Curr Osteoporos Rep DOI 10.1007/s11914-014-0214-3

BONE QUALITY IN OSTEOPOROSIS (MD GRYNPAS AND JS NYMAN, SECTION EDITORS)

Collagen Modifications in Postmenopausal Osteoporosis: Advanced Glycation Endproducts May Affect Bone Volume, Structure and Quality Thomas L. Willett & Julia Pasquale & Marc D. Grynpas

# Springer Science+Business Media New York 2014

Abstract The classic model of postmenopausal osteoporosis (PM-OP) starts with the depletion of estrogen, which in turn stimulates imbalanced bone remodeling, resulting in loss of bone mass/volume. Clinically, this leads to fractures because of structural weakness. Recent work has begun to provide a more complete picture of the mechanisms of PM-OP involving oxidative stress and collagen modifications known as advanced glycation endproducts (AGEs). On one hand, AGEs may drive imbalanced bone remodeling through signaling mediated by the receptor for AGEs (RAGE), stimulating resorption and inhibiting formation. On the other hand, AGEs are associated with degraded bone material quality. Oxidative stress promotes the formation of AGEs, inhibits normal enzymatically derived crosslinking and can degrade collagen structure, thereby reducing fracture resistance. Notably, there are multiple positive feedback loops that can exacerbate the mechanisms of PM-OP associated with oxidative stress and AGEs. Anti-oxidant therapies may have the potential to inhibit the oxidative stress based mechanisms of this disease.

T. L. Willett (*) : J. Pasquale : M. D. Grynpas Musculoskeletal Research Laboratory, Lunenfeld-Tanenbaum Research Institute at Mount Sinai Hospital, 60 Murray Street, Box 42, Toronto, Ontario, Canada M5T 3L9 e-mail: [email protected] T. L. Willett Division of Orthopaedic Surgery, Mount Sinai Hospital, Toronto, Ontario, Canada T. L. Willett Division of Orthopaedic Surgery, Department of Surgery, University of Toronto, Toronto, Ontario, Canada J. Pasquale : M. D. Grynpas Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada

Keywords Bone . Bone quality . Osteoporosis . Postmenopausal . Collagen . Mineral . Oxidative stress . Cross-linking . Advanced glycation endproducts . Fracture

Introduction Osteoporosis is recognized as a disease of reduced skeletal structural integrity, particularly of the spine, caused by loss of bone volume and resulting in skeletal fractures. Multiple forms of osteoporosis exist including primary forms associated with ageing and menopause and secondary forms associated with pharmacologic treatments, etc. This review focuses on postmenopausal osteoporosis (PM-OP), which results from bone remodeling imbalance favoring resorption and, therefore, net bone loss. Clinically, PM-OP is recognized by 1 or more vertebral or other fragility fractures, which can result from a combination of degradation of both structure and material properties. The classical model of PM-OP recognizes the decrease of estrogen at menopause as the major biological stimulus for increased bone remodeling and net loss of bone volume due to unopposed osteoclast activity (resorption). Ovariectomy (OVX) in animal models, such as rodents, replicates menopause with estrogen depletion and is an established animal model of PM-osteopenia, a precursor to PM-OP. OVX in the rat is followed by a rapid loss of trabecular bone in the ends of long bones (metaphyses) and in the spine. This reflects the situation found in postmenopausal women where most of the osteoporotic fractures are vertebral compression, Colles’ or hip fractures due to the loss of structure. An increased production of reactive oxygen species (ROS), termed oxidative stress, and associated decreases in antioxidants occur subsequent to the depletion of estrogen in menopause [1•, 2, 3] and OVX [4]. Oxidative stress is increased in various diseases involving inflammation, including ageing

