2016 HOFSTRA SEAS SUMMER FELLOWSHIP

REPORT

Megan MurphyDr.GhorayebJuly 22nd, 2016

SUMMARYThis summer, I worked as a lab assistant in the Ultrasound Research Lab with Dr. Ghorayeb for

eight weeks. The goal of my position was to be introduced to the instrumentation and techniques used

in the Ultrasound Research Lab. This position involved the following tasks:

1) Learn how to use the instruments in the Lab

2) Learn Matlab (Mathworks, R2015b, Natick, MA.)

3) Analyze fetal cardiac scans

4) Perform literature reviews of previously performed research experiments

5) Assist in Senior Design Experiments

6) Develop a protocol for a new osteoporosis experiment

I have learned how to use the scanning acoustic microscope (SAM) systems, the biomechanical

testing system, and their associated acquisition programs. My work also involved assisting other

bioengineering students working on their Senior Design component. Before using the instruments

myself, I read about the physics of ultrasound in order to understand how the SAM systems function.

Another aspect of my fellowship included the use of Matlab to analyze fetal cardiac scans

provided by the Center of Maternal Fetal Medicine at Northwell Health Hospital.

Moreover, numerous literature reviews were part of this research fellowship. For instance, I

read and reported on previous research works such as:

1) Frequency Specific Ultrasound Attenuation is Sensitive to Trabecular Bone Structure1 by Dr. Lin

2) Ultrasonic Evaluation of Bone Quality in Cadaver Ilia2 by Dr. Ghorayeb

3) Microarchitecture, the Key to Bone Quality3 by Dr. Brandi

4) Mechanical Properties of a Single Cancellous Bone Trabeculae Taken from Bovine

Femur4 by Dr. Enoki

5) Contact Stress Distribution on the Femoral Head of the Emu5 by Dr. Troy

In addition, I wrote a new protocol regarding the osteoporosis project in preparation for future

Senior Design endeavors.

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ULTRASOUNDThe first day in the lab, Dr. Ghorayeb taught me how to use the Sonix SAM system and software.

I took several practice scans of a piece of plexiglass containing four machine-drilled holes. I learned how

to adjust the resolution, the dimensions, and location of the transducer for the scans. It is very

important to take the scan at the focal length, which is the point where the lens is most focused and

receives the clearest return signals. Focal length is transducer specific. Another important guideline is to

be very careful with the scan window in which the data is collected (Figure 3). Depending on the size of

the window, the program may be collecting too little of the scan and missing data, or it may be

collecting too much of the scan and skewing the results.

After the practice scans, I read and took detailed notes on a packet about the terminology and

physics behind the system. Learning the terminology was very helpful because it made it easier for me

to communicate with the other students in the lab when performing ultrasound scans of bone samples.

Also, knowing about the science made it easier for me to decide what type of transducer to use in the

protocol I wrote at the end of the summer.

While working with students on their Senior Design component, I used both low-frequency and

high frequency ultrasonic SAM systems in the lab (Figure 1 and Figure 2). The systems are very similar

and relatively easy to use. Both the OKOS and SONIX machines are very delicate and are connected to

computers each set up with OKOS acquisition software which displays the A-scan wave forms, scan

specifications, transducer height adjustment arrows, and the results after the scan.

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Figure 1: Low Frequency Ultrasonic Scanning Acoustic Microscope system.

A)

B) C)

MATLAB

I spent the first week reacquainting myself with Matlab. When analyzing medical scans, medical

ultrasonic images are analyzed using a Matlab program developed by Dr. Ghorayeb. The program

performs texture analysis and determines the homogeneity level over a specific area of the scan. Dr.

Ghorayeb wanted me to be able to understand the function and purpose of each line of the code so I

would be able to design a similar code for future experiments if necessary. I went through the file and

figured out what resulted from each line of the code.

I spent many hours trying to adjust the program to automatically input the data found in Matlab

into an excel spreadsheet. The way the file works is when the file is run, a window pops up which asks

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Figure 2: High Frequency Ultrasonic Scanning Acoustic Microscope system.

