Richard Magin, Chair and Advisor Mrignayani Kotecha Dieter Klatt

Transcription

1 Proteoglycans Quantification in Tissue Engineered Cartilage using Sodium MRI at 11.7 T BY DAN YU B.S., Tianjin University, 2011 THESIS Submitted as partial fulfillment of the requirements for the degree of Master of Science in Bioengineering in the Graduate College of the University of Illinois at Chicago, 2013 Chicago, Illinois Defense Committee: Richard Magin, Chair and Advisor Mrignayani Kotecha Dieter Klatt

2 ACKNOWLEDGEMENTS I would like to thank my advisor Dr. Richard Magin, who guided and supported me all the time. Many thanks to my dear labmates who helped me in our group, Allen Ye, Kaya Yasar, Yifei Liu and especial thanks to Ziying Yin for supporting me as always. I would also like to thank Dr. Mrignayani Kotecha and Dr. Dieter Klatt for being my committee, as well as for providing interesting advice. In addition, I like to acknowledge RRC microimaging facility at UIC, especially support from Dr. Rob Kleps, Director of RRC facility. DY ii

3 TABLE OF CONTENTS Chapter 1. INTRODUCTION Background Motivation Research Goal Method Why Sodium Magnetic Resonance Imaging is useful in Osteoarthritis? Osteoarthritis Proteoglycans Why Choose Sodium Magnetic Resonance Imaging? Why not choose 1 H Magnetic Resonance Imaging? Challenges of Sodium Magnetic Resonance Imaging EXPERIMENTAL DESIGN Hypothesis Sample Preparation Step I: Phantoms Step II: Bovine Chondrocyte Pellets Pellets with Phosphate Buffer Saline Pellets with Fluorinert Oil Magnetic Resonance Imaging Acquisition RESULTS AND ANALYSIS Step I: Results of Phantoms Proton and Sodium MR Images Validation of Hypothesis Step II: Results of Pellets Week 1: Proton and Sodium MR Images Week 4: Proton and Sodium MR Images Validation of Hypothesis CONCLUSION AND DISCUSSION Concerns of Experimental Design Discussions How to Develop Image Contrast? iii

4 TABLE OF CONTENTS (continued) Improve Magnetic Resonance Imaging Protocol Future Work CITED LITERATURE VITA iv

5 LIST OF TABLES TABLE I. COMPARISON BETWEEN 1H AND 23NA... 6 II. SODIUM CONCENTRATION OF WEEK 1 AND WEEK 4 SAMPLE III. COMPARISON OF SODIUM CONCENTRATION IN WEEK 1, WEEK 4 PELLETS, CHONDROGENIC AND OSTEOGENIC SCAFFOLDS v

6 LIST OF FIGURES FIGURE 1. Evolution of Osteoarthritis Collage and proteoglycans in extra cellular matrix Proteoglycans and GAGs Phantoms with sodium concentration of 150 mm, 300 mm and 500 mm, respectively The process of culturing chondrocyte pellets T Bruker Avance spectrometer mm proton-sodium double tuned RF coil Proton and Sodium MR images of phantoms The correlation between sodium image intensity and sodium concentration Proton MR image for week 1 sample acquired with RARE protocol Sodium MR image for week 1 sample acquired by GEFC protocol Proton MR image for week 4 sample acquired with RARE protocol Sodium MR image for week 4 sample acquired with GEFC protocol The operating interface of ImageJ vi

7 15. Two pellets have significant different signal intensity, indicating water quantities are different vii

8 LIST OF ABBREVIATIONS OA Osteoarthritis MRI Magnetic Resonance Imaging CT Computed Tomography PG Proteoglycan ECM Extra Cellular Matrix GAG Glycosaminoglycan NMR Nuclear Magnetic Resonance PBS Phosphate Buffered Saline RARE Rapid Acquisition with Relaxation Enhancement FlASH Fast Low Angle Shot Magnetic Resonance Imaging GEFC Gradient Echo Flow Compensated viii

