Animal Models for Osteoporosis

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Animal Models for Osteoporosis Urszula T. Iwaniec1, Russell T. Turner2 1School 2Professor

of Biological and Population Health Sciences, Oregon State University Corvallis, OR, USA, and Director, Skeletal Biology Laboratory, College of Public Health and Human Sciences, Oregon State ­University, Oregon, USA

INTRODUCTION Animal models have aided our understanding of the pathophysiology and treatment of osteoporosis. They have been an especially useful tool for investigating interactions between multiple risk factors, such as aging, hormonal interactions, and genetic predisposition toward bone fragility [1–3]. Animals are indispensable for preclinical evaluation of the efficacy and safety of interventions intended to prevent and/or reverse bone fragility, and they have successfully identified detrimental [4] as well as the beneficial [5] skeletal actions of a wide variety of drugs. Without animal models, it is questionable whether current osteoporosis therapies would have reached clinical practice. Osteoporosis is a pathological condition that is characterized by skeletal fragility and increased fracture incidence. Osteoporotic fractures are due, in part, to a suboptimal bone architecture resulting from underlying severe bone loss. Pathological bone loss can result from numerous causes. Examples of individual risk factors for osteoporosis include gonadal hormone insufficiency, skeletal disuse, anti-inflammatory and immunosuppressant drug therapy, alcohol abuse, tobacco use, and advanced age. In some forms of osteoporosis (e.g., postmenopausal), increased bone remodeling contributes to the poor bone quality. Gross defects in the composition of bone matrix and mineralization are not recognized as contributing to osteoporosis but do contribute to other metabolic bone diseases (e.g., osteitis fibrosa) that may coexist with osteoporosis and result in further deterioration in bone quality [6,7]. This review focuses on the strengths and weaknesses of the most commonly used animal models for osteoporosis. Limitations inherent to the models as well as common errors in their application are emphasized to spur continued efforts to improve the available animal models. Furthermore, methods for analyzing bone mass, architecture, and turnover in animal models are evaluated. Osteoporosis. http://dx.doi.org/10.1016/B978-0-12-415853-5.00039-X

GOALS OF ANIMAL MODELS FOR OSTEOPOROSIS Animal models are used for multiple purposes in osteoporosis research. They include (1) investigation of signaling pathways that regulate bone growth, turnover, and repair; (2) characterization of the cellular, biomechanical, biochemical, and molecular mechanisms for osteoporosis; and (3) as preclinical models for prevention and reversal of bone loss. Animal models are also useful for investigating bone repair following an osteoporotic fracture. The requirements of the animal model differ markedly, depending on the objectives of the study. The ideal laboratory animal model would replicate the human condition with an absolute degree of fidelity. Unfortunately, this goal is seldom achieved. The defining fractures in human osteoporosis have not been reproduced in animals to date. In addition, it is often difficult to ascertain the true degree of correspondence between the mechanisms that lead to bone loss in the animal model and its human counterpart. The extent to which the underlying signaling pathways that regulate bone mass are conserved among species can rarely be known with certainty. It is, however, possible to examine the usefulness of an animal model objectively by evaluating the extent to which similar events, such as hormonal deficiency or aging, lead to similar metabolic, cellular, and architectural changes in humans and the animal model. This approach is usually straightforward when applied to a single risk factor (e.g., hormonal deficiency) but becomes much more difficult when trying to model complex processes such as aging. The ultimate test of an animal model’s utility is its ability to predict an outcome in people successfully. A final consideration for evaluating animal models is practicality. High cost and limited availability will prevent the widespread adoption of an otherwise promising model. Rodents, dogs, and monkeys are the principal animals used to model osteoporosis. Each species has strengths

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and weaknesses, and no laboratory animal is suited to model all of the risk factors that are associated with osteoporosis.

SPECIFIC ANIMAL MODELS Rat General The rat is a commonly used laboratory animal for studying osteoporosis, and its popularity as a model has increased in recent years. Rats are chosen because they are widely available, inexpensive to purchase and maintain, grow rapidly, have a relatively short life span, and have a well-characterized skeleton. In addition, the ovariectomized (OVX) skeletally mature rat is proven to have predictive value as a preclinical model for therapies to prevent and treat postmenopausal osteoporosis. The benefits just mentioned must be weighed against the disadvantages. The small size of a rat is a mixed blessing. The size of a skeleton does not scale linearly with body size. Compared to a human, the skeleton of a rat contributes a smaller fraction of the animal’s total body mass. Perhaps of even greater importance, cancellous bone in the rat contributes a smaller fraction of the total bone mass. As is discussed in detail later, differences in the relative amounts of cancellous and cortical bone can be important when interpreting biomarkers and total bone mass changes. The small bone mass of a rat is a major limitation for orthopedic studies directed toward improving surgical procedures following an osteoporotic fracture. The reader is directed to a review discussing animal models for osteoporosis from an orthopedic perspective [8]. There are striking similarities as well as fundamental differences between rats and humans in bone growth and remodeling that affect the use of the rat as an animal model for human osteoporosis. First, we discuss the similarities. There is compelling evidence for basic multicellular unit (BMU)-based endocortical and cancellous bone remodeling in rats as in humans [9,10]. Also, the crosssectional bone area of rats and humans increases slowly throughout life, resulting in life-long periosteal expansion in both species [11–13]. In humans, the epiphyses fuse shortly after sexual maturation, resulting in cessation of longitudinal bone growth. There is conventional x-ray imaging-based evidence for epiphyseal closure in rats, the precise timing of which was found to be bone- and growth plate-dependent [14]. Using fluorochrome labeling, a sensitive method to measure the rate of bone growth, we have not been able to detect longitudinal bone growth in hindlimb long bones of rats over 8 months

old consistently. Bone elongation appears to be confined to the initial one-quarter to one-thord of a rat’s expected life span, a relative growth period that is longer than in humans, but not markedly so. Highresolution microcomputed tomography (micro-CT) of growing and aged male and female rats has verified the fluorochrome-based results by clearly demonstrating bone bridging between the metaphysis and epiphysis [15], rendering the residual growth plate cartilage incapable of mediating longitudinal bone growth (Fig. 39.1). These findings support use of the rat as a model for the adult human skeleton provided that either animals with fused growth plates are used, or it is demonstrated experimentally that growth does not influence interpretation of the data. There are species differences between rats and humans that affect the use of the rat as a model for human skeletal biology. The human skeleton undergoes extensive intracortical bone turnover due to Haversian remodeling. In contrast, rats normally undergo very limited endocortical bone turnover and do not have a well-developed Haversian remodeling system. As a consequence, the rat is generally a poor model for investigating the role of cortical bone remodeling in the etiology and treatment of osteoporosis. This is a significant limitation of the rat model because Haversian remodeling is likely to play a role in bone loss associated with development of many, if not all, common forms of osteoporosis. There are exceptions to the generalization that rats do not undergo intracortical bone turnover. Severe periosteal resorption and cortical porosity can be induced in the rat. For example, elevated serum parathyroid hormone (PTH) causes cortical bone loss in rats as in humans [16,17]. Thus, the rat has the potential to be used in the safety screening of drugs to rule out increased cortical porosity as a detrimental side effect of therapy (Fig. 39.2). The Growing Rat Model for Investigating Peak Bone Mass A low peak bone mass is considered a risk factor for development of osteoporotic fractures later in life. The growing rat has potential as a model for evaluating the effects of genetics, gender, endocrine, and environmental factors on peak bone mass [18–21]. Specifically, growing rats can be used to investigate radial and longitudinal bone growth, replacement of primary spongiosa by secondary spongiosa, and endocortical bone modeling and remodeling. As in humans, there are gender differences in bone growth in rats that are temporally associated with body weight gain; female rats grow more slowly than males and reach a smaller peak bone mass (Fig. 39.3) [22–24]. The temporal relationship between sexual and skeletal maturation in rats and humans is also similar [18,19]. As in the human iliac crest (unpublished

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FIGURE 39.1  Computer-generated voxel gradient displays of a frontal section of a proximal rat tibia from a 24-month-old male rat. (A) A frontal cutaway view of the tibia, proximal edge pointing up. The red arrows in the box point to cancellous bone that has fused completely across the proximal epiphyseal growth plate (bridges). (B) Computer-generated projection image showing all of the highlighted bridges projected onto the proximal epiphyseal growth plate of the tibia, viewed face on. Red spots indicate location of highlighted bridges. Only the frontal half of the growth plate is shown. (C) Computer-generated projection image shown in the same orientation as in part (A) (showing a frontal view of all of the highlighted bridges of the proximal tibial growth plate, proximal edge pointing up). Only the frontal half of the growth plate is shown. Source: reproduced with permission from Martin et al. (2003) [15].

FIGURE 39.2  Periosteal resorption and cortical porosity at the tibial diaphysis in a rat model for chronic hyperparathyroidism. Compared to the control (panel A), parathyroid hormone treatment (Panel B) resulted in a dramatic increase in cortical porosity.

data), cancellous bone turnover in skeletally mature rats is higher in females than males [25]. The Sprague Dawley rat has been the most carefully studied strain, but limited data suggest similar patterns of growth in other commonly studied rat strains. The rapid growth phase of the growth curve occurs during the first 3 months of life. Not only are rats growing rapidly during this interval, but the growth rate changes continuously with time. The rate of weight gain reaches a peak shortly after puberty (6 weeks of age) and then declines rapidly with increasing age [26].

Bone growth parallels the changes in body weight during the rapid growth phase. The rapid growth phase is followed by much slower weight gain and bone growth. Bone growth in the rat is under genetic and hormonal regulation. As in humans, growth hormone and sex steroids mediate the pronounced sexual dimorphism of the rat skeleton by influencing the rates of bone elongation, cancellous bone modeling and remodeling, radial bone growth, and timing of epiphyseal closure [27–31]. Thus, the growing rat is a useful model for investigating the effects of endogenous ­ factors, nutrition, disease, drugs, and other

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FIGURE 39.3  Sexual dimorphism in tibia of Sprague Dawley rats resulting from gender differences in radial and longitudinal bone growth.

environmental factors on bone growth and acquisition of peak bone mass. The Growing Rat Model for Juvenile and Adolescent Onset Osteoporosis Osteoporosis is most common in the aged, but earlyonset juvenile forms of the disease also occur. Idiopathic osteoporosis, although uncommon, has been recognized in children. In contrast, juvenile osteoporosis is most often associated with an underlying pathology (e.g., beta-thalassemia) or occurs as a side effect of treatment for another condition (e.g., high-dose steroids for asthma) [32–37]. Animals are infrequently used to model juvenile osteoporosis. However, there is a great need for such research. Results of clinical trials in adults may not apply to children, and performing trials in children is often difficult [38]. When there is a known cause, growing rats may be useful to model the mechanisms of bone loss as well as the efficacy and side effects of potential treatments for juvenile onset osteoporosis. Rats are especially useful for modeling nutritional and endocrine deficiencies but may be valuable as models for druginduced osteoporosis as well [39–42].

The Growing Rat as a Preclinical Model for ­ Adult-Onset Osteoporosis The use of growing rats as a preclinical model for adult onset bone loss is strongly discouraged. The young, rapidly growing rat is a very poor model for the adult human skeleton because skeletal growth in growing rats is mediated by cellular processes that are less active (e.g., secondary intramembranous ossification) or not present (e.g., endochondral ossification) in adult humans. The limitations associated with using growing animals as models of adult-onset osteoporosis are discussed in detail in the next section. The appropriate age range of the animals to be investigated in the model depends on the goals of the study. Rapidly growing OVX rats are a valuable model for studying sexual dimorphism of the skeleton as well as interaction of sex steroids and other factors contributing to peak bone mass. There is much to be learned about regulation of the growth plate, transformation of primary to secondary spongiosa, and secondary intramembranous ossification that is amenable to the use of growing rats as a model. Estrogens, selective estrogen receptor modulators (SERMs), bisphosphonates, calcitonin, and receptor activator of nuclear factor kappaB (RANK) antagonists have in common inhibition of bone resorption. Not surprisingly, agents that suppress bone resorption, whether by inhibiting osteoclast differentiation, inducing osteoclast apoptosis, or suppressing osteoclast activity, frequently result in impressive increases in cancellous bone volume in rapidly growing animals (Fig. 39.4) [43–46]. This phenomenon is unlikely to represent bone anabolism. Many, if not all, of the factors that control bone resorption perturb vascular invasion of the growth plate. The extent of vascular invasion controls the length and area of the primary spongiosa, which in turn serves as the template for bone formation. It should be noted that this effect occurs exclusively in the growing skeleton. Thus, this mechanism for increasing bone mass is not relevant to osteoporotic adults. Moreover, drugs that inhibit bone resorption in growing animals result in cancellous bone with a histological appearance similar to osteopetrosis. In contrast to normal bone, calcified cartilage is surrounded by a layer of bone matrix of variable thickness. This condition results from the impaired ability to replace the calcified cartilage that forms the trabecular backbone. As a consequence of suppressed growth-related cancellous modeling, bone quality is degraded and, in extreme cases, hematopoiesis is compromised. Ovariectomized Rat Models for Postmenopausal Osteoporosis The observation that acute ovarian hormone deficiency leads to elevated cancellous bone turnover dramatically increased interest in the rat as a model for

