A Practical Manual for Musculoskeletal Research

The following sections are included:

• Foreword by Qian Chen

• Foreword by Shu-Xun Hou

• Foreword by Stephan M. Perren

• Foreword by L. K. Hung

• Preface

• Contents

• List of Contributors

DNA microarray, also known as DNA chip or gene chip, is a powerful tool that allows the measurement of tens of thousands of genes in parallel for gene expression and many other aspects of genome research. With the availability of increasing numbers of completely sequenced organisms, genome-wide microarrays are becoming more and more popular in various biological areas. DNA microarray, like other hybridization-based techniques such as Southern and Northern blots, is based on the principle that every nucleic acid strand carries the capacity to recognize its complementary sequences through base pairing. DNA microarray has been intensively used in various areas of human disease studies. It has also been recently applied by a number of investigators to elucidate molecular programs that define osteoblast differentiation. Several cellular models have been used, including committed osteogenic precursors of murine and human origin, immortalized human cells at various stages of differentiation, and uncommitted mesodermal progenitor cells. We believe that the potential of DNA microarray in human bone studies has yet to be explored, and may dramatically expand our scope of understanding molecular programs underlying the physiological and pathological conditions of human bone. This chapter will focus primarily on detailed protocols of DNA microarrays, in particular expression arrays.

The most exciting discoveries in the last two decades have been the mapping, sequencing, and understanding of genes in our body. In this chapter, we will discuss how to specifically map expression profiles of gene products — mRNA, protein, and reporter gene (X-gal) — using in situ hybridization, immunohistochemistry, and X-gal staining, respectively.

In this chapter, bone marrow harvest and isolation as well as mesenchymal stem cell (MSC) culture are described, including sources from human, rabbit, and rat. The biological activity changes of MSCs in the pathogenesis of bone disorders are exemplified by the steroid-associated osteonecrosis rabbit model. As a cell model, the value of MSCs in screening candidate agents against bone disorders is exemplified by icariin, a single-molecule component purified from epimedium, a famous bone-tonifying herb in traditional Chinese medicine. Besides their role as a cell model for the underlying mechanisms, exploration, and corresponding molecular drug screening of bone disorders, MSCs are also a very important source of cell seeds for bone-related tissue engineering repair, which needs a large scale of stem cells. We therefore introduce the concept of MSC in vitro expansion in microcarrier by bioreactor, and the magnetic resonance imaging (MRI) dynamic tracking technique after superparamagnetic iron oxide (SPIO) particle labeling after in vivo transplantation.

In recent years, mesenchymal stem cells (MSCs) have become more widely used in the treatment of bone fractures. Typically, MSCs are isolated from the bone marrow, expanded in cell culture, and implanted back into the subject using a polymeric material as the seeding scaffold. In the literature, there are a myriad of papers on the topic of MSC extraction, proliferation ability, and multipotency. As such, the characteristics of MSCs are not completely understood and their pluripotency is still being explored. The purpose of this chapter is to discuss the role of MSCs in bone remodeling and to highlight several methods for extracting, proliferating, and implanting MSCs into a defect.

Many musculoskeletal diseases and trauma can potentially be alleviated or cured by stem cell and tissue engineering approaches. Most musculoskeletal tissues are derived from mesenchymal stem cells (MSCs), which natively differentiate into chondrocytes, osteoblasts, adipocytes, fibroblasts, myocytes, etc. Although MSCs are assisted by other cell populations such as the hematopoietic lineages, there is no doubt that MSCs are the progenitors of the building blocks of the musculoskeletal system during development. The roles of MSCs in musculoskeletal regeneration have been demonstrated numerous times in various musculoskeletal tissues, and yet are not completely understood. Nonetheless, musculoskeletal regeneration often, but not always, requires biomaterial scaffolds that need to accommodate various cellular functions such as adherence, proliferation, and differentiation. Musculoskeletal scaffolds must also provide the structural similarity, mechanical strength, diffusion, and gas exchange needs of the tissues or organs to be regenerated. These conflicting needs of musculoskeletal scaffolds for cellular function and physical attributes of the regenerating tissue remain a challenge. Although a great deal can and should be learned from in vitro systems, follow-up in vivo studies are needed as a testing bed for cell-scaffold constructs. Additionally, there is a need to test the efficacy of musculoskeletal constructs in frequently inflammatory and diseased models. Furthermore, musculoskeletal regeneration often requires the engineering of two or more cell types, such as osteochondral, fibro-osseous, fibromyogenic, and myo-osseous tissues. This review provides a glimpse of selected approaches for the engineering of complex musculoskeletal tissue.