9 SUMMARY Musculoskeletal disorders affect nearly 33% Americans annually, with injuries to cartilage due to osteoarthritis and sports injuries, accounting for a large fraction of these afflictions. There are currently no effective long-term treatments and cartilage tissue engineering is expected to play a leading role in cartilage treatment. Cartilage tissue engineering relies on the production of collagen and proteoglycans (PG), the two major extracellular matrix components of cartilage, as biomarkers for success. The current characterization and quantification methods for these biomarkers, such as histology and biochemical analyses, are invasive and cannot be performed in vivo. As sodium is known to bind with negatively charged PG, sodium MRI has potential to be used for PG quantification in tissue engineered cartilage. In this study, we present preliminary results of sodium MRI using a) Phantoms with 150 mm, 300 mm, and 500 mm sodium concentration prepared in 1% agarose gel and b) Scaffold free bovine chondrocyte pellets grown in culture medium for four weeks. Sodium MRI experiments were performed at room temperature on an 11.7 T Bruker Avance spectrometer (23Na freq = MHz) using a 5 mm proton-sodium double tuned rf coil. Our preliminary results show that the sodium concentration changes correlate with sodium image intensity, which suggests a potential use of sodium MRI for PG quantification for tissue engineered cartilage. ix

10 1. INTRODUCTION 1.1. Background Osteoarthritis (OA) is one of the most common causes of pain and disability among older people. It affects approximately 27 million people in United States, and predicted to affect over 70 million Americans in 2030 [1]. OA is mainly caused by sports injuries, and aging. Even eliminating the cases of aging and mechanical wear, abnormal joint shape, muscle innervation, and lacking of muscle strength are also reported to be causes of OA [2]. OA is known to associated with a progressive degeneration of cartilage, which leads to narrowing of joint gap, as shown in the Figure 1 [3]. Figure 1. Evolution of Osteoarthritis 1

11 1.2. Motivation There are currently no effective long-term treatments for OA, and the current qualification methods, such as histology and biochemical analyses are invasive and cannot be performed in vivo. X-rays and micro-computed tomography are two commonly used techniques to visualize the cartilage morphology, but the cartilage signal is weak because cartilage does not sensitive to attenuating X-rays [4]. Magnetic resonance imaging (MRI) is emerging as leading noninvasive characterization technique for tissue engineered cartilage, because it is especially good providing good contrast between different soft tissues. This makes MRI a better choice compared to Computed Tomography (CT) and X-rays while imaging the cartilage. That is why we consider applying MRI to cartilage monitoring rather than other imaging techniques. Because particular physiology reasons, which will be explained in the next chapter, we chose sodium MRI as our target technique Research Goal The goal of our research is design experiments to test the sensitivity of using sodium MRI to monitor the changes of cartilage. Thus, to validate the possibility of using sodium MRI as early diagnosis tool of OA. 2

12 2. METHOD 2.1. Why Sodium Magnetic Resonance Imaging is useful in Osteoarthritis? Osteoarthritis Although the process of how Osteoarthritis (OA) happened is still poorly understood, we do know that every kind of OA is associated with loss of cartilage. The main components of articular cartilage are water, type II collagen and proteoglycans (PG). Water accounts for approximately 80% of the weight of articular cartilage. Type II collagen and proteoglycans account for the other 20% [5] Proteoglycans The extra cellular matrix (ECM) of articular cartilage is composed of proteoglycans attached to the hyaluronic acid and the hyaluronic acid is attached to the collagen fibrils, shown in Figure 2 [6]. Figure 2. Collagen and proteoglycans in extra cellular matrix 3

13 Proteoglycans have both chondroitin-sulfate- and keratinsulfate-rich regions showed in the blue dashed circle. They both make PG to be negatively charged. Proteoglycans are proteins covalently bound to a special class of long-chain polysaccharides called glycosaminoglycans (GAGs). GAGs are composed of a disaccharide repeat, shown in Figure 3. From the repeating units of GAGs, we can see that GAGs are negatively charged [7]. Figure 3. Proteoglycans and GAGs Because PG is negatively charged, it can bind to the sodium ions in the cartilage, which provide us a way to measure the quantity of PG. By measuring the quantity of sodium ions, we will get an estimate of the quantity of PG. Also, because PG is the main component of articular cartilage, it is resonable to use PG as a biomarker of cartilage. 4