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(A)

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(B)

FIGURE 39.4  Effects of estrogen on cancellous bone volume in a rapidly growing rat. Note that treatment with the hormone increases cancellous bone volume by (1) inhibiting the resorption of calcified cartilage, thereby increasing the template for deposition of new bone, and (2) suppressing the resorption of primary spongiosa. These cellular mechanisms for altering cancellous bone volume are not active in adults.

postmenopausal osteoporosis [47]. Subsequent studies, showing that OVX results in cancellous and cortical osteopenia, have led to the wide-scale adoption of the OVX rat as a preclinical model [48]. Similarly, bone loss is induced in female rats by luteinizing hormonereleasing hormone (LHRH) agonists and antagonists, aromatase inhibitors, and estrogen receptor antagonists [49,50]. These alternatives to OVX are reversible and are useful for modeling the bone loss associated with endocrine therapy for endometriosis and breast cancer. The initial identification in rats and subsequent confirmation in humans of the tissue selective actions of tamoxifen illustrate the predictive value of the rat model [5]. The development of SERMs for prevention of osteoporosis was a direct result of the initial animal observations. More recently, the OVX rat played an important role as a preclinical model in the development of intermittent PTH as a therapy for treating established osteoporosis [51–55]. OVX growing rats lose bone at selected skeletal sites. However, the cancellous osteopenia in the young rat is primarily due to altered bone growth and thus is mediated by a mechanism that differs fundamentally from postmenopausal bone loss [56]. Specifically, OVX in growing rats results in increased resorption of growth plate calcified cartilage during vascular invasion, resulting in a decrease in the amount of calcified cartilage (primary spongiosa) to serve as a template for future bone apposition. Additionally, increased resorption of the primary spongiosa located distal to the growth plate further decreases the potential for cancellous bone acquisition by prematurely destroying the calcified cartilage template. As a result of the deficits incurred by these two growth-related processes, OVX reduces the net addition of cancellous bone to the growing skeleton. Disturbed bone remodeling similar to that observed in postmenopausal women may also occur in the growing rat skeleton. However, the bone loss attributable to a bone remodeling imbalance is small compared to the

loss of primary spongiosa described in the preceding paragraph. As bone growth slows with age, the contribution of altered endochondral ossification to the skeletal effects of OVX diminishes, and the contribution of altered bone remodeling increases and eventually becomes the predominant mechanism for alteration of cancellous bone mass. OVX of skeletally mature rats is similar to menopause in that the surgery leads to cancellous and endocortical bone loss by (1) increasing the overall rate of bone remodeling and (2) altering the balance between bone formation and bone resorption, such that the latter predominates at selected skeletal sites. In addition to the differences in the mechanisms for the cancellous and endocortical bone loss between growing rats and postmenopausal women that have been discussed, OVX results in an acceleration of longitudinal and periosteal bone growth in growing rats [5,47]. As a consequence, the bones grow longer and have an increased cross-sectional area. Cancellous bone mass is decreased, but there can be, depending on age at castration, an overall increase in bone mass. If the goal of the research is to model human postmenopausal osteoporosis closely, OVX must be performed in skeletally mature rats. OVX of 16-month-old rats results in bone loss and closely approximates the relative timing of the onset of menopause in humans [57]. However, there are disadvantages associated with using aged rats. In addition to the increased costs and limited availability of the aged 16-month-old rats, the older animals are subject to a variety of age-related pathologies. Nevertheless, the advantages of using a model that more closely mimics the human disease often outweigh the disadvantages. An alternative to aged rats is to OVX rats at 8 months of age. This compromise retains the use of skeletally mature animals and is highly recommended when a long-duration study would prevent the use of older rats. Eight-month-old or older retired breeder rats are often available from animal suppliers. In general, we do

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not recommend the use of retired breeders as models for postmenopausal osteoporosis because multiple pregnancies and lactations result in osteopenia of a variable magnitude [58]. Retired breeders experience less bone loss following OVX and have more variable indices of bone mass and turnover than age-matched virgins. Male Gonadal Insufficiency Approximately 20% of all vertebral fractures and 30% of all hip fractures occur in men [59]. In men, low testosterone concentrations are a risk factor for osteoporosis. Orchiectomy in growing rats results in a reduced peak bone mass, whereas the surgery results in increased bone turnover and pronounced cancellous bone loss in adults. As in humans, there is evidence that the skeletal changes associated with orchiectomy are due to deficiencies in estrogens as well as androgens. These findings suggest that growing and adult orchiectomized rats are useful for investigation of factors related to the actions of sex steroids on accrual and maintenance of peak bone mass in males.

are easily controlled, and permit unlimited access to the skeleton for invasive procedures while retaining a response to alcohol that is similar to that of humans. The major uses of rat models have been to understand the etiology and severity of alcohol-induced bone loss better [72]. Indeed, the histological changes in the skeleton of alcohol-dependent rats [63] were subsequently identified in alcoholics, providing further evidence that the rat is useful for predicting human outcomes. Since the pattern of alcohol consumption differs among abusers, rats have been used to model chronic alcohol consumption [73] as well as binge drinking [74,75]. In the future, the rat may prove valuable for modeling interactions between alcohol and therapeutic drugs, including drugs designed to treat osteoporosis. In this regard, recent studies suggest that the bone anabolic response to PTH therapy is antagonized by chronic alcohol consumption [76]. Not all alcohol use is detrimental to the skeleton. There is evidence that moderate alcohol consumption may have positive effects on bone balance in postmenopausal women and elderly men [77–79]. The mechanisms that mediate this apparent beneficial effect of alcohol are poorly understood. There have been limited attempts to model the skeletal response to moderate alcohol consumption using rat models [73,80]. Considering the success of rat models designed to investigate chronic alcohol abuse, further research using rats to model moderate drinking appears to be warranted.

Disuse Osteoporosis The rat has been extensively used as a model for disuse osteoporosis; disuse has been induced in male and female rats by several methods, including unilateral sciatic nerve section, tendonotomy, unilateral limb casting, hindlimb unloading, and spaceflight [60–64]. These seemingly dissimilar methods result in similar skeletal changes, implying that the effects on bone are primarily due to skeletal unloading. Rat models have been used to study the etiology of disuse osteoporosis in growing and mature rats as well as to evaluate the efficacy of potential interventions. Disuse models may also be relevant to postmenopausal osteoporosis because reduced physical activity is a comorbidity factor for the latter condition [7,65]. Studies in OVX skeletally unloaded and exercising rats demonstrate that physical activity can impact the pattern of bone loss associated with gonadal insufficiency [66–69]. The finding that hindlimb unloading in rats blunts the bone anabolic response to a therapeutic dose of PTH in the unloaded limb suggests that physical activity may influence the skeletal response to bone therapeutic agents [70].

Senile Osteoporosis Generalized age-related bone loss begins in men and women by their fifth decade, continues unabated through the remainder of their life, and ultimately is responsible for senile osteoporosis. There is no compelling evidence that bone loss of the magnitude observed in aging humans occurs in aging rats. However, agerelated localized cancellous and endocortical bone loss is observed in intact rats [81,82], and the magnitude and rate of bone loss are greatly accentuated following gonadectomy [83]. Age-related bone loss in humans is multifactorial, making it very difficult to determine whether the molecular mechanisms that underlie the skeletal changes in aging rats and humans are similar [84,85].

Alcohol Use and Abuse Alcohol abuse is an important ‘lifestyle’ risk factor for osteoporosis. Human studies in alcoholics are generally difficult to perform and interpret. It is often hard to distinguish the specific skeletal effects of ethanol from comorbidity factors such as poor nutritional status, weight loss, decreased physical activity, cigarette smoking, and nutrient malabsorption related to chronic pancreatitis [71]. In comparison to clinical studies, the rat models for moderate drinking and alcohol abuse

Glucocorticoid-Induced Osteoporosis The usefulness of the rat as a model for glucocorticoidinduced osteoporosis is unclear. The transient increase in bone resorption and rapid severe bone loss that characterizes the pathogenesis of glucocorticoid-induced osteoporosis in humans is generally not apparent in rats. It is well established that glucocorticoids inhibit bone growth and turnover in the rat [86,87]. As a result, young rats treated with glucocorticoids become osteopenic relative to normal growing controls. However, this relative

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osteopenia due to an overall suppression of cortical bone growth does not accurately model glucocorticoidinduced bone loss in adult humans. In fact, there are reports of increased cancellous bone mass in growing rats treated with glucocorticoids [87,88]. Some studies in adult rats have reported increased bone resorption and reduced bone mineral density (BMD) in glucocorticoidtreated rats [89,90]. Other studies report an inhibition of bone resorption [86,87]. In addition to the interstudy variation, interpretation of rat studies is complicated by the inhibitory effects of high doses of glucocorticoids on reproductive hormones [86]. We have performed numerous short- and long-duration dose-response studies investigating the skeletal response to glucocorticoids in growing and adult rats. Glucocorticoid treatment suppressed bone growth in growing rats and suppressed bone turnover in adult rats. In no case did we observe a loss in bone mass. The many discrepancies in the published literature and our own experience suggest that the effects of glucocorticoids on bone turnover in the rat are inadequately understood to recommend existing models using this species for studying glucocorticoidinduced osteoporosis confidently. Unfortunately, no other animal model is clearly better. In contrast to the variable effects of glucocorticoids on bone resorption discussed earlier, the inhibition of bone formation that is observed in humans treated with glucocorticoids is reproducibly observed in the rat. Therefore, the rat appears to be an appropriate model for investigating the mechanism and potential countermeasures for this important detrimental effect of glucocorticoids. It is possible that the transient increase in bone resorption that is often observed in patients is related to an interaction between glucocorticoid therapy and the underlying pathology. Therefore, it may be fruitful to investigate the actions of glucocorticoids on bone in rats with inflammatory diseases. Inflammation-Induced Osteoporosis Immune cells may play a role in osteoporosis [91,92]. Local inflammation, induced by a variety of agents, results in systemic bone loss in rats, a finding that suggests that the rat may be a useful model for investigating the etiology and treatment of inflammation-induced bone loss [93–95]. Parathyroid Hormone Altered parathyroid function contributes to osteoporosis. Mild primary hyperparathyroidism (HPT), generally considered to be asymptomatic, is associated with increased fracture risk [96]. Mild secondary HPT is thought to contribute to age-related increases in bone turnover and bone loss [97,98]. Severe HPT results in multiple skeletal abnormalities, including periosteal resorption, osteomalacia, increased cancellous turnover,

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osteitis fibrosa, bone pain, and an increased fracture risk [99]. In contrast to postmenopausal osteoporosis, patients with severe parathyroid bone disease generally undergo cortical bone loss but maintain normal cancellous bone mass. Primary HPT has been modeled in the rat by continuous infusion of PTH using subcutaneously implanted osmotic pumps [52]. Secondary HPT has been modeled by reducing dietary calcium or by inducing renal failure [100]. The latter has been accomplished with dietary adenine or by 5/6 nephrectomy [101,102]. As in humans, the specific pathologies and severity of the resulting parathyroid bone disease observed in the animal models are closely associated with the circulating levels of PTH. Continuous infusion of PTH, at a rate of 40 μg/kg/ day, results in blood levels of the hormone (>500 pg/ mL) and skeletal manifestations comparable to patients who have severe parathyroid bone disease. The renal failure models, in contrast, result in lower PTH concentrations and mild-to-moderate bone disease. The models also differ in the interval of time required to induce skeletal abnormalities; the renal failure models require weeks to months, whereas continuous infusion of PTH requires less than 1 week of time for the bone disease to develop [55]. Under some conditions, transient increases in PTH levels can be highly bone anabolic. This so-called intermittent PTH treatment approach was first described [52] and then refined in rats [55,103–105]. As in rats, intermittent PTH treatment with teriparatide (PTH 1–34) in humans is bone anabolic and has been approved by the FDA for treatment of osteoporosis [106]. However, an increased risk for osteosarcoma was noted in Fisher 344 rats. As a result, recommended treatment with intermittent PTH is restricted to patients having a high risk for fractures. It should be noted that osteosarcoma does not appear to be more prevalent in patients with primary HPT or in patients receiving teriparatide [107]. The dose rate of PTH associated with increased cancer incidence in rats was much higher than the human therapeutic dose, and the cancer in these whole life studies occurred late in life [108]. Nevertheless, it will be important to monitor patients carefully receiving PTH therapy for cancer and other detrimental side effects of this potent hormone. Intermittent PTH therapy increases bone mass in many, but not all, patients [106]. The rat may be useful for understanding the underlying causes for PTH resistance. In this regard, recent studies suggest that disuse [70,109] and alcohol abuse [76] may impair the skeletal response to PTH. In the process of performing dose response studies, it became clear that bone formation is increased in rats treated with a human therapeutic dose rate of PTH (<1 μg/hg/day) and that the high dose rates commonly administered to rats (40–80 μg/hg/day) may obscure the interactions of the hormone [109].

