1. Chapter 18 Chapter 18 On the Evolution and Contemporary Roles of Bone Remodeling Copyright © 2013 Elsevier Inc. All rights reserved.

2. FIGURE 37.1 Basic multicellular unit (BMU) remodeling on an endosteal surface (trabecular or endocortical) begins with osteoclasts resorbing mineralized bone matrix. Initiation of remodeling requires activation of osteoclast receptors called receptor activator of nuclear factor kappa (RANK) by its lone ligand called receptor activator of nuclear factor kappa B ligand (RANKL). RANKL can alternatively bind to a natural decoy receptor called osteoprotegerin (OPG) or to a therapeutic antibody called denosumab, which prevents RANK activation and blocks the initiation of BMU remodeling. When osteoclasts resorb bone matrix they mobilize skeletal calcium and leave behind a scalloped surface. Osteoblasts then come in and refill the resorption space with osteoid that draws in extracellular calcium as it mineralizes. Source: From Dempster et al. [378] with permission. 2

3. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 37.2 (A) deep scalloped resorption lacunae on an indi­vidual human trabecula illustrating the initial resorptive phase of surface-based basic multicellular unit (BMU) remodeling. Source: From Mosekilde [61], with permission. (B) Haversian-based pores created by osteoclast tunneling of cortical bone. (See color plate.) Source: From Zebaze et al. [60], with permission. 3

4. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 37.3 Increased trabecular and cortical basic multicellular unit (BMU) remodeling after 12 months of limb disuse in dogs. Upper panel: Fluorescent photomicrographs of trabecular bone from a normally loaded dog (left) or a dog subjected to 12 months of limb disuse (right). Increased fluorochrome labeling indicates greater BMU remodeling and increased refilling of resorption spaces. Source: Original images courtesy of Dr. Chaoyang Li. From Li et al. [54], with permission. Lower panel: Photomicrographs of cortical bone from a normally loaded dog (left) and a dog subjected to 12 months of limb disuse (right) indicate greatly activated BMU remodeling, including extensive cortical porosity and endocortical erosion. (See color plate.) Source: From Li et al. [32], with permission. 4

5. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 37.4 Linear regressions between bone mineral density (BMD) or bone mineral content (BMC) versus bone strength. (A) Proximal femurs from adult human cadavers. Source: From Bouxsein et al. [306], with permission. (B) Radius from adult human cadavers. Source: From Augut et al. [379], with permission. (C) Lumbar vertebrae from mature cynomolgus monkeys. Source: From Ominsky et al. [98], with permission. 5

6. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 37.5 Conceptual framework describing the typical relationship between calcium, remodeling and bone mass. The “calcium” elephant can reflect calcium demand, or extracellular calcium concentrations in blood, serum, urine, feces, or breast milk. The elephant on the right represents bone mass (volume, mineral content, or density) and often bone strength. The central “remodeling” elephant consistently faces the same direction as the calcium elephant, impacting the apparent weight of the “bone mass” elephant as it moves backward (increased remodeling) or forward (decreased remodeling). For example, increased calcium demand (e.g. lactation) increases remodeling while decreasing bone mass; decreased remodeling (e.g. antiresorptive agents) decreases blood or urine calcium while increasing bone mass; activation of remodeling (e.g. parathyroid hormone (PTH), receptor activator of nuclear factor kappa B ligand (RANKL)) increases extracellular calcium while decreasing bone mass. 6

7. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 37.6 Receptor activator of nuclear factor kappa B ligand (RANKL) administration to rats for 6 weeks caused significant bone loss, including the thinning of individual trabecular elements as shown by longitudinal in vivo micro-computer tomography (micro-CT), and increased serum calcium. Discontinuation of RANKL injections after week 6 led to partial recovery of trabecular architecture, and the return of serum calcium to baseline levels. *p < 0.05 vs. day 0. Source: From Campbell et al. [57], with permission. 7

8. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 37.7 Receptor activator of nuclear factor kappa B ligand (RANKL) administration to rats increased serum levels of the bone resorption marker TRACP-5b (A) and the bone formation marker osteocalcin (B). Increased basic multicellular unit (BMU) remodeling with RANKL was accompanied by decreased trabecular bone mineral density (C), decreased trabecular bone volume at the proximal tibia (D), and decreased bone strength at the femur shaft and neck (E). *p < 0.05 vs. vehicle control. (See color plate.) Source: From Yuan et al. [79], with permission. 8

9. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 37.8 Opposing effects of basic multicellular unit (BMU) remodeling on calcium and bone mass. Ovariectomized (OVX) monkeys were treated with vehicle or denosumab (DMAb) (25 or 50 mg/kg/month) for 16 months starting 1 month after OVX; Sham operated controls were treated with vehicle. (A) Fluorochrome labeling of mineralizing surfaces in the lumbar vertebrae at month 16 indicates that BMU remodeling was increased by OVX and decreased with denosumab. (B) Serum calcium was increased by OVX and decreased by denosumab after 3 months of treatment (and throughout most of the study). (C) Lumbar spine bone mineral density (BMD) was decreased by OVX and increased by denosumab at month 16 (and throughout the study). (D) Structural strength of L3 and L4 vertebral bodies (averaged for each animal) was nonsignificantly decreased by OVX and significantly increased by denosumab at month 16. *p < 0.05 vs OVX control; ^p < 0.05 vs. Sham control. Source: From Ominsky et al. [98] and Kostenuik et al. [27], with permission. 9

10. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 37.9 Bone loss patterns in lactation versus surgical or natural menopause. Upper panel: Proximal tibial metaphysis from a normal female control rat, a lactating rat, and an ovariectomized (OVX) rat. Source: From Miller et al. [177], with permission. Middle panel: Femur cortex from a nonlactating control dog and a lactating dog indicate increased endocortical and intracortical remodeling and porosity with lactation. Source: From Vajda et al. [171], with permission. Bottom panel: Femur cortex from an ovary-intact 29-year-old woman (left) and a 67-year-old postmenopausal woman (right) indicate increased endocortical and intracortical remodeling and porosity associated with age and menopause. Source: Original images courtesy of Dr. Ego Seeman. From Zebaze et al. [60], with permission. 10

11. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 37.10 Rats were sacrificed at the end of lactation (gray bars) or 8 weeks after weaning (black bars), along with unmated age-matched controls. Upper panel: Increased vertebral trabecular bone formation rate per bone surface area indicates significantly greater basic multicellular unit (BMU) remodeling with lactation, which returned to normal levels after weaning. Lower panel: Compressive strength of lumbar vertebral bodies was 62% lower at the end of lactation compared to unmated controls. Eight weeks after weaning bone strength exhibited substantial recovery that was aligned with reduced BMU remodeling. Source: From Vajda et al. [179], with permission 11

12. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 37.11 Trabecular bone sections from the vertebra of a control dog (A) and a lactating dog (B) reveal fluorochrome labels taken up by mineralizing osteoid. The lactating sample exhibits significant increases in labels indicating an increased number of resorption spaces being refilled. (See color plate.) Source: Original images courtesy of Dr. Scott Miller. From Miller et al. [189], with permission. 12

13. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 37.12 Neuroendocrine changes with human menopause and lactation. Font sizes crudely approximate relative differences in levels, activity or function between conditions. Boxes around “ovary” and “anterior pituitary” indicate the initial intervention points in menopause and lactation. With menopause estrogen slowly declines with ovarian function, leading to feedback-related increases in luteinizing and follicle-stimulating hormones (LH, FSH). Osteoclasts and bone remodeling increase, leading to gradual irreversible decreases in bone mass. With lactation, the anterior pituitary releases prolactin that acts upon the hypothalamus to reduce gonadotropin-releasing hormone (GnRH). Then the anterior pituitary reactively releases less LH and FSH, leading to decreased ovarian production of estrogen. The decline in estrogen with lactation is rapid and profound, starting from highly elevated pregnancy levels, leading to increased remodeling and rapid bone loss that provides much of the calcium in breast milk. Parathyroid hormone-related protein (PTHrP) and prolactin might further increase remodeling rate to mobilize more calcium and cause greater bone loss. PTHrP also plays a key role in promoting renal calcium reabsorption. 13

14. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 37.13 Left: no correlation between skeletal microcracks in lumbar vertebrae versus normalized vertebral toughness in dogs treated for 1–3 years with vehicle, alendronate, risedronate, or raloxifene. Source: From Allen and Burr [234], with permission. Right: Tensile fracture toughness of cortical bone from the femur and tibia of elderly men and women versus microdamage density at the same site. A modest inverse relationship was seen for the femur but not the tibia. Fracture toughness assessed by shear testing indicated no significant relationship with microdamage (data not shown). Source: From Norman et al. [247], with permission. 14

15. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 37.14 Upper panel: A theoretical relationship between bone remodeling rate and bone strength suggests reduced bone strength with very low or very high remodeling rate. Source: From Weinstein [380], with permission. Middle and lower panels: Experimentally determined relationships between remodeling rate and bone strength in mature cynomolgus monkeys. Trabecular remodeling is expressed as the percentage of trabecular bone in L2 exhibiting fluorochrome labels (mineralizing surface/bone surface, MS/BS). Strength of trabecular cores from L5 and L6 (averaged) was assessed in compression. As trabecular remodeling approached zero, trabecular strength generally increased. Cortical remodeling is expressed as percentage of Haversian systems exhibiting fluorochrome labels in the tibial cortex. There was no relationship with toughness of prismatic beams milled from the humerus of the same animal. Source: From Kostenuik et al. [27], with permission. 15

16. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 37.15 Structural strength increases in denosumab-treated ovariectomized (OVX) monkeys versus reductions in the risk of related fracture types in a clinical trial of denosumab in postmenopausal women with osteoporosis. Left: Increase in structural bone strength in OVX monkeys treated for 16 months with denosumab, relative to vehicle-treated OVX controls. Increases were calculated from average strength values from two dose levels of denosumab. Strength improvements were greatest for yield load of L3–4 vertebrae (+55% vs. OVX-Vehicle), intermediate for peak load of the femur neck (+34%), and lowest for peak load of the purely cortical femur shaft (+14%). Source: From Ominsky et al. [98] with permission. Right: Percent decrease in fracture risk in postmenopausal women treated for 36 months with denosumab. Compared to placebo controls, women in the denosumab group exhibited 68% fewer spine fractures, 40% fewer hip fractures, and 20% fewer nonvertebral fractures that occur primarily at cortical sites. Source: From Cummings et al. [317], with permission. 16