Osteoblast isolation from neonatal rat calvariae and human trabecular bone, as well as osteoblast coculture with proximal tubular epithelial cells, is described in this chapter. These cells also serve as cell models for pharmaceutical evaluation in anabolic drug screening, thus providing a technical platform for the regulation of bone turnover and potentially aiding the development of new agents for prevention and treatment of metabolic bone disorders.

Osteoblast harvest from rat bone and bone marrow by mechanical isolation and induction culture methods, respectively, is described in this chapter. Osteoclasts act as a cell model for pharmaceutical evaluation on anti-bone resorption drug screening by providing a technical platform to study the regulation of bone resorption and to evaluate the agents developed for prevention and treatment of metabolic bone disorders.

Enhancing fracture healing and other related conditions (like delayed union or nonunion) can help to shorten hospitalization, reduce complications, and improve rehabilitation. Understanding the underlying mechanism of fracture healing will help to explore new treatment approaches in this aspect. Periosteal cells, as a cell type with differentiation ability to become bone-forming cells, are crucial in the fracture healing process. Research on the biology of periosteal cells or understanding the response of the cells to external stimulation may be useful to open up a new area to treat fracture healing. This chapter describes the isolation of human periosteal cells for research on biophysical interventions or other stimulations developed to enhance fracture healing.

Although osteocytes account for over 90% of bone cells, little progress has been made in their study partly due to the limitations of methodology. In this chapter, we will describe a few much improved techniques to visualize their morphology using both light microscope and electron microscope approaches. These include (1) fluorochrome labeling in conjunction with 4″-6-diamidino-2-phenylindole (DAPI) nuclear counterstaining, (2) imaging of the osteocyte canalicular system with Procion red, and (3) resin -casted scanning electron microscopy (SEM).

The interplay between neoplastic cells and multinucleate osteoclast-like giant cells found in giant cell tumor has been considered as a model of the cellular interactions that occur during bone resorption in both primary and metastatic neoplasms. This chapter describes the tissue culture techniques of giant cell tumor of bone. The main proliferating cells maintained in culture are the spindle-shaped stromal-like mononuclear cells, which represent the neoplastic component of this tumor.

Chondrocytes in the growth plate undergo a relatively linear differentiation process. The progression of a chondrocyte from the proliferative stage to the hypertrophic stage is governed by complex interactions with the extracellular matrix within which it resides. A network of peptides, ion channels, and second messengers affects the transcription of certain genes that are ultimately translated into peptides which control cellular activity. Much effort has been invested into replicating this environment under in vitro conditions. It has been found that the three-dimensional (3D) cell culture is a more accurate representation of the in vivo environment in comparison to the traditional monolayer culture. It has also been found that a variety of stimuli may be used to induce the proliferation and differentiation of chondrocytes; one such stimulus is the mechanical stimulation of chondrocytes embedded in a 3D Gelfoam sponge. Chondrocytes are obtained from the chicken sternum. After the cells are cultured and cyclically loaded, mRNA levels of various mechanosensitive genes are quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR). Mechanical stimulation has been shown to upregulate the expression of type X collagen mRNA in early hypertrophic chondrocytes. The entire process, beginning with the obtainment of chondrocytes and ending with the quantification and interpretation of gene expression, is detailed in the following chapter.

Cartilage damage is irreversible due to its properties of avascularity and lack of undifferentiated cells. Studying the biology of chondrocytes and cartilage is therefore critical to understand the underlying mechanism and to explore the potential repair approaches. It has been reported that three-dimensional (3D) chondrocyte culture behaves very differently from two-dimensional (2D) monolayer culture. Therefore, a native 3D culture of chondrocytes is valuable for such a research purpose, and may also be an option for repairing cartilage defects. This chapter describes a detailed protocol for high-yield chondrocyte isolation and the modified pellet culture technique (using a 3D chondrocyte culture), which can synthesize a bioengineered tissue up to 8 mm in diameter. The authors aim at providing a platform for researchers to study chondrocyte behavior in a 3D environment and to explore the application of scaffold-free tissue for transplantation into a large cartilage defect model.

Perhaps the most difficult task in bone histology work is the preparation of a good specimen. It is impossible to obtain a good image of bone or cartilage without careful preparation and processing of specimens. This chapter will describe very basic skills in the preparation of specimens. These techniques include the paraffin method, plastic method, and frozen method.

Decalcification is a technique used to process bone specimens for histopathology or to produce surgical graft material. However, it usually takes a long period of time for a bone specimen, especially large bone samples, to be completely decalcified. The long decalcification process could cause delay in obtaining experimental results and loss of signals as well as reduce staining qualities. This chapter describes the use of a new invention that incorporates ultrasonic waves into the decalcification process so as to shorten the period required for decalcification. The effectiveness of ultrasonic decalcification is also demonstrated. The application of such a technique would help both basic and clinical research scientists and technicians to accelerate their routine work in dealing with bone decalcifications.