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MOUSE General The mouse is under investigation as a model for osteoporosis. The advantages and disadvantages of small size discussed earlier for the rat are even more pronounced in the mouse. As is elaborated on in the succeeding paragraphs, the small quantities of bone, especially cancellous bone, create a challenge to investigators attempting to study bone turnover in the mouse skeleton. Mice have similar growth characteristics to the rat. The two rodents, however, differ radically in their physiologies. Mice and rats diverged from a common ancestor 12–24 million years ago [110,111]. In contrast, humans and chimpanzees split from a common ancestor less than eight million years ago. As a consequence of the species differences, rats and mice offer unique advantages as laboratory animal models for osteoporosis research.

Genetic Risk Factors for Osteoporosis The mouse is an exquisite laboratory animal model for studying the genetic contribution to peak bone mass and age-related bone loss [112]. It has also successfully replicated the skeletal phenotypes related to several genetic disorders in humans [113–116]. The presence of a skeletal system that in many ways is similar to humans provides obvious advantages over the most commonly used alternative animal genetic models, Caenorhabditis elegans and Drosophila. There are numerous well-characterized mouse strains with differences in bone mass and response to comorbidity factors. Additionally, transgenic technology allows the purposeful manipulation of specific gene expression [117]. As a consequence, there is a long and growing list of transgenic mice with perturbed bone metabolism. These genetic manipulations are not without pitfalls when applied to osteoporosis. Demonstration that a gene is associated with bone mass in the mouse does not assure that it has any role in the pathogenesis of osteoporosis. Similarly, a plethora of novel factors that ‘regulate’ bone mass and turnover have been identified based on bone phenotypes observed in gene knockouts, knockins, and loss and gain of function mutations in mice. Regulation of bone metabolism implies a response to changes in the levels of a gene product that are within the physiological range. Only in rare cases has the physiological relevance of genetic manipulations been established. Future improvements in the ability to regulate genes dynamically in specific cell types are necessary to clarify the significance of putative osteoporosis genes.

Postmenopausal Osteoporosis The value of the mouse as a genetic model is well established. In contrast, the use of this species as a preclinical model for postmenopausal osteoporosis is unproven and cannot be enthusiastically recommended. To date, the results have been disappointing. In general, OVX results in accelerated cancellous bone turnover in mice [118–121]. However, the magnitude of the change is highly straindependent and much less consistent than in rats [122–124]. The minute amount of cancellous bone that is present in mice, especially in long bones, is also a major disadvantage. This limitation is exacerbated by age-related cancellous bone loss, which begins in some commonly studied mouse strains shortly after peak bone mass is achieved at ~4 months of age [123]. The net result is that there is too little starting cancellous bone at many important skeletal sites to accurately measure changes in indices of bone mass and turnover following OVX. Unfortunately, one of the most challenging mice to evaluate is C57BL/6, a strain that is commonly used in genetic manipulations. This strain exhibits an especially low cancellous bone volume. For example, the mean cancellous bone volume at the distal femur of 7-month-old ovary-intact C57BL/6 mice is often as low as 3% (Fig. 39.5A–C). Measurement of dynamic histomorphometry and osteoblasts and osteoclasts is problematic because of the paucity of bone surface. To detect bone loss following OVX in the skeletally mature C57BL/6 mice reliably would require reducing the bone volume to near 0%, making it even more unlikely that measurement of fluorochrome labeling and bone cells will accurately reflect changes in bone turnover. Other than agents that induce de novo bone formation, it is unlikely that a model system in which there is so little cancellous bone can be reliably used to evaluate the efficacy of pharmacological interventions to restore bone mass in an osteopenic skeleton. The limitations just discussed can be at least partially addressed by investigating skeletal sites such as lumbar vertebrae, which have a higher cancellous bone volume (Fig. 39.5D–F). Although significant, the vertebral bone loss 3 months following OVX is modest, averaging about 27% (change in bone volume from ~12 to ~9%). There are clear differences between human and mouse physiology regarding the actions of estrogens and estrogen analogs such as tamoxifen [125]. Tamoxifen shows far less tissue discrimination in mice than in humans and administration of estrogens to mice induces endocortical bone formation (Fig. 39.6) [126]. This pathological condition does not occur in humans that overproduce estrogen or are treated with high levels of the hormone. These species differences suggest that the mouse model should be avoided in studies involving activation of estrogen receptors. Based on these limitations, use of the OVX mouse as a preclinical model should be approached with extreme caution.

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Mouse

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Lumbar Vertebra

Distal femur

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Baseline (4-month-old mouse)

Sham-operated (7-month-old mouse)

Ovariectomized (7-month-old mouse)

FIGURE 39.5  Effects of ovariectomy on cancellous bone volume in the distal femur (A–C) and lumbar vertebra (D–F) of C57BL/6 mice. Mice were ovariectomized or sham-operated at 4 months of age and left untreated for 3 months. Note the low bone (black) volume in the distal femur of both 4- and 7-month-old mice (Von Kossa/tetrachrome stain; photographs courtesy of T. J. Wronski).

Control

Estrogen-treated 1.0 mm

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FIGURE 39.6  Effects of estrogen on bone in mice. A weanling mouse was ovariectomized and administered estradiol for 6 months. Treatment was then discontinued. When analyzed 8 months later by microcomputer tomography, much of the estrogen-induced endocortical bone in the mid-shaft of the femur was still present. The pronounced osteosclerosis induced by estrogen in mice has no parallel in humans, contraindicating the use of mice as a preclinical model for pharmacological agents that may act through estrogen receptors.

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Disuse Osteoporosis The mouse has potential as a useful animal model for investigating disuse osteoporosis. In addition to depressed bone formation, disuse results in an increase in osteoclast number in unloaded mouse bone [127,128]. Uncoupling of bone formation and bone resorption is a hallmark of disuse osteoporosis in humans. Although a similar uncoupling has been observed in skeletally mature rats, the potential for genetic manipulation in mice provides an unmatched resource for investigating the role of specific genes in skeletal adaptation to mechanical usage [129]. Senile Osteoporosis Peak bone mass occurs ~4 months of age [112]. Decreases in whole body BMD [130] and cancellous BV/TV, an index of regional cancellous bone mass, are observed in aging mice. Bone loss occurs earlier in senescence accelerated mice (SAM) than in wild-type (WT) mice [131]. The mechanisms that lead to multiorgan senescence in SAM may not be the same as in WT mice, which in turn may differ from humans. Even in WT mice, cancellous bone loss occurs earlier and is more extreme than in humans of the same relative age. In this regard, cancellous bone virtually disappears at some important skeletal sites (e.g., distal femur and proximal tibia) by the time the animals are 1 year old (early middle age). In spite of our concern that the mechanisms for age-related bone loss in mice and humans may not be parallel, it is likely that mice will prove to be very useful for modeling genetics and environment as they interact to influence the aging skeleton. Glucocorticoid-Induced Osteoporosis There have been several studies with mixed results using the mouse as a model for glucocorticoid-induced bone loss. In one study, increases in osteoclast number and a decrease in BMD were observed [132]. However, in a subsequent study by the same research team, neither increased osteoclast number nor bone loss was observed [133]. Other studies in mice report inhibition of bone growth and turnover but no compelling evidence for a reduction in bone mass [134,135]. Thus, it is uncertain whether the mouse will prove to be an animal model that is superior to the rat for investigating glucocorticoidinduced bone loss. Parathyroid Hormone A limited number of studies have investigated the bone anabolic response of mice to intermittent PTH. Even fewer studies have investigated mice as a model for HPT-induced parathyroid bone disease. Results to date suggest that the mouse is relatively resistant to both the anabolic and catabolic skeletal actions of PTH [136–138]. Daily dose rates of the hormone in great

excess of those used in human subjects appear to be required to illicit a bone anabolic response in mice. In contrast to humans, intermittent PTH administered to mice appears to have a more pronounced bone anabolic response on cortical than cancellous bone [139]. Continuous administration of high-dose PTH to mice for 1 and 2 weeks resulted in increased bone turnover in mice. However, the increased cortical porosity, periosteal resorption, osteomalacia, and osteitis fibrosa that characterize parathyroid bone disease in humans and rats were absent in mice [140]. One of the most conspicuous species differences between mice and rats (and mice and humans) is the virtual absence of mature mast cells in bone marrow of the mouse strains evaluated to date. The significance of this finding is unclear, but mast cells have been implicated in mediating the skeletal responses of other species to PTH [141,142].

Dog Rodents are of limited value for investigating intracortical bone remodeling. Larger animals such as the dog are more appropriate for these studies because, similar to humans, dogs have well-developed Haversian remodeling. This large animal also has major advantages as a model for highly localized bone fragility such as that associated with stress shielding by orthopedic implants [143]. The dog is also well established as a laboratory animal model for generalized disuse. In contrast, the dog is not widely used as a model for postmenopausal osteoporosis. Whereas some investigators have detected bone loss following ovariectomy, with or without concurrent hysterectomy, other investigators have detected no changes [48,144,145]. The relative insensitivity and inconsistent response of the dog skeleton to decreased gonadal hormones may be due to the 6-month interval between periods of luteal activity. The large size and relatively long life span also discourage the use of the dog model because of the increased cost of maintaining the animals as well as administration of larger quantities of expensive and/or dangerous chemicals. An additional consideration is the reduced availability of molecular probes specific to dogs compared to rats and mice.

Primates Several species of monkeys have been used as models for osteoporosis. Monkey physiology is generally more similar to human physiology than the more commonly used animal models for osteoporosis. The most compelling evidence for generalized age-related osteopenia in an animal model is in monkeys [146]. Unfortunately, extensive bone loss has not been reported in monkeys

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until after their third decade of life, severely limiting the practical application of what otherwise would be an excellent model for aging. Monkeys are an established model for disuse, but they do not offer many significant advantages over other large domesticated animal species, such as dogs, which are more readily available [147]. One advantage that monkeys have over most alternative large animals is the availability of molecular probes and biochemical assays. Because of the species similarity, many human probes are suitable for use in monkeys. OVX results in elevated bone turnover and bone loss in monkeys raised in captivity [148]. However, studies in captive monkeys reared primarily in the wild failed to demonstrate bone loss following OVX. The use of monkeys as a model for osteoporosis is greatly limited by their expense, long life span, limited availability, and ethical concerns. In the absence of a consistent, robust response following OVX, the use of monkeys for largescale experiments designed to prove drug efficacy needs additional justification.

Other Large Animals Large domesticated animals such as sheep and goats are popular models for orthopedic research. As is the case for dogs, there are typically long intervals between luteal activity, suggesting that these species are not ideal models for postmenopausal bone loss. The large size, long life span, and specialized animal husbandry discourage the use of these animals for studies in which smaller animals can be substituted.

EVALUATION OF THE OSTEOPENIC SKELETON IN ANIMAL MODELS Experimental Design The experimental design and choice of methods to evaluate bone mass, architecture, and metabolism are as important as the choice of the animal model itself. Bone loss is the seminal characteristic of adult-onset osteoporosis. Evidence of bone loss is best obtained by demonstrating that the ‘osteoporotic’ animals have less bone than a baseline control group sacrificed at the start of the study. Demonstrating a difference between the ‘osteoporotic’ group and an age-matched control, although important, does not distinguish between bone loss and failure to acquire as much bone. The skeletons of laboratory animals are highly sensitive to housing conditions. As a consequence, great care must be taken in maintaining uniform conditions, especially when the study requires the use of multiple small cohorts of animals. It is recommended that studies be performed using a single cohort of animals of uniform age, weight, and source. Sometimes this is not possible. Limitations in the availability of adequate space, equipment, and trained personnel may require the use of multiple cohorts. It is not uncommon for only one or two mice in a litter to possess the desired genetic traits. As a consequence, many cohorts, each consisting of a few animals, may be required. Cohort differences are not limited to mice and can present a significant problem in rats as well. Figure 39.7 illustrates differences in cancellous

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FIGURE 39.7  Cancellous bone area in the proximal tibia metaphysis for four cohorts (with two groups/cohort, baseline and control) of male 6-month-old Fisher 344 rats. The controls were sacrificed 2 weeks following the baseline groups (data are mean ± standard error of the mean (SE)). Although identical in age, strain, and source, mean cancellous bone area varied from 20% to 28%, a difference large enough to obscure treatment effects in multiple cohort studies.

