Three-Dimensional Modeling of the Osteocyte Network by Chao Huang

Advisor: Ted S. Gross, Ph.D. University of Washington Department of Bioengineering

Senior Capstone Project 6 June 2008

Table of Contents
ABSTRACT ................................................................................................................................ 2 INTRODUCTION...................................................................................................................... 3 DEFINITION OF PROJECT ............................................................................................................... 3 MEDICAL AND SCIENTIFIC SIGNIFICANCE ..................................................................................... 4 SOCIAL, ETHICAL, AND ECONOMIC ISSUES.................................................................................... 5 TECHNICAL BACKGROUND ........................................................................................................... 7 Theory ..................................................................................................................................... 7 Review of literature................................................................................................................. 9 Previous relevant work in advisors laboratory ................................................................... 14 Outstanding technical issues at outset of project ................................................................. 17 INITIAL DESIGN OF EXPERIMENTS, TOOLS, AND DEVICES........................ 19 MATERIALS AND METHODS ........................................................................................................ 19 COSTS ........................................................................................................................................ 26 INITIAL RESEARCH PLAN ............................................................................................................ 27 RESULTS .................................................................................................................................. 29 FINAL TIMELINE ......................................................................................................................... 29 DATA ......................................................................................................................................... 30 EXPERIMENTAL/DESIGN DECISIONS ............................................................................................ 38 ANALYSIS AND CONCLUSIONS .................................................................................................... 39 SUGGESTIONS FOR FUTURE WORK .............................................................................................. 43 ACKNOWLEDGEMENTS.................................................................................................... 45 REFERENCES ......................................................................................................................... 46

Abstract
Mechanical loading stimulates enhanced bone formation, and intercellular communication through the gap junctions that join osteocytes (the mechanotransducers in bone) may play an important role in bones response to external stimuli. Osteocytes are believed to be responsible for maintaining the bone matrix by transmitting chemical signals induced by exogenous forces. Assuming that such signals can travel in both transverse and longitudinal directions within bone, I hypothesized that the three-dimensional (3-D) organization of the osteocyte network must be heterogeneous in space in order to steer these signals in specific directions. To investigate this theory, a MATLAB program that uses serial cross-sectional light microscope images of hematoxylin and eosin-stained murine tibia cortical bone was designed to quantify the spatial locations of the osteocytes and produce a 3-D representation of the osteocyte network morphology. Each cross-sectional plane is oriented such that the positive x-direction runs lateralmedial and the positive y-direction runs anterior-posterior; the positive z-direction runs distalproximal. The program automatically detects the xy-coordinates of cells in each image, and then compares the coordinates in successive images to identify matching cells present in multiple layers. By determining which cells are identical and which are unique in each layer, the zcoordinates of the cells can be extrapolated, thus allowing the 3-D osteocyte network model to be constructed. This model has shown that on average, osteocytes are closer to cells in a different serial layer than the layer it currently resides in. Because osteocytes that are closer together are more likely to be connected and transmit signals, my preliminary results suggest that the longitudinal component of the 3-D osteocyte network may play a vital role in determining the direction of signaling pathways.

Introduction
Definition of project
There is a need to develop a simple and fast method for identifying the three-dimensional (3-D) locations of osteocytes within a piece of bone. I have designed a MATLAB program that uses serial transverse images to automatically construct a 3-D model of the osteocyte network in murine cortical bone. The program takes the images as inputs and automatically detects the osteocyte locations in each cross-section through filtering means. A graphical user interface is provided to allow the user to manually correct any mistakes that the program has made in detecting cell locations. The user enters the current layer number for each cross-section in the ordered series of images, and saves this information along with the xy-coordinates as separate MATLAB files that can be loaded and accessed later. Once the necessary coordinate information has been collected for each image, the user may then choose the desired files to be used to construct the network model (the files must be contiguous cross-sections and in order). The model is a 3-D scatterplot that shows the spatial locations of the osteocytes in the user-selected layers and may be rotated in any direction to provide many different views. Upon saving the model, the user may select a number of parameters to be calculated, including the number of cells in each layer, the number cells in predefined sectors of each cross-section (to determine uniformity), and the minimum distance between neighboring cells for both planar and z-directions. Being able to perform morphological analysis by quantifying the 3-D organization of osteocytes within the section of bone is an important step for tracing the direction of their signaling pathways.

Medical and scientific significance

In humans, cortical (outer layer) and trabecular (spongy bone within the joint ends) bone begin to degrade after peak bone mass is reached at approximately 30 years of age [6]. This degradation is the result of elevated bone resorption (breakdown of bone and release of minerals) and decreased bone formation [3]. By age 40, bone loss is estimated to be 0.3% to 0.5% per year, and in women, menopause may elevate annual bone loss to up to 3% [6]. Based on these estimates, an average 70-year-old woman may experience a decrease of 25% to up to 40% in bone mass from her peak bone mass while a like-aged man may expect a decline of 10% to 15% [7]. In addition, diseases such as osteoporosis may further reduce the bone mineral density, therefore increasing the risk of bone fracture and posing a significant health concern for senior citizens [8]. Given these concerns, it is essential to develop novel techniques for sustaining or augmenting bone mass throughout an individuals lifetime. One of the signals for bone cells to form new bones is mechanical stress, such as that experienced during exercise [9]. Previous studies have shown that bone mass increases with exercise, and so it is apparent that mechanical components of bones functional environment have an effect on bone mass and morphology [10]. By studying how loads are anabolic to bone and identifying specific signals responsible for inducing new bone formation, nonpharmacologic methods using mechanical loading may be developed to inhibit osteoporosis and promote osteogenesis [11]. A model of the osteocyte network in bone must be constructed in order to visualize where bone cells are in relation to each other and understand how their organization dictates the transmission of mechanical signals to induce bone formation. As will be highlighted in the proceeding literature review, previous methods for analyzing signaling pathways in osteocyte

networks have either focused on osteocytes in a two-dimensional plane or used complicated and expensive techniques and equipment to construct a three-dimensional (3-D) model. Thus, there is a need to design a simple, easy, and fast computer program that will find the 3-D locations of osteocytes fairly accurately and perform basic morphological analysis in a short amount of time. Although there are already 3-D reconstruction programs available (e.g. ImageJ), such programs do not offer the same capabilities of a program designed specifically for analysis of a network of cells within bone. For example, traditional imaging methods would require the user to manually locate where cells are, which is both laborious and time-consuming. The program that I have designed automatically finds the locations of cells, constructs the 3-D model, and then performs calculations related to the organization of the network of cells. Gathering such data will yield substantial insight into how osteocytes are organized in bone and how the architecture of these networks might play a role in the complex process of bone formation in response to mechanical loading. Constructing an accurate model of the osteocyte network is therefore essential for tracing the mechanotransduction signaling pathways that induce bone growth.