Bone or cartilage cells are essentially colorless, and it is difficult to distinguish their morphologies under a light microscope. To better visualize these structures, various staining techniques have been developed; due to page constraints, we will only describe the most common methods for readers. These staining assays include hematoxylin and eosin (H&E) stain, used for general histology of the cell; Safranin O stain, used for the cartilage; and von Kossa and van Gieson stains as well as modified Goldner's trichrome stain, used for nondecalcified tissues.

This chapter introduces a Scion Image based on contact microradiography for evaluating the degree of secondary mineralization in basic structure units (BSUs) of cortical bone. The effects of long-term bis-phosphonate (incadronate disodium) administration on the degree of secondary mineralization in osteons in Beagle dogs are evaluated as an example of the application of this technique. The relevant evaluation parameters used and validated for comparison include the mean degree of secondary mineralization in osteons and the distribution curves of mineralization frequency. Scion Image based on contact microradiography is a simple and precise method that can accurately evaluate the mean degree of secondary mineralization in BSUs of bone. Experimental findings suggest that long-term incadronate administration significantly increases the degree and uniformity of secondary mineralization of osteons in a dose-dependent manner, but does not cause hypermineralization of bone tissue.

For the analysis of calcified tissues, nondestructive bioimaging techniques such as X-ray, peripheral quantitative computed tomography (pQCT), and micro-CT are becoming popular to measure their structural architecture and degree of mineralization. However, these techniques do not provide details on the biological changes of tissues. Thus, histology is still an important technique to demonstrate the interaction of mineralized tissue with other soft tissues. A special histological technique known as undecalcified histology or hard tissue histology is used to preserve the calcification information of tissues. Moreover, calcium phosphate-based ceramics has recently been applied in orthopedic research and clinics. Undecalcified histology involving bioceramics is described and discussed, using an experimental rabbit spinal model as an example, to illustrate how to attain good histology.

The use of micro-computed tomography (micro-CT) in orthopedic research has flourished in recent years. It has been shown to be an objective measurement of the trabecular bone structure and a useful tool in the study of osteoporosis. With its application base in osteoporosis, the present chapter introduces a protocol for work on other orthopedic conditions such as fracture healing, spinal fusion, and anterior cruciate ligament tunnel healing. In addition to the quantification of structural parameters of mineralized tissue, protocols for the quantification of blood vessels and porous structure of biomaterials are also presented.

In order to understand the mechanisms of fracture healing, especially the neovascularization of the callus, we have established a closed femoral fracture model in rats. This chapter describes a microangiography technique that has been adopted to investigate temporal changes in the three-dimensional (3D) vasculature of the healing callus. Quantitative evaluation protocols for vessel size distribution, total vessel volume, and volume fraction have also been established for comparative studies.

Noninvasive three-dimensional imaging of live animals is a powerful research tool that has become prevalent in many biomedical fields including cancer, aging, cardiovascular disease, and cognitive behavior. Micro-computed tomography (micro-CT) distinguishes itself from other imaging techniques in its ability to acquire high-resolution images based on the physical density of the material, facilitating precise assessments of tissue density and morphology of physiological systems such as bone, muscle, vasculature, and fat. To this end, in vivo micro-CT can measure temporal changes in tissue morphometry, under the influence of genetic and epigenetic factors during development, homeostasis, or repair, for testing the efficacy of pharmacological and nonpharmacological treatments or for evaluating the mechanical behavior of the tissue. Here, an in vivo micro-CT protocol is described with specific examples for bone, muscle, and fat.

Molecular imaging is a relatively new field in which a variety of imaging modalities are used to evaluate cellular and molecular process in vivo. From within this multidisciplinary field, this chapter focuses on the methodology of positron emission tomography (PET) imaging of small animals. While clinical nuclear medicine imaging of the musculoskeletal system primarily encompasses MDP bone scans and FDG PET scans for the evaluation of osseous malignancy, the tracer of choice for research applications in small animals (usually mice or rats) is fluorine-18–fluoride ion (F⁻). This tracer is incorporated into the hydroxyapatite matrix, and is therefore a preferential bone-imaging agent. It is otherwise nonspecific and has been used for a variety of applications including imaging fractures, microdamages, and bone cancers. After detailing the materials and methods necessary to perform PET imaging of small animals using F⁻, a review of the current literature in this area is provided with comprehensive examples of the types of images that can be obtained for both visual and quantitative representations of various musculoskeletal processes. Pitfalls for this type of imaging are also discussed. Future applications of this powerful modality are expected to grow as the technology improves.