14 2.2. Why Choose Sodium Magnetic Resonance Imaging? We chose sodium MRI because the following reasons. Sodium is the second most abundant Nuclear Magnetic Resonance (NMR) active nuclei found in body fluids and living tissues, especially have a high concentration in cartilage (~ mm) [8]. Sodium ion gradient between intra and extra cellular space is sign of cell viability, which could be used as indication of diseases. Sodium concentration is sensitive to changes in metabolic state of tissues. Contrast agent is not necessary in sodium MRI Why not choose 1 H Magnetic Resonance Imaging? Although proton MRI is the most widely used MRI, and proton is the most abundant nuclei in the body, we do not choose proton MRI as our main method in our research. As we know, about 80% of the cartilage is water, if we use proton MRI, what we actually see the water instead of others. Also, as many factors and interactions can contribute to the relaxation rates or diffusion maps of water, we are not able to get quantitative results. Besides, proton MRI does not give information about metabolic process directly, because it is nonspecific about cellular processes. 5

15 Challenges of Sodium Magnetic Resonance Imaging Relatively low sensitivity compared to proton MRI Low Signal to Noise ratio as compared to proton Short relaxation times Table I. Comparison between 1 H and 23 Na [9] 6

16 3. Experimental Design 3.1. Hypothesis The one of most abundant proteins in cartilage tissues is proteoglycan (PG), and it proves to be negatively charged because of the GAG structure. This characteristic makes PG be able to bind with free Na+ particles in cartilage tissues, which means it is possible to get a quantitative estimate of PG quantity based on the Na+ particle concentration in cartilage tissues. Thus comes the question, how can we know the Na+ particle concentration in cartilage tissues. Because image intensity directly affected by the sodium particle concentration, higher concentration will result in a higher image intensity. So we use sodium MRI image intensity to calculate the concentration of Na+. In our hypothesis, sodium MRI image intensity should have a strong and positive correlation to PG concentration in cartilage tissues. In order to validate this positive correlation, we design our experiments in two steps. First step, we want to design a phantom to test this hypothesis, which consists of known different Na+ concentration samples, and measure the image density of the different samples. If it validates our hypothesis, we thus move to the step two, in which we use cartilage tissue 7

17 samples with different PG concentrations to test whether the sodium MRI image intensity still has a strong positive correlation with the PG concentrations. If two steps are both successful, it proves the practicability of using sodium MRI to monitor the changes of PG in cartilage tissues, and which is promising in early diagnosis of cartilage losing related diseases Sample Preparation We prepared two steps to test and validate the relationship between sodium image intensity and the PG concentration. In step 1, we use phantoms with different Na+ concentration to test and compare the intensity. In step 2, we compare two cultured chondrocyte pellets samples to test the relationship between image intensity and PG concentrations Step I: Phantoms I made phantoms with 150 mm, 300 mm and 500 mm Sodium concentration prepared in 1% agarose gel. Agarose gel is a commonly used medium in cell culturing, and here used to mimic the extra cellular matrix (ECM) in the cartilage tissue. To efficient test the three different samples at one time, I prepared them in one phantom, which is a 5 mm tube consisting 150 mm sample with two different sized capillaries filled with 300 mm sample and 500 mm, shown in Figure 4. By this way, we can see the clearly intensity differences of the three samples 8

18 at a glance of the image. We also expect linear relation be proved according to analysis of the intensities of different circles. Figure 4. Phantoms with sodium concentration of 150 mm, 300 mm and 500 mm, respectively Step II: Bovine Chondrocyte Pellets We chose to use bovine chondrocyte pellets, because bovine chondrocyte are responsible for production of proteoglycan and collagen in bone cartilages. The longer cultured the chondrocyte pellets, the more they expected to product proteoglycans. Our bovine chondrocyte pellets were formed by centrifuging chondrocytes in tissue culture medium at 1000 g for 10 min, shown in Figure 5. One of the sample we used bovine is chondrocyte pellets cultured for 1 week and the other one cultured for 4 weeks. The latter one expected to have more proteoglycans, thus, should have higher image intensity. 9

19 Figure 5. The process of culturing chondrocyte pellets As we want to get the exact concentration out of image intensity, we added a reference to each sample. For week 1 pellets, we put the pellets in phosphate buffered saline (PBS), which is a standard phosphate buffer used in many biochemical procedures. We know the Na+ concentration of PBS is 154 mm. For week 4 pellets, we use a capillary filled with 300 mm sodium chloride phantom as reference Pellets with Phosphate Buffer Saline We chose phosphate buffer saline (PBS) to surround the pellets not only because we want to use it as a reference to quantitatively calculate the sodium concentration in week 1 chondrocyte pellets, but also good for increasing the total sodium particles in this sample to increase the sodium MRI signal. As the 23 Na physiological concentration is much lower than that of 1 H, sodium MRI images are normally much more blurry than hydrogen MRI images. Thus increasing the 10

