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Bone Histomorphometry in Renal Osteodystrophy
Bone Histomorphometry in Renal Osteodystrophy Susan M. Ott, MD Summary: On bone biopsies from patients with chronic kidney disease, measurements are made of the turnover, mineralization, and volume. Turnover depends on the bone formation rate and bone resorption rate; the former can be measured using tetracycline labelling. The osteoid width and bone apposition rate determine the mineralization rates. Bone volume includes both mineralized and unmineralized bone and is directly related to the porosity. Using these measurements, biopsies can be separated into the classic types of renal osteodystrophy: normal, adynamic, high-turnover, mixed, and osteomalacia. Fracture rates among these types are not consistent, but several studies have found high fracture rates with adynamic or osteomalacia. The bone density tests cannot distinguish between different types of bone histology. Semin Nephrol 29:122-132 © 2009 Published by Elsevier Inc. Keywords: CKD, renal osteodystrophy, Improving Global Outcomes , turnover, mineralization, volume (TMV), osteoclasts, osteoblasts, osteocytes
he kidney plays a critical role in the complex regulation of mineral metabolism, so it is not surprising that chronic kidney disease (CKD) causes bone disorders. Multiple factors influence the bone in patients with CKD, resulting in a variety of abnormalities seen on bone biopsies (Fig. 1). It is appropriate to discuss the microscopic appearance in a journal issue about the spectrum of renal osteodystrophy, especially because the derivation of the term is from the Latin specere, meaning “to look.” A spectrum suggests that something can be classified in terms of its position on a scale between 2 opposite points. This aspect of renal osteodystrophy applies to the bone formation rate. Superimposed on this spectrum are variations in the degree of mineralization of the bone matrix and the bone volume per bone tissue, which combined determine the bone density. An international committee sponsored by Kidney Disease: Improving Global Outcomes concurred that the most important pa-
T
Department of Medicine, University of Washington Medical Center, Seattle, WA. Address reprint requests to Susan M. Ott, MD, Associate Professor, Department of Medicine, University of Washington Medical Center, Box 356426, 1959 Northeast Pacific St, Seattle, WA 98195-6426. E-mail: smott@u. washington.edu 0270-9295/09/$ - see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.semnephrol.2009.01.005
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rameters to describe renal osteodystrophy are turnover, mineralization, and volume (TMV).1 Thus, instead of a 1-dimensional axis, the TMV system defines a more comprehensive 3-dimensional space. This article focuses on the new TMV classification. OVERVIEW OF BONE REMODELING Healthy bone is a dynamic tissue, continually resorbing bone and replacing it with new bone in discrete areas know as basic multicellular units, also called bone metabolic units (BMUs).2 A BMU is not a permanent structure. It forms in response to signal or stimulus, performs its function, and disbands, leaving a few residual lining cells and osteocytes. Each BMU undergoes its functions in the same sequence: origination and organization of the BMU, activation of osteoclasts, resorption of old bone, recruitment of osteoblasts, formation of new bone matrix, and mineralization. In solid cortical bone, a BMU drills a tunnel and then refills it. On the cancellous bone surface the BMU can channel like a river flowing, or can spread over the surface like warm fudge flowing over ice cream. The lifespan of a BMU is not well defined. Cortical BMUs can wander for months,
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Figure 1. Pathophysiologic pathways resulting in abnormal bone turnover. CKD directly results in decreased bone morphogenic protein (BMP 7), Klotho, 1,25-dihydroxyvitamin D, serum calcium, and acid, as well as increased serum phosphate and oxalate. Malnutrition, hypogonadism, corticosteroid use, and heparin treatment frequently are associated with CKD. Accumulation of metals occurs in those who are exposed. The dashed lines indicate feedback loops that are disrupted because of the kidney damage. Secondary changes are seen in several hormones, including PTH, fibroblast growth factor 23 (FGF-23) (the newly defined hormone secreted from bone cells in response to high phosphate), and insulin-like growth factor. These factors have differing and sometimes competing effects on the bone formation rate and the bone resorption rate.
usually in a straight line. Parfitt3 estimates the duration is 2 to 8 months. This is harder to measure in cancellous bone because the 2-dimensional sections do not capture the entire serpentine course of the BMU. Thus, the turnover of bone is different from the turnover on the skin, where the entire surface continuously is forming skin and shedding it. Some BMUs originate when the bone has been damaged; others may originate at random surfaces on the bone, under the influence of local or systemic hormones. The initiating events in a BMU cannot be detected by histomorphometry. Marrow stromal cells near the bone are under tonic inhibition by sclerostin from the interior osteocytes, which detect the
need for new bone formation.4 The osteocytes then stop secreting the sclerostin and start secreting other factors such as prostaglandins, nitric oxide, and growth factors. The stromal cells respond by secreting macrophage colonystimulating factor, which helps promote preosteoclasts. Under the appropriate conditions, the stromal cells generate pre-osteoblasts, which express receptor activator of nuclear factor (NF)kappaB (RANK) ligand on the cell surface. The pre-osteoclasts have RANK receptors, and when these are activated they fuse and form mature osteoclasts that will resorb bone. These eroded surfaces are the first events detected by light microscopy. At a given spot on the bone surface, resorption is rapid for the first 10 days,
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and continues for about a month. The osteoclasts undergo apoptosis; the lifespan and activity of the osteoclasts determine the depth of the resorption cavity. Meanwhile, the pre-osteoblasts that were generated by the marrow stem cells have proliferated, and when the osteoclasts retire the osteoblasts line the cavity. The team of osteoblasts forms a matrix, and after about 14 days directs mineralization of the matrix. The osteoblasts continue to form and to mineralize osteoid until the cavity is filled or nearly filled. The time to fill in the cavity at any given place on the surface is 124 to 168 days in normal individuals.5 After formation is concluded, that place on the surface is quiescent. The duration of time for the cycle on one point of the surface is called the total period (resorbing plus forming plus quiescent periods), which varies from 2 to 5 years. This period should not be confused with the lifespan of the BMU, which also would include the entire area covered by the BMU. The inverse of the total period is the activation frequency. After restoration of the bone volume, the newly formed bone undergoes further mineralization for about 3 years. Older bone has more densely packed crystals, and microradiographic studies have shown that newly formed bone may be 25% less dense than older bone.6 A dynamic review of bone remodeling is available online.7
determined by the bone resorption rate, and the bone balance would be positive. This unusual condition could be seen, for example, during recovery from lactation or treatment with an anabolic medication. During growth, the bone is modeling as well as remodeling, and the concept of turnover would apply only to that bone that is remodeling. Therefore, bone formation rate (BFR) and bone turnover are not the same, and either low turnover or high turnover can be associated with loss of bone volume and reduction in bone strength. Because bone resorption usually is similar to or greater than bone formation, the bone turnover rate is usually the bone formation rate, but it is important to remember that this is not always the case, and that the rates of both formation and resorption are necessary to describe the bone physiologic picture.
