salkar (University of Georgia); Tamara Kaplan, and Clinton Rubin, Department of Biomedical Engineering, Stony Brook University
Recent experimentation on animal models suggests that low magnitude (0.3 g), high frequency (30 Hz) mechanical vibration may encourage bone and muscle growth. Such a treatment could be a valuable intervention in battling osteoporosis and curbing the bone and muscle loss associated with long-term spaceflight. The application of this non-invasive, non-pharmacological treatment as a possible anabolic stimulus to bone and muscle was examined in an eight week pilot study in support of a project which is currently in the "select for flight" phase at NASA. Twenty-six normal, healthy individuals, ranging from 19 to 53 years of age, were recruited from the university to participate in the study. The fifteen men and eleven women received treatment on the left leg, while the right leg served as a contra-lateral control. Each subject was exposed to localized mechanical vibration of 0.3 g and 30 Hz delivered via a floor mounted oscillating platform for 10 minutes per day, 5 days per week, for 8 weeks. A treatment log documented subject compliance and provided insight into the plausibility of the treatment. The intra-subject control acted as a means to eliminate error due to any variation in individual response to the treatment. Bone ultrasound measurements and muscle performance testing using the Cybex 6000 was conducted before and after the eight week treatment period. Although a mid-point muscle test was conducted, mid-point bone scanning was not administered because of an anticipated lack of response of bone tissue at four weeks. Currently, the study is in progress and approaching completion. A baseline comparison between the left and right knee extensor at 180° per second revealed an average difference in peak torque of 8.33 ± 6.34 ft·lbs in men and 7.64 ± 6.39 ft·lbs in women. At four weeks, the left knee extensor at 180° per second showed an average increase of 17.18 ± 13.50% for men and 23.41 ± 30.35% for women, whereas the right knee showed an increase 23.93 ± 27.54% for men and 40.22 ± 47.75 % for women. Application of the Wilcoxon signed-rank test comparing pre- and mid-point muscle assessments showed no significant changes in muscle performance between the treated and untreated limbs (p values of 0.382 and 0.61 for men and women respectively). Although the study shows no physiological or functional response in subjects at this point, future vibration-based experiments may determine if such a measure will be beneficial in preventing or reducing bone and muscle loss, especially in osteoporotic individuals and astronauts. Funding for this study was provided by grants from the National Institutes of Health (NIGMS Grant # R25-GM62492).
NONLINEAR ANALYSIS OF CA2+ TRANSIENT SIGNALS FROM CARDIAC CELL CONTRACTION.
Laura Shih ( Princeton University);Sheng Lu, Ki Chon, Emilia Entcheva, Department of Biomedical Engineering, Stony Brook
In this study, linear and nonlinear data analysis methods were used to analyze patterns of calcium ion concentration transients representing cardiac cell contraction to show that certain fluctuations in calcium ion concentration levels lead to abnormal contraction patterns such as alternans. Ventricular myocytes from neonatal rats were cultured on polydimethylsiloxane scaffolds of various topographies. Cells were not paced at all or were paced with electrical stimulation at frequencies of 0.5-6 Hz to produce responses including spontaneous contraction, alternans and fibrillation. Contractions were optically measured using the fluorescence intensities of Fura-2, a calcium indicator, under UV light. Nonlinear analysis was used for purposes of characterizing nonlinear dynamics of the system. Two data series were extracted from the original signal and examined-time interval between peaks and peak height. Peak height was defined as the distance from the maximum transient value to the minimum value falling between the peak in question and the previous peak. A second-order, nonlinear autoregressive model was used to fit the peak height data. In normal signals that followed pacing, the model was a poor fit and spectral analysis of the peak height data series showed a broadband spectrum, often with a low-frequency component. In this case, the variability in peak height was likely a result of noise and was not of a physiological nature. In signals with second order alternans, however, the autoregressive model fit the data well, leading to the conclusion that the alternans signal is deterministic. Spectral analysis showed a time-invariant, high-frequency spectrum in these signals. The autoregressive method used could not accurately predict higher order alternans signals, whose peak height data series had a time-varying frequency spectrum. Comparison of the phase maps of peak height data showed a bifurcation from one cluster of points in the normal, paced case to two or more clusters in the alternans case, depending on order of alternans. The point spread of each cluster was minimal, likely due to noise in the original signal. Phase maps and nonlinear, autoregressive modeling of peak height in non-paced data were comparable to results in normal, paced data. Peak interval data series from non-paced data also did not fit autoregressive modeling well. Frequency analysis revealed a low-frequency component, though, possibly of physiological nature. This study was supported by a grant from GM62492.
TEMPORAL AND SPECTRAL CHARACTERISTICS OF INSPIRATORY MOTOR OUTPUT IN ADULT C57BL/6 MICE
Marvin H. O'Neal III, Evan T. Spiegel (Stony Brook University), Ki Chon, Irene C. Solomon, Departments of Biomedical Engineering, and Physiology and Biophysics, Stony Brook University
Temporal and spectral analyses were performed on diaphragm electromyogram (EMG) activity recorded from 36 urethane-anesthetized adult C57BL/6 mice to identify the temporal characteristics and frequency components inherent in inspiratory motor activity under hyperoxic conditions (60% O2 in a balance N2). Diaphragm EMG data were collected using a bipolar electrode; The EMG signal was band-pass filtered from 20 Hz to 250 Hz and sampled at 2 KHz. Both a raw and integrated trace were recorded. Temporal characteristics were quantified using four different measures: duration of burst (TI), duration between bursts (TE), frequency of bursts (fR), and shape of bursts. Temporal analyses (n=36) demonstrate that TI was 107 ±4 ms, TE was 266 ±18 ms, and fR was 161 ±7.8 bursts per minute. The bursts exhibit a eupneic or ramp-like pattern of discharge. Spectral analyses were performed using a Fast Fourier Transform (FFT) to determine spectral composition from 1 to 10 minutes of EMG recording. The FFT, a time invariant measure of spectral composition, revealed three dominant peaks over inspiratory activity (n=36). Medium frequency oscillations (MFO) in the range of 20-50 Hz, with an average peak at 38 ±1.5 Hz, and high frequency oscillations (HFO) in the range of 75-150, with an average peak at 107 ±3.2 Hz were observed. Additionally, significant activity was detected in the range of 160-234 Hz, with an average peak at 198 ±3.0 Hz and was designated as ultra-high frequency oscillations (UHFO). UHFO activity was generally of higher relative power when compared to the MFO and HFO and exhibited several distinct peaks. FFT analyses over 10 minutes of EMG from a subject (n=6) demonstrate consistency in the relative power and location of peaks. Despite the benefits of an FFT, it assumes that the given signal is stationary or lacks any spectral dynamics over time, however previous work demonstrates variability of such neuronal signals. Therefore, a smoothed pseudo Wigner-Ville distribution (SPWD) Short Time Fourier Transform algorithm was applied to explore the time-varying properties of inspiratory activity during hyperoxic conditions. SPWD analysis (n=36) confirms the frequency ranges and relative power provided by the FFT and suggests that frequency composition is highly varying in time. Total power over the inspiratory burst resembles an augmenting pattern, with peak spectral activity occurring from 70-100% TI. Temporal and spectral analyses suggest there are no differences between genders. The temporal data are consistent with previous literature on adult mice, and spectral analyses share similarities with previous work with respect to the MFO and HFO as reported in other adult mammals. This study was supported by a training grant from the National Institutes of General Medical Sciences (R25-GM62492), and a grant from the National Heart Lung and Blood Institute (R01-HL63175).