In this chapter, the humane use of animals in surgical research is described, with reference to Russell and Burch's The Principles of Humane Experimental Technique (1992) — commonly known as the 3R's of replacement, reduction, and refinement — as well as the ethical need for researchers to justify the experiment and take responsibility for the well-being of animals in their care. The basic role of animal ethics committees is also discussed. The chapter then describes in practical terms the preparation of the experimental animal for surgery; the techniques for anesthesia, including knock-down, intubation, and maintenance; and the drugs used for premedication before anesthesia, maintenance of anesthesia, and, most importantly, pre-emptive and postoperative pain relief. The monitoring of the experimental animal under anesthesia and during recovery is also discussed.

Wear debris–induced periprosthetic disease is a major concern after total joint replacement. The wear debris stimulates a cascade of inflammation, resulting in peri-implant osteoclastogenesis and osteolysis. Although in vitro studies have contributed considerably to our understanding of wear debris-induced adverse biological reactions, animal experiments are necessary to understand the more complex mechanisms in vivo. In this chapter, we describe a mouse model that allows the quantification of osteoclastogenesis and osteolysis. Ultrahigh molecular weight polyethylene (UHMWPE) particles are implanted onto calvariae in C57BL/J6 mice, which then develop greater levels of active inflammatory osteolysis than do the control species. The particles are washed in ethanol to remove surface-adherent endotoxin, thus reducing endotoxin interference. Osteolysis can be found in the middle sagittal suture and the adjacent region in mouse calvaria 1 week after implantation. Decalcified hematoxylin and eosin (H&E)-stained sections are used to quantify the osteolysis area. Osteoclastogenesis regions are identified and quantified in tartrate-resistant acid phosphatase (TRAP)-stained sections. Larger areas of osteolysis and TRAP-stained osteoclastic activity are found to be induced by UHMWPE particles than developed by the sham group.

Distraction osteogenesis refers to bone regeneration under tensile stress with axial rhythmic distraction after osteotomy. It is applied on limb lengthening to treat dwarfism, bone transport, correction of limb deformity, and arthrodiastasis. However, clinical complications have been reported, including delayed consolidation, pin tract infection, and muscle wasting. It is essential to generate animal models for investigating the enhancement of bone formation, the design of bone lengthening fixation, and the distraction protocol. This chapter describes and discusses a rabbit tibial distraction osteogenesis model established by the authors for various applications.

Nonunion of fracture has presented an overall therapeutic challenge in clinical practice. Selection of an adequate nonunion model is the basis for testing effective prevention or treatment of nonunion by new intervention techniques. This chapter describes two atrophic nonunion models in rabbits for simulating the clinical conditions that cause nonunion: by the creation of a critical-sized bony defect, and by the interposition of soft tissue. Both nonunion models are evaluated radiographically and histomorphologically at the end of the experiment. Atrophic nonunion characteristics are clearly present in all animals in the defect and interposition of soft tissue models 12 weeks after operation, and persist until 22 weeks. Both the critical-size defect and the tissue interposition techniques are therefore regarded as effective methods to develop atrophic nonunion models.

Fixation of osteoporotic fracture is always a challenging procedure in orthopedic surgery. Loosening of fixation implants from osteoporotic bone is not uncommon. A large osteoporotic animal model that resembles human osteoporotic changes is therefore essential in osteoporosis research. It can be used to test for better fixation techniques and biomaterials developed for the enhancement of osteoporotic fractures. However, many factors should be considered in developing osteoporotic animal models; these include the comparability to human skeletal physiology, the risk of zoonotic disease, the ease of handling, as well as the possibility of regional and climatic accommodation of the animals. Among the large animals, goat is the most suitable one to fulfill the requirements in the authors' institution and region. This chapter describes the methodologies developed and adopted for developing an osteoporotic goat model. Ovariectomy (OVX), one of the common methods to induce osteoporosis in animals, is systemically described. The importance of a low-calcium diet for accelerating bone loss in OVX goats is incorporated and discussed. The chapter also illustrates different methods of monitoring the development of osteoporosis as well as the relative results. These include the monitoring of serum estradiol concentration, monitoring of changes in bone mineral density (BMD) using peripheral quantitative computed tomography (pQCT) on iliac crest biopsies and calcanei, analysis of the trabecular microarchitecture of iliac crest biopsies using micro-computed tomography (micro-CT), microradiography of iliac crest biopsies, and biomechanical indentation test on calcanei and humeral heads.