Definition of Turnover
Measurement of Bone Formation Rate
Although the term bone turnover is used frequently, it is not clearly defined. If the bone resorption rate and bone formation rate were equal, then the turnover could be defined as either rate. The bone volume would not be changing, and the bone balance would therefore be zero. This situation is generally seen in healthy young adults. If the bone resorption rate is greater than the formation rate, because the resorbed cavities are not completely filled with new bone, then the turnover would be reflected by the bone formation rate, and the bone balance would be negative. This is seen frequently with aging and postmenopausal osteoporosis. On the other hand, if the bone formation rate is greater than the bone resorption rate in an adult skeleton, the turnover would be
Tetracycline will deposit in the bone where calcium is being deposited, and can be seen under a fluorescent microscope (Fig. 2). A small dose will therefore label the surface of bone that is actively mineralizing. The BFR can be directly measured with 2 labels administered about 10 days apart. The length of the tetracycline labels multiplied by the distance between the labels is the area of new bone formed during the label interval. In other words, the rate of new bone formation depends both on the rate of apposition at the surface and the total surface involved in forming bone. The BFR can be expressed in reference to the bone surface, bone volume, or tissue volume. The appropriate referent depends on the situation. The most easily understood concept of turnover is the BFR as a
TURNOVER IN RENAL OSTEODYSTROPHY In most cases of renal osteodystrophy, those with high bone formation rates also have high resorption rates, and those with adynamic bone disease have low rates of both formation and resorption. Thus, the bone formation indices are used to describe turnover because they can be measured with much more accuracy than the bone resorption rate.
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Figure 2. Tetracycline labeling. (A) Adynamic bone without label, (B) normal labels.
percentage per year of the bone volume. The absolute amount of bone formed (BFR per tissue volume) theoretically would provide the best correlation with a serum marker of bone formation. The tetracycline labels usually are seen clearly and measurements are straightforward. Sometimes the edges taper gradually into the inactive bone surface, making the exact end of the label indefinite, but this causes only minor measurement difficulties. More commonly, when there is very rapid bone formation, the labels become blurry and diffuse, making them difficult to measure. Conversely, when the bone is forming very slowly the labels do not show separation and it is difficult to tell if a label is a double label or a single label. The use of 2 different kinds of tetracycline (demeclocycline and tetracycline) is helpful in these cases because they have different colors when viewed using fluorescent microscopy.
Activation Frequency The activation frequency is related to the BFR, but it is not a BFR because the activation frequency also depends on the amount of bone formed during each remodeling cycle. This amount of bone formed during an average remodeling cycle is represented by the wall thickness, which is the thickness of new bone made in one cycle. Activation frequency is the BFR divided by the wall thickness. Activation frequency can increase even though the BFR is unchanged, merely from a decrease in wall thickness. With aging, the activation frequency increases; this is owing to a combination of increased bone formation and decreased wall thickness.8 Some people think that an increased activation frequency causes bone loss. They confuse bone balance with bone turnover. It is the imbalance between resorption and formation that causes osteoporosis, not the increased activa-
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tion frequency. There is a relationship between activation frequency and bone loss, however, because if each BMU is in negative balance, then a greater number of active BMUs will result in a greater bone loss. The activation frequency also is confused with the frequency of originating new BMUs (BMU birthrate). Activation frequency refers to just one spot on the bone surface. When a traveling BMU reaches a spot, that surface becomes active. The birthrate of new BMUs depends on how long they live. A bone with many short-lived BMUs can have the same activation frequency, wall thickness, and BFRs as a bone with a few long-lived BMUs. The concept of BMU lifespan is simple to understand but cannot be measured on a 2-dimensional section of bone because BMUs wander in and out of the plane of the section. In bone biopsies from patients with CKD, it is often difficult to measure the wall thickness. This must be measured on a quiescent surface, and many patients with hyperparathyroidism
have limited, if any, quiescent surfaces. The measurement depends on identifying a change in the orientation of lamella, but patients with CKD often have abnormal woven bone. When the BFR is very low, the completed BMUs may remain for years, and thus measurements do not reflect the current physiology. For these reasons, many histomorphometrists do not calculate the wall thickness or activation frequency in patients with CKD.
Other Measurements Related to Bone Turnover Other measurements that relate to the formation aspects of bone turnover include the osteoblastic surface, the number of active osteoblasts, and the osteoid surface. None of these gives the same accurate information as the tetracycline labeling. Bone resorption is related to the number of osteoclasts and the depth of resorption cavities, but there is no direct way to
Figure 3. Example of osteomalacia.