Social, ethical, and economic issues

Bone formation induced by mechanical stress is studied with the ultimate goal of adapting and using mechanical loading in a clinical setting. If the ways in which bone perceives and responds to mechanical stimuli are well understood, then such information may be used to design novel exercise regimens that provoke a similar response in bone as mechanical loading. Exercise is a socially accepted activity in all cultures of the world that is widely considered to promote good health. Therefore, it is not expected that introducing a new exercise regimen specially designed to induce bone growth will engender substantial fear or mistrust among the public. While the public may be much more hesitant to use a new device or undergo a surgical procedure, a novel 5

exercise regimen should not be a difficult medical technique to encourage the public to try. Previous studies have shown that athletes who play racquet sports have enhanced bone mass in their serving arms [12], and these visible and tangible results should encourage subjects to choose exercise over other pharmacological means of inducing bone formation. Because exercise regimens present a non-pharmacological intervention for bone loss, it is also expected that such treatments will not be as difficult to gain FDA approval for as pharmaceutical treatments such as estrogens and bisphophonates. In addition, when the biochemistry and mechanics of bone formation are better understood, exercise regimens will be specially designed for the elderly to induce maximal bone formation from minimal strain magnitudes, lessening any ethical concern for causing injury during clinical trials. Furthermore, it may be more beneficial socially and ethically for the public to adopt a non-pharmacological technique for enhancing bone formation since it is a tangible activity that can be controlled by the individual, unlike reading directions and blindly taking medication without knowing what is happening inside ones body. A final advantage of mechanically induced bone formation over pharmacological treatments is that, beyond experimental and clinical testing, there is little to no cost involved. In 1995, the national healthcare cost of osteoporosis was estimated to be $13.8 billion and may increase to as much as $240 billion over the next 50 years [13]. Most of these costs can be attributed to either medication, which may be avoided if the exercise alternative is chosen, or surgery, which may also be avoided if individuals begin their exercise regimens earlier in life. With the potential social, ethical, and economic benefits of a possible low-magnitude exercise regimen, mechanical loading holds the potential to be an effective and safe technique for inducing new bone formation to counter the effects of aging.

Technical background
Theory
The primary mechanical function of bone is to provide support for muscles to act against and hold the body in an upright position [14]. Bone is constantly subjected to a dynamic loading environment through a persons daily movements, and therefore must adapt responsively to maintain its structure and withstand physical activity [15]. The basis for bones adaptation to exogenous forces is through bone remodeling, which consists of bone resorption and new bone formation. Bone resorption is the breaking down of bone into its minerals (such as calcium) by osteoclasts, resulting in the transfer of such minerals and proteins to a different location via blood [16]. New bone is then formed by osteoblasts that synthesize osteoid, an unmineralized bone matrix composed of organic components. Some osteoblasts will become trapped within the matrix that they lay down and differentiate into osteocytes, the most abundant cells in bone (each spanning approximately 10 m) [17]. In addition to being responsible for maintaining the bone matrix, osteocytes are believed to be the mechanosensors and mechanotransducers in bone [18]. Bone adaptation requires cellular mechanotransduction, and osteocytes are affected by a variety of mechanical factors generated by loading. These factors include strain generated across the cells substrate, pressure within intramedullary cavities, and shear forces through the canaliculi that connect the cells [11]. The conversion of these forces into a cellular response consists of four phases: mechanocoupling, biochemical coupling, transmission of signaling, and effector cell response. During mechanocoupling, the applied mechanical forces are transduced into local mechanical signals perceived by osteocytes [14]. Such signals may be sensed by a number of mechanoreceptors including mechanosensitive channels that induce membrane hyper or depolarization, integrin proteins that span the membrane to couple the cell to its extracellular environment, connexins that form regulated channels allowing direct exchange of small 7

molecules between adjacent cells, and membrane structure proteins that facilitate transmembrane communication as well as provide docking positions for signaling complexes [11]. After these local mechanical signals are sensed, biochemical coupling occurs, when the mechanical signals are transduced into biochemical signals that lead to gene expression or protein activation [14]. Biochemical signaling may occur through G-proteins, calcium transients, MAPK activation, and release of nitric oxide [11]. Cell-to-cell transmission of biochemical signals occurs intracellularly through the processes that join osteocytes as well as through the extracellular fluid in which osteocytes are immersed [19]. Gap junctions at the tip of the cell processes that contain hemichannels provide a direct and efficient mechanism for intracellular signaling pathways [20], although osteocytes also remain in contact via their common environment in the contiguous bone fluid space. Such fluid acts as a coupling medium through which mechanochemical signals may still be transmitted by hydraulic conductivity, pressure and osmotic gradients, as well as electromechanical and acoustic energy effects [15]. The combination of the intracellular and extracellular transmission of signals induces an effector cell response at the tissue level, at which point bone remodeling commences [14]. The degree of bone adaptation in response to external loading depends on strain magnitude, distribution, duration, frequency, bone history, and type of stress created (compression, tension, or shear). Over the last century, numerous experiments have been conducted to test each of these factors effect on bone adaptation and many common threads have emerged [21]. The following literature review highlights some of these findings and ends with a summary of the research on bone adaptation being carried out in my current advisors laboratory.

Review of literature
The notion that the form and function of bone is produced and maintained by mechanical forces was first popularized by anatomist Julius Wolff in his 1892 treatise, The Law of Bone Remodeling [22]. In what has now become known as Wolffs Law, Wolff states that Every change in the form and function of bone [] is followed by certain definite changes in their internal architecture, and equally definite alteration in their external conformation, in accordance with mathematical laws [23]. Through demonstrating a definite link between trabecular bone architecture and the functional stresses placed upon it, Wolff was able to assert that the stresses surrounding bone forces the architecture inside living bone to continuously adapt through remodeling. Later, embryologist Wilhelm Roux generalized the notion of functional adaptation, suggesting that mechanical stimuli govern effector cells that regulate bones formation and adaptation locally in a self-organizational process [22]. In the early 20th Century, biologist Darcy Thompson proposed that the condition of strain, caused by the stress induced by mechanical forces, is a direct stimulus for bone growth and thus is the source of functional adaptation. In the 1960s, orthopedist Harold Frost asserted that not only was mechanical strain the primary determinant of bone adaptation, but that a minimum strain threshold must be reached before bone adaptation occurs [21]. The combination of all these findings provided a foundation for other investigators to study the relationship between bones mechanical environment and its effect on bones development of mass and architecture. Early investigations that have used approaches such as exercise, disuse, and stress protection employed loading regimens superimposed on existing effects of normal load-bearing, and thus an accurate, discernable remodeling response to the newly applied mechanical situation could not be extracted [1]. Such difficulties led to the design of novel experimental setups that could elucidate bones functional adaptation to mechanical forces. A new technique in which 9