EARLY BLADDER CANCER DETECTION WITH OPTICAL COHERENCE TOMOGRAPHY (OCT)
Victor Sutan (Stony Brook University); Yingtian Pan, TuQiang Xie, ZhenGuo Wang, MeiFang Lee, and WeiChieh Huang, Department of Biomedical Engineering, Stony Brook University
The ability of optical coherence tomography as a diagnosis tool has been recognized and proven in previous studies (Fujimoto et al). It has also been demonstrated that OCT is able to produce images that correlate with their corresponding histology images (Pan et al). We are developing the endoscopic OCT system for early bladder cancer detection. Bladder cancer, at its earlier stages, grows in the thin lining of urothelial cells. Bladder Cancer is treatable if detected at its earlier stages while current diagnostic technologies do not have high enough resolution to detect the early cancer. For statistical data, we have designed a systematic study of 54 rats. All rats were induced with carcinogen Methyl-Nitroso-Urea (MNU). The instillations were done in the week 2, 4, 6, and 8. Bladder diagnoses were performed starting at week 10 assuming there was enough time for the artificial cancer to grow. Prior to bladder dissection, rats were instilled with 5% Aminolevulinic-acid (ALA) as a fluorescence dye. The OCT imaging was then focused on fluorescence positive area. After the imaging was done, the samples were placed in formaldehyde for histology preparation. Finally, histology images were compared with OCT images. The results showed that OCT has enough sensitivity to detect abnormality in urothelial cells. These included the identification of hyperplasia or thickening of the urothelial cells which was confirmed using histology. The OCT is also able to detect neoplasia or early cancer cells. Another interesting finding was that the OCT is able to reduce errors introduced by fluorescence dye. The OCT verified the fluorescence positive areas which are normal under OCT and histology. Moreover, the OCT also verified the fluorescence negative areas which have hyperplasia under OCT. This study was supported by National Institutes of Health R25-GM61492, R01-DK59265, and Whitaker grant 00149.
Reference:
1. M. E. Brezinski and J. G. Fujimoto, "Optical Coherence Tomography: High-Resolution Imaging in Nontransparent Tissue," IEEE J. Sel. Top. Quantum Electron. Vol 5, No. 4 1185-1192 (1999).
2. T. -Q. Xie, M. L. Zeidel, and Y. -T. Pan, "Detection of tumorigenesis in urinary bladder with optical coherence tomography," Optic Express Vol 10, No. 24, page 1431.CRYSTALLIZATION OF THE C-TERMINAL DOMAIN OF THE KEY NUCLEOTIDE EXCISION REPAIR PROTEIN UvrC
UvrC is an endonuclease that is responsible for the incision of damaged DNA within the nucleotide excision repair pathway. The C-terminal domain of UvrC, was cloned into the pTxb1 vector of the Intein-CN System and subsequently overexpressed and purified. Our goal was to crystallize this fragment, which is responsible for incising the DNA 7 bases 5' of the DNA lesion. The technique of "hanging drop" vapor diffusion was used in which 1?l of protein and 1?l of a selected precipitant are mixed together on a cover slide. The cover slide is inverted and sealed over a well containing 1ml of precipitant solution. Hundreds of these experiments were set up varying the composition of the precipitant as well as the concentration of the protein (10mg/ml to 40mg/ml). Each precipitant solution contained a unique mixture of salt, buffer, and organic molecules. These wells were placed in a 22 C incubator and checked daily for the formation of crystals. Initially there will be a higher concentration of water in the protein/precipitant solution compared to the well. Over a period of a few days the water will diffuse down its concentration gradient from the drop to the well, thereby concentrating the protein and precipitant simultaneously. It is the hope of the crystallographer that under the proper conditions the protein will no longer be able to stay in solution and will fall out in the form of crystals rather than an amorphous precipitate. Through extensive screening, the C-terminal domain of UvrC crystallized in a solution of 1.5M Li2SO4, 100mM Hepes pH 7.0. These crystals were too small to be used for diffraction experiments. At this point various additives (other small molecules; salts, reducing agents, etc…) were combined with this condition in an attempt to improve the size of the crystals. Currently, variations such as additives, temperature, and pH are still under way in hope to produce larger crystals.This work was supported by a training grant from R25-GM62492
Puiyee Tong (Stony Brook University); James Truglio and Caroline Kisker, Department of Pharmacological Sciences, Stony Brook
GENE THERAPY USING HYALURONAN MICROSPHERES TO INDUCE ANGIOGENESIS
Man Tsz (Mandy) Yau (University at Buffalo); William Chen, Department of Biomedical Engineering, Stony Brook.
Diseases related to ischemia affect over 6 million people in the United States are directly linked to the leading cause of death in the United States. The delivery of growth factors such as vascular endothelial growth factor (VEGF) has been shown to stimulate the formation of new blood vessel and to minimize the poor circulation caused by the narrowing of the artery. The major disadvantages of using protein therapy are their short half lives and the cost of the therapy. Gene therapy could address this problem because the cells can be injected to over-express these proteins.
Previous studies have shown that HA microspheres can successfully deliver reporter plasmid DNA and transfect the targeted tissue in vivo. The aim of the study was to determine the effects of therapeutic gene on the growth of blood vessels in hamsters using hyaluronan (HA, 1.6 to 3.3x106 Daltons) microspheres encapsulated DNA encoding for VEGH was delivered to hamster cheek pouches in an attempt to induce angiogenesis. This project was in collaboration with Dr. Molly Frame who analyzed the effect of neo-angiogenesis and the function of microvascular in cheek pouch by the VEGF released from HA micropspheres. Our project was to validate if this effect was from the exogenous VEGF delivered from HA microspheres.
The hamsters were anesthetized and injected with HA microspheres containing 100 ?g of plasmid DNA encoding for VEGF regulated by cytomegalovirus immediate early promoter with V5 reporter sequence. Injection was followed by 10 days incubation. On the 10th day, the injected tissue was examined for enhanced vascularization and micro-vascular network communication. Afterward, the hamster was terminated and the cheek pouch, heart, and the liver were harvested and immediately frozen in liquid nitrogen. The RNA was isolated using Trizol, any residual DNA was digested using DNAseI enzyme, and the RNA was purified using Rneasy kit. PCR was performed to detect DNA contamination using RNA templates. After initial denaturation at 95 C for 15 minutes, the purified RNA template was amplified for 30 cycles using the following steps: 1) denatured at 95 C for 1 minute, 2) annealing at 55 C for 30 seconds, and 3) extension at 68 C for 1 minute/ RT-PCR was subsequently performed using purified RNA templates. The reverse transcription parameters were reverse at 95 C for 30 minutes using a one step RTT-PCR using a kit. The PCR parameters were as previously stated.