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calculate the bone resorption rate. Some formulas that have been proposed require an assumption that the bone is in balance, which is not valid in patients with CKD. The amount of marrow fibrosis is related to high parathyroid hormone (PTH) levels, which usually increase the bone formation and resorption rates. High PTH levels also cause blurry tetracycline labels, woven bone, and intratrabecular tunneling. Some histologic findings in bone document causes of bone disease, such as aluminum, amyloid, or iron deposition. Other tests not routinely performed include the number and condition of osteocytes, number of apoptotic cells, the structural microarchitecture, the cortical porosity, the mineralization density, the mechanical stiffness of the bone material, the volume of the fat cells, the thickness of the lamella, and the characteristics of the tetracycline labels. Immunohistochemical measurements of bone proteins may be used in the future. Serum biochemical markers of bone formation and resorption potentially could be very
helpful in determining these rates. They have not yet been validated in patients with CKD. In osteoporotic patients, the markers relate to BFRs determined by tetracycline in some situations but not in others, and there is considerable variability. More research is needed. MINERALIZATION IN RENAL OSTEODYSTROPHY The second axis is mineralization, which reflects the amount of unmineralized osteoid. The classic disease with an abnormality of mineralization is osteomalacia, in which the BFR is low and osteoid volume is high. Some patients have excess osteoid, which is a result of high BFRs; they do not actually have a problem with the mineralization process. Patients with low BFRs and normal osteoid have adynamic disease (they do not even form the osteoid matrix, so they do not manifest a problem with mineralization). Mineralization is measured by the osteoid maturation time or the mineralization lag time, both of which depend heavily on the
Figure 4. Example of mixed lesion.
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osteoid width as well as the distance between tetracycline labels. After the osteoblasts lay down new collagen, they direct mineralization of the matrix. This is normally a regulated and orderly process, but in patients with CKD the mineralization can be delayed or disorganized. This results in thickened osteoid. Rapid bone formation also can result in thick osteoid, but in that case the tetracycline labels also are more widely separated. The osteoid maturation time is the osteoid width divided by the distance between labels per day. The mineralization lag time is the osteoid maturation time adjusted for the percentage of osteoid surface that has a tetracycline label. This adjustment, however, assumes that the osteoid surface without a label is in a resting phase, but there is no evidence to support a resting phase. Osteomalacia has been defined by different investigators as increased osteoid volume, increased osteoid maturation time, or increased mineralization lag time. Parfitt9 defined osteomalacia in patients with mal-
absorption if they had a combination of osteoid volume/bone volume greater than 10%, osteoid thickness greater than 12.5 m (note that the thickness is a measurement that requires a correction for section obliqueness, which is the width/1.2), and mineralization lag time greater than 100 days. There is currently no consensus about the exact definition of abnormal mineralization in patients with CKD. BONE VOLUME IN RENAL OSTEODYSTROPHY The final axis is bone volume, which has not been included in previous schemes of describing renal osteodystrophy. Bone volume contributes to bone fragility and is separate from the other parameters. The bone volume is the end result of changes in bone formation and resorption rates. If overall BFR is greater than overall bone resorption rate, the bone is in positive balance and the bone volume will increase. If mineralization remains constant, an increase in
Figure 5. Example of adynamic bone disease.
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Figure 6. Example of osteitis fibrosa.
bone volume also would result in an increase in bone mineral density and should be detectable by radiographic densitometry. Although both cortical and cancellous bone volume decrease in typical idiopathic osteoporosis, these compartments frequently are different in patients with CKD. For example, with high PTH levels, cortical bone volume is decreased but the cancellous volume is increased.10 The concept of bone volume is the easiest to understand. It is direct and reproducible to measure within a sample. There is a large error, however, from one biopsy to the next in the same person. From contiguous iliac crest biopsies there is an average of 29% difference.11 Bone volume is related to bone strength, but the same volume can have different microarchitecture. The trabecular thickness can be calculated from measurements of the bone surface per volume relationship, and this is an index related to bone strength. Other methods of measuring the architecture include the strut length, the star volume, and the number of nodes connecting
trabeculae. These 2-dimensional measurements are inferior to newer 3-dimensional measures of connectivity that are made with microcomputed tomography. There is consensus that bone volume is expressed as bone volume per tissue volume, which is the same as bone area per tissue area when expressed in 2 dimensions. This can be measured in cortical or in cancellous bone. RELATIONSHIP BETWEEN TMV SYSTEM AND TYPES OF RENAL OSTEODYSTROPHY Most studies of renal osteodystrophy have classified biopsies based on the BFR or presence of fibrosis, as well as the degree of mineralization (Figs. 3-6). Bone volume has not been used to place patients into categories. Thus, those with low turnover and normal mineralization have adynamic bone disease or low-turnover bone disease. Patients whose bone biopsies show high bone turn-
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over, particularly with other signs of high PTH, have high bone turnover or hyperparathyroid bone disease or osteitis fibrosa. Those with abnormal mineralization and low bone formation are osteomalacia, and those with abnormal mineralization and high bone formation have mixed disease (Fig. 7). There is no consensus on the exact measurements to define these categories. A large review of the prevalence of these types of renal osteodystrophy in hemodialysis patients showed osteitis fibrosis in 34%, mixed lesions in 32%, adynamic disease in 19%, osteomalacia in 10%, and normal or mild disease in 5%. Patients receiving peritoneal dialysis had adynamic bone disease more frequently (50%) with fewer cases of mixed
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lesion or osteitis fibrosis (Kidney Disease: Improving Global Outcomes guidelines, unpublished data).
NATURAL HISTORY The natural history of bone histomorphometry is variable. The placebo groups from randomized clinical trials12-19 and one longitudinal study20 show an overall trend toward worsening turnover (either getting too high or too low) and stable mineralization and volume. The wide variability in the natural history of bone histology reflects the complex pathophysiology of chronic kidney disease-mineral and bone disorder (CKD-MBD).
Figure 7. Relationship between TMV and classic types of renal osteodystrophy. The bone volume in this diagram is hypothetical; actual studies show variability in the bone volume for each type of disease.
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CLINICAL CORRELATIONS
In patients with advanced stages of CKD, the bone density does not predict fractures as well as in patients with normal renal function. This is probably owing to abnormalities in bone structure and quality that are common in CKD patients, as well as a greater likelihood of nonskeletal causes of fractures.