bones were externally loaded in vivo over a period of weeks was first used by Hert et al. in early 1970s studies with rabbits. Hert found that dynamic strains increased the amount of bone formation in the rabbits while static strains did not, and therefore suggested that dynamic strains are primarily Figure 1: Schematic of template used for responsible for bone adaptation [21]. In 1984,
parallel osteotomies and holding the ulna in place as loads were administered [1].

Rubin and Lanyon refined Herts loading approach and confirmed his findings by functionally isolating the external ulnae in turkeys and applying known intermittent loads over a period of six weeks. As Figure 1 illustrates, a template was clamped and pinned to the ulna, allowing two parallel transverse osteotomies to be performed and leaving the entire midshaft of the ulna undisturbed. The ends of the pins were connected to a loading apparatus that imposed a 0.5-Hz intermittent compressive load cycle to engender strains in the bone. The number of consecutive load cycles that was applied each day was varied to determine the bones response to different load magnitudes. The strains were measured at the surface of the bone using strain gauges and could be directly correlated to the remodeling induced by the loading. Photon-absorption densitometry and postmortem histological methods were used to comparatively assess bone mineral content and determine the course of remodeling in response to the strain stimulus [1]. In comparisons of unloaded, control bone and dynamically loaded bone with a variable number of consecutive load cycles, it was found that bone mass decreased in ulnae that were not subjected to any external loading, which was the predicted response to disuse. As Figure 2 shows, ulnae subjected to four loading cycles a day showed a slight increase in bone mineral content, and as the number of consecutive cycles was increased to 36 cycles of an identical strain regimen, substantial subperiosteal and endosteal new-bone formation was measured [1]. 10

Interestingly, ulnae subjected to 360 and 1800 cycles of loading did not have significantly higher bone mineral content that those subjected 36 cycles, demonstrating that an increased duration of loading does not yield a proportional increase in bone mass, but rather causes the bone formation response to saturate [21]. A likely

explanation for this behavior is that bone cells are triggered to form new bone by a strain-specific stimulus instead of responding
Figure 2: Percentage change in bone mineral content to a non-specific (reparative) effect, and a produced by a variable number of loading cycles per day [1].

small exposure to this stimulus appears to be

enough to induce engender new bone formation. This result was important, especially clinically, as it suggested that the adaptive response of bone can be engendered by short, infrequent loads rather than requiring a long period of repetitive activity [1]. In a later study, Rubin and Lanyon modified their experiment to apply 100 consecutive 1Hz load cycles daily over eight weeks to engender variable strain magnitudes within the ulna bone. By comparing the cross-sectional areas of the loaded and unloaded ulna midshafts, it was found that newly formed bone area was proportional to the applied strain magnitude, as illustrated by Figure 3. These results suggested that the main function of bones adaptive remodeling is to produce a mass and orientation of bone tissue that is optimal for withstanding the strain that the bone is subjected to. Mechanical strain therefore appears to be a natural stimulus that induces an increase in bone mass not normally countered by bone resorption 11

activity [2]. In 1994, Rubin and McLeod then studied the effect that strain frequency has on bone adaptation by employing three different loading protocols over eight weeks: (1) no loading (disuse), (2) disuse followed by 100 seconds of 1-Hz low-magnitude mechanical stimulus daily, and (3) disuse followed by 100 seconds of 20-Hz mechanical stimulus at a similar strain magnitude. It was found that Figure 3: Change in cross-sectional area of the ulna
midshaft from comparison between loaded and unloaded

disuse alone caused a mean 8.3% loss of bone bone [2]. as there was no longer a stimulus to induce bone formation to negate resorption activity. The daily 1-Hz low-amplitude mechanical strain stimulation, however, caused a mean 28% increase in area, and importantly, the 20-Hz protocol caused bone growth to increase to 69%. These results suggested that not only can a brief and low-magnitude stimulus induce bone formation, but also that bone adaptation and formation are sensitive to loading frequency [1]. The dynamic loading experiments conducted by Rubin et al. to elucidate the effects that strain rate, frequency, and magnitude have on inducing new bone formation paved the way for others to study the physiology and biochemistry behind bones adaptive response to mechanical stimuli. The most important aspect of studying bones adaptive response is to understand how osteocytes communicate with each other to induce new bone formation, and the most fundamental need for achieving this goal is to image where osteocytes are in relation each other. However, osteocytes are encased in a hard, mineralized matrix in the inner cavity of bone, thus making the task of imaging them and describing cell-to-cell signaling pathways in threedimensions (3-D) extremely difficult [24]. Therefore, previous methods of imaging have only 12

been capable of showing the osteocyte distribution in a two-dimensional (2-D) plane. For example, Krempien et al. revealed the non-viable cell network in 2-D by staining lacunae (small spaces in the bone that contain one osteocyte each) with fuchsin stain [25]. Such a network model is not sufficient for analyzing the direction of signaling pathways as the longitudinal component along the length of the bone is not included. In efforts to move towards 3-D imaging, Palumbo et al. were able to gather morphological data of the 3-D structure of osteocytes by cutting transverse sections of bone serially in thin slices and visualizing with a transmission electron microscope [26]. Although such methods accurately depict the locations of osteocytes, they do not offer the means to perform morphometric analysis on osteocyte processes, the primary means of intercellular communication in bone. Recently, Sugawara et al. conducted extensive analyses of the osteocyte network morphology by using 3-D reconstructed fluorescent images [4]. As shown in Figure 4,
Figure 4: 3-D reconstruction of osteocyte network shown from periosteal side of bone. Top: binds to the actin filaments present in osteocyte processes [27]. arrows show osteocytes, bar = 20 m. Bottom: zoomed-view, bar = The 3-D osteocyte structure was imaged on a confocal laser 10 m [4].

the osteocytes were stained with Texas-Red phalloidin, which

scanner microscope (CLSM), which allowed for the construction of a model to calculate the length of processes, volume, and surface area of osteocytes. This was the first time that 3-D morphology and morphometry analysis was performed on bone [4]. However, given the amount of time needed to prepare the bone sections for staining and imaging with an expensive and complex piece of machinery such as the CLSM, in addition to the

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laborious task of manually collecting morphological data, it is evident that there is a need to develop a simpler and faster way of analyzing the 3-D osteocyte network.