20 sodium particles in total will be crucial for increasing the signal-to-noise ratio Pellets with Fluorinert Oil Fluorinert oil is an electrically insulating, stable fluorocarbonbased fluid, which is used in various cooling applications. Because it contains no sodium particles, we put a capillary filled with 300 mm sodium chloride as a reference. We expect to see the circle of capillary and the pellets in one sodium MRI image, and then we can calculate the sodium concentration of pellets based on the intensity of the capillary circle. There is a possible concern about Fluorinert oil. Because of it contains no sodium ions, the total sodium particles may be too few to get a good signal Magnetic Resonance Imaging Acquisition All sodium MRI experiments were performed at room temperature on a 11.7 T Bruker Avance spectrometer (23Na freq = MHz), shown in Figure 6. Figure T Bruker Avance spectrometer 11

21 5 mm proton-sodium double tuned RF coil, shown in Figure 7. The standard Bruker GEFC protocol with 2ms/100ms (TE/TR) was used to acquire sodium images. Figure 7. 5 mm proton-sodium double tuned RF coil 12

22 4. Results and Analysis Every sodium image is complied with a proton MRI image, in order to make sure we selected the right slice of the sample. What we did is, taking several proton MRI slices and selected the best one, which contains the whole subjects we want to observe. Then we use that slice as the geometry to scan for the sodium image. This is especially useful when sodium concentration is low and does not have a good resolution. After we get both proton MRI image and sodium MRI image with the same slice, we are able to compare and to analyze Step I: Results of Phantoms Proton and Sodium MR Images In proton image, we can clearly see three different sized circles. In the same slice, we got the sodium image, which shows that the blue circle (500 mm NaCl) is brighter than the red circle (300 mm NaCl), and the rest (150 mm NaCl) is the darkest, shown in Figure 8. At a glance, we can tell that higher the sodium concentration, higher the image intensity. Figure 8. Proton and Sodium MR images of phantoms 13

23 Validation of the Hypothesis Furthermore, in order to have the quantitative relation between the sodium concentration and the intensity, we selected 3 areas (yellow dashed circles in Figure 8. Sodium Image) in each phantom circle and calculate the average intensity of each area, which is represented by a point in the fitting curve below, which shows that sodium MRI intensity has a linearly correlation with sodium concentration indeed. This proves that it is reasonable to use sodium MRI image intensity as a parameter to describe the sodium concentration. Figure 9. The correlation between sodium image intensity and sodium concentration 4.2. Step II: Results of Pellets Week 1: Proton and Sodium MR Images 14

24 The week 1 sample contains two pellets, which are cultured in medium for one week, and are surrounded by PBS. We scanned the sample with TriPilot and Rapid Acquisition with Relaxation Enhancement (RARE) MRI protocols. They both are commonly used proton MRI protocols. TriPilot is typical rapid protocol based on Fast Low Angle Shot Magnetic Resonance Imaging (FLASH) to get 3 orthogonal reference images, the axial, coronal and sagittal views of the sample. It is helpful to select the best slice according to three-dimensional views. After we decided the target slice, which contains the pellets, we scan this particular slice with RARE protocol. After comparing the results generated from FLASH and RARE, the latter seemed to give the better resolution in this case. So we also chose RARE to acquire proton MR images in the week 4 case. Below is the proton MR image captured by the RARE protocol, which clearly shows two pellets. Figure 10. Proton MR image for week 1 sample acquired with RARE protocol 15

25 Based on this same geometry, we scanned with Gradient Echo Flow Compensated (GEFC) protocol for sodium images. In result, we can see boundaries of 5 mm MRI tube, but no significant signs of pellets. As we actually take this sodium image based on the same geometry of previous proton MR image, the sodium image should have the pellets shown in the exact same position. So we drew the boundaries of the pellets area in the sodium image, and calculate the average intensity based on that. The parameters we used in GEFC protocol is: 2D imaging, TE/TR=2 ms/100 ms, average of numbers=300, slice thickness=2 mm. Figure 11. Sodium MR image for week 1 sample acquired by GEFC protocol Week 4: Proton Sodium MR Images 16