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bone volume measured by the same individual in four cohorts of male Fisher rats. The rats did not differ from one another in age and weight and were obtained from the same source. Chemicals are often delivered to experimental animals at dose rates that greatly exceed the levels that could be achieved in humans. In preclinical studies, the dose range of the agent to be tested and the method of delivery should be carefully considered to take into account how the agent will be used in a patient. Administration by a subcutaneous route is not ideal for delivery if the agent is intended to be given orally. The benefits of potential therapeutic agents can be greatly exaggerated if they exceed the levels that can be achieved in humans. For example, the putative beneficial effects of phytoestrogens present in food may be exaggerated when the active agent(s) is delivered subcutaneously or in a concentrated form. The qualitative effects of a chemical as well as how it interacts with other factors can be influenced by dose rate. PTH is often delivered to animals at dose rates two orders of magnitude greater than the human therapeutic dose. Studies have demonstrated that therapeutic and high-dose PTH show substantial differences in their relative effects on osteoblast number and activity in weight bearing and unloaded rats [70,109]. End points that are routinely studied in human subjects are useful for evaluating the fidelity of the animal model. The principal purpose of the animal model, however, is to extend knowledge beyond that which can be obtained in humans by employing more sophisticated and/or invasive methods than are generally available for human studies. One of the most important applications of animal models is to identify and characterize the precise role of individual molecules and signaling pathways that regulate bone mass and quality. Changes in bone mass occur as a result of an imbalance between bone formation and bone resorption. At the end of the day, the near infinite number of combinations of bone ‘regulating’ factors affect only four cellular end points: osteoblast number and activity and osteoclast number and activity. Why do so many molecules ‘regulate’ so few processes? In part, the reason is due to our failure to distinguish between permissive and regulatory molecules. A permissive molecule/pathway is either on or off (light switch model) because the molecule in question is produced above or below a threshold level. A regulatory molecule exerts a dose-dependent action over a physiological range (rheostat model). Some permissive factors play essential roles in the development of the skeletal system. The temporal and spatial expressions of these factors are highly regulated. Many permissive factors, however, are much less specific. Because they play a role in the metabolism of many cells, targeting this type of factor is likely to have many undesirable side effects. Similarly, permissive factors that are critical for

normal bone development may have minimal impact in adults. For these reasons, molecules and pathways that are relatively specific to bone are the more promising targets for pharmacological interventions.

Biochemical Markers of Bone and Mineral Homeostasis Analysis of mineral homeostasis can be performed in laboratory animals more easily than in humans, using a variety of in vivo and ex vivo approaches. The mineral (Ca, P,  Mg) content of blood and urine is easily measured, and radioisotopes can be administered as tracers. Additionally, ex vivo studies can be used to extend the capabilities of human studies to evaluate transport of minerals across the intestinal mucosa. In contrast, the availability of biochemical markers of bone metabolism is generally more limited for animal models than for humans. As a consequence, human assays have frequently been adapted to animals with a potential for loss of specificity and sensitivity. Markers for osteoblast differentiation and activity (e.g., alkaline phosphatase and osteocalcin) and collagen breakdown products (e.g., C- and N-telopeptide of collagen cross-links, deoxypyridinolines) are the most common biomarkers of bone metabolism [149]. These markers are useful for indirect detection of changes in bone metabolism and mineral homeostasis at the level of the whole organism. Theoretically, since the same measurements are routinely performed in humans, a direct comparison between the human and animal model can be made. Also, repetitive collection of blood and urine to establish a time course can be made in most laboratory animals. There are several important limitations of biochemical markers. Biochemical markers provide no information regarding bone mass and strength. They do not distinguish between the appendicular and axial portions of the skeleton or between cortical and cancellous bone. Compared to humans, a disproportionately large amount of the skeleton of small animals such as mice and rats is cortical bone. As a result, biomarkers may not detect important localized changes in bone metabolism. Finally, interpretation of biochemical markers must be made with great caution in rapidly growing animals as well as in severely osteopenic animals because changes in age and bone volume will influence levels of biomarkers. Because of these limitations, biochemical markers are best used as an adjuvant to methods that directly evaluate bone mass and regional bone turnover.

Densitometry Formerly, bone mass and density were evaluated ex vivo by using Archimedes’ principle to calculate bone

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density. Densitometry has largely replaced the use of these simple but reliable methods. Single and dual photon densitometers designed for small animals are especially useful tools to investigate longitudinal changes in bone at multiple skeletal sites. Densitometry has two additional advantages over Archimedes’ principle: (1) The procedure is not destructive, and (2) it allows for disassociation of cancellous from cortical bone. Bone mineral content (BMC) can be determined very accurately by densitometry because bone mass is inversely proportional to the attenuation in intensity of the x-ray beam passing through the specimen. The crosssectional area of the bone can be estimated from the projection of the attenuated x-ray beam onto the detector array. Unfortunately, as is discussed later, the method may have inadequate sensitivity when applied to bones of small animals. In humans, BMC is generally divided by the apparent bone cross-sectional area in order to adjust for differences in bone size. The derived value, called areal bone mineral density (aBMD), is the most commonly reported densitometry end point in animal models as well as in humans. aBMD should not be used as a surrogate for bone mass or density. In point of fact, aBMD has no physical significance. In contrast to mass and volumetric density, aBMD can change markedly depending on the orientation of the bone. Furthermore, bone mass and the projected bone area need not change proportionally during normal growth and aging or in response to a disease or treatment. Because bone area and mass are each changing rapidly, interpretation of BMD data is especially problematic in growing animals. Without supporting evidence, aBMD data cannot be interpreted unambiguously. The value of BMD is also limited by the inherent limitations of using a two-dimensional (2D) x-ray projection to estimate bone area. The choice of aBMD as the near universal skeletal endpoint for investigating the genetics of bone mass seems unwarranted because aBMD obscures differences in bone size, an important determinant of bone strength. BMD measurements should not be reported, unless supporting evidence (e.g., histomorphometry or micro-CT) is available. Instead, BMC is the preferred reported endpoint of DXA analysis of bones from small animals.

In Vivo Three-Dimensional Imaging: Experiments in Radiation Biology? The application of peripheral quantitative computed tomography (pQCT) and high-resolution microCT to assess bone changes in living animals provides the investigator with powerful imaging techniques [150]. These instruments are capable of significantly higher spatial resolution than traditional densitometry

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methods, with micro-CT providing the greater resolution. They also have the capability of dynamic imaging of tissues in three dimensions (3D). Theoretically, the 3D architecture of bone can be studied over time in small animals at sufficient resolution to visualize individual trabeculae. The ability to perform longitudinal studies could reduce the number of subjects that are required for study, an important consideration when dealing with hard-to-generate genetically modified animals. However, radiation exposure and scanning interval place serious constraints on the spatial resolution of micro-CT. Reconstructing images using voxel (3D equivalent of a pixel) dimensions that are similar in dimension to the trabecular thickness in a mouse (~40 μm) can cause serious errors, due to partial volume effects (discussed in more detail later), greatly limiting the amount of architectural information that can be derived from these reconstructions. Having a larger trabecular thickness (~100 μm), more accurate bone architectural information can be obtained in rats than in mice. To obtain meaningful architectural information in small rodents, a voxel size <30 μm on a side is required. A 30-μm voxel size will result in a radiation exposure of ~0.1 Gy/scan. Reducing the voxel dimensions to 15 μm to obtain higher-quality architectural data will require eight times as many voxels and, as a consequence, increase the radiation exposure to ~1 Gy/scan. Further increments in resolution would result in a corresponding exponential increase in radiation exposure. It should be emphasized that these high levels of radiation may be confined to the region scanned and thus are unlikely to be detrimental to the overall health of the animal. However, as discussed later, the scans are clearly invasive. An exposure to 1 Gy will have serious local consequences. This conclusion is supported by an extensive literature related to the biological effects of ionizing radiation. An acute exposure to 1 Gy will kill all proliferating cells, and this will be followed by a rebound effect during which cell proliferation is increased [151– 153]. Dose rates of 0.1–1.0 Gy will have major effects on immune cells and influence signaling by many factors known to participate in regulation of bone metabolism (Table 39.1). Thus, studies using high-resolution in vivo scanning should not be performed without due consideration of the side effects. The detrimental effects of radiation can be minimized by using the largest voxel size consistent with the goals of the study and by including nonirradiated controls at all time points that the test animals are irradiated. Unfortunately, the latter limitation diminishes one of the most important advantages of the technique, reducing the number of animals required to perform an experiment by performing repeat measurements.

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TABLE 39.1  Signaling Pathways Influenced by 0.1–1.0 Gy P53 Connexin 43 Nuclear factor kappabeta (NF-κB) interleukin (IL)-10, IL-12 Transforming growth factor (TGF)-β Insulin-like growth factor (IGF)-I c-kit

Ex Vivo Microcomputed Tomography Two general versions of ex vivo micro-CT have been developed: synchrotron and bench top. The synchrotronbased instruments are capable of greater spatial resolution than bench top instruments because of the high yield of parallel monochromatic x-rays that can be generated using a synchrotron source. Because the radiation is monochromatic, the x-ray attenuation coefficients of the micro-CT images are proportional to density. Thus, synchrotron micro-CT can be used to measure bone density distribution at the subtrabecular level [154–156]. The major disadvantages of synchrotron micro-CT are (1) the very limited access to synchrotron radiation and (2) the small volumes that can be reasonably analyzed at high resolution. With the availability of commercial instruments, bench top micro-CT is becoming increasingly common as a tool to evaluate animal bone. Compared to in vivo micro-CT, much higher resolution can be obtained when micro-CT is applied to tissues ex vivo because the high radiation exposure is no longer a concern. Detailed 3D architectural measurements as well as bone density can be obtained from the reconstructions [157,158]. Less information is gained from bench top micro-CT than from instruments that utilize synchrotron radiation because the light from the former is not monochromatic. At present, this is a limiting factor because x-ray attenuation is influenced by wavelength as well as mass. Up to the present, bench top and synchrotron microCT have been primarily used to evaluate bone mass, density, and architecture. They are capable of much more. Using a specially designed cryostatic system, it was proven feasible to isolate RNA from iliac crest bone biopsies from dog and human for gene expression analysis following micro-CT analysis [159,160]. Mechanically loaded bone regions as small as individual trabeculae can be compared to the unloaded bone and the mechanical properties can be calculated after measuring the deformation, providing an alternative method to finite element modeling [161]. Micro-CT can be used to image ectopic mineralization [162]. Soft tissues can be imaged and structural details enhanced with the use of contrast agents [163]. The ability to image the vasculature within

as well as surrounding bone, muscles, and tendons has enormous potential for studies related to bone/soft tissue interactions. It may be possible to measure bone blood flow by imaging microspheres [164] and use heavy atoms that seek mineralizing bone as contrast agents to detect local changes in bone density at sites undergoing turnover. Micro-CT could then be used to measure bone mineralization rates in 3D using principles analogous to light-microscope-based fluorochrome labeling. Edge detection of bone by the micro-CT can be a limiting factor in measuring rodent bones. In particular, partial volume effects become a serious problem when the architectural components of the bone are very small. A partial volume effect occurs when a voxel overlaps the edge of the bone. Failure to include the voxel underestimates bone size, whereas including it results in bone size overestimation. A second limiting factor of a micro-CT is resolution. In this regard, the micro-CT shares some analogies with microscopy. Resolution, whether imaging with visible light or x-rays, is the ability to distinguish closely spaced objects as separate structures. It is critical to maximize spatial resolution for accurate structural analysis of complex structures such as cancellous bone. In the case of the conventional microscope, the wavelength of the light determines the theoretical resolution (~0.2 μm) of the instrument, but the actual resolution depends on the optics and material being visualized. In the case of the micro-CT, x-ray beam diameter and voxel dimensions are related to but are not equivalent to resolution. As is the case for microscopy, micro-CT resolution can only be determined experimentally [165]. However, it is reasonably certain that two separate structures with adjacent voxels cannot be resolved. As a consequence, the maximum resolution of the micro-CT is more than twice the voxel dimensions. As such, structural analyses of bones obtained from animals the size of adult mice will tax the capabilities of the bench top micro-CT. Although it is possible to further decrease beam diameter and voxel size, the improvement in resolution must be weighed against the accompanying reduction in the volume of bone that can be reasonably imaged and the dramatic increase in computing power and time required to reconstruct and analyze the images. For this reason, specimens are usually scanned at the largest voxel size compatible with the goals of the study. An important subjective variable for micro-CT analysis is the threshold value. The threshold is the value of the x-ray attenuation that is used to distinguish the boundaries separating structures differing in density (e.g., bone from marrow) and is determined empirically. For bone, changing the threshold value will change the absolute value for the measured end points such as bone volume/tissue volume, trabecular thickness, trabecular number, and trabecular separation to varying amounts (Fig. 39.8). Furthermore, the magnitude of change in

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commonly measured endpoints varies with bone mass and trabecular dimensions. In other words, the impact of underestimating or overestimating the threshold will vary with sample. Compared to a large bone, reducing the threshold value of a small bone can have a much greater effect on trabecular number. The importance of thresholding is not limited to rodent bone; the threshold value has been shown to influence measurements in bone from animals as large as pigs [166]. These considerations should not dissuade the investigator from performing micro-CT, but rather to encourage very careful validation of methods and end points.