Curr Osteoporos Rep

and diabetes [5, 6•]. ROS are often free radicals and, therefore, are highly reactive [6•]. Oxidative stress is known to result in various forms of damage to proteins including fragmentation and oxidative side chain modifications (especially carbonylation) [6•]. Furthermore, oxidative stress depletes anti-oxidants (such as ascorbate and pyridoxamine) [3, 4] and it contributes to the formation of glyco-oxidation products termed advanced glycation endproducts (AGEs) [6•]. AGEs are the result of spontaneous nonenzymatic glycation of proteins by glucose and the reactive products of sugar metabolism [7]. The products are many, heterogeneous, and involve the initial reaction of aldehyde groups with amine groups on lysine, hydroxylysine, and arginine [7]. Characterization is difficult but two types of AGEs are known—adducts and crosslinks [7]. Various adducts, such as carboxymethyllysine, and crosslinks, such as pentosidine, imidazolone and glucosepane, have been elucidated [8, 9]. Some require oxidation for formation and, therefore, oxidative stress increases their formation [10, 11]. For example, pentosidine formation is oxidation dependent, producing a fluorescent AGE crosslink with a stable structure [7]. Its stability and fluorescence have made it the established marker for AGEs [9]. Initially, AGEs were thought to accumulate in tissues with slow turnover, such as cartilage and bone with suppressed turnover, because remodeling was thought to be required to remove them from tissues [12–14]. However, increased AGE content in bone occurs in both low turnover and high turnover conditions and, therefore, may be more dependent on the presence of oxidative stress rather than the tissue half-life [15••]. The heightened presence of AGEs in osteoporotic bone is established in both clinical specimens [16, 17] and in the bones of animal models [18••, 19••]. Furthermore, the AGE content, measured in terms of pentosidine content, has been associated with the occurrence of hip fractures in human patients [20]. This review concerns advanced glycation endproduct collagen modifications associated with PM-OP and the mechanisms by which they may negatively affect both bone structure and bone material, leading to clinical fractures. We have chosen to focus on the intriguing roles of AGEs in PM-OP attributable to a growing body of work and growing interest in these spontaneously formed, insidious perpetrators of disease.

Part A—Collagen Modifications and Bone Loss Although there is a classical model of the bone loss observed in PM-OP, many recent studies are moving toward an oxidative stress related paradigm. This theory suggests that sex steroid depletion is not the direct cause of the increase in bone remodeling observed, but rather it works indirectly by increasing the ROS within the body, therefore, causing oxidative stress, which then affects the balance of

bone remodeling [21, 22]. This theory centers on the idea that estrogens have an antioxidant effect, which prevents the production of ROS beyond the levels that are needed for regulatory functioning within the cells [22]. Goettsch et al. looked at the role of intracellular NADPH oxidase 4 (NOX4), which is an enzymatic source of ROS within the body [23•]. They found that in middle-aged women, a mutation of the NOX4 gene was associated with altered parameters of bone metabolism, as well as finding an increased expression of NOX4 in the bones of patients with untreated osteoporosis. Finally, they looked at the effects of ovariectomy in female mice and found that with this depletion of sex steroids there was an increase in the levels of NOX4, and with mice that had acute NOX4 deletion there was an inhibitory effect on bone loss. This suggests that the loss of estrogen may lead to an increase in NOX4, therefore, increasing the amount of ROS in the body. Another study by Alemida et al. shows the effect of estrogen on p66shc, an adaptor proteins that amplifies mitochondrial ROS generation and leads to hydrogen peroxide (a common ROS)-induced apoptosis of osteoblasts [24]. P66shc becomes activated through the phosphorylation by protein kinase c (PKC)β, which is activated by ROS. They showed that estrogen blocks the ability of PKCβ to phosphorylate p66shc, which then does not increase the amount of ROS present, and both osteoblast apoptosis and NF-kB activation are reversed. The mechanisms by which this increase in oxidative stress might cause increased bone remodeling are still relatively unknown. One explanation is that oxidative stress plays a role in the production of oxidation dependent AGEs [25]. While AGEs form naturally with age, the oxidative stress associated with estrogen depletion may increase the amount of AGEs formed within the bone [10, 11]. The formation of AGEs is a multistep process that includes both glycation and oxidation reactions [7]. Reducing sugars and carbohydrate metabolites react with amino groups in proteins to form Schiff bases, which rearrange to form Amadori compounds, which then undergo oxidation to form AGE [7]. ROS are needed for the oxidation step to take place, therefore, when there are more ROS available, more AGEs, such as pentosidine, are formed [26]. Since both AGEs and ROS have been found to increase after menopause [1•, 2–4, 16, 17, 18••, 19••, 20], the increase in AGEs might be due to the oxidative stress resulting from estrogen depletion. This is observed in other diseases, such as diabetes, where oxidative stress is also high, and an increase in AGEs is also observed [25]. Many receptors for AGEs have been found, such as macrophage scavenger receptor (MSR) and AGE-1,-2, and −3 [8] but the most characterized is the Receptor for Advanced Glycation Endproducts (RAGE), which is a multi-ligand member of the immunoglobulin superfamily of cell surface receptors, which binds AGE adducts (eg, carboxymethyllysine) among other ligands [27•]. The RAGE receptor is found on a variety of cell