Fractures and Bone Histomorphometry Most of the studies of bone histomorphometry have not been designed to evaluate the relationship between fractures and types of renal osteodystrophy fully. One study of 31 dialysis patients found that those with low turnover osteodystrophy had a fracture rate of 0.2 per year compared with 0.1 per year in those with osteitis fibrosis; this was owing to a high number of rib fractures in the low turnover patients.21 A review of 2,340 biopsies performed in Brazilian patients for clinical indications found that the frequency of fractures was significantly higher in those with osteomalacia compared with other forms. There were no differences in fracture history between those with adynamic bone disease, high bone turnover, or mixed bone disease.22 A study that followed up 62 patients for 5 years after bone biopsy found a higher rate of fractures in those with adynamic bone disease.23
Bone Density The relationship between bone density and bone biopsy is not consistent. In patients with CKD, Lindergard10 measured bone volume/tissue volume on 71 biopsies from dialysis patients, and did not see a correlation with bone mineral density (BMD) at the radius. Similar results were seen by Gerakis et al23 in a study of 62 patients. Torres et al,24 on the other hand, found a correlation coefficient of 0.82 between bone volume/tissue volume and quantitative computed tomography of the spine, and Van Eps et al25 found lower dual-energy x-ray absorptiometry values in patients with low bone volume on biopsy. The relationship between BMD and type of renal osteodystrophy varies among reports. Four studies found similar BMD in all types.26-29 Lower BMD has been reported in both high and low bone turnover, with wide ranges.23,30,31 These findings emphasize that the older types of renal osteodystrophy do not provide enough information about bone strength. In addition, the radiographic tests cannot distinguish between unmineralized and mineralized bone.
SUMMARY Patients with CKD have complex abnormalities on bone biopsies, which change in inconsistent patterns. In addition to abnormally high or low BFRs, they show abnormal mineralization and/or bone volume. Patients throughout the spectrum have increased clinical fractures and bone pain, presenting challenges to physicians trying to improve the bone strength. REFERENCES 1. Moe S, Drueke T, Cunningham J, et al. Definition, evaluation, and classification of renal osteodystrophy: a position statement from Kidney Disease: Improving Global Outcomes (KDIGO). Kidney Int. 2006;69: 1945-53. 2. Ott S. Histomorphometric analysis of bone remodeling. In: Bilezikian J, Raisz L, Ga R, editors. Principles of bone biology. San Diego: Academic Press; 2002. p. 303-20. 3. Parfitt AM. Osteonal and hemi-osteonal remodeling: the spatial and temporal framework for signal traffic in adult human bone. J Cell Biochem. 1994;55:273-86. 4. Robling AG, Niziolek PJ, Baldridge LA, et al. Mechanical stimulation of bone in vivo reduces osteocyte expression of sost/sclerostin. J Biol Chem. 2008;283: 5866-75. 5. Eriksen EF, Gundersen HJG, Melsen F, Mosekilde L. Reconstruction of the formative site in iliac trabecular bone in 20 normal individuals employing a kinetic model for matrix and mineral apposition. Metab Bone Dis Rel Res. 1984;5:243-52. 6. Jowsey J. Age changes in human bone. Clin Orthop Rel Res. 1960;17:210-7. 7. Osteoporosis and Bone Physiology [cited 2009 Feb 5]. Available from: http://courses.washington. edu/bonephys. 8. Recker RR, Lappe JM, Davies KM, Heaney RP. Bone remodeling increases substantially in the years after menopause and remains increased in older osteoporosis patients. J Bone Miner Res. 2004;19:1628-33. 9. Parfitt AM. Osteomalacia and related disorders. In: Aviolo L, Krane SM, editors. Metabolic bone disease. Philadelphia: Saunders; 1990. p. 329-96. 10. Lindergard B, Johnell O, Nilsson BE, Wiklung PE. Studies of bone morphology, bone densitometry, and labora-
Bone Histomorphometry in Renal Osteodystrophy Susan M. Ott, MD Summary: On bone biopsies from patients with chronic kidney disease, measurements are made of the turnover, mineralization, and volume. Turnover depends on the bone formation rate and bone resorption rate; the former can be measured using tetracycline labelling. The osteoid width and bone apposition rate determine the mineralization rates. Bone volume includes both mineralized and unmineralized bone and is directly related to the porosity. Using these measurements, biopsies can be separated into the classic types of renal osteodystrophy: normal, adynamic, high-turnover, mixed, and osteomalacia. Fracture rates among these types are not consistent, but several studies have found high fracture rates with adynamic or osteomalacia. The bone density tests cannot distinguish between different types of bone histology. Semin Nephrol 29:122-132 © 2009 Published by Elsevier Inc. Keywords: CKD, renal osteodystrophy, Improving Global Outcomes , turnover, mineralization, volume (TMV), osteoclasts, osteoblasts, osteocytes
he kidney plays a critical role in the complex regulation of mineral metabolism, so it is not surprising that chronic kidney disease (CKD) causes bone disorders. Multiple factors influence the bone in patients with CKD, resulting in a variety of abnormalities seen on bone biopsies (Fig. 1). It is appropriate to discuss the microscopic appearance in a journal issue about the spectrum of renal osteodystrophy, especially because the derivation of the term is from the Latin specere, meaning “to look.” A spectrum suggests that something can be classified in terms of its position on a scale between 2 opposite points. This aspect of renal osteodystrophy applies to the bone formation rate. Superimposed on this spectrum are variations in the degree of mineralization of the bone matrix and the bone volume per bone tissue, which combined determine the bone density. An international committee sponsored by Kidney Disease: Improving Global Outcomes concurred that the most important pa-
T
Department of Medicine, University of Washington Medical Center, Seattle, WA. Address reprint requests to Susan M. Ott, MD, Associate Professor, Department of Medicine, University of Washington Medical Center, Box 356426, 1959 Northeast Pacific St, Seattle, WA 98195-6426. E-mail: smott@u. washington.edu 0270-9295/09/$ - see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.semnephrol.2009.01.005
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rameters to describe renal osteodystrophy are turnover, mineralization, and volume (TMV).1 Thus, instead of a 1-dimensional axis, the TMV system defines a more comprehensive 3-dimensional space. This article focuses on the new TMV classification. OVERVIEW OF BONE REMODELING Healthy bone is a dynamic tissue, continually resorbing bone and replacing it with new bone in discrete areas know as basic multicellular units, also called bone metabolic units (BMUs).2 A BMU is not a permanent structure. It forms in response to signal or stimulus, performs its function, and disbands, leaving a few residual lining cells and osteocytes. Each BMU undergoes its functions in the same sequence: origination and organization of the BMU, activation of osteoclasts, resorption of old bone, recruitment of osteoblasts, formation of new bone matrix, and mineralization. In solid cortical bone, a BMU drills a tunnel and then refills it. On the cancellous bone surface the BMU can channel like a river flowing, or can spread over the surface like warm fudge flowing over ice cream. The lifespan of a BMU is not well defined. Cortical BMUs can wander for months,