Previous relevant work in advisors laboratory

The laboratory group that I have joined, the Orthopaedics Science Laboratories (OSL)

directed by Professor Ted S. Gross, examines how bone responds to physical stimuli. Many of the aforementioned methods for loading and Figure 5: Schematic of the noninvasive murine loading
device. The mouses right tibia is secured at the

imaging have been adapted for investigation and metaphysis by a gripping cup attached to the adjustable
medial support. A computer-controlled linear force modeling of real-time cell-to-cell signaling in actuator applies small forces to the distal tibial metaphysic [3].

response to mechanical loading. To study the effects of loading, a novel, Gross et al. designed a noninvasive device that allows for controlled external loads of various waveforms to be applied to murine tibia (Figure 5). In previous experiments, the tibia of the mouse was positioned against the fixed lateral support, and the shielded linear electromagnetic actuator applied a load waveform via a digital analog interface that enables programmable waveforms. After the mice were euthanized using carbon dioxide, cross-sections of the loaded bone (approximately 30 m thick) were used to examine how the tibias cross-sectional area had changed. Results have shown that the loading elicits a significant augmentation of the bones midshaft area. Figure 6 shows a
Figure 6: Cross-sections of an unloaded tibia (left) and a 33.7% increase in cortical width for a loaded right tibia mechanically loaded tibia (right). The arrow indicates a 33.7% compared to the unloaded left tibia from the same mouse. In increase in cortical width of the loaded tibia [3].

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addition, Gross et al. confirmed Rubin and Lanyons observation that dynamic loading induced substantial new bone formation whereas static loading produced an absence of response. It was concluded that the mechanical loading device is capable of noninvasively inducing an anabolic response to mechanical load consistent with aforementioned models of bone adaptation [3]. More recent investigations of the OSL have focused on using low-magnitude mechanical loading with rest intervals inserted between each load cycle. As was explained previously, it is hypothesized that the fluid flow near osteocytes underlies mechano-

transduction in bone and that this flow past the cell bodies and processes is highly viscuous. If bone is repetitively loaded, then osteocyte level fluid flow may not have enough time to recover from inertial damping waveform and (B) rest-inserted waveform
with identical magnitude and total cycle Figure 7: First 12 s of (A) standard loading

effects between each load cycle, and therefore the number, differing only by the 10-s pause effects of loading-induced bone formation may be
inserted between each loading cycle of the rest inserted waveform.[5]

reduced after the first few cycles of repetitive loading. It was hypothesized that a non-loaded 10second rest interval inserted between each loading cycle (Figure 7) would allow sufficient recovery from inertial damping effects and elevate fluid flow near osteocytes, thus increasing the osteogenic potential of a low-magnitude mechanical loading regimen. To verify this hypothesis, three loading regimens were tested: (1) a low-magnitude 1-Hz waveform, (2) a high-magnitude 1-Hz waveform (twice as large as the low-magnitude waveform), and (3) a low-magnitude 1-Hz waveform with a 10-second interval of rest inserted between each load cycle. Results showed that while low-magnitude loading induced minimal bone-forming activity, the bone formation rate was significantly enhanced when a 10-second rest interval was inserted between each load 15

cycle, transforming the ineffective low-magnitude regimen into a potent anabolic stimulus. As Figure 8 illustrates, the rest-inserted loading significantly enhanced bone formation despite a 10fold reduction in the number of load cycles daily, and was also statistically comparable to the induced bone formation of rate of the high-magnitude loading regimen that was 10-fold greater in cycle number and 2-fold greater in load magnitude and rate. These results suggest great promise for rest-inserted loading
Figure 8: Periosteal bone formation rates for control (intact) and experimental bone induced to be used as a potential strategy to build bone mass by low-, high-, and rest-inserted mechanical loading [5].

via exercise [5].

To better understand the mechanotransduction that occurs during induced bone formation, the OSL has developed a computer simulation of real-time cellular signaling induced by mechanical stimuli using agent based modeling (ABM). ABM is a technique used to explore the wide range of consequences of introducing a stimulus to the agent or local level. Through a set of user-specified rules, ABM provides a method for multi-scale modeling of complex global behaviors. The OSLs current model is governed by three cellular functions: (1) threshold levels of bone tissue strain (the change in length of the bone due to a bending stimulus) required to activate osteocytes, (2) propagated activity to neighboring cells for strains above threshold, (3) modulated activity dependent on rates at which molecular stores are replenishedcaused by insertion of unloaded rest between load cycles. The model has confirmed that rest-inserted stimuli can induce enhanced and sustain signaling within the osteocyte network by enabling molecular stores to be replenished before the cells are activated again [28]. The OSL is now focused on fine-tuning and adding more depth to the ABMs capabilities. So far, the model has assumed that osteocytes are spatially arranged in an idealized 16

network within cortical bone [28]. Thus, the model would be greatly improved if a more accurate description of where exactly osteocytes are located within a volume of bone was provided. My project makes it possible for the 3-D locations of osteocytes to be found automatically and quickly, therefore introducing the opportunity of incorporating this 3-D network of cells into the ABM simulations.

Outstanding technical issues at outset of project

In my original capstone proposal, I aimed to not only find the 3-D locations of the osteocyte bodies, but also show the interconnectivity of their long, slender processes. I proposed to use a confocal laser scanning microscope to image bone sections stained with the fluorescent phallotoxin phalloidin that binds at the interface between F-actin subunits in the osteocyte processes [29], much like Sugawara et al.s method described previously [4]. However, when using the Differential Interference Contrast mode on the confocal laser scanning microscope (CLSM), I found the fluorescent stain was not concentrated in the osteocyte cell bodies, but rather was spread out throughout all regions of the imaging plane. I concluded that this result must suggest that the phalloidin was not binding specifically to the actin in osteocytes, and proceeded to spend the majority of the Summer and Autumn quarters of 2007 altering and testing my staining protocol. After employing many changes that still did not produce improved images, I contacted Invitrogen (the company that supplied the phalloidin) for product support. Invitrogen suggested that the reason why the phalloidin was non-specifically binding the osteocytes is because the actin filaments in the processes were being denatured by the incubation step in my procedure, and recommended that I try cryo-sectioning instead. After considering the amount of work left to finish and the decreasing amount of time left, I decided that attempting to learn, use, and potentially still be unsuccessful with cryo-sectioning was not an idea worth pursuing. 17