Curr Osteoporos Rep

types, including osteoclasts and their precursors [28], and osteoblasts [29]. Although expressed, RAGE is normally at low levels in a healthy adult, however, during chronic inflammation (increased oxidative stress) as in diabetes, RAGE expression is increased [11]. When AGEs bind to RAGE, intracellular signaling pathways are activated, which cause the release of transcription factor nuclear factor NF-κB via mitogen activated protein kinases [27•]. RAGE expression is upregulated by NF-κB, so as RAGE binds its ligands it continues to create NF-κB, which continues to increase the expression of RAGE, working in a positive feedback mechanism and accelerating the disease [27•]. Please see the left side of Fig. 1. The prolonged activity of intracellular signaling pathways, such as the ERK pathway, can lead to altered gene expression via NF-κB and the subsequent release of several inflammatory signals including as the interleukin-1 and interleukin-6 [27•, 31]. These can induce changes in bone cell activities, such as increased osteoclast-mediated resorption and osteoblastmedicated formation [30, 31]. Being expressed in both osteoblasts and osteoclasts, RAGE has effects on both of these cell types. The effects of AGE-RAGE binding on osteoclasts has been documented by Ding et al., who suggest that RAGE is directly involved in determining bone volume/density, bone quality, and osteoclast formation [30]. When testing the previously mentioned factors in RAGE deficient mice (RAGE −/−) relative to wild-type mice, the investigators found that RAGE (−/−) mice had significantly increased bone mineral density, bone volume, better bone biomechanical properties at the structural level including strength, stiffness, and energy-to-fracture, and decreased osteoclast formation. RAGE (−/−) mice were also completely resistant to bone loss after OVX. Studies by Miyata et al. also suggest that AGEs enhance osteoclast-induced bone resorption [32]. These results taken together suggest that RAGE expression plays a critical role in bone resorption and, therefore, bone remodeling and volume/density. However, it is known that healthy rodents typically do not have large quantities of pentosidine in their bones [33]. Therefore, the question of whether the RAGE-dependent resorption mechanism requires the existence of AGE ligands in the bone matrix prior to or during OVX induced remodeling remains unanswered. It is unknown whether RAGE expression and signaling are increased due to oxidative stress alone. In addition to the regulation of osteoclasts, AGE-RAGE binding can have direct effects on several processes involving osteoblasts. Franke et al. showed that AGE-RAGE interaction resulted in the uptake of AGEs into osteoblast cells leading to increased osteoclastogenesis and impaired matrix mineralization [34]. McCarthy et al. have also shown AGEs have an effect on osteoblast function by regulating both the proliferation and differentiation of osteoblasts as well as affecting the ability for these cells to attach to a type-I collagen matrix [35].

A study by Alikhani et al. also showed that AGE/RAGE binding has pro-apoptotic effects in osteoblastic cells, both in vivo and in vitro [36]. These results show that AGEs have an effect on osteoblasts, therefore potentially hindering their ability to form new bone, and possibly contributing to the bone volume decrease seen in PM-OP because of remodeling imbalance. The binding of AGEs to RAGE has been found to cause the release of ROS, ultimately leading to more oxidative stress through the positive feedback loop outlined above [25, 37, 38]. Yan et al. suggest that this increase of oxidative stress is due to the generation of NF-kB, which was found to increase when mice were injected with AGEs [37]. Another theory, proposed by Wautier et al., suggests that AGE-RAGE binding activates NADPH oxidases, which then generates reactive oxygen species [39]. Based on the literature, a complex of multiple positive feedback mechanisms is proposed for the loss of bone volume in PM-OP. Oxidative stress is heightened after the estrogen depletion observed during menopause attributable to the loss of estrogen’s antioxidant properties. This in turn increases the amount of reactive oxygen species present, possibly causing an increase in AGE formation within bone tissues. The increased numbers of AGEs bind to RAGE situated on bone cells, resulting in further generation of ROS, increased bone resorption and stagnant bone formation due to the release of inflammatory factors, and intercellular processes cause further upregulation of RAGE as well as oxidative stress within the bone.