Histomorphometry Histology provides a 2D assessment of bone mass and architecture [5,61,62,83,167,168]. The method has much greater resolution than densitometry and most x-raybased imaging techniques, including micro-CT. One of the most powerful applications of histology is the use of fluorochrome labeling techniques to estimate changes in bone turnover. This approach is called dynamic histomorphometry and is exquisitely sensitive because the fluorochromes act as time markers that can be used to limit the measurements to exclude bone that was formed prior to the treatment interval. Furthermore, histology is the only routine method for estimating bone cell number. Osteoclast, preosteoblast, osteoblast, lining cell, and osteocyte number can be measured directly in

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histological sections as can other cells (e.g., immune and fat cells) that are capable of influencing bone. Changes in osteoblast activity can be estimated from measurements of cell number and dynamic histomorphometry. Dynamic bone histomorphometry is the gold standard for evaluation of osteoblast function. The method involves the use of two closely spaced (in time) fluorochromes. The timing of administration of these labels is critical. Labels given too close together will be difficult to distinguish from one another, resulting in an underestimate of double-labeled perimeter and an inaccurate measurement of intralabel distance. Labels given too far apart will result in underestimation of bone formation because of resorption of one or both labels and failure to detect double labels due to label escape error (label escape). A disproportionate amount of single label is a good indicator of label escape. The proper timing requires a thorough knowledge of the model system. Bone turnover is age-, compartment- (cancellous vs. cortical), bone-, and species-dependent. We have successfully used three fluorochrome labels to be able to measure fast and slow bone formation in the same study [25]. Another timing error is administration of fluorochromes post adaptation such that bone turnover has returned to normal. To avoid misinterpretation of a negative result, it is important to have additional endpoints (e.g., bone mass measurements and/or biochemical markers of bone turnover). There is no well-established comparable dynamic index for bone resorption, but changes in the

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FIGURE 39.8  Microcomputed tomography images of cancellous bone in the distal femur of a mouse depicting changes in cancellous bone endpoints as a function of threshold. Note that bone volume/total volume is much more sensitive to threshold than trabecular number, which in turn is more sensitive than trabecular thickness.

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rate of bone resorption and osteoclast activity can be inferred from the net change in osteoclast number, bone volume, and osteoblast activity. In some cases, it is possible to estimate changes in the rate of bone resorption by measuring retention of a fluorochrome label [168]. Most histomorphometric measurements are normalized to a standardized tissue sampling area. This approach is valid when the sampling site is comparable in all of the control and experimental groups. This requirement is difficult to accomplish when comparing animals of differing ages or growth rates. Histomorphometry can be performed at any skeletal site, and theoretically, information on global changes to the skeleton could be obtained using this method. However, tissue preparation and analysis are sufficiently time consuming that, for practical reasons, histomorphometry is limited to evaluation of a relatively small number of sites. Many investigations focus on hindlimb long bones or lumbar vertebrae, which are representative of the appendicular and axial skeleton, respectively. Densitometry, pQCT, micro-CT, and ribonucleic acid (RNA) analysis are methods that complement bone histomorphometry for evaluating bone mass, architecture, and cell activity, respectively [112,148,150,155,168–170]. As indicated, the data obtained using the most powerful available methods for analyzing architecture (microCT) and cell numbers and activities (histomorphometry) are usually limited to small sampling sites. Observations confined to a small portion of the skeleton should not be generalized to the entire skeleton. The skeletal response to localized changes in mechanical loading is an obvious example of the importance of evaluating multiple sites; during prolonged spaceflight, astronauts exhibit profound site-specific changes ranging from decreased, no change, and increased bone mass. Systemic factors can also have distinct local effects. For example, the leptindeficient ob/ob mouse has a reduced femur bone mass, which is in part due to decreased bone length [171,172]. However, leptin deficiency may actually increase cancellous bone volume [173]. The opposing effects of leptin deficiency on cortical and cancellous bone in the mouse contrast with those of estrogen deficiency on the femur in rapidly growing rats. Estrogen deficiency increases longitudinal bone growth, bone length, and overall bone mass, but decreases cancellous bone mass. It is therefore recommended that studies be performed to characterize multiple skeletal compartments (cortical and cancellous) representing the axial and appendicular skeleton.

acids and nucleotides by radioautography. Immunohistochemistry and in situ hybridization provide important additional tools for localizing gene products and gene expression, respectively, to specific cells [174–176]. 3H-Thymidine radioautography and bromodeoxyuridine (BrDU) immunohistochemistry have proven invaluable for in vivo analyses of bone cell differentiation [57,177,178]. Each method has advantages, but in most circumstances the two methods provide comparable results. The BrDU method is much more rapid than radioautography and avoids the use of radioisotopes. However, BrDU is a carcinogen, and very high concentrations are required for immunohistochemical detection. In contrast, the concentration of high specific activity thymidine used for radioautography is so low that it does not change the concentration of intracellular thymidine pools. Because bone cells turn over slowly, thymidine labeling may be the superior method in continuous labeling experiments. Continuous labeling was developed in order to label sufficient numbers of preosteoblasts and preosteoclasts for kinetic studies [57,177,178]. In situ localization of molecular markers of bone cell differentiation and apoptosis has greatly expanded the ability to study bone cell kinetics in animals. Most studies have focused on a qualitative assessment of gene expression and protein localization. Quantitative analysis, however, has been performed [179]. Fluorescence-activated cell sorting (FACS), an ex vivo method in which cells isolated from bone can be identified and separated based on molecular cell surface markers, can be used in combination with in situ techniques to analyze bone cell differentiation [180]. A major, but hopefully temporary, limitation of the application of this technique to bone biology is the limited number of molecular and cell surface markers known for distinguishing early osteoblast lineage cells. Ex vivo methods can be used to measure whole tissue gene expression and protein analysis. Total tissue RNA has been analyzed by Northern blot, RNase protection assays, and various polymerase chain reaction (PCR)-based methods [181]. Protein is often measured by Western blot, but radioimmunoassay and bioassays are also performed [182,183]. Although generally more quantitative than in situ analyses, these ex vivo techniques are often less sensitive because of high backgrounds due to the same factors produced by other cells and less specific because they measure total tissue activity. A combination of in situ and ex vivo analyses is often more informative than either approach alone.

Molecular Histomorphometry

Mechanical Testing

Molecular histomorphometry couples resolution of individual cells within tissue sections and molecular techniques to detect the presence of specific molecules within a cell. Initial studies in molecular histomorphometry involved the localization of radiolabeled amino

Bone strength is rarely measured in humans but is likely to be an important risk factor for osteoporotic fractures. A relationship between bone strength and fracture risk has not been established in most animal models because of low fracture rates. Nevertheless, measurement

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Fracture Repair

of bone mechanical properties is an important tool for evaluating the functional significance of changes in bone mass and/or architecture. Three-point bending, four-point bending, and torsion testing are the most common methods of measuring bone mechanical properties. These measurements are performed at the midshaft diaphysis, a site at which osteoporotic fractures are uncommon. Compression testing of vertebrae and cantilever testing of the head of the femur have also been developed. These newer techniques are highly recommended because they more closely approximate the types of failures associated with osteoporotic fractures [184]. A limitation of mechanical testing procedures is that the applied force is directional and often does not accurately model skeletal loading leading to a fracture. Additionally, the contribution of soft tissues to fracture resistance is rarely considered. Thus, the development of nondestructive testing procedures on bone and attached tissues in which the direction of the applied forces could be varied would be very valuable.

Cell Biology Animal models for osteoporosis can provide a source of cells for in vitro studies designed to evaluate potential changes in the composition and/or proliferative capacity of bone cell populations. Isolated cells can also be profitably used for studies of disturbed signaling pathways.

FRACTURE REPAIR Fracture repair studies are not routinely performed in osteoporotic animal models, although well-characterized animal models for fracture healing have been developed

[185]. Such studies are urgently needed because impaired fracture repair can dramatically increase morbidity in elderly patients. Animal models can be used to investigate the effects of age, hormones, and lifestyle choices on fracture repair. Existing and future treatments may significantly reduce the risk of osteoporotic fractures. However, there is no immediate likelihood that any intervention will prevent all fractures. Therefore, it is imperative that interventions for osteoporosis be carefully investigated in animal models to evaluate their effects on fracture healing.

SUMMARY Animal models have proven to be essential tools in our quest to understand the etiology and treatment of osteoporosis. They will continue to aid in our understanding of the contribution of specific genes to establishment of peak bone mass and optimal bone architecture, as well as the genetic basis for a predisposition toward accelerated bone loss in the presence of comorbidity factors such as estrogen deficiency. Existing animal models will continue to be useful for modeling changes in bone metabolism and architecture induced by well-defined local and systemic factors. However, there is a critical unfulfilled need to develop and validate better animal models to allow fruitful investigation of the interaction of the multitude of factors that precipitate osteoporosis. Asymptomatic, until a defining fracture occurs, ­distal forearm, hip, and vertebral fractures are common in osteoporotic but rare in nonosteoporotic individuals. The etiology of osteoporotic fractures is complex (Fig. 39.9), in part because osteoporosis is generally due to multiple factors. An osteoporotic skeleton predisposes an individual to a fracture but generally does not

Etiology of musculoskeletal injuries Injury

Poor resistance to injury

Inadequate strength

Inadequate muscle tone Inadequate bone architecture

Poor physical environment Falls

Inadequate lighting Inadequate building construction

Poor balance Inadequate vision Inadequate motor coordination

FIGURE 39.9  The etiology of osteoporotic fractures. VI.  GENERAL PATHOPHYSIOLOGY OF OSTEOPOROSIS

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precipitate the fracture. The immediate cause is trauma, usually a fall. Contemporary estimates suggest that ~50% of vertebral fractures and more than 95% of distal forearm and hip fractures occur as a direct result of trauma [186]. Furthermore, some biomechanical studies suggest that the forces generated during a fall by an osteoporotic individual who suffers a fracture are often sufficient to result in a fracture in an individual with a normal bone mass [187–190]. A better understanding of falls and how to prevent or mitigate the effects of a fall would clearly contribute to reducing osteoporotic fractures. Bone density and architecture rather than falls have been the center of mass for osteoporosis research, including animal research. Expanding the scope of animal research to consider risk factors that contribute to a fall should be a future priority.

References [1]  Ferrari SL, Rizzoli R. Gene variants for osteoporosis and their pleiotropic effects in aging. Mol Aspects Med 2005;26(3):145–67. [2]  Raisz LG. Pathogenesis of osteoporosis: concepts, conflicts, and prospects. J Clin Invest 2005;115(12):3318–25. [3]  Shen H, Liu Y, Liu P, Recker RR, Deng HW. Nonreplication in genetic studies of complex diseases – lessons learned from studies of osteoporosis and tentative remedies. J Bone Miner Res 2005;20(3):365–76. [4]  Sogaard CH, Mosekilde L, Schwartz W, Leidig G, Minne HW, Ziegler R. Effects of fluoride on rat vertebral body biomechanical competence and bone mass. Bone 1995;16(1):163–9. [5]  Turner RT, Wakley GK, Hannon KS, Bell NH. Tamoxifen prevents the skeletal effects of ovarian hormone deficiency in rats. J Bone Miner Res 1987;2(5):449–56. [6]  Faibish D, Ott SM, Boskey AL. Mineral changes in osteoporosis: a review. Clin Orthop Relat Res 2006;443:28–38. [7]  Lane NE. Epidemiology, etiology, and diagnosis of osteoporosis. Am J Obstet Gynecol 2006;194(Suppl. 2):S3–11. [8]  Turner AS. The sheep as a model for osteoporosis in humans. Vet J 2002;163(3):232–9. [9]  Baron R, Tross R, Vignery A. Evidence of sequential remodeling in rat trabecular bone: morphology, dynamic histomorphometry, and changes during skeletal maturation. Anat Rec 1984; 208(1):137–45. [10]  Erben RG. Trabecular and endocortical bone surfaces in the rat: modeling or remodeling? Anat Rec 1996;246(1):39–46. [11]  Allen MR, Hock JM, Burr DB. Periosteum: biology, regulation, and response to osteoporosis therapies. Bone 2004;35(5): 1003–12. [12]  Seeman E. Periosteal bone formation – a neglected determinant of bone strength. N Engl J Med 2003;349(4):320–3. [13]  Turner RT, Spelsberg TC. Correlation between mRNA levels for bone cell proteins and bone formation in long bones of maturing rats. Am J Physiol 1991;261(3 Pt 1):E348–53. [14]  Joss EE, Sobel EH, Zuppinger KA. Skeletal maturation in rats with special reference to order and time of epiphyseal closure. Endocrinology 1963;72:117–22. [15]  Martin EA, Ritman EL, Turner RT. Time course of epiphyseal growth plate fusion in rat tibiae. Bone 2003;32(3):261–7. [16]  Abdelhadi M, Nordenstrom J. Bone mineral recovery after ­parathyroidectomy in patients with primary and renal hyperparathyroidism. J Clin Endocrinol Metab 1998;83(11):3845–51.