Figure 3: Output of ODIS acquisition system. A) Wave during the A-scan. B) Arrows used to adjust the position of the transducer on the machine before the scan. C) Measurements used to adjust the size, area, and resolution of the scan.

the user to choose a picture to analyze. After a picture is selected, a separate window with the chosen

picture opens. By clicking two points on the picture, the user creates a region of interest (ROI) for the

program to collect data from. Then, the program calculates the level of homogeneity in the ROI and

displays the maximum, minimum, and average homogeneity of the region.

Currently, when data is found, the data for the average is automatically put in the computer’s

clipboard (Figure 4) and that data can be inserted into a separate excel file by pasting. This is a

convenient feature when only the average is required: however, it is sometimes helpful to look at all

data values collected. I wanted to adjust the code so the maximum, minimum, and average data would

be automatically saved in respective columns in a specific excel file. I also wanted the program to be

able to shift down a row when a new picture is chosen so the new data would be input in its own row. I

could not find a code that would keep the program from continuously writing the newest data collected

into the same row, erasing the previously collected data.

Figure 4: File excerpt that copies the average data value directly to the clipboard.

FETAL CARDIOLOGYAs part of a joint collaboration between the Maternal Fetal Medicine Center at Northwell and

Dr. Ghorayeb, I got involved in the analysis of fetal cardiac ultrasound scans to see whether a correlation

existed between the tissue homogeneity and the heart conditions found in the fetuses. Prior research

has showed in other organs that levels of homogeneity or heterogeneity were an indication of certain

conditions. These levels can also show if the organ is well developed or not. For example, the more

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heterogeneous a fetal lung is, the more developed it is. We were curious to see if there would be a

similar trend in the fetal heart.

When running through the scans, I took separate homogeneity readings for the septum and the

lower left ventricular wall in each image and recorded all values in an excel file. We came to realize that

it was difficult to choose a good ROI to use to analyze the scans. Since each scan was in a different

orientation and slightly blurry, it was very hard to distinguish the parts of the heart. Fetal hearts are very

small and it is understandable that it is hard to get a clear scan, but clearer scans with a more uniform

orientation would have been more useful. It also would have been better if every heart had the same

condition. Each scan we received and analyzed had a different condition, so there was no way to

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Figure 5: Cardiac scan with ROIs on the septum (top) and left ventricle (bottom).

correlate a specific condition with a trend in homogeneity. We asked for more scans to find more

information, but we never received them.

LITERATURE REVIEWDuring my fellowship I was given two research papers about the effects of osteoporosis in

trabecular bone. The first research paper I received was by Dr. Lin et al.1, titled “Frequency Specific

Ultrasound Attenuation is Sensitive to Trabecular Bone Structure,” and the second paper was by Dr.

Ghorayeb et al. 2, titled “Ultrasonic Evaluation of Bone Quality in Cadaver Ilia.”

Currently, the most popular way to measure bone properties is dual energy x-ray

absorptiometry (DXA), but DXA uses ionizing radiation and there are limitations on how much

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Figure 6: Data found after scans analyzed with Matlab.

information the x-ray scan can reveal. Another system that measures bone properties is broadband

ultrasound attenuation (BUA), which is noninvasive, but it is not very sensitive because it can only work

in a frequency range between 300 and 700 kHz. Dr. Lin’s study investigated the possibility of using

another form of ultrasound called frequency modulated ultrasound attenuation (FMA) to evaluate

trabecular structural properties. FMA can function in a much wider range than BUA, so for this

experiment four different frequency bands in the range between 300 KHz and 1.9 MHz. Dr. Lin used

through transmission for the ultrasonic scans, and he was searching to see if there was a correlation

with the effectiveness of the band range and the orientation of the bone. It was found that there was a

connection with the orientation and frequency band in the anterio-posterial orientation, but in the end

it was concluded that FMA should be used to supplement BUA findings because FMA relies on the

linearity of the attenuation frequency relationship.

Dr. Ghorayeb’s study also looked into the possibilities of using quantitative ultrasound (QUS)

tools to measure and monitor bone density. In this experiment, information about the ilia samples was

gathered with ultrasound, the same information was found with DXA, and then the results were

compared. It was found that the information gathered with QUS had an error of 3.5% when compared

to the DXA measurements, but it was proportional to the trends in percent bone loss and bone mineral

density. The conclusion for this experiment was that ultrasound measurements of the bone properties

would be very helpful with the monitoring of progression or regression of osteoporosis in a living being.