The PCR analysis showed no DNA contamination, and the RT-PCR amplified only the messenger RNA isolated from the tissues of interest. The RT-PCR results for the hamster injected with plasmid DNA as the delivery vehicle showed the transfection of the cheek pouch, the heart, and the liver. In contrast, the HA-DNA microspheres RT-PCR showed the transfection ot the cheek pouch alone. The result suggested that only the treated tissue were transfected. Since localizaion of the gene therapy is a desirable trait for a delivery vehicle and delivered gene showed evidence of neo-angiogenesis that was verified by our collaborators, HA microspherse could be an ideal vehicle for applications in gene therapy. This work was supported by a training grant from the National Institutes of General Medical Sciences (R25-GM62492).
OXYGEN MONITORING IN CARDIAC CELLS USING OXYGEN SENSITIVE SCAFFOLDS
Hui Jing Yu (Stony Brook University), Melissa Farrell, Harold Bien, Yordan Kostov, Govind Rao and Emilia Entcheva, Department of Biomedical Engineering, Stony Brook University
Providing proper perfusion and oxygen supply are essential for engineering cardiac muscle in vitro. We present a non-invasive technique for continuous direct monitoring of oxygen gradients inside engineered 3D cardiac cell constructs, primary cardiomyocytes (CM) and fibroblasts (FB). Scaffold-embedded biocompatible optical oxygen sensors were prepared for optimized cell growth using long-lifetime luminescent dye, entrapped in polydimethysiloxane (PDMS), and fused to a textured Teflon surface. The sensing dye's lifetime is 3-27 microseconds depending on the oxygen concentration and was measured as a phase angle using frequency domain fluorometry. A miniaturized incubator-tailored device was used to monitor the sensing scaffold, employing blue Light-Emitting Diode (LED) excitation. We analyzed n=6 CM and n=4 FB cultures over the course of 1-12 days. Cell viability was examined using nuclear dye after termination of experiments and images of cell nuclei confirm excellent survival of cells under the conditions. Some cultures were treated with an actin-myosin inhibitor, 2, 3-butanedione monoxime (BDM), to assess the contribution of mechanical activity to total oxygen uptake. Results show that contractile CM have about 3 times faster oxygen uptake rate (phase angle per hour) as compared to not metabolically active FB (-0.208+0.110 vs. -0.068+0.027). A 65% and 54% reduction in the rates of oxygen consumption were found in CM and FB respectively when BDM treated, indicating a significant contribution of cell contractility to oxygen consumption. The proposed technique is unique and holds great potential for non-invasively assessing oxygenation and functionality in engineered cardiac tissue. This study was supported by a training grant from the National Institutes of Health (GM62492).
USING MICROARRAYS FOR GENE EXPRESSION
Matthew H. Zafran (University of Rochester); Anil Dhundale, Melissa Monaghan, and Emily Huang, Applied Biosciences Laboratory, Department of Biomedical Engineering, Stony Brook University
The purpose of this project was to prepare human cDNA microarrays from the sequence verified human cDNA library, purchased from Invitrogen (Resgen), for differential gene expression studies. DNA microarrays can be used in identification of complex genetic diseases, drug discovery and toxicology studies, mutation/polymorphism detection (SNP's), pathogen analysis, and differing expression of genes over time, between tissues, and disease states. The 41,472 known nucleic acid sequences (genes and EST's) are contained in the library in E.coli. The bacterial clones were cultured and processed for PCR amplification. The purified cDNA products were then spotted on glass slides. Genes may be up-regulated or down-regulated in different tissue types or conditions. By flourescently labeling two different types of RNA, microarrays can be used to examine the gene expression profiles. HeLa RNA, a cervical cancer cell line, was labeled with Cy3 and a control reference RNA with Cy5. These were then hybridized to the microarray and scanned to fluoresce in order to identify the differentially expressed genes. These genes may be of potential interest for further research or as pharmaceutical drug targets. Twenty-three plates containing 2,208 genes and EST's were purified and made ready for spotting. Seventy-three slides were printed, each containing 4,608 spots, of which 1,152 unique genes or EST's are ready for experimentation. Out of the 1,152 unique spots we found 74 HeLa genes that were highly expressed and had at least four-fold up-regulation. These genes need to be more closely looked at to examine function. This work was supported by a training grant from the National Institutes of Health, National Institutes of General Medical Sciences (R25-GM62492).
Signal Transmissibility through the Skeleton during Whole Body Vibration is dependent on posture.
Ben Adler (Stony Brook University), Beney J. Lee, Jesse Muir, Stefan Judex, Yi-Xian Qin and Clinton Rubin, Department of Biomedical Engineering, Stony Brook University
There is a growing body of evidence which suggests that skeletal mechanosensitivity may provide an avenue for the treatment of the chronic bone loss seen in osteoporosis. Specifically, it has been shown in numerous studies that low magnitude-high frequency (0.3g, 30Hz) whole body vibrations (WBV) provide an anabolic stimulus to bone growth. In order to apply this treatment methodology to a variety of unique situations i.e. weightlessness or bed rest, it is important to demonstrate the feasibility of skeletal signal transmission in these settings. The goal of this study was to determine the percent transmissibility of signal [(measured signal/input signal)*100%] in a mock bed rest situation, in preparation for future studies. WBV was induced in four standing subjects with a programmable vibration platform (Juvent Inc.). While standing on the vibrating plate (without shoes), the subjects were asked to stand with knees locked, relaxed, and bent (~20º). Signals were measured simultaneously from the plate surface and a bite bar via accelerometers (Crossbow Technology Inc.). The voltage signal from the accelerometers was recorded on a laptop (Dell) with the use of a homemade breadboard (J.M.), and BNC connector blocks and data acquisition cards (National Instruments). A Labview (National Instruments) program (Y.X.Q.) was used to record all data, while a graphic user interface (J.M.) written in MATLAB (The MathWorks) was used for calculations of transmissibility. One tailed-paired Student's t-tests with Bonferroni correction (?=0.05/3=0.0167) were performed on the transmissibility data of four subjects. In the relaxed position, the four subjects had an average transmissibility of 93.8±19.9% (mean ± s.d.), in the knees locked position this value increased to 130.02±21%, whereas in the knees bent position transmissibility dropped to 26.7±9.8%. T-tests revealed significant differences between the relaxed and bent positions (p=0.0079), and the locked and bent positions (p=0.00097). No significant difference was found between the locked and relaxed position (p=0.024, ?=0.0167). These data demonstrate the clear role that posture plays in the transmission of mechanical signals (i.e. vibrations) through the skeleton. Although exposure to WBV has been traditionally thought to be a risk factor for health problems such as lower back pain, vibrations of low magnitude and high frequency can be perceived as anabolic signals by the skeleton. This work has attempted to describe the way in which these signals are transmitted through the skeleton. Such information becomes important when the vibrations are applied in a novel manner. Bed rest experiments represent such a departure from the classic weight bearing stance used in this work. These experiments provide a disuse model of weightlessness in which bone loss can be studied. Although the experimental recumbent mockup was not ready at the time of writing, the work described here will be used in conjunction with future transmissibility measurements on recumbent individuals. By accurately describing how signals are transmitted in the standing individual, it will be possible to draw conclusions about the efficacy of signal transmission in the recumbent position. Such information is crucial in optimizing treatment parameters for the most effective retardation of bone loss. This work was kindly funded by grants from NASA.