Seminars in Nephrology, Vol 29, No 2, March 2009, pp 122-132
he kidney plays a critical role in the complex regulation of mineral metabolism, so it is not surprising that chronic kidney disease (CKD) causes bone disorders. Multiple factors influence the bone in patients with CKD, resulting in a variety of abnormalities seen on bone biopsies (Fig. 1). It is appropriate to discuss the microscopic appearance in a journal issue about the spectrum of renal osteodystrophy, especially because the derivation of the term is from the Latin specere, meaning “to look.” A spectrum suggests that something can be classified in terms of its position on a scale between 2 opposite points. This aspect of renal osteodystrophy applies to the bone formation rate. Superimposed on this spectrum are variations in the degree of mineralization of the bone matrix and the bone volume per bone tissue, which combined determine the bone density. An international committee sponsored by Kidney Disease: Improving Global Outcomes concurred that the most important pa-
T
Department of Medicine, University of Washington Medical Center, Seattle, WA. Address reprint requests to Susan M. Ott, MD, Associate Professor, Department of Medicine, University of Washington Medical Center, Box 356426, 1959 Northeast Pacific St, Seattle, WA 98195-6426. E-mail: smott@u. washington.edu 0270-9295/09/$ - see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.semnephrol.2009.01.005
122
rameters to describe renal osteodystrophy are turnover, mineralization, and volume (TMV).1 Thus, instead of a 1-dimensional axis, the TMV system defines a more comprehensive 3-dimensional space. This article focuses on the new TMV classification. OVERVIEW OF BONE REMODELING Healthy bone is a dynamic tissue, continually resorbing bone and replacing it with new bone in discrete areas know as basic multicellular units, also called bone metabolic units (BMUs).2 A BMU is not a permanent structure. It forms in response to signal or stimulus, performs its function, and disbands, leaving a few residual lining cells and osteocytes. Each BMU undergoes its functions in the same sequence: origination and organization of the BMU, activation of osteoclasts, resorption of old bone, recruitment of osteoblasts, formation of new bone matrix, and mineralization. In solid cortical bone, a BMU drills a tunnel and then refills it. On the cancellous bone surface the BMU can channel like a river flowing, or can spread over the surface like warm fudge flowing over ice cream. The lifespan of a BMU is not well defined. Cortical BMUs can wander for months,
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Figure 1. Pathophysiologic pathways resulting in abnormal bone turnover. CKD directly results in decreased bone morphogenic protein (BMP 7), Klotho, 1,25-dihydroxyvitamin D, serum calcium, and acid, as well as increased serum phosphate and oxalate. Malnutrition, hypogonadism, corticosteroid use, and heparin treatment frequently are associated with CKD. Accumulation of metals occurs in those who are exposed. The dashed lines indicate feedback loops that are disrupted because of the kidney damage. Secondary changes are seen in several hormones, including PTH, fibroblast growth factor 23 (FGF-23) (the newly defined hormone secreted from bone cells in response to high phosphate), and insulin-like growth factor. These factors have differing and sometimes competing effects on the bone formation rate and the bone resorption rate.
usually in a straight line. Parfitt3 estimates the duration is 2 to 8 months. This is harder to measure in cancellous bone because the 2-dimensional sections do not capture the entire serpentine course of the BMU. Thus, the turnover of bone is different from the turnover on the skin, where the entire surface continuously is forming skin and shedding it. Some BMUs originate when the bone has been damaged; others may originate at random surfaces on the bone, under the influence of local or systemic hormones. The initiating events in a BMU cannot be detected by histomorphometry. Marrow stromal cells near the bone are under tonic inhibition by sclerostin from the interior osteocytes, which detect the
need for new bone formation.4 The osteocytes then stop secreting the sclerostin and start secreting other factors such as prostaglandins, nitric oxide, and growth factors. The stromal cells respond by secreting macrophage colonystimulating factor, which helps promote preosteoclasts. Under the appropriate conditions, the stromal cells generate pre-osteoblasts, which express receptor activator of nuclear factor (NF)kappaB (RANK) ligand on the cell surface. The pre-osteoclasts have RANK receptors, and when these are activated they fuse and form mature osteoclasts that will resorb bone. These eroded surfaces are the first events detected by light microscopy. At a given spot on the bone surface, resorption is rapid for the first 10 days,
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and continues for about a month. The osteoclasts undergo apoptosis; the lifespan and activity of the osteoclasts determine the depth of the resorption cavity. Meanwhile, the pre-osteoblasts that were generated by the marrow stem cells have proliferated, and when the osteoclasts retire the osteoblasts line the cavity. The team of osteoblasts forms a matrix, and after about 14 days directs mineralization of the matrix. The osteoblasts continue to form and to mineralize osteoid until the cavity is filled or nearly filled. The time to fill in the cavity at any given place on the surface is 124 to 168 days in normal individuals.5 After formation is concluded, that place on the surface is quiescent. The duration of time for the cycle on one point of the surface is called the total period (resorbing plus forming plus quiescent periods), which varies from 2 to 5 years. This period should not be confused with the lifespan of the BMU, which also would include the entire area covered by the BMU. The inverse of the total period is the activation frequency. After restoration of the bone volume, the newly formed bone undergoes further mineralization for about 3 years. Older bone has more densely packed crystals, and microradiographic studies have shown that newly formed bone may be 25% less dense than older bone.6 A dynamic review of bone remodeling is available online.7
determined by the bone resorption rate, and the bone balance would be positive. This unusual condition could be seen, for example, during recovery from lactation or treatment with an anabolic medication. During growth, the bone is modeling as well as remodeling, and the concept of turnover would apply only to that bone that is remodeling. Therefore, bone formation rate (BFR) and bone turnover are not the same, and either low turnover or high turnover can be associated with loss of bone volume and reduction in bone strength. Because bone resorption usually is similar to or greater than bone formation, the bone turnover rate is usually the bone formation rate, but it is important to remember that this is not always the case, and that the rates of both formation and resorption are necessary to describe the bone physiologic picture.