[17]  Lotinun S, Evans GL, Bronk JT, Bolander ME, Wronski TJ, Ritman EL, et al. Continuous parathyroid hormone induces cortical porosity in the rat: effects on bone turnover and mechanical properties. J Bone Miner Res 2004;19(7):1165–71. [18]  Yingling VR, Khaneja A. Short-term delay of puberty causes a transient reduction in bone strength in growing female rats. Bone 2006;38(1):67–73. [19]  Sengupta S, Arshad M, Sharma S, Dubey M, Singh MM. Attainment of peak bone mass and bone turnover rate in relation to estrous cycle, pregnancy and lactation in colony-bred Sprague-Dawley rats: suitability for studies on pathophysiology of bone and therapeutic measures for its management. J Steroid Biochem Mol Biol 2005;94(5):421–9. [20]  Miller SC, Bowman BM. Rapid improvements in cortical bone dynamics and structure after lactation in established breeder rats. Anat Rec A Discov Mol Cell Evol Biol 2004;276(2):143–9. [21]  Iida H, Fukuda S. Age-related changes in bone mineral density, cross-sectional area and strength at different skeletal sites in male rats. J Vet Med Sci 2002;64(1):29–34. [22]  Zhang XZ, Kalu DN, Erbas B, Hopper JL, Seeman E. The effects of gonadectomy on bone size, mass, and volumetric density in growing rats are gender-, site-, and growth hormone-specific. J Bone Miner Res 1999;14(5):802–9. [23]  Turner RT, Hannon KS, Demers LM, Buchanan J, Bell NH. Differential effects of gonadal function on bone histomorphometry in male and female rats. J Bone Miner Res 1989;4(4):557–63. [24]  Rol de Lama MA, Perez-Romero A, Hermanussen M, Ariznavarreta C, Tresguerres JA. Sexual dimorphism in growth as measured by microknemometry: different responses to GH deficiency and exogenous GH administration. Neuroendocrinology 1998;68(3):210–9. [25]  Hefferan TE, Evans GL, Lotinun S, Zhang M, Morey-Holton E, Turner RT. Effect of gender on bone turnover in adult rats during simulated weightlessness. J Appl Physiol 2003;95(5): 1775–80. [26]  Turner RT, Maran A, Lotinun S, Hefferan T, Evans GL, Zhang M, et al. Animal models for osteoporosis. Rev Endocr Metab Disord 2001;2(1):117–27. [27]  Yeh JK, Liu CC, Aloia JF. Effects of exercise and immobilization on bone formation and resorption in young rats. Am J Physiol 1993;264(2 Pt 1):E182–9. [28]  Ohlsson C, Bengtsson BA, Isaksson OG, Andreassen TT, Slootweg MC. Growth hormone and bone. Endocr Rev 1998;19(1):55–79. [29]  Johansson AG, Engstrom BE, Ljunghall S, Karlsson FA, Burman P. Gender differences in the effects of long term growth hormone (GH) treatment on bone in adults with GH deficiency. J Clin Endocrinol Metab 1999;84(6):2002–7. [30]  Jansson JO, Eden S, Isaksson O. Sites of action of testosterone and estradiol on longitudinal bone growth. Am J Physiol 1983;244(2):E135–40. [31]  Isaksson OG, Jansson JO, Gause IA. Growth hormone stimulates longitudinal bone growth directly. Science 1982;216(4551):1237–9. [32]  Soejima K, Landing BH. Osteoporosis in juvenile-onset diabetes mellitus: morphometric and comparative studies. Pediatr Pathol 1986;6(2–3):289–99. [33]  Skowronska-Jozwiak E, Lorenc RS. Metabolic bone disease in children: etiology and treatment options. Treat Endocrinol 2006;5(5):297–318. [34]  Mahachoklertwattana P, Pootrakul P, Chuansumrit A, Choubtum L, Sriphrapradang A, Sirisriro R, et al. Association between bone mineral density and erythropoiesis in Thai children and adolescents with thalassemia syndromes. J Bone Miner Metab 2006;24(2):146–52.

VI.  GENERAL PATHOPHYSIOLOGY OF OSTEOPOROSIS

Summary

[35]  Lorenc RS. Idiopathic juvenile osteoporosis. Calcif Tissue Int 2002;70(5):395–7. [36]  Galusca B, Bossu C, Germain N, Kadem M, Frere D, LafageProust MH, et al. Age-related differences in hormonal and nutritional impact on lean anorexia nervosa bone turnover uncoupling. Osteoporos Int 2006;17(6):888–96. [37]  Adams RJ, Wilson DH, Taylor AW, Daly A, Tursan d’Espaignet E, Dal Grande E, et al. Coexistent chronic conditions and asthma quality of life: a population-based study. Chest 2006;129(2):285–91. [38]  Brown JJ, Zacharin MR. Attempted randomized controlled trial of pamidronate versus calcium and calcitriol supplements for management of steroid-induced osteoporosis in children and adolescents. J Paediatr Child Health 2005;41(11):580–2. [39]  Xian CJ, Howarth GS, Cool JC, Foster BK. Effects of acute 5-fluorouracil chemotherapy and insulin-like growth factor-I pretreatment on growth plate cartilage and metaphyseal bone in rats. Bone 2004;35(3):739–49. [40]  Wada C, Kataoka M, Seto H, Hayashi N, Kido J, Shinohara Y, et al. High-turnover osteoporosis is induced by cyclosporin A in rats. J Bone Miner Metab 2006;24(3):199–205. [41]  Cegiela U, Sliwinski L, Kaczmarczyk-Sedlak I, Folwarczna J. In vivo effects of high-dose methotrexate on bone remodeling in rats. Pharmacol Rep 2005;57(4):504–14. [42]  Brzoska MM, Moniuszko-Jakoniuk J. Low-level lifetime exposure to cadmium decreases skeletal mineralization and enhances bone loss in aged rats. Bone 2004;35(5):1180–91. [43]  Turner RT, Evans GL, Wakley GK. Mechanism of action of estrogen on cancellous bone balance in tibiae of ovariectomized growing rats: inhibition of indices of formation and resorption. J Bone Miner Res 1993;8(3):359–66. [44]  Pazzaglia UE, Andrini L, Leutner M, DiNucci A. The effects of bone resorption inhibitors on the growth plate and proximal tibial metaphysis of rats: clinical implications. Orthopedics 1998;21(2):195–9. [45]  Pataki A, Muller K, Green JR, Ma YF, Li QN, Jee WS. Effects of short-term treatment with the bisphosphonates zoledronate and pamidronate on rat bone: a comparative histomorphometric study on the cancellous bone formed before, during, and after treatment. Anat Rec 1997;249(4):458–68. [46]  Capparelli C, Morony S, Warmington K, Adamu S, Lacey D, Dunstan CR, et al. Sustained antiresorptive effects after a single treatment with human recombinant osteoprotegerin (OPG), a pharmacodynamic and pharmacokinetic analysis in rats. J Bone Miner Res 2003;18(5):852–8. [47]  Wronski TJ, Lowry PL, Walsh CC, Ignaszewski LA. Skeletal alterations in ovariectomized rats. Calcif. Tissue Int 1985;37(3): 324–8. [48]  Turner RT, Riggs BL, Spelsberg TC. Skeletal effects of estrogen. Endocr Rev 1994;15(3):275–300. [49]  Gallagher A, Chambers TJ, Tobias JH. The estrogen antagonist ICI 182,780 reduces cancellous bone volume in female rats. ­Endocrinology 1993;133(6):2787–91. [50]  Goulding A, Gold E. A new way to induce oestrogen-deficiency osteopaenia in the rat: comparison of the effects of surgical ovariectomy and administration of the LHRH agonist buserelin on bone resorption and composition. J Endocrinol 1989;121(2):293–8. [51]  Wronski TJ, Yen CF, Qi H, Dann LM. Parathyroid hormone is more effective than estrogen or bisphosphonates for restoration of lost bone mass in ovariectomized rats. Endocrinology 1993;132(2):823–31. [52]  Tam CS, Heersche JN, Murray TM, Parsons JA. Parathyroid hormone stimulates the bone apposition rate independently ­ of its resorptive action: differential effects of intermittent and ­continuous administration. Endocrinology 1982;110(2):506–12.

957

[53]  Meng XW, Liang XG, Birchman R, Wu DD, Dempster DW, ­Lindsay R, et al. Temporal expression of the anabolic action of PTH in cancellous bone of ovariectomized rats. J Bone Miner Res 1996;11(4):421–9. [54]  Hock JM, Gera I. Effects of continuous and intermittent ­administration and inhibition of resorption on the anabolic ­response of bone to parathyroid hormone. J Bone Miner Res 1992;7(1):65–72. [55]  Dobnig H, Turner RT. The effects of programmed administration of human parathyroid hormone fragment (1-34) on bone histomorphometry and serum chemistry in rats. Endocrinology 1997;138(11):4607–12. [56]  Turner RT, Evans GL, Wakley GK. Reduced chondroclast ­differentiation results in increased cancellous bone volume in estrogen-treated growing rats. Endocrinology 1994;134(1): 461–6. [57]  Dobnig H, Turner RT. Evidence that intermittent treatment with parathyroid hormone increases bone formation in adult rats by activation of bone lining cells. Endocrinology 1995;136(8): 3632–8. [58]  Miller SC, Shupe JG, Redd EH, Miller MA, Omura TH. Changes in bone mineral and bone formation rates during pregnancy and lactation in rats. Bone 1986;7(4):283–7. [59]  Amin S. Male osteoporosis: epidemiology and pathophysiology. Curr Osteoporos Rep 2003;1(2):71–7. [60] Li XJ, Jee WS, Chow SY, Woodbury DM. Adaptation of cancellous bone to aging and immobilization in the rat: a single photon absorptiometry and histomorphometry study. Anat Rec 1990;227(1):12–24. [61]  Morey ER, Baylink DJ. Inhibition of bone formation during space flight. Science 1978;201(4361):1138–41. [62]  Thompson DD, Rodan GA. Indomethacin inhibition of ­tenotomy-induced bone resorption in rats. J Bone Miner Res 1988;3(4):409–14. [63]  Turner RT, Aloia RC, Segel LD, Hannon KS, Bell NH. Chronic alcohol treatment results in disturbed vitamin D metabolism and skeletal abnormalities in rats. Alcohol Clin Exp Res 1988;12(1):159–62. [64]  Turner RT, Bell NH. The effects of immobilization on bone histomorphometry in rats. J Bone Miner Res 1986;1(5):399–407. [65]  Mosekilde L. Age-related changes in bone mass, structure, and strength – effects of loading. Z Rheumatol 2000;59(Suppl. 1):1–9. [66]  Westerlind KC, Wronski TJ, Ritman EL, Luo ZP, An KN, Bell NH, et al. Estrogen regulates the rate of bone turnover but bone balance in ovariectomized rats is modulated by prevailing mechanical strain. Proc Natl Acad Sci U S A 1997;94(8):4199–204. [67]  Peng Z, Tuukkanen J, Vaananen HK. Exercise can provide protection against bone loss and prevent the decrease in mechanical strength of femoral neck in ovariectomized rats. J Bone Miner Res 1994;9(10):1559–64. [68]  Mo A, Yao W, Li C, Tian X, Su M, Ling Y, et al. Bipedal stance exercise and prostaglandin E2 (PGE2) and its synergistic effect in increasing bone mass and in lowering the PGE2 dose required to prevent ovariectomized-induced cancellous bone loss in aged rats. Bone 2002;31(3):402–6. [69]  Iwamoto J, Takeda T, Sato Y. Effect of treadmill exercise on bone mass in female rats. Exp Anim 2005;54(1):1–6. [70]  Turner RT, Lotinun S, Hefferan TE, Morey-Holton E. Disuse in adult male rats attenuates the bone anabolic response to a therapeutic dose of parathyroid hormone. J Appl Physiol 2006;101(3):881–6. [71]  Turner RT. Skeletal response to alcohol. Alcohol Clin Exp Res 2000;24(11):1693–701. [72]  Sampson HW, Perks N, Champney TH, DeFee 2nd B. Alcohol consumption inhibits bone growth and development in young actively growing rats. Alcohol Clin Exp Res 1996;20(8): 1375–84.