Part B—Collagen Modifications and Bone Quality Degradation In Part A, we provided a brief overview of the rather complex cell-signaling mechanisms involving RAGE-AGE that may lead to bone remodeling imbalance and, therefore, net bone loss. In addition to bone mass/volume loss, which can lead to overall structural weakness, there are also mechanisms that lead to changes in the mechanical performance of the bone material itself by way of loss of material strength, stiffness, ductility, and toughness. These are referred to as changes in bone quality. A key factor in this loss of bone quality seems to be oxidative stress (Fig. 1). It is well established that estrogen depletion during menopause, and similarly in the animal models using ovariectomy, results in an increase in the presence of ROS and depletion of antioxidants (such as vitamin B6). As discussed above, this oxidative stress can cause a multitude of degrading changes to proteins such as collagen including fragmentation and amino acid side group modification. It also plays a role in the formation of some AGEs such as pentosidine [6•]. Perhaps most

Curr Osteoporos Rep Fig. 1 Oxidative stress associated intracellular pathways (left) and matrix modifications (right) leading to postmenopausal osteoporosis and fragility fractures

importantly, oxidative stress may play a key role in altering the formation of the enzymatically derived crosslinks in the new bone formed during the heightened turnover observed in PM-OP. Multiple studies of bone collagen from human PM-OP and in OVX models have shown that the concentration of enzymatically derived crosslinks is greatly reduced compared with controls [18••, 19••, 20, 40, 41]. This is most significant for the normally abundant immature crosslinks that outnumber mature crosslinks by at least two to one [15••]. They are thought to be relatively abundant in normal bone and to play an important role in providing bone with its strength [33]. The proposed importance of these crosslinks was further supported by studies of animal models treated with agents causing lathyrism (lysyl oxidase inhibition (copper deficiency [42], vitamin B6 deficiency [43], β-amino-propionitrile treatment [44])) demonstrating loss of strength and toughness. Similarly reduced concentrations of enzymatically derived crosslinks have been found in bone from OP patients suffering vertebral and hip fractures [20, 40, 41]. While enzymatic crosslinking occurs at the intra and intermolecular level in bone collagen, these changes associated with PM-OP are thought to alter the mechanical behavior at larger length scales including the strength and ductility of the mineralized fibrils. Altered crosslinking may in fact alter fibril formation, structure [45] and even mineralization [34]. Saito et al. have shown in the case of osteoporotic hip fractures in humans and in the study of ovariectomized monkeys that both collagen crosslinking and overall degree of mineralization of bone are different from otherwise normal

bone [18••, 20, 41]. This is consistent with many other studies showing decreased mineralization in OP [46]. Collagen crosslinks are known to play a role in stabilizing collagen fibril structure (packing) and this structured collagen acts as a template for mineralization [47]. Therefore, the altered crosslinking in PM-OP has the potential to disrupt normal mineralization. Osteogenesis Imperfecta is an illustrative, though extreme, model of this idea [48]. It is unclear at this time whether the mineralization of newly formed bone in PMOP differs measurably from the mineralization of newly formed bone resulting from normal remodeling. As in all composite materials, the relative amounts and the structures of the phases and the interfaces between the phases play key roles in determining the mechanical behavior of said composite material. This can include not only the strength but also ductility and fracture toughness of the material [49]. The specific mechanisms that affect the collagen fibril structure, the subsequent mineralization of these fibril templates, and the properties of the interfaces between the phases in PM-OP require further clarification. While enzymatically derived crosslink content is reduced in PM-OP bone, recent studies have demonstrated that pentosidine is increased but to a much smaller extent [18••, 20, 41]. Both effects seem to follow the presence of oxidative stress but the mechanisms are not entirely clear. One intriguing hypothesis, developed to explain the altered crosslinking in diabetes, but which may also work in PM-OP, is that the action of the lysyl oxidase co-factor vitamin B6 (pyridoxal/ pyridoxamine) is competitively inhibited by heightened oxidative stress [33]. Vitamin B6 is a potent free radical