Therefore, I settled on using light microscopy to image my bone sections with hematoxylin and eosin (H&E) staining, a simpler and faster protocol that the OSL had had success with in the past. Hematoxylin is a dye that colors basophilic structures containing nucleic acids blue while the alcohol-based acidic eosin is a dye that colors eosinophilic structures composed of intracellular or extracellular protein pink. Thus, in a bone-cross section, the nuclei of the osteocytes would appear as blue while its surrounding regions would appear pink, allowing the locations of the cells to be easily seen [30]. The disadvantage of using H&E stain is that it is not fluorescent and thus incompatible with the CLSM. Using the z-motor feature of the CLSM, serial transverse images could be taken at predetermined intervals of a thick section of bone stained with phalloidin. With H&E and light microscopy, however, these individual thin transverse sections must be physically cut in order to be stained and imaged. Such a procedure creates an image registration problem, as each imaged section will be misaligned relative to others, thus creating difficulties in comparing the coordinates of osteocyte locations between sections. Addressing this technical issue was the main challenge in writing my MATLAB program for creating the 3-D osteocyte network model, and the design process is detailed in the proceeding Methods section.

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Initial Design of Experiments, Tools, and Devices

Materials and methods
A section of bone taken from the midshaft of an unloaded (control) left murine tibia was decalcified and embedded in paraffin blocks. Five serial transverse sections, each 5 m thick, were machine-cut from the block, mounted on glass slides, and stained with a commercially available hematoxylin dye and a prepared eosin solution following the proceeding protocol. A stock solution of eosin was made from 1 g Eosin Y dye, 20 mL deionized water, and 80 mL 95% ethanol (EtOH), and the working solution of eosin was made from 25 mL stock solution, 75 mL 80% EtOH, and 0.5 mL Glacial Acetic Acid.

H&E Staining Protocol for Paraffin-Embedded Sections 1. De-paraffinization and rehydration a. Wash in 3 changes of xylene (5 min. each). Blot excess xylene. b. Wash in 100% EtOH (5 min.) c. Wash in 95% EtOH (3 min.) d. Wash in 80% EtOH (3 min.) e. Wash in 70% EtOH (3 min.) f. Wash in deionized water (5 min.) 2. Hematoxylin staining a. Stain with hematoxylin (5 min.) b. Wash in tap water to allow stain to develop (5 min.) c. Wash in deionized water (5 min.). Blot excess water. 3. Eosin staining and dehydration a. Stain with eosin working solution (30 sec.) b. Wash in 70% EtOH (5 min.) c. Wash in 2 changes of 95% EtOH (1 min. each) d. Wash in 2 changes of 100% EtOH (3 min. each). Blot excess EtOH. e. Wash in xylene (15 min.) 4. Coverslip slides using Permount (xylene-based).

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After H&E staining, the cross-sections were visualized on a light/epifluorescent microscope and individual images were saved as JPEGs. Figure 9 shows an example of a transverse section imaged stained with H&E and imaged on the light microscope.

Figure 9: H&E-stained transverse section of unloaded murine tibia imaged using light microscopy.

Because the image was saved in black and white, the hematoxylin-stained parts (osteocyte cell bodies) appear as small dark areas and the eosin-stained parts (the remaining regions of the section) appear as light gray. As was previously mentioned, each section that was stained and imaged was rotated at a different angle and therefore was not aligned with every other section. To address this issue, each section was rotated (in Adobe Photoshop) so that the periosteal surface joining the two sharpest 20

corners was 15 off of the vertical plane, as Figure 10 illustrates. This aligns each cross-section in an anatomically oriented position and makes consistent analysis possible. In addition, the background of the image was removed and replaced with a solid black background to isolate the bone and facilitate image-filtering steps later.

Figure 10: Transverse section rotated 15 from the vertical plane.

Next, a solid white mask of the bone area was created in Photoshop for each section. The H&Estained image and the mask for each section were then loaded and saved in MATLAB as intensity image matrices. Using the regionprops function, the center of mass of each of the masks could be calculated, as shown in Figure 11. Because each section was only 5 m apart, the bone morphology should not have changed significantly between sections, and therefore each section should have had a similar center of mass relative to the dimensions of its own image. To verify that all sections had approximately the same shape (and thus center of mass), the moments

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of area were calculated for each of the five sections and are listed in Table 1. The moment about the x axis was calculated as I x = y 2 dA and the moment about the y axis was calculated as

I y = x 2 dA . The moments were similar for all sections, with the coefficient of variation (mean
divided by standard deviation) for Ix being 0.80% and the coefficient of variation for Iy being 1.90%. This confirmed that each transverse section had a similar shape and was rotated correctly in alignment with the other sections.

Center of mass

Figure 11: Solid mask of transverse section showing the center of mass of its area (green dot).

Table 1: Moments of area of each transverse section.

Layer 1 2 3 4 5 Mean SD CV Ix (m4) 4.0262E+11 4.0704E+11 4.0264E+11 4.0490E+11 3.9834E+11 4.0311E+11 3.2351E+09 0.80% Iy (m4) 4.9522E+11 5.1149E+11 4.9051E+11 5.0130E+11 5.1173E+11 5.0205E+11 9.5289E+09 1.90%

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To automatically find the locations of osteocyte cell bodies in the H&E-stained images, each image was put through a series of filters. First, a threshold value was determined for the color (shade of black) differentiating the cell bodies (darkened nuclei) and the rest of the intracellular and extracellular protein (lighter gray colors). The edges of the cell bodies were then detected using the threshold value and the area of each osteocyte body was delineated. Finally, each outline of an osteocyte body was dilated and filled in, thus enlarging the osteocyte body area and facilitating the process of locating these areas. Figure 12 shows an example of the image after the final filtering step.

Figure 12: Transverse section after filtering steps to facilitate the automatic detection of its osteocyte locations. Each little area within the section represents the location of an osteocyte cell body.

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The center of mass of each of the osteocyte body areas was then automatically found, after which the user was allowed to manually double-check and fix any mistakes that the automatic celllocation detector may have made. Figure 13 shows the found cell locations each marked by a single red dot within each osteocyte lacuna.

Figure 13: Automatically detected osteocyte locations, each marked by red dots within the individual lacunae.