The Effect of Thresholding on Trabecular Bone Modulus as Determined by a Finite Element Method
Sailaja Akella, Suzanne Ferreri, Yi-Xian Qin, Department of Biomedical Engineering, Stony Brook University
Introduction: Micro-CT and the finite element method (FEM) are tools used to evaluate the microstructural and mechanical properties of bone. Micro-CT quantifies bone architecture by calculating bone volume fraction (BV/TV) and trabecular number (Tb.n). The original grayscale image needs to be binarized to differentiate bone from marrow to perform the evaluations. Threshold and Gaussian filter parameters are used for this segmentation process. Currently threshold values are determined by visually comparing the grayscale and binarized images. This is a limitation because the values that quantify bone micro structure largely depend on threshold. The purpose of this project is to study the processes used to evaluate micro-CT images and its affect on Young’s modulus.
Methods: Human calcaneus from a 70 year old male was scanned in the medial lateral direction by micro-CT 40 at 34?m resolution. A virtual cut of a 7mm cube was made. The base threshold was 114, sigma was 1.2, and support was 2 as recommended by SCANCO. The threshold was varied by ± 1, ± 2 while the Gaussian filter parameters remained constant. A FE model with linear elastic isotropic material properties was made and subjected to 1% compressive strain (? app) with frictionless contact. Boundary conditions were applied so that nodes in the x-y plane where z=0 were restricted from motion in the z-direction, all nodes where z=0 and x=0 were restricted in the y direction, and nodes where z=0 and y=0 was constrained in the x-direction. The apparent stress (? app) was calculated by the FEM and the apparent modulus was calculated as,
Results: Preliminary data shows that the bone volume fraction decreased linearly as threshold increased. The apparent modulus showed a similar trend, but decreased nonlinearly.Discussion: The role of threshold and Gaussian filter parameters on morphological and mechanical properties of bone is not clearly addressed by previous research. A study by Ito et al. looked at the influence of threshold on microstructural properties but did not look at small changes in threshold values or the effect of mechanical properties 1. Another study by Hara et al. looked at small changes in threshold and found that BV/TV and apparent modulus were most affected 2. However, they did not show how bone’s ability to withstand mechanical loading is affected. The goal of our study is to address these issues. The hypothesis is that the choice of threshold used will affect the bones ability to withstand loading as determined by a FEM. Preliminary analysis of our data shows a linear relationship between BV/TV and threshold which is consistent with previous studies 1. The apparent modulus decreases nonlinearly with increasing threshold value. This may be possible because the range of threshold values was too small to represent the trend. Also, changing the threshold could largely affect the BV/TV, Tb.n, and connectivity which could affect the apparent modulus. Further studies need to be done to make a valid conclusion. One limitation with the methodology is determining the correct threshold. Without knowing the theoretical value for BV/TV there is no way to verify the threshold. For future work Archimedes’ principle will be tested to check the accuracy of threshold.
Reference:
1. Ito M, Nakamura T, Matsumoto T, Tsurusaki K, Hayashi K. Analysis of Trabecular Microarchitecture of Human Iliac Bone Using Microcomputed Tomography in Patients with Hip Arthrosis with or without Vertebral Fracture. Bone 1998; 23: 163-9.
2. Hara T, Tanck E, Homminga J, Huiskes R. The influence of microcomputed tomography threshold variations on assessment of structural and mechanical trabecular bone properties. Bone 2002; 31: 107-109
IN-SITU ANALYSIS AND CORRELATION OF THE CHEMICAL, MECHANICAL AND STRUCTURAL PROPERTIES IN DEVELOPING MOUSE BONE.
Theodore Feldman, William Little, Bhavin Busa, Russell Garman, Lee Rae Donahue*, Stefan Judex and Lisa Miller**, Department of Biomedical Engineering, Stony Brook University, Stony Brook, New York 11794, The Jackson Laboratory*, Bar Harbor, ME 04609 and The National Synchrotron Light Source**, Brookhaven National Laboratory, Upton, NY 11973.
Introduction: As the prevalence of degenerative bone diseases continues to increase at a rapid rate, so does the need to accurately assess the status of a skeleton that is a risk of fracture. Current clinical assessments of bone, such as dual-energy x-ray absorptiometry (DEXA) measure only the quantity of bone present1. However, bone quality is just as important in determining the likelihood of fracture2. To that end, before bone quality can be used in clinical assessments of bone strength it is necessary to define this rather elusive concept3. To facilitate this definition, the effect of the, morphological, material and chemical properties on the mechanical properties of the bone matrix must be understood3. This study examined the variation of the chemical components of cortical mouse bone during the mineralization process, for the future purpose of determining the apparent effect of these chemical changes on the material properties, namely nano-mechanical strength and morphology of the bone matrix.Methods:
The tibia of four female BALB/cByJ mice (Jackson Laboratory, Bar Harbor, ME) ranging from 10 to 20 days of age were harvested and dehydrated for ten days in ethanol solutions. High-resolution micro-computed tomography was then performed on all specimens at the mid-diaphysial region. The samples were cut at the mid-diaphysis (defined as 50% of the total length of the bone) using a low-temperature titanium-sapphire laser ablation system (Spectra-Physics, CA). One-half of the bone was then embedded in a low-viscosity epoxy (Buehler, IL) and polished for future nano-indentation while the remaining half of the tibia was embedded in poly-methyl-methacrylate (PMMA) to facilitate thin-sectioning. Synchrotron-based infrared micro-spectroscopy (SIRMS) was performed at beamline U10B of the National Synchrotron Light Source (Brookhaven National Laboratory) on the samples embedded in epoxy to determine the protein and mineral composition of the bones. Data collection was performed in reflection mode using an aperture size of 20?m×20?m and 256 scans per pixel. Three to four 5?m sections were then sectioned by microtome (Leica, Germany) from each specimen embedded in PMMA and SIRMS was repeated to facilitate a more comprehensive chemical analysis. Data collection was conducted in transmission mode using a 15?m×15?m aperture with 128 scans per pixel.Results
Analysis indicated a significant degree of mineralization (i.e. high phosphate to protein ratios) in both the 10 and 20-day-old samples. The normalized phosphate-to-protein ratio of the 20-day-old sections was approximately 20% greater than that of the 10-day-old specimens while the normalized global carbonate-to-protein ratio of the 20-day-old sample was 30% greater than that of the 10-day-old specimens. The spatial non-uniformity of the phosphate-to-protein ratio of 10-day-old bone was approximately 40% compared to 20% in 20-day-old bone. Additionally, the coefficient of variation of the carbonate-to-protein ratio computed for the 10-day-old bone was 50%, twice that of the 20-day-old bone. This variation in mineralization as well as inverse relationship between age of the bone, heterogeneity of apparent mineral content and porosity was confirmed by the high-resolution computer tomography imaging.Discussion
Data indicate that mineralization of the mid-diaphysial region of cortical bone in mice occurs rapidly during the first twenty days of life as apparent by the two-fold differences in heterogeneity among data points from the same bone as well as the noticeable differences in the levels of organic and inorganic chemical species present in the bone matrix. A direct correspondence between apparent morphology from high-resolution computed-tomography and chemical analysis using synchrotron-based infrared micro-spectroscopy has been demonstrated. The rapid rate of mineralization as well as the substantial mineral content of 10-day-bone emphasizes the need to study specimens of a wider age range, including several time points between 10 and 20 days of age as well as younger and older specimens. In summary, results indicate the direct relationship between the chemical and morphological properties of 10 and 20-day-old mouse tibia. In the future, additional quantitative morphological evaluation as well as nano-identation of the samples will be performed to examine how chemical and morphological changes impact the strength of the bone matrix.