Curr Osteoporos Rep

scavenger. Saito et al. suggested that B6 might be, in a sense, distracted from catalyzing enzymatic crosslinking by instead engaging as an anti-oxidant and perhaps as an AGE inhibitor [15••, 33]. If B6 is distracted from catalyzing lysyl oxidase mediated crosslinking, greater amounts of lysine, hydroxylysine and arginine residues would be available and then nonenzymatic formation of AGEs would have a competitive advantage. With further research, this hypothesis may be shown to explain the higher amounts of AGEs detected in PM-OP and OVX specimens. There may also be a role for hyperhomocysteinemia in the AGE-related mechanisms of PM-OP. Recent work by Saito et al., using a rabbit model combining ovariectomy and a methionine rich diet that promotes hyperhomocysteinemia, demonstrated the expected loss in enzymatic crosslinks caused by OVX [19••]. This effect was independent of the methionine rich diet [19••]. Interestingly, pentosidine was only increased (by two fold) in the group fed the methionine rich diet [19••]. Hyperhomocysteinemia leads to elevated oxidative stress, an important contributor to the elevated levels of pentosidine detected in PM-OP [15••]. In earlier work by the same group that examined cancellous bone from cases of intracapsular femoral neck fractures, the enzymatic crosslinks were decreased and pentosidine was increased along with slightly increased plasma homocysteine and greatly reduced plasma pyridoxal (vitamin B6) levels [44]. The increased presence of AGEs may feedback into the RAGE dependent cell signaling as reviewed above (Fig. 1 left side). They may also affect bone quality. The relative importance of this increase in AGE content in terms of bone quality is unknown. Many studies, particularly ageing studies, of both human bone and bone from animal models have demonstrated negative correlations between pentosidine content and bone ductility, toughness and sometimes strength [50–53]. However, this is only correlation, not causation, and other factors, such as the aforementioned loss of enzymatic crosslinks and collagen degradation due to oxidative stress are also in play. One study showing a negative correlation between pentosidine content and bone toughness also showed a positive correlation between the strength of the collagen network and the toughness of the bone [53]. The strength of the collagen network is a function of its connectivity, which is a function of molecular weight or chain length between crosslinks, fragmentation/degradation, and total crosslinking content. The same group found a negative correlation between bone toughness and collagen degradation measured in terms of % collagen solubilized by trypsin [54]. Collagen degradation may result from oxidative stress driven fragmentation and would contribute to loss of connectivity. Another group found strong positive correlations between bone toughness/fracture toughness

and collagen connectivity measured using a form of hydrothermal isometric tension testing [55]. This again was an ageing study where bone strength and toughness decreased with age, as did the measures of collagen network connectivity [55]. AGE accumulation is widely suspected of contributing to the deterioration of the mechanical properties of bone. This is mainly due to a few in vitro studies that reported that increased AGE content in otherwise normal bone can lead to loss of ductility and therefore, loss of toughness (not detectably affecting strength) [56–58]. These in vitro models differ from bone in ageing and PM-OP in many ways; perhaps most importantly the bone has normal levels of enzymatic crosslinking and it lacks the other forms of damage [54]. The bone collagen becomes hypercrosslinked [59•]. The relative importance of the presence of AGEs in determining bone quality in the case of PM-OP deserves further examination for at least two reasons. Firstly, the small increase in AGE crosslink concentration in PM-OP, measured in terms of the convenient biomarker pentosidine, does not compare to the great loss of enzymatically derived crosslinking content. They differ by approximately two orders of magnitude. See Table 1. Second is the concept of the connectivity of the bone collagen network. Crosslinking increases connectivity by tying protein chains together. In bone, as a mineral-filled polymer composite [49], increased connectivity should increase collagen strength and therefore, bone’s ultimate strength and toughness. Therefore, the large decrease in enzymatic crosslinks in PM-OP sensibly reduces bone strength and toughness. Following the same logic, a concurrent increase in AGE crosslinks might be expected to partly compensate for the loss in strength rather than contribute to it. And yet, the amount of pentosidine measured is approximately two orders of magnitude smaller than the loss of enzymatically derived crosslinks due to estrogen withdrawal meaning the potential compensatory effect is likely negligible. Consistently, there is mention of another AGE crosslink, termed glucosepane, which is structurally similar to pentosidine but far more difficult to measure because it is labile (lost during preparative steps such as acid hydrolysis) [60, 61]. Glucosepane has been measured in skin and kidney membrane and found to be more abundant than pentosidine in those tissues [60, 61]. However, its presence in bone has not be shown and, again, it would be expected to increase the connectivity and the strength of the collagen and, therefore, the bone quality. One must note that AGE crosslinks are thought to form nonspecifically, linking two intra-helical sites rather than linking between one intra-helical site and one site in the collagen telopeptide as immature enzymatic crosslinks are known to do [7]. Given sufficient concentration, particularly when superimposed on normal levels of enzymatic crosslinking, AGE crosslinks degrade fibril ductility while