26 Week 4 sample contains two pellets cultured for 4 weeks. Compared to week 1 sample, week 4 sample is supposed to product more proteoglycans than that of week 1 sample. Thus, week 4 sample should have a higher sodium concentration. In order to calculate the sodium concentration in pellets, we put a reference capillary filled with 300 mm NaCl. The same with previous week 1 experiment; we used TriPilot protocol to locate the best slice containing both the view of pellets and the reference capillary circle. Then we scan this particular slice with same RARE protocol with same parameters to get the ideal proton MR image, shown below. The left corner is the reference capillary, and the right bright area is the pellets. Figure 12. Proton MR image for week 4 sample acquired with RARE protocol 17

27 Then we move on to sodium image. We applied the GEFC protocol with same parameters to get week 4 sodium image, which is shown in Figure 13. From this image, we can hardly see the boundaries as we see in the week 1 sodium image, that is because in this case week 4 pellets are surrounding with Fluorinert Oil, which has no sodium at all. This decreases the sodium MRI signal-to-noise ratio. However, we still can see a little bright area in the left corner, which is the sign of reference capillary. In the same way, we drew the pellets area according to the proton image, and then calculate the sodium concentration based on the image intensity and the reference concentration. Figure 13. Sodium MR Image for week 4 sample acquired with GEFC protocol 18

28 Validation of Hypothesis In order to test the hypothesis that week 4 pellets should have more proteoglycans, thus have a higher sodium concentration, we calculated the target pellets areas in sodium MR images based on the references. In week 1 sample, the reference is the surrounding PBS, which has a 154 mm sodium concentration. In week 4 sample, the reference is the capillary, which has a 300 mm sodium concentration. The way we did it is, first get intensity values of the selected areas by using software ImageJ (Figure 14). Then convert intensity values to sodium concentration values based on references. Figure 14. The operating interface of ImageJ 19

29 In result, we do get a higher sodium concentration in week 4 pellets comparing to week 1, which is consistent with our hypothesis. Table II. Sodium concentration of week 1 and week 4 sample * Concentration (Pellets) = Intensity (pellets)/intenity (reference) Concentration (reference) 20

30 5. Conclusion and Discussion Our results of phantoms show that the sodium concentration changes correlate with sodium image intensity, which suggests a potential use of sodium MRI for PG quantification for tissue engineered cartilage. Results of bovine cartilage chondrocyte pellets also show there is a correlation between sodium concentration and imaging intensity. We plan to apply the sodium imaging technique to quantify proteoglycans in different cartilage tissue engineering constructs. Our result show that the sodium concentration is higher (~189 mm) in chodrogenic scaffolds as compared to osteogenic scaffolds (~151 mm).we also found that the sodium image intensity in chondrogenic ECM scaffolds is higher than osteogenic scaffolds, which is consistent with biochemical analyses of these samples showing higher amount of proteoglycans in chondrogenic samples (Table III)[10]. Table III. Comparison of sodium concentration in week 1, week 4 pellets, chondrogenic and osteogenic scaffolds All of these results are consistent with our hypothesis, and all prove that sodium MRI is a promising technique to apply to PG quantification in lots of proteoglycan-related cartilage diseases. 21

31 5.1. Concerns of Experimental Design However, we do have concerns about about our chondrocyte pellets results. Due to limited available bovine cartilage chondrocyte pellets supply, we only tested on two samples, which contain two pellets in each. All data came from the total four pellets, and if one pellet is significant different from others, it will shift the result far from correct. For instance, in the proton image of week 4 sample, we see that the two pellets have a different intensity, which may suggest the two pellets contain significant different amount of water molecules (Figure 15). Figure 15. Two pellets with red circle and blue circle respectively have significant different signal intensity, indicating water quantities are different 22