VI.  GENERAL PATHOPHYSIOLOGY OF OSTEOPOROSIS

958

39.  ANIMAL MODELS FOR OSTEOPOROSIS

[73]  Turner RT, Kidder LS, Kennedy A, Evans GL, Sibonga JD. Moderate alcohol consumption suppresses bone turnover in adult female rats. J Bone Miner Res 2001;16(3):589–94. [74]  Sampson HW, Gallager S, Lange J, Chondra W, Hogan HA. Binge drinking and bone metabolism in a young actively growing rat model. Alcohol Clin Exp Res 1999;23(7):1228–31. [75]  Callaci JJ, Juknelis D, Patwardhan A, Wezeman FH. Binge alcohol treatment increases vertebral bone loss following ovariectomy: compensation by intermittent parathyroid hormone. Alcohol Clin Exp Res 2006;30(4):665–72. [76]  Sibonga JD, Iwaniec UT, Shogren KL, Rosen CJ, Turner RT. Effects of parathyroid hormone (1-34) on tibia in an adult rat model for chronic alcohol abuse. Bone 2007;41:175–80. [77]  Williams FM, Cherkas LF, Spector TD, MacGregor AJ. The effect of moderate alcohol consumption on bone mineral density: a study of female twins. Ann Rheum Dis 2005;64(2): 309–10. [78]  Rapuri PB, Gallagher JC, Balhorn KE, Ryschon KL. Alcohol intake and bone metabolism in elderly women. Am J Clin Nutr 2000;72(5):1206–13. [79]  Cawthon PM, Harrison SL, Barrett-Connor E, Fink HA, Cauley JA, Lewis CE, et al. Alcohol intake and its relationship with bone mineral density, falls, and fracture risk in older men. J Am Geriatr Soc 2006;54(11):1649–57. [80]  Sampson HW, Shipley D. Moderate alcohol consumption does not augment bone density in ovariectomized rats. Alcohol Clin Exp Res 1997;21(7):1165–8. [81]  Wang L, Banu J, McMahan CA, Kalu DN. Male rodent model of age-related bone loss in men. Bone 2001;29(2):141–8. [82]  Wronski TJ, Dann LM, Scott KS, Cintron M. Long-term effects of ovariectomy and aging on the rat skeleton. Calcif Tissue Int 1989;45(6):360–6. [83]  Sibonga JD, Zhang M, Ritman EL, Turner RT. Restoration of bone mass in the severely osteopenic senescent rat. J Gerontol A Biol Sci Med Sci 2000;55(2):B71–8. [84]  Fitzpatrick LA. Secondary causes of osteoporosis. Mayo Clin Proc 2002;77(5):453–68. [85]  Hertoghe T. The “multiple hormone deficiency” theory of aging: is human senescence caused mainly by multiple hormone deficiencies? Ann N Y Acad Sci 2005;1057:448–65. [86]  King CS, Weir EC, Gundberg CW, Fox J, Insogna KL. Effects of continuous glucocorticoid infusion on bone metabolism in the rat. Calcif Tissue Int 1996;59(3):184–91. [87]  Shen V, Birchman R, Liang XG, Wu DD, Lindsay R, Dempster DW. Prednisolone alone, or in combination with estrogen or dietary calcium deficiency or immobilization, inhibits bone formation but does not induce bone loss in mature rats. Bone 1997;21(4):345–51. [88]  Jowell PS, Epstein S, Fallon MD, Reinhardt TA, Ismail F. 1,25-dihydroxyvitamin D3 modulates glucocorticoid-induced alteration in serum bone Gla protein and bone histomorphometry. Endocrinology 1987;120(2):531–6. [89]  Liao JM, Zhu QA, Lu HJ, Li QN, Wu T, Huang LF. Effects of total coumarins of Cnidium monnieri on bone density and biomechanics of glucocorticoids-induced osteoporosis in rats. Zhongguo Yao Li Xue Bao 1997;18(6):519–21. [90]  Nitta T, Fukushima T, Nakamuta H, Koida M. Glucocorticoidinduced secondary osteopenia in female rats: a time course study as compared with ovariectomy-induced osteopenia and response to salmon calcitonin. Jpn J Pharmacol 1999;79(3): 379–86. [91]  Clowes JA, Riggs BL, Khosla S. The role of the immune system in the pathophysiology of osteoporosis. Immunol Rev 2005;208:207–27. [92]  Tamler R, Epstein S. Nonsteroid immune modulators and bone disease. Ann N Y Acad Sci 2006;1068:284–96.

[93]  Desimone DP, Greene VS, Hannon KS, Turner RT, Bell NH. Prostaglandin E2 administered by subcutaneous pellets causes local inflammation and systemic bone loss: a model for inflammationinduced bone disease. J Bone Miner Res 1993;8(5):625–34. [94]  Hanyu T, Chotanaphuti T, Arai K, Tanaka T, Takahashi HE. Histomorphometric assessment of bone changes in rats with type II collagen-induced arthritis. Bone 1999;24(5):485–90. [95]  Smith BJ, Lerner MR, Bu SY, Lucas EA, Hanas JS, Lightfoot SA, et al. Systemic bone loss and induction of coronary vessel disease in a rat model of chronic inflammation. Bone 2006;38(3):378–86. [96]  Khosla S, Melton 3rd LJ, Wermers RA, Crowson CS, O’Fallon W, Riggs B. Primary hyperparathyroidism and the risk of fracture: a population-based study. J Bone Miner Res 1999;14(10):1700–7. [97]  Eriksen EF. Primary hyperparathyroidism: lessons from bone histomorphometry. J Bone Miner Res 2002;17(Suppl. 2):N95–7. [98]  Miller PD. Treatment of osteoporosis in chronic kidney disease and end-stage renal disease. Curr Osteoporos Rep 2005;3(1): 5–12. [99]  Elder G. Pathophysiology and recent advances in the management of renal osteodystrophy. J Bone Miner Res 2002;17(12):2094–105. [100] Zhang Y, Lai WP, Wu CF, Favus MJ, Leung PC, Wong MS. Ovariectomy worsens secondary hyperparathyroidism in mature rats during low Ca diet. Am J Physiol Endocrinol Metab 2007;292:E723–31. [101] Miller MA, Chin J, Miller SC, Fox J. Disparate effects of mild, moderate, and severe secondary hyperparathyroidism on cancellous and cortical bone in rats with chronic renal insufficiency. Bone 1998;23(3):257–66. [102] Tamagaki K, Yuan Q, Ohkawa H, Imazeki I, Moriguchi Y, Imai N, et al. Severe hyperparathyroidism with bone abnormalities and metastatic calcification in rats with adenine-induced uraemia. Nephrol Dial Transplant 2006;21(3):651–9. [103] Hock JM, Fonneca J, Gunness-Hey M, Kemp BE, Martin TJ. Comparison of the anabolic effects of synthetic parathyroid hormone-related protein (PTHrP) 1-34 and PTH 1-34 on bone in rats. Endocrinology 1989;125:2022–7. [104] Liu CC, Kalu DN. Human parathyroid hormone-(1-34) prevents bone loss and augments bone formation in sexually mature ovariectomized rats. J Bone Miner Res 1990;5:973–82. [105] Shen V, Birchman R, Xu R, Otter M, Wu D, Lindsay R, et al. Effects of reciprocal treatment with estrogen and estrogen plus parathyroid hormone on bone structure and strength in ovariectomized rats. J Clin Invest 1995;96:2331–8. [106] Rosen CJ. What’s new with PTH in osteoporosis: where are we and where are we headed? Trends Endocrinol Metab 2004;15:229–33. [107] Harper KD, Krege JH, Marcus R, Mitlak BH. Osteosarcoma and teriparatide? J Bone Miner Res 2007;22:334. [108] Vahle JL, Sato M, Long GG, Young JK, Francis PC, Engelhardt JA, et al. Skeletal changes in rats given daily subcutaneous injections of recombinant human parathyroid hormone (1-34) for 2 years and relevance to human safety. Toxicol Pathol 2002;30:312–21. [109] Turner RT, Evans GL, Lotinun S, Lapke PD, Iwaniec UT, Morey-Holton E. Dose-response effects of intermittent PTH on cancellous bone in hindlimb unloaded rats. J Bone Miner Res 2007;22:64–71. [110] Adkins RM, Gelke EL, Rowe D, Honeycutt RL. Molecular phylogeny and divergence time estimates for major rodent groups: evidence from multiple genes. Mol Biol Evol 2001;18(5):777–91. [111] Springer MS, Murphy WJ, Eizirik E, O’Brien SJ. Placental mammal diversification and the Cretaceous-Tertiary boundary. Proc Natl Acad Sci U S A 2003;100(3):1056–61. [112] Beamer WG, Donahue LR, Rosen CJ, Baylink DJ. Genetic variability in adult bone density among inbred strains of mice. Bone 1996;18(5):397–403.

VI.  GENERAL PATHOPHYSIOLOGY OF OSTEOPOROSIS

Summary

[113] Forlino A, Piazza R, Tiveron C, Della Torre S, Tatangelo L, Bonafe L, et al. A diastrophic dysplasia sulfate transporter (SLC26A2) mutant mouse: morphological and biochemical characterization of the resulting chondrodysplasia phenotype. Hum Mol Genet 2005;14(6):859–71. [114] Gong Y, Slee RB, Fukai N, Rawadi G, Roman-Roman S, Reginato AM, et al. LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 2001;107(4):513–23. [115] Kuivaniemi H, Tromp G, Prockop DJ. Mutations in collagen genes: causes of rare and some common diseases in humans. FASEB J 1991;5(7):2052–60. [116] Lazner F, Gowen M, Pavasovic D, Kola I. Osteopetrosis and osteoporosis: two sides of the same coin. Hum Mol Genet 1999;8(10):1839–46. [117] Clark S, Rowe DW. Transgenic animals. In: Bilezikian JP, editor. Principles of Bone Biology. San Diego: Academic Press; 1996. [118] Modder UI, Sanyal A, Kearns AE, Sibonga JD, Nishihara E, Xu J, et al. Effects of loss of steroid receptor coactivator-1 on the skeletal response to estrogen in mice. Endocrinology 2004;145(2): 913–21. [119] Sims NA, Clement-Lacroix P, Minet D, Fraslon-Vanhulle C, Gaillard-Kelly M, Resche-Rigon M, et al. A functional androgen receptor is not sufficient to allow estradiol to protect bone after gonadectomy in estradiol receptor-deficient mice. J Clin Invest 2003;111(9):1319–27. [120] Usui M, Yoshida Y, Tsuji K, Oikawa K, Miyazono K, Ishikawa I, et al. Tob deficiency super enhances osteoblastic activity after ovariectomy to block estrogen deficiency-induced osteoporosis. Proc Natl Acad Sci U S A 2004;101(17):6653–8. [121] Yoshitake H, Rittling SR, Denhardt DT, Noda M. Osteopontindeficient mice are resistant to ovariectomy-induced bone resorption. Proc Natl Acad Sci U S A 1999;96(14):8156–60. [122] Bouxsein ML, Myers KS, Shultz KL, Donahue LR, Rosen CJ, Beamer WG. Ovariectomy-induced bone loss varies among inbred strains of mice. J Bone Miner Res 2005;20(7):1085–92. [123] Iwaniec UT, Yuan D, Power RA, Wronski TJ. Strain-dependent variations in the response of cancellous bone to ovariectomy in mice. J Bone Miner Res 2006;21(7):1068–74. [124] Li CY, Schaffler MB, Wolde-Semait HT, Hernandez CJ, Jepsen KJ. Genetic background influences cortical bone response to ovariectomy. J Bone Miner Res 2005;20(12):2150–8. [125] Turner RT. Mice, estrogen, and postmenopausal osteoporosis. J Bone Miner Res 1999;14(2):187–91. [126] Crandall TL, Joyce RA, Boggs DR. Estrogens and hematopoiesis: characterization and studies on the mechanism of neutropenia. J Lab Clin Med 1980;95(6):857–67. [127] Iwaniec UT, Wronski TJ, Amblard D, Nishimura Y, van der Meulen MC, Wade CE, et al. Effects of disrupted beta1-integrin function on the skeletal response to short-term hindlimb unloading in mice. J Appl Physiol 2005;98(2):690–6. [128] Sakai A, Mori T, Sakuma-Zenke M, Takeda T, Nakai K, Katae Y, et al. Osteoclast development in immobilized bone is suppressed by parathyroidectomy in mice. J Bone Miner Metab 2005;23(1):8–14. [129] Sawakami K, Robling AG, Ai M, Pitner ND, Liu D, Warden SJ, et al. The Wnt co-receptor LRP5 is essential for skeletal mechanotransduction but not for the anabolic bone response to parathyroid hormone treatment. J Biol Chem 2006;281(33): 23698–711. [130] Hamrick MW, Ding KH, Pennington C, Chao YJ, Wu YD, Howard B, et al. Age-related loss of muscle mass and bone strength in mice is associated with a decline in physical activity and serum leptin. Bone 2006;39:845–53. [131] Silva MJ, Brodt MD, Uthgenannt BA. Morphological and mechanical properties of caudal vertebrae in the SAMP6 mouse model of senile osteoporosis. Bone 2004;35(2):425–31.