Spinal fusion is commonly used in spinal surgery to treat spinal deformity and degenerative disease. In animal models, two commonly used posterior spinal fusion surgeries are intertransverse process and interbody spinal fusion. These experimental spinal fusions could provide clues for the medical treatment of patients by stem cell therapy, tissue engineering, or gene therapy. They also serve as study models for testing new biomaterials and their derived composites with bioactive factors or cells. In this chapter, the procedure for rabbit experimental posterior intertransverse process spinal fusion and the method to assess the success of this animal model are described.

Musculoskeletal diseases, especially osteoporosis, are serious health threats to many individuals including the aging elderly, astronauts, and long-duration-functional-resting (e.g. spinal cord injury) patients. The degree of bone loss and muscle atrophy due to disuse is closely associated with increased fracture risk, affecting the morbidity and mortality of the population. Many appropriate animal models have been developed to fully investigate the mechanisms responsible for musculoskeletal adaptations under disuse environment; more importantly, these models are the key in discovering new interventions for osteoporosis. Hindlimb suspension (HLS) is a well-accepted functional disuse model employed on rodents. In this model, the animal's hindlimbs are lifted and suspended for a period of time (days to weeks), thus removing daily weight-bearing activities to the hindlimbs. This chapter will mainly focus on the technical aspects of HLS as a functional disuse model for studying musculoskeletal tissues. Detailed materials and methods are provided for investigators to easily design and efficiently set up a HLS study. The limitations of HLS and other alternative functional disuse models (i.e. casting and neurectomy) are also discussed for further consideration.

Since disuse can be induced by or associated with nerve injury in human beings, it is necessary to develop a neurogenic disuse model for studying the potential beneficial effects of both pharmaceutical and nonpharmaceutical interventions on the prevention and treatment of disuse-related problems, such as disuse-induced osteoporosis. Bone innervation plays an important role in the local modulation of bone metabolism in both intact bone and fracture healing. The establishment of an animal fracture model associated with denervation will facilitate further study of the effect of reinnervation on bone healing. The aim of this chapter is to describe the method for establishing and evaluating (1) the neurogenic disuse model by sciatic nerve resection and (2) the fracture model associated with sciatic nerve resection. The effects of sciatic nerve resection on the responses of both trabecular and cortical bones are usually studied in the tibia of the rat. Peripheral quantitative computed tomography (pQCT) and bone histomorphometry are frequently used methods for evaluating decrease in bone mass and deterioration of the bone microarchitecture in disused bone. The process of fracture healing can be evaluated by radiography, mechanical testing, pQCT, and histological methods. The related key findings are also included in this chapter, and will help in our understanding of the strengths and weaknesses or limitations of these methods for creating and evaluating the two relevant animal models. Practical tips for performing the experiments are provided, illuminating potential confounding factors and enhancing our awareness of potential limitations.

Steroid-associated osteonecrosis (ON), also known as avascular necrosis (AVN), and subsequent subchondral joint collapse are clinically reported, since pulsed steroids are frequently prescribed as a life-saving agent for serious infectious diseases and chronic autoimmune diseases. However, the postsurgical prognosis of total joint replacement is especially poor in steroid-associated ON patients. It is necessary to establish an appropriate animal model(s) in order to test the efficacy of agents developed for clinical applications. This chapter mentions three classical, published induction protocols for steroid-induced or steroid-associated ON and their drawbacks, leading to a new approach for establishing a more effective model with a detailed description of the induction protocol. Multiple bioimaging methods are established for the evaluation of the pathogenic pathway related to decreased blood flow to bone and the endpoint of ON, i.e. histopathological evidences of ON lesion formation. Applications of this model are proposed for future applications involving efficacy studies of agents developed for the prevention of steroid-associated ON.

Anterior cruciate ligament (ACL) reconstruction is a significant clinical problem. Clinical studies reveal that 11%–32% of patients show an unsatisfactory prognosis and that up to 10% may require surgical revision. Graft-tunnel healing is one of the major factors affecting the outcome of ACL reconstruction. Given that there is no analog like tendon insertion in the bone tunnel in animal or human, it is necessary to establish an appropriate animal model for a better understanding of the biology of graft-tunnel healing; in particular, the structure of the tendon insertion site in the bone tunnel should be known in prior. Recently, a protocol for establishing ACL reconstruction animal models has been developed after a critical review of the literature in the past decades. The assessment protocol consists of three-dimensional (3D) structural analysis of bone ingrowth by micro-computed tomography (micro-CT), two-dimensional (2D) structural analysis of newly formed tendon insertion in the bone tunnel by routine histology, and mechanical testing of the strength of the graft–tunnel complex as the endpoint evaluation. Densitometric evaluation is also used to assess the changes of pre-existing bone with graft–tunnel healing by peripheral quantitative computed tomography (pQCT). In this chapter, we also discuss several aspects on the application of this experimental model for developing therapeutic strategies to enhance graft–tunnel healing.