References
1Cummings, S. R., Bates, D., Black, D. M. (2002) Clinical Use of Bone Densitometry. Journal of the American Medical Association 288(15). 1889-1897.
2Judex, S., Boyd, S., Qin, Y-X, Miller, L., Müller, R., Rubin, C. (2003) Combining High-Resolution Microct with Material Composition to Define the Quality of Bone Tissue. Current Osteoporosis Reports 1(1). 11-19.
3Cefalu, C. A. (2004) Is Bone Mineral Density Predictive of Fracture Risk Reduction? Current Medical Research and Opinion 20(3). 341-349.Acknowledgements
This work was supported by a training grant from the National Institutes of Health (NIGMS) R25-GM62492. The National Synchrotron Light Source is supported by the U.S. Department of Energy, Division of Materials Sciences and Division of Chemical Sciences, under Contract No. DE-AC02-98CH10886.
DIFFUSION THROUGH NANOTUBES
Sara Goldgraben (Stony Brook University) and Mary D. Frame, Department of Biomedical Engineering, Stony Brook University
The purpose of this study is to measure flow into and through nanotubes. We want to test the flow through nanotubes to determine whether diffusion or electro kinetic flow is playing a role. We hypothesize that the flux of solutes through nanotubes is predicted by diffusion theory. We used fluorescent compounds to evaluate diffusion. To test the limit of our intensified camera system (Dage Gen/Sys II Intensifier, shutter1.0, gain 0-250, coupled to a CCD 72) used to detect fluorescence, we performed a calibration with known FITC-dextran (4000 MW) concentrations. The camera signal was converted to grayscale (1-255), and plotted v.s. concentration. The limit of our detection is 10 µm (FITC) at a gain of 150, and 100 pm (XRITC) at a gain of 200. The calibration was done using the dye with a 100 µm optical path length. Initially, we performed a proof of methods experiment to detect diffusion of dye using a 100 µm (inner dia.) glass tube (eg. the same optical path length as for the calibration). This glass tube was imbedded with access to inflow and outflow chambers. One chamber was filled with water and the other with FITC-dextran. Diffusivity was determined using Fick's law of diffusion (J=D*A*dc/dx). Our expected value of diffusivity is 1.58x10-6 cm2/s in bulk fluid for this molecular weight compound. Next we imbedded nanotubes in sylgard to test our ability to detect fluorescent compounds within this test system. The percent by mass concentration of nanotubes in the sylgard was 1%. We tested the samples to see if a 200 µl sample of 10 µm XRITC dye would saturate the material when it is placed next to it on a glass slide. Then using fluorescence microscopy we took readings at a gain of 150. In general as time increased so did the fluorescence of the material. This indicates that the nanotubes are patent since they are allowing the dye to diffuse in. However, if the XRITC dye is making its way underneath the sylgard-nanotube material, or if the sylgard is adsorbed to the dye, the results are inconclusive. The future goal of this project is to measure fluid movement in nanotubes. We want to test quantum dots to see how they flow through the nanotubes; quantum dots are coated with a polymer, are water soluble, and are 20-50nm in diameter. The ultimate goal of this basic research includes constructing novel drug delivery vehicles, such as a dermal patch. This work was supported by grants from the NIGMS R25-GM62492, the NIH MARC program, grant #T34- GM08655, and NIH #HL55492 (MF).
THE TRANSMEMBRANE HELICES IN THE THROMBOPOIETIN RECEPTOR HAVE A RIGHT-HANDED CROSSING ANGLE
Amanda Gorecki (Sacred Heart University), Gabriella Reubins and Steven O. Smith, Center for Structural Biology, Stony Brook University
The thrombopoietin receptor is the main regulator of megakaryocytic differentiation and platelet formation. The receptor has a large extracellular domain, a single transmembrane helix and a large cytosolic domain. The receptor forms a dimer in cell membranes as a result of transmembrane helix-helix interactions. Binding of thrombopoietin leads to activation of the dimeric receptor by changing the orientation of transmembrane helices. This study sought to determine the structure of the transmembrane helix dimer in order to understand how thrombopoietin binding is coupled to reorientation of the cytosolic domain. Specifically, the goal was to establish whether the thrombopoietin transmembrane helix dimerizes with a right-handed or left-handed crossing angle. Computational studies were undertaken to evaluate experimental data recently obtained (unpublished) by Constantinescu and coworkers showing which amino acids are found in helix interfaces of inactive and active receptor dimers. Six different computer simulations were run on dimers of the thrombopoietin receptor transmembrane helix in order to identify stable low-energy structures. Simulations were run using protein sequences corresponding to the murine and human receptors. The only variable in the simulation was the axial separation of the helices which was chosen to be either 9.5Å, 10Å or 10.5Å. These values bracket the average helix separation (9.6 Å) found in proteins. Low energy structures were identified in each simulation having both right-handed and left-handed crossing angles. In each simulation one left-handed and one right-handed structure was selected that best fit the experimental data on the inactive receptor, and one left-handed and one right-handed structure was selected that best fit the experimental data on the active receptor. Overall, the right-handed crossing geometry was found to best fit the experimental data. There was a better correspondence between the experimental and calculated results on the amino acids that occur in the dimer interfaces for active and inactive receptors for the right-handed structures than for the left-handed structures. This work was supported by grant to S.O.S. from the National Institutes of Health (GM46732).
EFFECTS OF DIET AND RADIATION ON RAT BONE GROWTH AND MORPHOLOGY
Lamya Karim (Stony Brook University), Stefan Judex and Anne Schirmer, Department of Biomedical Engineering, Stony Brook UniversityIntroduction. Achieving maximal peak bone mass is a major prophylaxis for preventing the onset osteoporosis, a disease in which bones loose their density and become prone to fracture. Principal influences on bone mass are genetics and environmental stimuli.1 One of the environmental factors involves a diet including a composition of fatty acids and fibers. Previous data indicate that fish oil can enhance bone formation.2 Little is known, however, about the interaction between specific fatty acids and fiber type and its influence on bone growth in the presence of radiation. Here, we hypothesized that fish oil in the presence of cellulose can enhance bone growth and prevent the detrimental effects of radiation.
Materials and methods. Sixty four-week old male rats were fed diets consisting of an 85% basal mix of cellulose and 15% fish oil or corn oil for four weeks. Half of the rats were radiated during the final week of the diet. Left femurs were harvested after sacrifice at eight weeks of age. The metaphysis and mid diaphysis of the femur was analyzed at medium resolution (20 micron) using micro computed tomography. Two-paired t-tests were used to compare the different diets within the radiated group and to compare between control and radiated groups.