Definition of Turnover
Measurement of Bone Formation Rate
Although the term bone turnover is used frequently, it is not clearly defined. If the bone resorption rate and bone formation rate were equal, then the turnover could be defined as either rate. The bone volume would not be changing, and the bone balance would therefore be zero. This situation is generally seen in healthy young adults. If the bone resorption rate is greater than the formation rate, because the resorbed cavities are not completely filled with new bone, then the turnover would be reflected by the bone formation rate, and the bone balance would be negative. This is seen frequently with aging and postmenopausal osteoporosis. On the other hand, if the bone formation rate is greater than the bone resorption rate in an adult skeleton, the turnover would be
Tetracycline will deposit in the bone where calcium is being deposited, and can be seen under a fluorescent microscope (Fig. 2). A small dose will therefore label the surface of bone that is actively mineralizing. The BFR can be directly measured with 2 labels administered about 10 days apart. The length of the tetracycline labels multiplied by the distance between the labels is the area of new bone formed during the label interval. In other words, the rate of new bone formation depends both on the rate of apposition at the surface and the total surface involved in forming bone. The BFR can be expressed in reference to the bone surface, bone volume, or tissue volume. The appropriate referent depends on the situation. The most easily understood concept of turnover is the BFR as a
TURNOVER IN RENAL OSTEODYSTROPHY In most cases of renal osteodystrophy, those with high bone formation rates also have high resorption rates, and those with adynamic bone disease have low rates of both formation and resorption. Thus, the bone formation indices are used to describe turnover because they can be measured with much more accuracy than the bone resorption rate.
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Figure 2. Tetracycline labeling. (A) Adynamic bone without label, (B) normal labels.
percentage per year of the bone volume. The absolute amount of bone formed (BFR per tissue volume) theoretically would provide the best correlation with a serum marker of bone formation. The tetracycline labels usually are seen clearly and measurements are straightforward. Sometimes the edges taper gradually into the inactive bone surface, making the exact end of the label indefinite, but this causes only minor measurement difficulties. More commonly, when there is very rapid bone formation, the labels become blurry and diffuse, making them difficult to measure. Conversely, when the bone is forming very slowly the labels do not show separation and it is difficult to tell if a label is a double label or a single label. The use of 2 different kinds of tetracycline (demeclocycline and tetracycline) is helpful in these cases because they have different colors when viewed using fluorescent microscopy.
Activation Frequency The activation frequency is related to the BFR, but it is not a BFR because the activation frequency also depends on the amount of bone formed during each remodeling cycle. This amount of bone formed during an average remodeling cycle is represented by the wall thickness, which is the thickness of new bone made in one cycle. Activation frequency is the BFR divided by the wall thickness. Activation frequency can increase even though the BFR is unchanged, merely from a decrease in wall thickness. With aging, the activation frequency increases; this is owing to a combination of increased bone formation and decreased wall thickness.8 Some people think that an increased activation frequency causes bone loss. They confuse bone balance with bone turnover. It is the imbalance between resorption and formation that causes osteoporosis, not the increased activa-
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tion frequency. There is a relationship between activation frequency and bone loss, however, because if each BMU is in negative balance, then a greater number of active BMUs will result in a greater bone loss. The activation frequency also is confused with the frequency of originating new BMUs (BMU birthrate). Activation frequency refers to just one spot on the bone surface. When a traveling BMU reaches a spot, that surface becomes active. The birthrate of new BMUs depends on how long they live. A bone with many short-lived BMUs can have the same activation frequency, wall thickness, and BFRs as a bone with a few long-lived BMUs. The concept of BMU lifespan is simple to understand but cannot be measured on a 2-dimensional section of bone because BMUs wander in and out of the plane of the section. In bone biopsies from patients with CKD, it is often difficult to measure the wall thickness. This must be measured on a quiescent surface, and many patients with hyperparathyroidism
have limited, if any, quiescent surfaces. The measurement depends on identifying a change in the orientation of lamella, but patients with CKD often have abnormal woven bone. When the BFR is very low, the completed BMUs may remain for years, and thus measurements do not reflect the current physiology. For these reasons, many histomorphometrists do not calculate the wall thickness or activation frequency in patients with CKD.
Other Measurements Related to Bone Turnover Other measurements that relate to the formation aspects of bone turnover include the osteoblastic surface, the number of active osteoblasts, and the osteoid surface. None of these gives the same accurate information as the tetracycline labeling. Bone resorption is related to the number of osteoclasts and the depth of resorption cavities, but there is no direct way to
Figure 3. Example of osteomalacia.
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calculate the bone resorption rate. Some formulas that have been proposed require an assumption that the bone is in balance, which is not valid in patients with CKD. The amount of marrow fibrosis is related to high parathyroid hormone (PTH) levels, which usually increase the bone formation and resorption rates. High PTH levels also cause blurry tetracycline labels, woven bone, and intratrabecular tunneling. Some histologic findings in bone document causes of bone disease, such as aluminum, amyloid, or iron deposition. Other tests not routinely performed include the number and condition of osteocytes, number of apoptotic cells, the structural microarchitecture, the cortical porosity, the mineralization density, the mechanical stiffness of the bone material, the volume of the fat cells, the thickness of the lamella, and the characteristics of the tetracycline labels. Immunohistochemical measurements of bone proteins may be used in the future. Serum biochemical markers of bone formation and resorption potentially could be very
helpful in determining these rates. They have not yet been validated in patients with CKD. In osteoporotic patients, the markers relate to BFRs determined by tetracycline in some situations but not in others, and there is considerable variability. More research is needed. MINERALIZATION IN RENAL OSTEODYSTROPHY The second axis is mineralization, which reflects the amount of unmineralized osteoid. The classic disease with an abnormality of mineralization is osteomalacia, in which the BFR is low and osteoid volume is high. Some patients have excess osteoid, which is a result of high BFRs; they do not actually have a problem with the mineralization process. Patients with low BFRs and normal osteoid have adynamic disease (they do not even form the osteoid matrix, so they do not manifest a problem with mineralization). Mineralization is measured by the osteoid maturation time or the mineralization lag time, both of which depend heavily on the
Figure 4. Example of mixed lesion.