After I read both papers, I searched for similar studies online. The third paper I read,

“Microarchitecture, the Key to Bone Quality,” by Dr. Brandi et al. 3, which stressed the importance of

knowledge of the microarchitecture in the trabecular bone. Osteoporosis weakens and breaks down the

trabecular bone connections, and as the disease progresses, the bone becomes hollower, which makes

it harder for the bone to resist fracture. Dr. Brandi concluded that the way to best diagnose and treat

osteoporosis is by measuring the spatial distribution of the microarchitecture and the bone mass. She

mentioned both DXA and CT scanning as a means to provide measurements of bone mass and actual

images of the bone’s actual internal architecture.

Another paper I read that focused on microarchitecture was “Mechanical Properties of a Single

Cancellous Bone Trabeculae Taken from Bovine Femur,” by Dr.Enoki et al. 4, which focused on both

cancellous and trabecular bone and elasticity in different anatomical orientations. Cancellous and

trabecular samples were obtained from the bovine femur, and all tests were performed in each

anatomical orientation. Compression tests were performed on the cancellous bone samples and three

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point bending tests were performed on the trabecular samples. The tests lead to the conclusions that

trabecular bone structure has influence over the mechanical properties of cancellous bone, and that

orientation affects the elasticity in both types of bone.

Finally, I read a paper slightly outside of what I spent the summer researching called, “Contact

Stress Distribution on the Femoral Head of the Emu,” by Dr. Troy et al. 5 which focused on the loading at

the hip and the femoral head of the emu. The study was testing emu femurs to see if the emu was a

viable candidate for simulations of osteonecrosis in human hips. The data found that the emu would in

fact be ideal for such simulations.

SENIOR DESIGN ASSISTANCEAs mentioned previously, part of this fellowship was to be involved with assisting a student

working on the biomedical engineering Senior Design component. The aim of the project was to study

levels of osteoporosis induced in bovine femoral head samples. The study followed the protocol

described by Lin et al. 5. Seven trabecular bone samples were cut out of a bovine femur head with the

dimensions of 1 cm X 1cm X .5cm. The bone marrow was cleaned off the samples with a high pressure

washer, and the samples were then wrapped in gauze soaked in X10 phosphate buffer saline.

Seven samples were required because each sample was an example of a different degree of

osteoporosis, which was simulated by timed exposure to acid. Two samples served as controls, and five

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samples were exposed to 1.8% formic acid at 20 minute time intervals (20 min., 40 min., 60 min., 80

min., 100 min.).

After two days of being immersed in a buffer saline solution, we demineralized the samples

were then demineralized by placing them in five beakers that were filled halfway with 1.8% formic acid

and placed on a shaker. The shaker was set to 120 rotations per minute. Each beaker was labeled to

show how long each sample was exposed to the acid. After the first twenty minutes passed, we took the

20 min. sample off the shaker, removed the sample from the beaker, and placed it in ammonia for thirty

minutes to stop the demineralization process. Every twenty minutes after that, we repeated the same

steps for the 40 min., 60 min., 80 min., and 100 min., samples. After the demineralization process was

finished for all samples, we placed the trabecular bones in new beakers filled with water and put them

in the fridge.

The following day we performed ultrasound scans of each sample with a 75 MHz transducer

using the OKOS SAM system. After we took the scans of each sample, we sent the scan pictures to Dr.

Ghorayeb. Once received, the scans were analyzed with Matlab to find the porosity in each sample. The

data was close to what we expected: however, we believe the data was being skewed bysome of the

cortical bone that was not removed in the 80 min. and 100 min. samples because both samples should

have been much more porous than what the analysis showed.

The next day, we found the wet and dry mass of each sample. The following week, we

performed mechanical testing on the samples with an Instron biomechanical testing machine. After

adjusting the settings of the machine, a sample was placed on a metal plate. Force was then applied to

each sample until the bone broke, which allowed us to find the Young’s Modulus for each sample.

PROTOCOL DEVELOPMENTAfter assisting with the Senior Design, I was asked to write a more detailed and specific protocol

of a similar experiment with a larger sample size based on Dr. Lin’s and Dr. Ghorayeb’s papers. While

working on the Senior Design, I noticed a few problems that could have altered the end results.