Results. Within the radiated rats, those fed fish oil instead of corn oil had a 5% (p = 0.03) greater cortical bone area in the mid-diaphysis (figure 1). Bone morphology was not significantly altered by diet in the trabecular and cortical metaphysis. When comparing radiated to control groups, radiated rats had a greater endocortical envelope area in the metaphyseal region (4%, p = 0.02) than the control rats in the presence of corn oil, but no effects were seen in the presence of fish oil. Radiated rats also had significantly greater cortical bone area than the controls in both metaphysis (23%, p = 0.00 and 13%, p = 0.04) and mid-diaphysis (11%, p = 0.00 and 6%, p = 0.02) in fish oil and corn oil, respectively.
SYNTHESIS OF NOVEL FILTER MATERIALS WITH MONODISPERSE NANOPORES.
Kerry Lanigan and Helmut Strey, Department of Biomedical Engineering, Stony Brook University
Bioseparation, particularly protein bioseparation, is an integral component of the billion dollar bioprocess industry. Bioseparations comprise a significant portion of the costs associated with bioprocessing, and in some cases even constitute the majority of the costs. For this reason, it is of extraordinary interest to develop a better filter with optimized throughput and selectivity. Surfactants have been found to self-assemble into ordered nanostructures when added to systems of a polar solvent, such as water, and a nonpolar solvent, such as an oil. This project was designed to capitalize on this principle of self assembly to create monodisperse filters by using a polymerizable oil in these systems, and initiating polymerization after the ordered structures were formed, to preserve that structure. By these means, there is potential for the creation of an ideal filter with finely tuned pore size and good structural integrity. We investigated and successfully prepared several self-assembling phases, including long-range ordered hexagonal microemulsion phases, L3 sponge phases, and bicontinuous cubic phases in the laboratory containing a polymerizable monomer. Following the nanostructure self-assembly, we successfully polymerized some of the ordered phases, yielding slightly compressible and elastic but thermoset solids. Sample characterization has been performed using small angle x-ray scattering and scanning electron microscopy, but additional analysis must be performed prior to drawing conclusions about the success of these efforts in creating nanoporous filter materials. This work was supported by a training grant from the National Institutes of General Medical Sciences (R25-GM624
AN IN VITRO MOUSE BRAIN BASED IN VIVO MOUSE BRAIN ATLAS
Karen Law (Massachusetts Institute of Technology) and Helene Benveniste, Medical Department, Brookhaven National Laboratory and Stony Brook University
The goal of this project is to attain good quality in vitro (in skull) mouse brain images using magnetic resonance microscopy for use in building an in vitro mouse brain atlas. Two C57BL6/J male mice 12-14 weeks old were deeply anaesthetized with 1.5 cc of Nembutal and were then perfusion fixed with formalin. The head was isolated and skin was stripped away so that only the skull and brain remained and was stored in formalin overnight. The specimen was imbedded in fomblin and scanned with a Bruker 9.4T MR machine and a commercial volume rf-coil initially using parameters developed on a 17T machine at the University of Florida. Modification of these parameters was performed to try to attain the best SNR and CNR. Resulting parameters for the 9.4T images from our T2* scan are as follows: TE = 9 ms, TR = 140 ms, flip angle = 40 degrees, NEX = 16, spatial resolution = 55 µm³, acquisition time ~ 40 hours. A comparison of our image and the image taken from the 17T machine in Florida are below. Ghosty artifacts and bright spots on our images may result from interference from the skull as well as the possible inhomogeneity of the magnetic field due to inaccurate placement of the specimen. Future work will strive to attain better SNR and spatial resolution. These images will contribute to the creation of the in vitro mouse brain atlas, which will serve as a bridge to transfer anatomical templates from excised mouse brains to future in vivo images to create an in vivo mouse brain atlas. This study was supported by a grant from NIH RO1 EB 00233-04.
REGIO- AND STEREO-SELECTIVE RH-CATALYZED C-H INSERTION OF A CARBAMATE NITROGEN
Diana Leung (SUNY Binghamton) and Kathlyn A. Parker, Department of Chemistry, Stony Brook UniversityThe experiments carried out were to determine if it was possible to synthesize D-Angolosamine Glycal, one of the building blocks needed for synthesizing kidamycin. Kidamycin is an antitumor antibiotic. To produce D-Angolosamine Glycal C-H insertion of a carbamate nitrogen must be done to form the precursor of D-Angolosamine Glycal. A four-step scheme was done to produce a chiral carbamate 4 for C-H insertion of a carbamate nitrogen. After each reaction was completed a flash chromatography was done to obtain the product, and the use of thin layer chromatography and 1H-NMR were used to confirm the production of the desired compound in each step.
From previous studies done by a lab member, C-H insertion of a carbamate nitrogen 2 occurred next to the double bond. It was hypothesized that C-H insertion of a carbamate 4 would occur next to the triple bond because the carbamate 2 insertion occurred next to the double bond. Instead of producing the precursor for the production of D-Angolosamine, an interesting discovery was made about the stereo-selectivity of carbamate nitrogen for the oxygen instead of the triple bond. With such an interesting discovery, further studies need to be conducted on why the Rh-catalyst favored the insertion on the C-H attacted to -OTBDPS, instead of the C-H next to the triple bond. Also, experiments need to be carried out to find a better catalyst for producing a C-H insertion of a carbamate nitrogen to the C-H next to the triple bond. This study was supported by grants from Chemistry REU, IBRP, NSF, NIH, and the National Cancer Institute.
PREDICTING THE RATE OF CREEP OF THE LUMBAR SPINE FROM ITS STRESS RELAXATION RATE DURING FLEXION USING THE QUASI-LINEAR MODEL OF FUNG
Amadeus Lopez (Rensselaer Polytechnic); Jesse Little, Allsyon Ianuzzi, and Partap Khalsa, Department of Biomedical Engineering, Stony Brook UniversityThe purpose of this study was to determine the moment relaxation function of the lumbar spine and predict a creep rate using the quasi-linear model. For the moment relaxation experimental set up, intact lumbar spines (T12-S1, n =5) were dissected and potted at the sacrum. The lumbar spines were then fixed to the testing surface. Moment arms for each spine were measured. An actuator was coupled to T-12 and set up collinearly with the lumbar spine. Spine specimens were actuated in flexion displacements (10mm-40mm). The peak forces developed were used to find the peak moments and the times that they occurred through out the trial. The log [moment (Nm)] – log [time (seconds)] were calculated and plotted. The data were then linearly fit with a regression line. The slope of the linear regression line was recorded as the corresponding moment relaxation rate. The average moment relaxation rate for the spines at each displacement was plotted, and fitted using an exponential rise to max, whose equation defined the moment relaxation function. The moment relaxation function was determined to be: M(x) = -.4582+.4415(1-exp-.2242x). The creep experimental setup was similar. The actuator applied a velocity controlled force to 10Nm then back to 0 Nm at the same velocity on the intact lumbar spines (n=7) for ten cycles. Peak displacements of the spine and the times they occurred were recorded and plotted. The data were then linearized by taking the log (displacement) and log (time) and plotting the results. The plot was then fit with a linear regression line whose slope was recorded as the corresponding creep rate. The average displacement during the creep experiment was calculated to be 66.8 mm. This displacement was then plugged into the previously determined moment relaxation function to extrapolate a moment relaxation value on the graph, and it was determined to be -.0167Nm/sec. The predicted creep rate using the quasi-linear model was the inverse of the moment relaxation rate, and it was determined to be .0167 mm/sec. The data for creep rate at 10 Nm were not normal; therefore we could not use the mean and standard deviations to test the predicted rate statistically. A box plot using the median and the 25th and 75th percentiles was used and showed that the predicted relaxation rate fell within the box plot. The quasi-linear model was sufficient to model the behavior of moment relaxation and creep.