32 Such things happen in the cell culturing process. We can never be sure that things like this excluded from our experiments, but it can be controlled by increasing the number of pellets in each sample to test. Furthermore, we also consider monitoring PG changes in a longer time. In our research, we compared the week 1 sample and the week 4 sample. Although it gives out a convincing result, it is better to test on more samples, cultured in week 1, week 2,, week 5 etc Discussions Except to increase the number of samples to control uncertainties, it is also essential to improve the quality of sodium MR images How to Develop Image Contrast? In our experiments, we see boundaries of reference PBS in week 1 sodium MR image, and we only see reference capillary in week 4 sodium MR image. Both of them show no sign of pellets, but only the reference. This may due to two reasons. One is the overall sodium concentration is too low to have a good sodium signal (week 4 sample with Fluorinert Oil), so there are much noise. The other possible reason is the sodium concentration in chondrocyte pellets are actually very close to the sodium concentration with surrounding references (154 mm PBS). 23

33 As we know, Fluorinert Oil has a 0 mm Na+ concentration, comparing to PBS having a 154 mm Na+ concentration. If Fluorinert Oil is proved to make the signal too weak, and the PBS in the opposite has a close sodium concentration to pellets, we should consider using a reference with a sodium concentration between 0 mm and 154 mm Improve Magnetic Resonance Imaging Protocol In our experiment, we chose GEFC protocol for sodium MR imaging. The TE and TR we used are 2 ms and 100 ms. We selected parameters by trying. As GEFC is a T 2 -weighted sequence, the TE should be around T 2, and TR should be around T 1. Guided by T 1 and T 2 to adjust the protocol parameters may increase the contrast of image. It is also possible that we should test other sequences to see whether there is more suitable protocols to increase the image contrast Future work After we can successfully see pellets in the sodium images, and calculated data based on different intensities validates the hypothesis, it is promising to move to the next step, clinical trials. It is helpful to test two groups of volunteers for quantity of PG in cartilage, one group is healthy subjects, and the other is patients. 24

34 It is the ultimate goal of this research that sodium MRI can be used as a PG quantification method in cartilage loss diseases, like Osteoarthritis (OA). Thus, the quantity of cartilage can be monitored, and further be used as early-stage diagnose of OA, which may possible prevent the disease from developing. 25

35 CITED LITERATURE 1. Osteoarthritis, NIH Fact Sheets, 2. Buckwalter, J.A. and Mankin, H.J.: Articular cartilage: degeneration and osteoarthritis, repair, regeneration, and transplantation. Instr Course Lect. 47: , Simon Meadows.: Osteoarthritis Treatment and Genetics Cockman M.D., and Blanton, C.A.: Quantitative imaging of proteoglycan in cartilage using a gadolinium probe and microct. Osteoarthritis Cartilage. 14(3):210-4, Andrew D. P. and Russell F. W.: Basic Science of Articular Cartilage and Osteoarthritis. Clin Sports Med. 24: 1 12, Larry W. M.: Intra-articular hyaluronan (hyaluronic acid) and hylans for the treatment of osteoarthritis: mechanisms of action. Arthritis Res Ther. 5:54-67, The Organization of Cells in Tissue- The Extracellular Matrix, Cell Junctions and Cell Adhesion,, Penn State WikiSpaces, 2009, 8. Shapiro E.M. and Borthakur A.: Sodium visibility and quantitation in intact bovine articular cartilage using high field (23)Na MRI and MRS. J Magn Reson. 142(1):24-31,

36 CITED LITERATURE (continued) 9. Kotecha, M., Ziying Y and Magin, R.L.: Monitoring Tissue Engineering and Regeneration by Magnetic Resonance Imaging and Spectroscopy. J Tissue Sci Eng. S11: 007, Yu, D., Ziying Y and Kotecha, M.: Proteoglycans Quantification in Tissue Engineered Cartilage using Sodium MRI at 11.7 T. MBECC

37 VITA NAME: Dan Yu EDUCATION: - B.S., Applied Physics, Tianjin University, M.S., Bioengineering, University of Illinois at Chicago, 2013 EXPERIENCE: - MRI Lab, University of Illinois at Chicago, Center of Bioinformatics Tianjin University (TUBIC), Laboratory of Condensed Matter Physics, Tianjin University, HONORS: - Teaching Assistantship, Research Assistantship, PUBLICATIONS: - Application of Nuclear Magnetic Resonance in Porous Media, UBSJ, Proteoglycans Quantification in Tissue Engineered Cartilage Using Sodium MRI at 11.7 T, MBECC,