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osteoid width as well as the distance between tetracycline labels. After the osteoblasts lay down new collagen, they direct mineralization of the matrix. This is normally a regulated and orderly process, but in patients with CKD the mineralization can be delayed or disorganized. This results in thickened osteoid. Rapid bone formation also can result in thick osteoid, but in that case the tetracycline labels also are more widely separated. The osteoid maturation time is the osteoid width divided by the distance between labels per day. The mineralization lag time is the osteoid maturation time adjusted for the percentage of osteoid surface that has a tetracycline label. This adjustment, however, assumes that the osteoid surface without a label is in a resting phase, but there is no evidence to support a resting phase. Osteomalacia has been defined by different investigators as increased osteoid volume, increased osteoid maturation time, or increased mineralization lag time. Parfitt9 defined osteomalacia in patients with mal-
absorption if they had a combination of osteoid volume/bone volume greater than 10%, osteoid thickness greater than 12.5 m (note that the thickness is a measurement that requires a correction for section obliqueness, which is the width/1.2), and mineralization lag time greater than 100 days. There is currently no consensus about the exact definition of abnormal mineralization in patients with CKD. BONE VOLUME IN RENAL OSTEODYSTROPHY The final axis is bone volume, which has not been included in previous schemes of describing renal osteodystrophy. Bone volume contributes to bone fragility and is separate from the other parameters. The bone volume is the end result of changes in bone formation and resorption rates. If overall BFR is greater than overall bone resorption rate, the bone is in positive balance and the bone volume will increase. If mineralization remains constant, an increase in
Figure 5. Example of adynamic bone disease.
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Figure 6. Example of osteitis fibrosa.
bone volume also would result in an increase in bone mineral density and should be detectable by radiographic densitometry. Although both cortical and cancellous bone volume decrease in typical idiopathic osteoporosis, these compartments frequently are different in patients with CKD. For example, with high PTH levels, cortical bone volume is decreased but the cancellous volume is increased.10 The concept of bone volume is the easiest to understand. It is direct and reproducible to measure within a sample. There is a large error, however, from one biopsy to the next in the same person. From contiguous iliac crest biopsies there is an average of 29% difference.11 Bone volume is related to bone strength, but the same volume can have different microarchitecture. The trabecular thickness can be calculated from measurements of the bone surface per volume relationship, and this is an index related to bone strength. Other methods of measuring the architecture include the strut length, the star volume, and the number of nodes connecting
trabeculae. These 2-dimensional measurements are inferior to newer 3-dimensional measures of connectivity that are made with microcomputed tomography. There is consensus that bone volume is expressed as bone volume per tissue volume, which is the same as bone area per tissue area when expressed in 2 dimensions. This can be measured in cortical or in cancellous bone. RELATIONSHIP BETWEEN TMV SYSTEM AND TYPES OF RENAL OSTEODYSTROPHY Most studies of renal osteodystrophy have classified biopsies based on the BFR or presence of fibrosis, as well as the degree of mineralization (Figs. 3-6). Bone volume has not been used to place patients into categories. Thus, those with low turnover and normal mineralization have adynamic bone disease or low-turnover bone disease. Patients whose bone biopsies show high bone turn-
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over, particularly with other signs of high PTH, have high bone turnover or hyperparathyroid bone disease or osteitis fibrosa. Those with abnormal mineralization and low bone formation are osteomalacia, and those with abnormal mineralization and high bone formation have mixed disease (Fig. 7). There is no consensus on the exact measurements to define these categories. A large review of the prevalence of these types of renal osteodystrophy in hemodialysis patients showed osteitis fibrosis in 34%, mixed lesions in 32%, adynamic disease in 19%, osteomalacia in 10%, and normal or mild disease in 5%. Patients receiving peritoneal dialysis had adynamic bone disease more frequently (50%) with fewer cases of mixed
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lesion or osteitis fibrosis (Kidney Disease: Improving Global Outcomes guidelines, unpublished data).
NATURAL HISTORY The natural history of bone histomorphometry is variable. The placebo groups from randomized clinical trials12-19 and one longitudinal study20 show an overall trend toward worsening turnover (either getting too high or too low) and stable mineralization and volume. The wide variability in the natural history of bone histology reflects the complex pathophysiology of chronic kidney disease-mineral and bone disorder (CKD-MBD).
Figure 7. Relationship between TMV and classic types of renal osteodystrophy. The bone volume in this diagram is hypothetical; actual studies show variability in the bone volume for each type of disease.
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CLINICAL CORRELATIONS
In patients with advanced stages of CKD, the bone density does not predict fractures as well as in patients with normal renal function. This is probably owing to abnormalities in bone structure and quality that are common in CKD patients, as well as a greater likelihood of nonskeletal causes of fractures.
Fractures and Bone Histomorphometry Most of the studies of bone histomorphometry have not been designed to evaluate the relationship between fractures and types of renal osteodystrophy fully. One study of 31 dialysis patients found that those with low turnover osteodystrophy had a fracture rate of 0.2 per year compared with 0.1 per year in those with osteitis fibrosis; this was owing to a high number of rib fractures in the low turnover patients.21 A review of 2,340 biopsies performed in Brazilian patients for clinical indications found that the frequency of fractures was significantly higher in those with osteomalacia compared with other forms. There were no differences in fracture history between those with adynamic bone disease, high bone turnover, or mixed bone disease.22 A study that followed up 62 patients for 5 years after bone biopsy found a higher rate of fractures in those with adynamic bone disease.23
Bone Density The relationship between bone density and bone biopsy is not consistent. In patients with CKD, Lindergard10 measured bone volume/tissue volume on 71 biopsies from dialysis patients, and did not see a correlation with bone mineral density (BMD) at the radius. Similar results were seen by Gerakis et al23 in a study of 62 patients. Torres et al,24 on the other hand, found a correlation coefficient of 0.82 between bone volume/tissue volume and quantitative computed tomography of the spine, and Van Eps et al25 found lower dual-energy x-ray absorptiometry values in patients with low bone volume on biopsy. The relationship between BMD and type of renal osteodystrophy varies among reports. Four studies found similar BMD in all types.26-29 Lower BMD has been reported in both high and low bone turnover, with wide ranges.23,30,31 These findings emphasize that the older types of renal osteodystrophy do not provide enough information about bone strength. In addition, the radiographic tests cannot distinguish between unmineralized and mineralized bone.