The bone-to-tendon (B-T) or osteotendinous junction is a unique structure within the musculoskeletal system that connects both bone and tendon through the transitional fibrocartilage zone. Injuries to the B-T junction occur as a result of trauma, sports injury, or local chronic inflammation. B-T repair is, however, inferior compared to repair taking place within homogeneous tissues such as bone fracture or tendon repair, as B-T repair involves regeneration of the transitional fibrocartilage zone. Delay in B-T junction healing may often occur, thus preventing early mobilization and rehabilitation. How to accelerate B-T healing is challenging. This chapter describes an experimental partial patellectomy model established for studying both normal and delayed B-T junction healing, with the aim of providing a platform in order to evaluate potential biological and biophysical interventions developed or to be developed for acceleration and/or enhancement of B-T junction repair.

There are no gold standard experimental models for osteoarthritis (OA). In recent years, around 25 different OA models have been reported, including surgically induced, enzymatically or chemically induced, spontaneous, genetically modified, and drug- or supplement-induced models using different animal species. Each model has its advantages and disadvantages. This chapter introduces an anterior cruciate ligament transection (ACLT)-induced OA model in rats, and the relevant histological evaluation methods for confirmation of the successful establishment of this model.

Physeal injury is not uncommon in pediatric orthopedics, with Salter–Harris type II (SH II) fracture being the most common type that may lead to growth arrest and eventually limb shortening. Therefore, research on SH II fracture will hold great potential to benefit children with such an injury. This chapter outlines the creation of a partial growth plate defect model in rabbits that mimicks a SH II fracture for applications in various growth plate or articular cartilage research topics. The establishment of an SH II rabbit model described in this chapter provides some relevant and applicable evaluation methods. This model will be helpful for research on the biology of premature physeal closure during injuries or the exploration of new biomaterials for physeal reconstruction.

Nondestructive three-dimensional (3D) micro-computed tomography (CT) image analysis used in orthopedic research needs to be accompanied by adequate tools for the numerical assessment of experimental systems. Such quantitative tools should be user-friendly and intuitive, not too complex for the orthopedic researcher to implement, as well as accurate and repeatable in order to be suitable for laboratory application. Here, two experimental systems are examined and straightforward micro-CT analysis methods are described, allowing the experimental outcomes to be accurately quantified in a flexible and multidimensional manner. These systems include the study of osteointegration around a metal implant in bone, and the study of porosity of a biocompatible scaffold matrix for tissue engineering (specifically, the study of scaffolds for permeability to cellular ingrowth) Both studies involve a number of standard image analysis techniques applied in a 3D manner, such as erosion and dilation (applied flexibly to both the image and the region of interest), distance transforms, and novel techniques like “shrink wrap”. Applied in combination in an easily programmable “task list” (otherwise known as “scripting”), these functions provide a powerful and versatile range of 3D structural analyses.

Dual-energy X–ray-based technology has developed special software to automatically identify areas overlapped by metal-containing orthopedic implants so that the surrounding bone can be isolated and monitored for follow-up over time. A body of literature has documented the scan parameters that can be used for studies involving hip arthroplasty, knee arthroplasty, and bone lengthening. The software has been demonstrated to be effective in describing patient response to different types of implants, how bone in different regions of the implant change with time, and the response of bone along the implant to treatment. High-density detection software with operator-placed regions of interest (ROIs) has demonstrated usefulness in describing changes over time, response to treatment, and the relationship between bone loss and muscle condition in knee arthroplasty. Similarly, using high-density detection software with operator-set ROIs has allowed the objective quantification and monitoring of gap mineralization in patients undergoing bone lengthening. With care taken to insure proper X-ray flux, patient positioning, and ROI positioning in analysis, this special software has proved to be an effective tool in dual-energy X-ray absorptiometry (DXA) studies of conditions involving orthopedic implants.

Callotasis or distraction osteogenesis is a well-established orthopedic treatment method in the correction of limb length inequality or bone defect. Plain radiography is commonly used to monitor the progress of distraction osteogenesis, with the known limitation that it can only provide qualitative measurements. Dual-energy X-ray absorptiometry (DXA) is well accepted as the standard clinical equipment for studying bone mineral density in osteoporosis quantitatively with reasonable precision. It has also been successfully used to quantify and monitor bone regeneration in callus distraction, and is able to provide valuable information for clinicians in determining the distraction rate and the timing for frame removal. This chapter summarizes the essential technical details of DXA measurement of the bone mineralization status of callus distraction osteogenesis and its clinical indications.