The x and y coordinates of these locations were saved after subtracting the respective x and y coordinates of the particular sections calculated center of mass. Saving the coordinates with respect to the sections center of mass ensured that the coordinates from each section were aligned. 24

Once the x and y coordinates of the osteocyte locations were found for each transverse section, the last remaining step in creating the 3-D osteocyte network model was to find the z coordinates. Given that osteocytes are approximately 10 m thick and the transverse sections are each 5 m thick, it was assumed that any osteocyte would appear in at most two contiguous transverse section images. If such is the case, then the z coordinate of the osteocyte is, in reality, somewhere in between the two layers. Based on this assumption, the z coordinates of the osteocyte locations could be approximated (extrapolated) by viewing two contiguous images at a time, finding the osteocytes that appear in both images (defined as within a distance threshold of 30 m), and moving these osteocyte locations into a layer that was halfway between the two current layers. This was repeated for all subsequent groups of contiguous layers. At the end, the topmost and bottommost layers out of the entire group were removed because they could have contained cells that were in the previous or the next layer, which were not imaged. Figure 14 illustrates the entire procedure of automatically extrapolating the z coordinate of the cell locations.

Figure 14: (A) Start with 5 layers; (B) find the osteocytes that appear in two contiguous layers, remove them from those layers, and place them in a layer halfway in between; (C) remove the topmost and bottommost layers to end with a total of 7 layers.

Once all three coordinates of the osteocyte locations were found, the 3-D osteocyte network model could be constructed by simply creating a scatterplot of the coordinates. Such a model 25

provided the basis for morphological analysis to be performed and this data is presented in the proceeding Results section.

Costs
Table 2 summarizes the approximate cost of materials used for this project. Some equipment such as the light microscope or software such as Photoshop and MATLAB were already present in the laboratory and free to use, so their costs are not included.

Table 2: Approximate cost of materials. Solutions used for current project: - H&E stain - Alcohols - Xylenes - Other misc. solutions Solutions previously used: - Texas-Red phalloidin - Mouse-on-mouse blocking reagent Lab materials: - Gloves - Pipettes - Glass slides - Expenses for bone sectioning - Other misc. materials Total:

$100

$400

$300

$800

26

Initial research plan

My capstone is a design-based project and therefore involves very little research elements. Rather, the MATLAB program that I have designed to construct a model of the 3-D osteocyte network in bone is a tool that can be used for research purposes in the future. For example, the model may be used to compare and contrast the osteocyte network of different regions of bone or bones that underwent different loading regimens, and such possibilities are discussed later in the Suggestions for future work portion of the Results section. Despite the lack of research focus in my project, I designed my MATLAB program to meet three general criteria: accuracy, usability, and efficiency. The model must identify the 3-D locations of osteocytes in a section of bone as accurately as possible, and this required me to develop a method to not only find the transverse (x, y) coordinates of the cells in each section, but also to extrapolate a longitudinal (z) coordinate for each cell. I decided to write my program using five transverse section images for a total thickness of 25 m as this made up a substantial portion of bone while still comprising a small number of sections for easier testing and troubleshooting. However, the final program allows for more transverse sections to be added and included in the model, therefore meeting my second criteria of usability. A graphical user interface (Figure 15) was also implemented in the program for ease of use, insuring that all users may be able to construct the 3-D model without being proficient in MATLAB. Finally, the use of simple H&E-stained images and almost fully-automated processes of the program creates a quick and efficient technique for visualizing the 3-D locations of osteocytes. The establishment of these spatial locations and the added capability of performing simple morphological analysis on the network model also improve the usability and efficiency of the program, and provide the basis for collecting data for future research studies.

27

Figure 15: Graphical user interface for constructing the 3-D osteocyte network model. The user may load the H&Estained image and solid mask, find and save its osteocyte location coordinates, and pick the contiguous layers to include in the network.

28

Results
Final timeline

29

Data
Figure 16 shows the constructed 3-D osteocyte network model with its seven distinctive layers as described in the Methods section. Each scatterplot point represents the spatial location of an ostoecyte in the section of bone.

Figure 16: 3-D osteocyte network model showing seven layers constructed from five transverse section images. The axes of the model are in micrometers.

30

From the initial five layers of transverse sections, the mean surface area of a layer was calculated to be 0.579 0.006 mm2. The number of osteocytes present in each of the seven constructed layers is listed in Table 3 along with their respective osteocyte densities. There was a variable range in the number of osteocytes for each layer, with the first (bottom) layer containing significantly more osteocytes than the other layers. Therefore, the standards of deviation for number of osteocytes and osteocyte density that were calculated in each layer were relatively high.

Table 3: Number of osteocytes and osteocyte density in each layer of the model.
Layer 1 2 3 4 5 6 7 Mean # of Osteocytes 319 119 149 152 262 137 187 189.3 73.9 Density (osteocytes/mm2) 550.7 205.4 257.2 262.4 452.3 236.5 322.8 326.8 127.7

Simple morphological analysis was performed on the constructed 3-D osteocyte network model to get a better understanding of the networks architecture. For each osteocyte in each layer, the distance to its closest neighboring cell was calculated, taking into account only cells that were in the same layer as the current one. Figure 17 shows a histogram of these closestneighbor distances for each of the seven layers in the network. The mean and the median distances were calculated for each layer, and the mean distance across all layers was calculated as well.

31

Figure 17: Histograms for the distance of the closest neighboring cell calculated for each osteocyte in each of the seven layers. The distances were calculated taking into account only other cells in the same layer as the current one. Mean 2-D distance for all layers = 31.9 14.9 m.

32

The closest neighboring cell distances were then calculated again, this time taking into account cells in the two layers directly above and below the current layer as well. Figure 18 shows histograms of these new distances for each of the seven layers, with the mean and median distances calculated for each layer as well as the mean distance across all layers.

Figure 18: Histograms for the distance of the closest neighboring cell calculated for each osteocyte in each of the seven layers. The distances were calculated taking into account cells in the current layer as well as those in the layers directly above and below the current one. Mean 3-D distance for all layers = 21.1 8.1 m.

33

The results from Figures 17 and 18 suggest that an osteocyte, on average, is closer to cells in a different layer than cells in its current layer because of the discernibly smaller 3-D distance (21.1 m) compared to the 2-D distance (31.9 m). To verify that this was a consistent trend throughout all regions of the bone section, each transverse section was divided into eight equal sectors as shown in Figure 19. Morphological analysis was performed for each of the separate sectors to compare and contrast any differences between them that may possibly be skewing the preceding data.