SUMMARY Patients with CKD have complex abnormalities on bone biopsies, which change in inconsistent patterns. In addition to abnormally high or low BFRs, they show abnormal mineralization and/or bone volume. Patients throughout the spectrum have increased clinical fractures and bone pain, presenting challenges to physicians trying to improve the bone strength. REFERENCES 1. Moe S, Drueke T, Cunningham J, et al. Definition, evaluation, and classification of renal osteodystrophy: a position statement from Kidney Disease: Improving Global Outcomes (KDIGO). Kidney Int. 2006;69: 1945-53. 2. Ott S. Histomorphometric analysis of bone remodeling. In: Bilezikian J, Raisz L, Ga R, editors. Principles of bone biology. San Diego: Academic Press; 2002. p. 303-20. 3. Parfitt AM. Osteonal and hemi-osteonal remodeling: the spatial and temporal framework for signal traffic in adult human bone. J Cell Biochem. 1994;55:273-86. 4. Robling AG, Niziolek PJ, Baldridge LA, et al. Mechanical stimulation of bone in vivo reduces osteocyte expression of sost/sclerostin. J Biol Chem. 2008;283: 5866-75. 5. Eriksen EF, Gundersen HJG, Melsen F, Mosekilde L. Reconstruction of the formative site in iliac trabecular bone in 20 normal individuals employing a kinetic model for matrix and mineral apposition. Metab Bone Dis Rel Res. 1984;5:243-52. 6. Jowsey J. Age changes in human bone. Clin Orthop Rel Res. 1960;17:210-7. 7. Osteoporosis and Bone Physiology [cited 2009 Feb 5]. Available from: http://courses.washington. edu/bonephys. 8. Recker RR, Lappe JM, Davies KM, Heaney RP. Bone remodeling increases substantially in the years after menopause and remains increased in older osteoporosis patients. J Bone Miner Res. 2004;19:1628-33. 9. Parfitt AM. Osteomalacia and related disorders. In: Aviolo L, Krane SM, editors. Metabolic bone disease. Philadelphia: Saunders; 1990. p. 329-96. 10. Lindergard B, Johnell O, Nilsson BE, Wiklung PE. Studies of bone morphology, bone densitometry, and labora-
Bone Histomorphometry in Renal Osteodystrophy Susan M. Ott, MD Summary: On bone biopsies from patients with chronic kidney disease, measurements are made of the turnover, mineralization, and volume. Turnover depends on the bone formation rate and bone resorption rate; the former can be measured using tetracycline labelling. The osteoid width and bone apposition rate determine the mineralization rates. Bone volume includes both mineralized and unmineralized bone and is directly related to the porosity. Using these measurements, biopsies can be separated into the classic types of renal osteodystrophy: normal, adynamic, high-turnover, mixed, and osteomalacia. Fracture rates among these types are not consistent, but several studies have found high fracture rates with adynamic or osteomalacia. The bone density tests cannot distinguish between different types of bone histology. Semin Nephrol 29:122-132 © 2009 Published by Elsevier Inc. Keywords: CKD, renal osteodystrophy, Improving Global Outcomes , turnover, mineralization, volume (TMV), osteoclasts, osteoblasts, osteocytes
he kidney plays a critical role in the complex regulation of mineral metabolism, so it is not surprising that chronic kidney disease (CKD) causes bone disorders. Multiple factors influence the bone in patients with CKD, resulting in a variety of abnormalities seen on bone biopsies (Fig. 1). It is appropriate to discuss the microscopic appearance in a journal issue about the spectrum of renal osteodystrophy, especially because the derivation of the term is from the Latin specere, meaning “to look.” A spectrum suggests that something can be classified in terms of its position on a scale between 2 opposite points. This aspect of renal osteodystrophy applies to the bone formation rate. Superimposed on this spectrum are variations in the degree of mineralization of the bone matrix and the bone volume per bone tissue, which combined determine the bone density. An international committee sponsored by Kidney Disease: Improving Global Outcomes concurred that the most important pa-
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Department of Medicine, University of Washington Medical Center, Seattle, WA. Address reprint requests to Susan M. Ott, MD, Associate Professor, Department of Medicine, University of Washington Medical Center, Box 356426, 1959 Northeast Pacific St, Seattle, WA 98195-6426. E-mail: smott@u. washington.edu 0270-9295/09/$ - see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.semnephrol.2009.01.005
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rameters to describe renal osteodystrophy are turnover, mineralization, and volume (TMV).1 Thus, instead of a 1-dimensional axis, the TMV system defines a more comprehensive 3-dimensional space. This article focuses on the new TMV classification. OVERVIEW OF BONE REMODELING Healthy bone is a dynamic tissue, continually resorbing bone and replacing it with new bone in discrete areas know as basic multicellular units, also called bone metabolic units (BMUs).2 A BMU is not a permanent structure. It forms in response to signal or stimulus, performs its function, and disbands, leaving a few residual lining cells and osteocytes. Each BMU undergoes its functions in the same sequence: origination and organization of the BMU, activation of osteoclasts, resorption of old bone, recruitment of osteoblasts, formation of new bone matrix, and mineralization. In solid cortical bone, a BMU drills a tunnel and then refills it. On the cancellous bone surface the BMU can channel like a river flowing, or can spread over the surface like warm fudge flowing over ice cream. The lifespan of a BMU is not well defined. Cortical BMUs can wander for months,
Seminars in Nephrology, Vol 29, No 2, March 2009, pp 122-132