STUDY OF TECHNIQUES APPLICABLE TO THE CONCERTED ROTATIONS OF PROTEIN BACKBONES
Isaac Pflaum and Carlos Simmerling, Department of Chemistry, State University of New York at Stony Brook
This study evaluated the applicability of several methods to the concerted rotation of protein backbones for a hybrid molecular dynamics, monte carlo simulation. A detailed analysis of previously employed techniques, such as concerted rotation with angles (Jorgensen 2003) and chain re-growth, (Escobedo 2000) led to the conclusion that these techniques were not appropriate for this application due to limitations on the size of individual moves. Further study of chain geometry lead to the understanding that portions of the protein backbone could be treated using robot actuator kinematic equations, and that this method had no apparent limitations on the size of individual movements. Also, this study revealed that this method is more elegant, intuitive, and more easily communicated to a general audience than previously proposed techniques. This treatment utilizes the equations of motion for a single atom, the effector; one equation describing the movement of the atom with respect to the three dihedral backbone angles to the right, and the other with respect to the three dihedral angles to the left. Each equation is decomposed into a linear system of equations, which gives rise to two transition matrices, A(r) and A(l). When multiplied by a column vector containing the magnitudes of the appropriate angles, each transition matrix yields as the product the Cartesian coordinates of the effector. Setting the systems equal to each other and inverting one of the transition matrices gives and expression for all possible combinations of the dihedral angles in question that satisfy this relation. This work was supported by a training grant from the National Institures of Healh, R25-GM62492
ADVANTAGES IN THE USE OF SYNCHROTRON-GENERATED X-RAY MICROPLANAR BEAMS (MICROBEAMS).
Anna Ruvinskaya, Silmilly Toribio, Danielle Williams, Ajantha Nithi, John W. McDonald and F. Avraham Dilmanian , Medical Department, Brookhaven National Laboratory, Upton, New York 11973, USA.
Despite recent advances in conventional radiation therapy, including Intensity Modulated Radiation Therapy (IMRT) and Stereotactic Radiosurgery, the method still fails in treating highly malignant brain tumors such as high grade gliomas (e.g., glioblastoma multiforme, GBM), and pediatric tumors of the central nervous system (CNS). The main limitations stem from the damage the therapy produces in the surrounding normal brain tissue. It has been shown in studies of the rat brain tumors that microbeam radiation therapy (MRT) addresses these limitations by a) sparing the normal brain tissue (Slatkin et al., PNAS 1995), and b) preferentially damaging the tumor, even when used from a single direction (Laissue et al., Int. J. Cancer 1998). The previous studies of MRT in the rat brain used the tumor model 9L gliosarcoma (9LGS), which is not a true representation of the human high grade gliomas. In particular, 9LGS tumors are encapsulated and have limited invasiveness, emulating more metastatic brain tumors rather than high grade gliomas. In a recent experiment, we applied MRT to a new rat tumor model, namely the F98 glioma. This tumor very closely simulates the characteristics of the human GBM due to its invasive, highly proliferative nature with an infiltrative pattern of growth within the brain. Furthermore, intracranial F98 tumors are reproducible. In the first part of my work we irradiated rats with F98 tumors bilaterally (anteroposteriorly and laterally) with single-exposure microbeams of 45 Gy and 35 Gy, and single-exposure broad beams of 35 Gy and 25 Gy. The results are being analyzed.
In the second part of my work we studied the rat spinal cord with microbeams. The current method of producing demyelination in the rat spinal cord, used to study remyelination, involves injection of gliotoxins into the spinal cord. The method is not only invasive (it requires surgery), but also can damage axons and the vasculature. However, due to the properties of microbeams mentioned above, it can be used as a novel tool for producing targeted, selective axon-sparing demyelination much more efficiently. In the earlier studies conducted to follow the processes of myelination, the spinal cords of adult rats were transaxially irradiated at the level of the T9 vertebra using a vertical microplanar beam 270-ìm wide and 12 mm high with a high incident dose of 1000 Gy (750 Gy in depth doses). Evaluation by immunohistochemistry studies after two weeks from irradiation revealed loss of mature astrocytes, oligodendrocytes and selective demyelination while sparing axons. After a three month period from irradiation, substantial recovery of myelination, preservation of axons and repopulation of cells were observed through the use of immunohistochemistry and electron microscopy. The sensorimotor function was also measured through the use of the accelerating rotor rod. Results indicated that motor function was substantially disrupted after the initial irradiation and improved over a three month period in concert with remyelination. In the studies I participated in different dosages (500-1250 Gy), new time points, and new beam geometries were used to provide additional information on the process of recovery of the spinal cord from microbeam irradiation. Results are being analyzed using immunohistochemical methods. Research supported by NIH and by the U.S. Department of Energy, and the IBRP program at SUNY Stony Brook
CHARACTERIZATION OF MOLECULES TRANSPORT IN AN ENGINEERED EXTRACELLULAR MATRIX.