XtremeCT is a three-dimensional (3D) high-resolution peripheral quantitative computed tomography (HRpQCT) device that enables the acquisition of both volumetric bone mineral density (BMD) and bone structure. This chapter provides general information on the specific features of XtremeCT, and a guide to perform XtremeCT measurements using standard measurement protocols for in vivo human application. Its potential for studying large animals in vivo and ex vivo is also mentioned.

This chapter describes quantitative computed tomography (QCT) image processing techniques for bone mineral density (BMD) and structure assessments of the proximal femur and vertebrae, as well as their applications in clinical trials for assessing osteoporosis therapy efficacy, in studying bone loss due to weightlessness, in understanding age-related changes in bone, and in examining how BMD and geometric characteristics correlate to fracture risk. With intrasubject rigid registration, we observe improved longitudinal measurement precision by reducing operator errors, and directly visualize bone loss of astronauts associated with long-duration spaceflight. To develop a general framework for three-dimensional (3D) bone modeling and population comparison, we adapt intersubject registration techniques to transform groups of hip QCT scans into a common reference space. We apply this technique to study the spatial distribution of microgravity-induced bone loss in the proximal femur.

Micro-finite element (micro-FE) analysis is a numerical technique to obtain the mechanical properties of bone or artificial bone-like structures as they relate to their microstructures. The micro-FE approach was developed for use in cooperation with images obtained from micro-computed tomography (CT), peripheral quantitative computed tomography (pQCT), or magnetic resonance imaging (MRI). This chapter describes the establishment of the micro-FE model and the factors that may influence the numerical results. Our study on the microstructural and mechanical parameters of a trabecular bone from a vertebral body is used as an example to illustrate the process of building the micro-FE model and the analysis of results. Some recent developments and applications of this technique are also introduced. The computational data provided by the micro-FE technique may help us to better understand the bone mechanical properties as well as the failure mechanisms associated with osteoporosis, osteoarthritis, loosening of implants, and cell-mediated adaptive bone remodeling processes.

A nuclear magnetic resonance (NMR) spin-spin (T₂) relaxation technique is described for determining water distribution changes in cortical bone tissue. The advantages of using the NMR T₂ relaxation technique for bone water distribution are illustrated. The Carr-Purcell-Meiboom-Gill (CPMG) sequence-based T₂ relaxation data can be used to determine the porosity, and its inversion T₂ relaxation spectrum can be transformed to a pore-size distribution with the longer relaxation times corresponding to larger pores. The free induction decay (FID)-based T₂ relaxation data can be inverted to T_2-FID relaxation distribution, and this distribution can then be transformed to bound and mobile water distribution with the longest relaxation time corresponding to mobile water and the middle relaxation time corresponding to bound water. The technique is applied to quantify apparent changes in porosity as well as bound and mobile water in cortical bone age, microdamage, and disuse. Overall bone porosity is determined using the calibrated NMR fluid volume from the proton relaxation data divided by overall bone volume. The NMR bound and mobile water changes are determined and found from cortical bone specimens obtained from different-aged donors and functional disused turkey. It is also demonstrated that the NMR T₂ relaxation data is sensitive to changes resulting from the creation of microdamage in cortical bone, which can be interpreted as an effective increase in bone porosity; the result indicates that the detection of cortical bone microdamage is possible by this technique. The obtained information may be used as a measure of bone quality describing porosity and water content, both of which may be important determinants of bone strength and fracture resistance.

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is the rapid acquisition of serial MRI images before, during, and after the administration of an MR contrast agent. Unlike conventional enhanced MRI, which simply provides a snapshot of enhancement at one point in time, DCE-MRI permits a fuller depiction of the wash-in and wash-out contrast kinetics, and thus provides insight into the microcirculation of the studied tissues or lesions. With the dynamic signal intensity change post-contrast agent injection, empirical measures such as maximal signal intensity enhancement and initial enhancement slope can be easily obtained. Such data are also amenable to two-compartment pharmacokinetic modeling, from which parameters based on the rates of exchange between the compartments can be generated. DCE-MRI can be used to characterize masses, stage tumors, and noninvasively monitor therapy. Measures of contrast uptake by dynamic MRI have demonstrated a convincing ability to aid in diagnosing the presence of viable tumors and to measure the response for arange of human tumors. While questions remain about how to best extract noninvasive pharmacokinetic measures of drug access from these novel dynamic imaging methods, scientists and clinicians are optimistic that these methods can provide important new clinical measures which reflect the range of biological variations within and between naturally occurring solid tumors. Efforts to standardize DCE-MRI acquisition, analysis, and reporting methods will allow wider dissemination of this useful functional imaging technique.