Figure 19: Transverse bone section divided into eight equal sectors.

34

The mean number of osteocytes per sector was calculated across all layers and is shown in a bar graph in Figure 20. On average, some sectors had more osteocytes than others, so it was essential to compare the architecture of each sector.

Figure 20: Mean number of osteocytes per sector across all layers of the network = 23.9 5.8 osteocytes.

To examine the architecture of each sector, the distance to the closest neighboring cell was again calculated for each osteocyte in each layer, first taking into account only cells in the current layer. Figure 21 shows a bar graph of the mean closest-neighbor distance calculated for all sectors in each of the seven layers. Although there was some variability in distances among individual layers, when the mean distance was calculated for each sector across all layers, the red bar graph in Figure 21 shows that the closest-neighbor distances were fairly consistent in each sector. The mean distance across all sectors was calculated to be 33.6 m, a very similar distance to the 31.9 m distance calculated in Figure 17.

35

Figure 21: Bar graphs of the mean closest-neighbor distances calculated in each sector for each of the seven layers in the network. The distances were calculated taking into account only other cells in the same layer as the current one. The red bar graph shows the mean distance across all layers. The mean distance across all sectors was 33.6 1.6 m.

The closest neighboring cell distances per sector were then calculated taking into account cells in the two layers directly above and below the current layer as well. Figure 22 shows a bar graph of the new mean closest-neighbor distance calculated for all sectors in each of the seven layers. The red bar graph once again shows that the mean nearest-neighbor distances across all

36

layers were fairly consistent, and the mean distance across all sectors was calculated to be 21.9

m, a very similar distance to the 21.1 m distance calculated in Figure 18. These results
confirm that the trend of osteocytes being closer on average to cells in a different layer than its own layer is pervasive throughout all regions of the bone.

Figure 22: Bar graphs of the mean closest-neighbor distances calculated in each sector for each of the seven layers in the network. The distances were calculated taking into account cells in the current layer as well as those in the layers directly above and below the current one. The red bar graph shows the mean distance across all layers. The mean distance across all sectors was 21.9 2.1 m.

37

Experimental/design decisions
As previously mentioned, an experimental decision was made to forego phalloidin staining in favor of H&E as nearly two quarters worth of testing the phalloidin and confocal laser scanning microscopy (CLSM) method proved to be unsuccessful. It was determined that an easier and faster histology protocol would better serve the purposes of the project, and therefore the H&E protocol was used. If images were obtained from CLSM, then the computer programming portion of the project would have been less complex since there would no longer be a need to account for the misalignment of bone transverse sections. However, it was decided that the faster and simpler method of constructing a 3-D osteocyte network model using H&E-stained images was worth the tradeoff of having to design an additional technique/algorithm to align the images before constructing the model. The method used to make the 3-D osteocyte network model was also simple in design, therefore improving the usability of the computer program as it only requires users to have a minimal amount of computer/programming skills and engineering background knowledge. The concepts of center of mass and moment of area (second moment of inertia) are relatively straightforward and easily grasped by most users with any basic mathematics background, so they may use the computer program with a good understanding of how it operates instead of as a black box. Despite the simplicity of the program, it has proven to be fairly accurate. Calculating the moments of area of the transverse sections (Table 1) showed that they were correctly aligned with little error, and there was also agreement between the morphological data generated by the model and those in the literature, as will be discussed in the next section. Thus, the decision to employ simple experimental/design techniques for this project has provided a usable and efficient method for constructing a 3-D osteocyte network model without sacrificing accuracy. 38

Analysis and conclusions

Overall, my computer program for constructing a 3-D osteocyte network model using H&Estained transverse section images performed well and met my three general criteria of efficiency, usability, and accuracy. The H&E staining protocol only took approximately half the time to execute as the phalloidin protocol, and the light microscope imaging system was also much more straightforward to use than the confocal laser scanning microscope. Although using the light microscope introduced the problem of misalignment between images, the images were re-aligned in Photoshop and checked in MATLAB by calculating and comparing the moment of area of each section. As Table 1 shows, the error in alignment (coefficient of variation) was found to be less than 2%, and thus it was concluded that my manual re-alignment method was acceptably accurate. With no user manipulation, the program took less than one minute in total running time to go from loading the five individual transverse section images to constructing the full 3-D model. However, as was expected for a simple automated program, there were several errors in the found osteocyte locations (usually around 50 errors per image). Therefore, to improve the accuracy of the model, the user had to manually double-check and fix these mistakes that were made by the automatic cell-locating program, which took up to 10 minutes per image. Although this method is significantly less laborious than locating every cell by hand, the amount of time it takes to fix the mistakes generated by the automated program can still be reduced if the imagefiltering algorithm can be improved in the future. This would in turn improve the efficiency and accuracy of the program and model. The 3-D osteocyte network shown in Figure 16 has significantly more osteocytes in its first (bottom) layer compared to its other layers. This discrepancy is unlikely a systematic error as all layers were put through the same network-construction program to extrapolate the z 39

coordinates of the osteocytes. Therefore, it is likely that there were simply more osteocytes towards that particular end of the bone, and this could be verified by cutting, imaging, and analyzing more contiguous transverse sections from that end. Despite the variability in the number of osteocytes per layer in the network, the average osteocyte density (326.8 127.7 osteocytes/mm2) calculated in Table 3 is within the same range as the density found by other investigators (an estimated 400-500 osteocytes/mm2 by Power et al. [31]), albeit a little lower. More transverse sections need to be imaged and analyzed to obtain a better comparison of osteocyte density, although this preliminary agreement in values between my models calculations and those found in the literature suggests that the imaging and cell-locating techniques used in this project were fairly accurate. For each osteocyte in each layer in the model, the distance to its nearest neighboring cell calculated taking into account only cells in the same layer as the current one (referred to as the 2D distance) averaged to be 31.9 14.9 m, as shown in Figure 17. The distribution of distances around this mean was generally normal, although there were some layers that had almost as many distances near the minimum and maximum as around the mean. However, the mean and median distances were very similar (the largest difference being 15.7%), indicating that the values at the extremes were not significantly skewing the mean value. When the nearest neighboring cell distances were calculated also taking into account cells in the two layers directly above and below the current one (referred to as the 3-D distance), the mean distance was calculated to be 21.1 8.1 m as shown in Figure 18. In addition to having a smaller standard deviation than that of the 2-D distance, the 3-D distances also show a much more normal distribution around the mean. The differences between the mean and median 3-D distance in each layer were also smaller on average than those for the 2-D distance, with the largest difference being only 6.7%. 40