An Sung, Kaustabh Ghosh, Fubao Lin, Molly Frame, Xiao Zheng Shu, Glenn Prestwich, and Richard Clark, Department of Biomedical Engineering, Stony Brook University
The viability of engineered tissue constructs is compromised by inadequate exchange of nutrient and waste. The knowledge of molecule transport kinetics is therefore crucial to the design and optimization of engineered tissue replacements. One of such engineered tissue construct is our HA-based crosslinked hydrogel containing the fibronecting functional domains necessary for cell migration. This study examined the diffusion of molecules through the polymer network of our HA-based hydrogel. The experiment was setup as an unsteady-state system, and a list of protein standards and model drugs were selected as diffusants with molecular weight ranging from 0.18 kDa to 66.0 kDa. Solutions of HA hydrogel mixed with the diffusant were molded in modified 3ml syringes. These crosslinked hydrogels were incubated at 4Co for 24hr. The syringes containing the hydrogel were separately immersed in and leveled with 1X PBS solution in a glass beaker. At the end of each experimental time point, absorbance of the PBS solution was measured by a spectrometer, which was then used to calculate the amount of diffusant released. The results showed that larger molecular weight molecules diffused slower, and the molecules larger than 36.0 kDa did not diffuse freely out of the hydrogel. Minute traces of the released diffusant were detected with the larger molecules, but they were likely to be impurity or degraded fragments of the molecule. The results also suggested that there was an interaction between the diffusants and the hydrogel since not all molecules were able to diffuse or wash out. As the molecular size of the diffusant increases, the interaction becomes greater because the equilibrium appeared at smaller fraction of diffusant release. Another limitation of this unsteady-state experiment setup was the alteration in crosslinking kinetics of the hydrogel caused by the addition of diffusant. Due to these limitations it is desirable to compare the current setup with a different system. This has never been previously addressed, and the comparable systems should overcome the limitations and compare the validity of the results. It was proposed in this study that simple modifications of current unsteady-state setup could enable it to model steady-state diffusion. In one such modification the volume of hydrogel was decreased to a thin film at the bottom of syringe, and the diffusant solution was poured on top of the hydrogel instead of incorporating in the hydrogel. A timed-activating motor was positioned at the top of the system to prevent the formation of stagnant layer at the interface between the hydrogel surface and the diffusant solution. The preliminarily results showed that this modified system works well and it is a very simple and reproducible design that could improve current diffusion studies. This work was supported by a training grant from the National Institutes of General Medical Sciences (R25-GM62492).THE CHARACTERIZATION OF BLADDER MOTION AND URINARY INCONTINENCE. Christopher Q. Trinh (University of California, Berkeley); Qi Wu, Zhenguo Wang, Yingtian Pan, Department of Biomedical Engineering, Stony Brook University
Urinary incontinence is the lack of control of the bladder that ultimately leads to the leakage of urine. The functional mechanism of the bladder is altered when a patient is suffering from incontinence. The details of the mechanism of incontinence are not well understood. The mathematical characterization of the pattern of motion exhibited by the urinary bladder would be extremely seminal in the diagnosis and treatment of urinary incontinence. It will provide a method of comparing and contrasting normal bladders and bladders with incontinence. This preliminary study involved high-resolution imaging of the motion of the bladder. The effects of aging and temperature on the motion of the bladder were examined using optical coherence tomography (OCT). Data was collected by scanning animal bladders with OCT. Living conditions are simulated by regulating temperature at 37°C, using Ringer solution, and bubbling clinical blood gas to maintain the proper pH. Each time a part of the bladder was scanned, 50 sequential cross-sectional scans were taken at approximately 1-2 fps. In each set of 50 scans, folds at different regions of the bladder were chosen and their movement was traced through all 50 scans. This data was used to analyze the motion of a single fold in the lateral direction. The data was also used to analyze the relative lateral distance between two folds. This provided a quantitative method of studying how aging and temperature affect the motion of the bladder. To study the effect of aging, young and geriatric rat bladders were studied. Data was collected from the neck, middle, and bottom regions of the bladder. Groups were formed, each consisting of one young and one geriatric rat. The results were analyzed from all three regions within each group and then compared for consistency. When studying the lateral displacement of a single fold, no trend for the amplitude of motion could be made when analyzing the graphs of the lateral displacement with the mean subtracted. However, young rat bladders appear to oscillate at a higher frequency than geriatric rat bladders due to the concentration of low frequency components in the graphs of the power spectrum of the geriatric rat bladders. The same results were obtained when analyzing the relative lateral distance between two folds. Rabbit bladders were used to study the effect of temperature. One region was scanned at four different temperatures: 25.8°C, 30.0°C, 33.0°C, and 37.5°C. Again the lateral displacement of a single fold was analyzed. From the graphs of the lateral displacement with the mean subtracted and the amplitude spectrum, it appears that as temperature increases the amplitude of motion decreases while the frequency of motion increases. The study suggests that aging and temperature definitely affect the motion of the bladder. Furthermore, this suggests that studying bladder motion with OCT has the potential to provide insight into the mechanism of urinary incontinence. However, more rigorous experimentation including the analysis of more bladder samples and better signal processing is necessary. The study was supported by the National Institutes of General Medical Sciences (R25-GM62492).
The ability to design and control cellular interactions on a micrometer scale is essential for tissuegenesis and tissue engineering. Here we present an inexpensive and simple approach to engineer patterns of excitable heart cells: primary cardiac myocytes (CM) and cardiac fibroblasts (FB). Two specific aspects were examined in this study: 1) determining optimized conditions for micropatterning of proteins and cells; 2) quantifying the effects of protein patterning on cardiac cell arrangement and cell function. Pattern templates were designed by acoustic micromachining or pattern laser printing on acetate surfaces. Molding of a versatile biocompatible elastomer, polydimethylsiloxane (PDMS), was then used to obtain stamps for deposition of attachment factors for cell growth, such as fibronectin. The stamps were utilized for protein patterning by microcontact printing onto polystyrene surfaces. Cell adhesion to non-coated areas was prevented by blocking agents. Transfer of proteins was verified by imaging the deposition of fluorescein labeled collagen on surfaces. Cell morphology was studied by labeling and imaging the cytoskeletal protein arrangement (F-actin) using phalloidin stain. Optical mapping of propagating intercellular calcium waves was performed by fast camera system and calcium-sensitive dye (Fluo-4) to assess cellular viability and cell communication in the patterned networks. Optimization of cell micropatterning was attempted by using bovine serum albumin and Pluronic as blocking agents. Empirically, it was found that 0.5% w/v Pluronic was most effective in blocking undesired attachment for patterning of neonatal rat cardiomyocytes. Several patterned networks were prepared and examined (n=6 CM and n=9 FB), with parallel patterns having line widths from 7 to 400?m. Random organization was found in wider cell patterns as opposed to uniformly stretched cell lining of thinner patterns, indicating the importance of protein environment to cell orientation and intracellular protein arrangement. Cross-bridges between neighborhood patterns were also found in FB possibly due to their higher cell motility. Using laser printing allowed for more flexibility in designing patterns of arbitrary geometries for cell growth. Optical mapping of calcium waves revealed highly anisotropic conduction with slower electrical propagation across lines of excitable CM as compared to normal cell culture with random cell distribution. The micropatterning method used in this study demonstrated precise spatial control of cellular orientation through selective attachment to proteins. Different cellular behavior resulted from different cell organization design, suggesting the ability to regulate cellular function. This in vitro system can serve as a model for heart disease, esp. if the two cell types are combined into inter-dispersed patterns, such as seen in fibrosis. Furthermore, micropatterning of excitable cells (CM) can help guide the way to design biological circuits analogues for solving computational problems. This work was supported by funding from the Whitaker Foundation (RG-02-0654) and by a training grant from the National Institutes of Health for the Interdisciplinary Biomedical Research Program (IBRP).
MICROPATTERNING OF HEART CELLS BY MICROCONTACT PRINTING OF ATTACHMENT PROTEINS.
Hui Jing Yu (Stony Brook University), Shanza Malik and Emilia Entcheva, Department of Biomedical Engineering, State University of New York at Stony Brook
Guivion Zumbado (Stony Brook University) and Michael Hadjiargyrou, Department of Biomedical Engineering, Stony Brook University
Many genes and proteins are expressed inside the fracture callus including growth factors. Fibroblast growth factor 7 (FGF7) gene expression results from transcriptional and translational synthesis as the FGF7 protein, a polypeptide that binds to specific surface receptors of fibroblast and keratynocyte membranes. To determine FGF7 upregulation within the bone callus, post fracture as well as intact bone RNA templates were amplified using RT-PCR. This temporal study of different post fracture days, intact bone and embryogenic RNA pools demonstrates the role in signaling as it specifically pertainsto the FGF7 gene during endochondral ossfication processes. This study is funded by the Interdiciplinary Biomedical Research Program at Stony Brook University.
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