959

[132] Weinstein RS, Chen JR, Powers CC, Stewart SA, Landes RD, Bellido T, et al. Promotion of osteoclast survival and antagonism of bisphosphonate-induced osteoclast apoptosis by glucocorticoids. J Clin Invest 2002;109(8):1041–8. [133] Weinstein RS, Jia D, Powers CC, Stewart SA, Jilka RL, Parfitt AM, et al. The skeletal effects of glucocorticoid excess override those of orchidectomy in mice. Endocrinology 2004;145(4):1980–7. [134] McLaughlin F, Mackintosh J, Hayes BP, McLaren A, Uings IJ, Salmon P, et al. Glucocorticoid-induced osteopenia in the mouse as assessed by histomorphometry, microcomputed tomography, and biochemical markers. Bone 2002;30(6):924–30. [135] Rooman R, Koster G, Bloemen R, Gresnigt R, van Buul-Offers SC. The effect of dexamethasone on body and organ growth of normal and IGF-II-transgenic mice. J Endocrinol 1999;163(3): 543–52. [136] Ferrari SL, Pierroz DD, Glatt V, Goddard DS, Bianchi EN, Lin FT, et al. Bone response to intermittent parathyroid hormone is altered in mice null for {beta}-Arrestin2. Endocrinology 2005;146(4):1854–62. [137] Iida-Klein A, Lu SS, Kapadia R, Burkhart M, Moreno A, Dempster DW, et al. Short-term continuous infusion of human parathyroid hormone 1–34 fragment is catabolic with decreased trabecular connectivity density accompanied by hypercalcemia in C57BL/ J6 mice. J Endocrinol 2005;186(3):549–57. [138] Tanaka S, Sakai A, Tanaka M, Otomo H, Okimoto N, Sakata T, et al. Skeletal unloading alleviates the anabolic action of intermittent PTH(1-34) in mouse tibia in association with inhibition of PTH-induced increase in c-fos mRNA in bone marrow cells. J Bone Miner Res 2004;19(11):1813–20. [139] Iwaniec UT, Wronski TJ, Liu J, Rivera MF, Arzaga RR, Hansen G, et al. Parathyroid hormone stimulates bone formation in mice deficient in LRP5. J Bone Miner Res 2006;22:394–402. [140] Lotinun S, Evans G, Zhang M, Turner RT. Induction of peritrabecular fibrosis following continuous infusion of PTH in mice requires mature mast cells. J Bone Miner Res 2006;21(Suppl. 1):S315. [141] Blair HC, Dong SS, Julian BA. Expression of stem cell factor by osteoblasts in normal and hyperparathyroid bone: relation to ectopic mast cell differentiation. Virchows Arch 1999;435(1):50–7. [142] Lotinun S, Sibonga JD, Turner RT. Triazolopyrimidine (Trapidil), a platelet-derived growth factor antagonist, inhibits parathyroid bone disease in an animal model for chronic hyperparathyroidism. Endocrinology 2003;144(5):2000–7. [143] Moyen BJ, Lahey Jr PJ, Weinberg EH, Harris WH. Effects on intact femora of dogs of the application and removal of metal plates. A metabolic and structural study comparing stiffer and more flexible plates. J Bone Joint Surg Am 1978;60(7):940–7. [144] Kimmell DB. Animal models for in vivo experimentation in osteoporosis research. In: Marcus R, Feldman D, Kelsey J, editors. Osteoporosis. San Diego: Academic Press; 1996. [145] Geddes AD. Animal models of bone disuse. In: Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of Bone Biology. San Diego: Academic Press; 1996. p. 671–90. [146] Pope NS, Gould KG, Anderson DC, Mann DR. Effects of age and sex on bone density in the rhesus monkey. Bone 1989;10(2): 109–12. [147] Young DR, Niklowitz WJ, Brown RJ, Jee WS. Immobilizationassociated osteoporosis in primates. Bone 1986;7(2):109–17. [148] Binkley N, Kimmel D, Bruner J, Haffa A, Davidowitz B, Meng C, et al. Zoledronate prevents the development of absolute osteopenia following ovariectomy in adult rhesus monkeys. J Bone Miner Res 1998;13(11):1775–82. [149] Leeming DJ, Alexandersen P, Karsdal MA, Qvist P, Schaller S, Tanko LB. An update on biomarkers of bone turnover and their utility in biomedical research and clinical practice. Eur J Clin Pharmacol 2006;62(10):781–92.

VI.  GENERAL PATHOPHYSIOLOGY OF OSTEOPOROSIS

960

39.  ANIMAL MODELS FOR OSTEOPOROSIS

[150] Lane NE, Haupt D, Kimmel DB, Modin G, Kinney JH. Early estrogen replacement therapy reverses the rapid loss of trabecular bone volume and prevents further deterioration of connectivity in the rat. J Bone Miner Res 1999;14(2):206–14. [151] Heissig B, Rafii S, Akiyama H, Ohki Y, Sato Y, Rafael T, et al. Low-dose irradiation promotes tissue revascularization through VEGF release from mast cells and MMP-9-mediated progenitor cell mobilization. J Exp Med 2005;202(6):739–50. [152] Li W, Wang G, Cui J, Xue L, Cai L. Low-dose radiation (LDR) induces hematopoietic hormesis: LDR-induced mobilization of hematopoietic progenitor cells into peripheral blood circulation. Exp Hematol 2004;32(11):1088–96. [153] Nothdurft W, Kreja L. Hemopoietic progenitor cells in the blood as indicators of the functional status of the bone marrow after total-body and partial-body irradiation: experiences from studies in dogs. Stem Cells 1998;16(Suppl. 1):97–111. [154] Borah B, Dufresne TE, Ritman EL, Jorgensen SM, Liu S, Chmielewski PA, et al. Long-term risedronate treatment normalizes mineralization and continues to preserve trabecular architecture: sequential triple biopsy studies with microcomputed tomography. Bone 2006;39(2):345–52. [155] Ritman EL, Bolander ME, Fitzpatrick LA, Turner RT. Micro-CT imaging of structure-to-function relationship of bone microstructure and associated vascular involvement. Technol Health Care 1998;6(5–6):403–12. [156] Tzaphlidou M, Speller R, Royle G, Griffiths J. Preliminary estimates of the calcium/phosphorus ratio at different cortical bone sites using synchrotron microCT. Phys Med Biol 2006;51(7):1849–55. [157] Laib A, Barou O, Vico L, Lafage-Proust MH, Alexandre C, Rugsegger P. 3D micro-computed tomography of trabecular and cortical bone architecture with application to a rat model of immobilisation osteoporosis. Med Biol Eng Comput 2000;38(3): 326–32. [158] Laib A, Kumer JL, Majumdar S, Lane NE. The temporal changes of trabecular architecture in ovariectomized rats assessed by microCT. Osteoporos Int 2001;12(11):936–41. [159] Jaecques SV, Van Oosterwyck H, Muraru L, Van Cleynenbreugel T, De Smet E, Wevers M, et al. Individualised, micro CT-based finite element modelling as a tool for biomechanical analysis related to tissue engineering of bone. Biomaterials 2004;25(9): 1683–96. [160] Maran A, Khosla S, Riggs BL, Zhang M, Ritman EL, Turner RT. Measurement of gene expression following cryogenic mu-CT scanning of human iliac crest biopsies. J Musculoskelet Neuronal Interact 2003;3(1):83–8. [161] Muller R, Gerber SC, Hayes WC. Micro-compression: a novel technique for the nondestructive assessment of local bone failure. Technol Health Care 1998;6(5–6):433–44. [162] Fitzpatrick LA, Turner RT, Ritman ER. Endochondral bone formation in the heart: a possible mechanism of coronary calcification. Endocrinology 2003;144(6):2214–9. [163] Den Buijs JO, Bajzer Z, Ritman EL. Branching morphology of the rat hepatic portal vein tree: a micro-CT study. Ann Biomed Eng 2006;34(9):1420–8. [164] Marxen M, Paget C, Yu LX, Henkelman RM. Estimating perfusion using microCT to locate microspheres. Phys Med Biol 2006;51(1):N9–16. [165] Ritman E, Jorgensen S, Beighley P, Thomas P, Dunsmuir J, Romero J, et al. Synchrotron-based micro-CT of in situ biological basic functional units and their integration. Proc SPIE Dev X-ray Tomogr 1997;3149:13–24. [166] Hara T, Tanck E, Homminga J, Huiskes R. The influence of microcomputed tomography threshold variations on the assessment of structural and mechanical trabecular bone properties. Bone 2002;31(1):107–9.

[167] Wronski TJ, Morey ER. Skeletal abnormalities in rats induced by simulated weightlessness. Metab Bone Dis Relat Res 1982; 4(1):69–75. [168] Cavolina JM, Evans GL, Harris SA, Zhang M, Westerlind KC, Turner RT. The effects of orbital spaceflight on bone histomorphometry and messenger ribonucleic acid levels for bone matrix proteins and skeletal signaling peptides in ovariectomized growing rats. Endocrinology 1997;138(4):1567–76. [169] Gluer CC. Monitoring skeletal changes by radiological techniques. J Bone Miner Res 1999;14(11):1952–62. [170] Rosen HN, Tollin S, Balena R, Middlebrooks VL, Beamer WG, Donohue LR, et al. Differentiating between orchiectomized rats and controls using measurements of trabecular bone density: a comparison among DXA, histomorphometry, and peripheral quantitative computerized tomography. Calcif Tissue Int 1995;57(1):35–9. [171] Hamrick MW, Della-Fera MA, Choi YH, Pennington C, Hartzell D, Baile CA. Leptin treatment induces loss of bone marrow adipocytes and increases bone formation in leptin-deficient ob/ob mice. J Bone Miner Res 2005;20(6):994–1001. [172] Steppan CM, Crawford DT, Chidsey-Frink KL, Ke H, Swick AG. Leptin is a potent stimulator of bone growth in ob/ob mice. Regul Pept 2000;92(1–3):73–8. [173] Ducy P, Amling M, Takeda S, Priemel M, Schilling AF, Beil FT, et al. Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell 2000;100(2):197–207. [174] Blumer MJ, Schwarzer C, Perez MT, Konakci KZ, Fritsch H. Identification and location of bone-forming cells within cartilage canals on their course into the secondary ossification centre. J Anat 2006;208(6):695–707. [175] Min H, Chen CZ. Methods for analyzing microRNA expression and function during hematopoietic lineage differentiation. Methods Mol Biol 2006;342:209–27. [176] Nomura S, Takano-Yamamoto T. Molecular events caused by mechanical stress in bone. Matrix Biol 2000;19(2):91–6. [177] Barou O, Palle S, Vico L, Alexandre C, Lafage-Proust MH. Hindlimb unloading in rat decreases preosteoblast proliferation assessed in vivo with BrDU incorporation. Am J Physiol 1998;274(1 Pt 1):E108–14. [178] Turner CH, Owan I, Alvey T, Hulman J, Hock JM. Recruitment and proliferative responses of osteoblasts after mechanical loading in vivo determined using sustained-release bromodeoxyuridine. Bone 1998;22(5):463–9. [179] Zaman G, Jessop HL, Muzylak M, De Souza RL, Pitsillides AA, Price JS, et al. Osteocytes use estrogen receptor alpha to respond to strain but their ERalpha content is regulated by estrogen. J Bone Miner Res 2006;21(8):1297–306. [180] Szczepanski T, van der Velden VH, van Dongen JJ. Flowcytometric immunophenotyping of normal and malignant lymphocytes. Clin Chem Lab Med 2006;44(7):775–96. [181] Copland JA, Davies PJ, Shipley GL, Wood CG, Luxon BA, Urban RJ. The use of DNA microarrays to assess clinical samples: the transition from bedside to bench to bedside. Recent Prog Horm Res 2003;58:25–53. [182] Cho CH, Nuttall ME. Emerging techniques for the discovery and validation of therapeutic targets for skeletal diseases. Expert Opin Ther Targets 2002;6(6):679–89. [183] Young MF. Bone matrix proteins: their function, regulation, and relationship to osteoporosis. Osteoporos Int 2003;14(Suppl. 3):S35–42. [184] Sogaard CH, Wronski TJ, McOsker JE, Mosekilde L. The positive effect of parathyroid hormone on femoral neck bone strength in ovariectomized rats is more pronounced than that of estrogen or bisphosphonates. Endocrinology 1994;134(2):650–7. [185] Barnes GL, Kostenuik PJ, Gerstenfeld LC, Einhorn TA. Growth factor regulation of fracture repair. J Bone Miner Res 1999;14(11):1805–15.

VI.  GENERAL PATHOPHYSIOLOGY OF OSTEOPOROSIS

Summary

[186] Roy DK, Pye SR, Lunt M, O’Neill TW, Todd C, Raspe H, et al. Falls explain between-center differences in the incidence of limb fracture across Europe. Bone 2002;31(6):712–7. [187] Hayes WC, Myers ER. Biomechanical considerations of hip and spine fractures in osteoporotic bone. Instr Course Lect 1997;46:431–8. [188] Robinovitch SN, Hayes WC, McMahon TA. Distribution of contact force during impact to the hip. Ann Biomed Eng 1997;25(3):499–508.

961

[189] Schwartz AV, Nevitt MC, Brown Jr BW, Kelsey JL. Increased falling as a risk factor for fracture among older women: the study of osteoporotic fractures. Am J Epidemiol 2005;161(2): 180–5. [190] Wainwright SA, Marshall LM, Ensrud KE, Cauley JA, Black DM, Hillier TA, et al. Hip fracture in women without osteoporosis. J Clin Endocrinol Metab 2005;90(5):2787–93.

VI.  GENERAL PATHOPHYSIOLOGY OF OSTEOPOROSIS