One issue dealt with cortical bone that was left on some of the samples. Cortical bone is much

stronger than trabecular bone and it would not be as affected by the demineralization treatment as the

trabecular bone. Since the cortical layer was still strong during the ultrasound and mechanical tests, the

results were skewed. If there was no cortical on the samples, the samples would have appeared more

porous on the ultrasound scans, and the samples might have broken faster during mechanical testing.

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Also the presence of cortical bone did not allow ultrasound waves to fully propagate into the trabecular

layer, thereby giving rise to a more homogeneous reading of the sample.

Additionally, the acid did not uniformly demineralize all sides of the samples because of the way

they were sitting in the beakers during the demineralization process shaking. Some of the samples were

lying flat against the bottom or the side of the beaker, which protected a side from the acid. The

protected side was then stronger and less porous than the rest of the sample, which skewed the

porosity results and the mechanical testing.

Finally, when immersing the bones in X10 phosphate buffer solution, the gauze was squeezed to

remove any excess solution before wrapping the bone. This caused the samples to dry out which made

the samples not well-prepared for the rest of the experiment.

When writing out the protocol for the new osteoporosis experiment, I was very specific and

detailed about how every step is to be done to make sure that the results would not be skewed. In this

new protocol, I decided that there are to be seven trabecular samples from five separate bovine femur

heads to increase the sample size and get more information out of the experiment as a whole. It is

expected that the samples from different bones will all react similarly, but there may be some slight

variation. I specified that pre-demineralization scans will be taken of every sample in a specific

orientation. This will give us an idea of how porous the samples are before the demineralization tests.

After we perform the demineralization, we will be able to find the exact percentage of porosity and the

difference between the initial sample and sample after treatment. The orientation the scan is taken in is

also important. If post-demineralization scans are taken with the same orientation as the pre-scans, we

will hopefully be able to see the same “landmarks” within the scans as the brightness of the scans

decreases with increased time in acid.

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After I finished writing the protocol, Dr. Ghorayeb contacted Dr. Lin at Stony Brook to see if he

would like to work with us on this experiment. Stony Brook lab has different lab equipment than

Hofstra, which would allow us to find more information from the experiment. If they used their diamond

cutter to cut out the trabecular samples from the femur heads, they would be able to be remove all

unwanted cortical bone. The diamond cutter would also expose the samples to less heat then a regular

band saw. Stony Brook also has a through transmission ultrasound machine that would allow us to find

the Young’s Modulus by finding the velocity at which the ultrasound signal passes through the samples

in each anatomical orientation. A collaboration could lead to a more accurate and informational

experiment, which has the potential to be very important and influential.

CONCLUSIONS

   

The Summer Research Fellowship

Program allowed me to learn many things that

I need to know to be able to contribute to the work

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Figure 7: Outlined steps of the Experiment Protocol described in this section.

done in the Ultrasound Research Lab, and I am eager to conduct research and work on future

experiments including my Senior Design. I really appreciated the opportunity to work with Dr. Ghorayeb,

and I believe that this summer position was educational and successful, and that it will lead towards

more important research in the near future.

REFERENCES

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1 Lin W., Serra-Hsu F., Cheng J., and Qin Y.-X., "Frequency specific ultrasound Attenuation is sensitive to

Trabecular bone structure," Ultrasound in Medicine & Biology, vol. 38, no. 12, pp. 2198–2207, Dec. 2012.

2

Ghorayeb S. R., Rooney D. M., "Ultrasonic evaluation of bone quality in cadaver Ilia," Annals of Biomedical

Engineering, vol. 41, no. 5, pp. 939–951, Jan. 2013.

3 Brandi M. L., "Microarchitecture, the key to bone quality," Rheumatology, vol. 48, no. suppl 4, pp. iv3–iv8,

Sep. 2009.

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Enoki S., Sato M., Tanaka K., Katayama T., "Mechanical Properties of a Single Cancellous Bone Trabeculae

Taken from Bovine Femur," International Journal of Modern Physics: Conference Series, vol. 06, pp. 349–

354, Jan. 2012.

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Troy K. L., Brown T. D., Conzemius M. G., "Contact stress distributions on the femoral head of the emu

(Dromaius novaehollandiae)," Journal of Biomechanics, vol. 42, no. 15, pp. 2495–2500, Nov. 2009.