The results in Figures 17 and 18 suggest that, on average, osteocytes are more likely to be closer to cells in a different layer than its own layer. Not only was the mean 3-D distance shorter than the 2-D distance, the 3-D standard deviations and differences between mean and median were also smaller, and the 3-D distance distributions were more normal in shape than that of the 2-D distances. This indicates that there was less variability in the calculated 3-D distances compared to the 2-D distances, thus reinforcing the perceived trend of osteocytes being closer to cells in a different layer than its own layer. To verify that such a trend was present in all regions, the transverse sections were divided into eight sectors as shown in Figure 19. The mean number of osteocytes per sector calculated across all layers was 23.9 5.8 osteocytes, indicating that there was some variability between layers, especially between Sectors 7 and 8 (a 200% difference). To determine if this relatively large variability had an effect on the previously calculated nearest-neighboring cell distances, the 2-D and 3-D distances were calculated in each separate sector of each layer. As Figures 21 and 22 show, there was some variability among the sectors in different layers for both the 2-D and 3D distances. However, when averaged across all sectors and layers, the distances showed much less variability, with a mean 2-D distance of 33.6 1.6 m and a mean 3-D distance of 21.9 2.1 m. The mean 2-D and 3-D distances averaged across sectors were very similar to the distances initially calculated from Figures 17 and 18 (a 5.1% difference for 2-D distance and a 3.8% difference for 3-D distance), suggesting that the nearest neighboring cell distance calculations were not significantly different between the different sectors of the transverse section. Therefore, these results verify that the tendency for an osteocyte to be closer to cells in a different layer than its own layer is true throughout all regions of the transverse section. To check the accuracy of the calculations made by my computer program, the mean 2-D and 3-D distances were compared to similar data found in the literature. Chakkalakal et al. 41

estimated that the length of canaliculi linking adjacent osteocytes in the same transverse plane was approximately 30 to 40 m [32], which is in the same range as my programs calculated mean 2-D distance of 31.9 13.9 m. Sugawara et al. were able to use their CLSM-generated osteocyte network to measure the point-to-point 3-D distance between the centers of mass of adjacent osteocytes and reported a mean distance of 24.1 2.8 m [4]. These values are also in the same range as my programs calculated nearest neighboring cell 3-D distance of 21.1 8.1

m. The approximate agreement in morphological data between those previously measured and
those generated by my model not only verifies the accuracy of my program, but also demonstrates its efficiency. My technique for constructing a 3-D osteocyte network model is therefore advantageous in that it uses significantly simpler (H&E staining and light microscopy) and faster (automatic cell detection and model construction) methods than those previously used to produce equally accurate data. An interesting result that was obtained from constructing the 3-D osteocyte network model is that osteocytes are seemingly closer to cells in layers different than their own layer. Because osteocytes that are closer together are more likely to be connected via their processes and transmitting signals to each other [33], this result suggests that the longitudinal component of cell-to-cell communication may have a larger influence on the direction of signaling pathways than previously recognized. Whether or not osteocytes signaling pathways tend to travel in the longitudinal direction rather than in the transverse direction remains to be validated in future investigations. The 3-D bone morphology in different parts along the bones long axis needs to be examined to determine the true complex nature of osteocyte signaling pathway directions. Such analysis may be performed using sections that are cut transversely as well as longitudinally to verify the accuracy of the z-dimension data. In addition, more detailed imaging methods such as those employed by Sugawara et al. [4] may be used to determine the exact connections that 42

each osteocyte has to its neighbors in both the transverse and longitudinal directions. These possible investigations are beyond the scope of this capstone project; however, this project has demonstrated that obtaining a solid understanding of the direction of signaling pathways requires the study of bones 3-D osteocyte network architecture and specifically the influence of the longitudinal component of cell-to-cell signaling.

Suggestions for future work

Although my computer program has proven to be quite efficient compared to previously used methods for analyzing osteocyte networks, improvements may still be made to increase its efficiency and accuracy. Specifically, if more time was devoted to understanding how to properly stain the bone with the fluorescent antibody phalloidin, then the images obtained through CLSM should not only show the spatial location of osteocytes, but also the processes that connect them. Visualizing exactly how osteocytes are connected is vital to understanding the direction of their signaling pathways, and is also much more accurate than only knowing the osteocytes spatial locations and inferring their activity based on their distances to each other. In addition, using phalloidin staining may possibly eliminate the need to cut serial transverse sections of the bone (and later re-align them) as CLSM is capable of taking serial transverse images within thick sections of bone. Despite these advantages of phalloidin staining and CLSM, there is a tradeoff between using complex and expensive techniques to construct a more detailed model and using faster and simpler techniques to construct a fairly accurate model. Such a decision must be carefully considered and will depend on the needs of future experiments. As my capstone project stands now, the technique designed to automatically construct a 3-D osteocyte network model provides the opportunity to analyze the morphology of any given section of bone in a quick, easy, yet accurate manner. The computer program may be used to 43

construct models of different parts of a section of bone to examine how the osteocyte network differs in these various sections. Alternatively, models could be constructed for bones that underwent different loading regimens to compare and contrast how the osteocyte network has changed in response to the loading. As long as there is a need to quickly examine the morphology of any type of bone, the computer program may easily be modified according to the bones specifications and then be used for analysis. The automatically obtained spatial locations of osteocytes may also be incorporated into the OSLs ABM to improve the accuracy of its real-time simulations of osteocyte signaling activity induced by artificially-introduced stimuli. Should it be decided that the ABMs simulations in 3-D need to be verified by in vivo experiments, then my computer program may need to be modified to include the capability of constructing the osteocyte network model using phalloidin-stained CLSM images that illustrate the physical interconnectivity of osteocytes. The development of a program that can perform fast and accurate analysis of bone morphology in addition to being able to show the detailed architecture of the osteocyte network may prove to be an invaluable tool for investigators to understand mechanotransduction signaling pathways in bone.

44

Acknowledgements
I would like to thank my advisor, Professor Ted S. Gross, for the opportunity to work at the OSL and for his time and effort in guiding me through my capstone project. I would also like to thank the rest of the OSL team, particularly DeWayne Threet for teaching me various immunohistochemistry and imaging techniques and Brandon Ausk for aiding me with MATLAB. Finally, I would like to acknowledge the additional help with programming that I received from my classmates Jason Padvorac and Yung-Chun Chen. This capstone project was funded by the Deans Undergraduate Research Award in the University of Washington College of Engineering and the NIH AR45565.

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