Attrition and eventual loss of articular cartilage are crucial elements in the pathophysiology of osteoarthritis. Preventing the breakdown of cartilage is believed to be critical in order to preserve the functional integrity of a joint. Magnetic resonance imaging (MRI) and advanced digital postprocessing techniques have opened novel possibilities for in vivo quantitative analysis of cartilage morphology, structure, and function in health and disease. Techniques of semiquantitative scoring of human knee cartilage pathology and quantitative assessment of human cartilage have recently been developed. Though cartilage represents a thin layer of material relative to the size of voxels typically used for MRI, cartilage thickness and volume have been quantified in human and in small animals. MRI-detected cartilage loss has been shown to be more sensitive than radiography-detected joint space narrowing. Progress made in MRI technology in the last few years allows longitudinal studies of knee cartilage with an accuracy good enough to follow disease-caused changes and to evaluate the therapeutic effects of chondroprotective drugs.

Cell traction force (CTF) is essential for controlling cell shape, enabling cell motility, and maintaining cellular homeostasis. As such, CTF plays a critical role in wound healing and angiogenesis of musculoskeletal tissues. Cell traction force microscopy (CTFM) — a modern technology to determine CTF — is briefly presented in this chapter, followed by a detailed description of the materials and methods necessary for its implementation. Finally, examples are given to illustrate many potential applications of CTFM technology in musculoskeletal investigation.

Nanoindentation is a relatively new technique that can achieve high-resolution characterization of material properties in musculoskeletal tissues. It has been shown to be an effective tool in describing changes in bone mineralization that result from disease and aging by quantifying the material's direct response to mechanical loading. Several studies have shown that the results of nanoindentation are highly sensitive to parameters such as sample preparation, loading protocol, indent location, and sample storage. This chapter describes the techniques for a successful nanoindentation protocol and addresses key technical considerations.

The protocols presented in this chapter focus on the micromechanical tensile testing of osteonal/interstitial bone, single trabeculae, and small animal bone specimens. Micromechanical testing of these small samples is technically challenging and often time-consuming. To alleviate the possible difficulties, the protocols for specimen preparation, apparatus and fixture design, data acquisition/interpretation, and associated techniques are provided for researchers in order to adapt these testing methodologies to their research needs.

Standard mechanical testing methodologies are typically conducted in line with ASTM (American Society for Testing and Materials) or BS (British Standards) guidelines. However, the nature of musculoskeletal tissues — which are generally inhomogeneous, anisotropic, porous, viscoelastic composite materials with widely varying mechanical properties — means that standard testing methodologies are often inappropriate. Depending on the nature of the specimen and the type of testing to be conducted, the equipment and methodology used in mechanical testing of musculoskeletal tissues vary widely. It is practically impossible to dictate an optimum approach that should be used in the mechanical testing of musculoskeletal tissues. Several approaches may be equally valid, and the strategy adopted will generally depend on the particular requirements of individual studies. As such, a “how to” guide to biomechanical testing is almost impossible to compile and of limited practical use. This chapter is therefore written from the opposite, cautionary perspective, indicating common approaches to the biomechanical testing of musculoskeletal tissues as well as considerations that need to be taken into account during each stage of testing. Once the aim of a biomechanical test or series of tests has been established, it is hoped that this chapter will help in the choice of an appropriate strategy and indicate areas where particular care should be taken.

Motion analysis is increasingly used to study the complex kinematics of the musculoskeletal system. In this chapter, both the theory and applications of motion analysis are presented. After the definition of a Cartesian coordinate frame is introduced, a description of transformations between multiple coordinate frames is given; the decomposition of a transformation matrix into anatomical joint motion parameters (e.g. Euler angles) is then explained. Kinematic analysis in musculoskeletal research is illustrated by several examples. The first example describes a reaching-and-grasping task in which mathematical transformations are applied to position the hand with respect to an object during grasping. The second example demonstrates the utility of motion analysis in revealing the coupling motion of the wrist between flexion-extension and radial-ulnar deviation. The third example shows the application of the motion analysis technique to the study of thumb kinematics, providing insight into the complex movements of thumb joints generated by individual muscles. The last example illustrates the study of knee biomechanics, including a description of knee joint kinematics during functional activities and determination of in situ ligament forces using robotic technology. It is hoped that the theoretical knowledge and biomechanical examples will help readers apply the motion analysis technique to various research problems associated with the musculoskeletal system.

The following sections are included:

• Index