19.1±12.5 (+406 %) 15.8±10.63 (+205 %)

Pentosidine controls (mol/mol collagen×10−3)

Female cynomolgus monkeys Female rabbits

Understanding the primary importance of oxidative stress in PM-OP supports the leveraging of anti-oxidant therapies in the battle against osteoporotic fractures. Raloxifene (an estrogen analog) and pyridoxamine (a B6 vitamer) are candidate drugs [19••, 33, 62••, 63, 64]. To date, Raloxifene therapy has been shown to improve bone mass, bone quality, and crosslinking profiles in humans and animals [19••, 33, 62••, 63, 64]. Some of the positive effects of Raloxifene on bone mechanical properties may be cell independent [62••]. This interesting idea requires further study to fully elucidate the mechanisms. Pyridoxamine-based therapies specifically for osteoporosis are not widely reported, even though it is an acknowledged anti-oxidant, anti-AGE, and anti-carbonyl agent [65, 66].

a

With a methionine rich diet that induces hyperhomocysteinemia

1.128±0.387 0.172±0.063 −25.7 % of Total 0.867±0.206a (−18.9 %) Total 0.480±0.028a (−24 %) Total 1.069±0.283 Total 0.633±0.059 Total Saito et al., 2011 Saito et al., 2010

4.7±1.8 7.7±2.4 Bailey, 1992 Oxlund et al., 1995

Oxlund et al., 1996 Satio et al., 2006

Low density mineral fraction High density mineral fraction

−20 % to −40 % Immature crosslinks −45 % pyridinolines (Presumably paralleled by immature crosslinks) −24 % to −30 % Immature crosslinks −10 % of Total

Enzymatic crosslinking in OP / OVX (mol/mol collagen)

Part C—Therapies

Enzymatic crosslinking in controls (mol/mol collagen) Study

Table 1 Studies linking changes in collagen crosslinks to degradation of bone quality

also strengthening the fibrils. But in the case of OP where the concentration of enzymatically derived crosslinks is greatly reduced and the increase in AGEs is very small, the effect of the changing crosslinking profile on bone mechanical properties is likely negligible. Pentosidine and some other AGEs require oxidation for their formation and, therefore, their increased presence in PM-OP is not surprising. With time and after more comprehensive studies, it may be concluded that pentosidine is simply an indicator of heightened oxidative stress and collagen damage rather than a causal contributor to the degraded mechanical properties of PM-OP bone. Oxidative stress driven inhibition of enzymatic crosslinking and collagen degradation leading to loss of network connectivity provides a more cohesive explanation, which requires further elucidation.

1.816±1.094 (+61 %)* 0.188±0.070 (+9.3 %) 0.342±0.090 (+98.8 %)+

Human femoral neck and head β-amino-propionitrile treated female rat femora Human vertebral trabeculae Human intercapsular hip fractures

Pentosidine In OP/OVX (mol/mol collagen×10−3)

Model

Curr Osteoporos Rep

Conclusions Postmenopausal osteoporosis starts with estrogen depletion and subsequently increased oxidative stress occurs. This leads to both imbalance in bone remodeling, with the net effect of bone volume loss, and degraded bone quality, through loss of collagen crosslinking. The combined effect can result in vertebral, hip, and other fragility fractures; clinical indicators of postmenopausal osteoporosis. It seems that there are multiple positive feedback loops originating with oxidative stress that interact and drive the progression of postmenopausal osteoporosis. Therapies with anti-oxidant potential have promise to slow these pathologic mechanisms. Further investigations are required to completely elucidate these mechanisms, and trials will be required to establish antioxidant-based therapies clinically.

Curr Osteoporos Rep Compliance with Ethics Guidelines Conflict of Interest TL Willett, J Pasquale, and MD Grynpas declare that they have no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

References Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance 1.• Cervellati C, Bonaccorsi G, Cremonini E, Bergamini CM, Patella A, Castaldini C, et al. Bone mass density selectively correlates with serum markers of oxidative damage in postmenopausal women. Clin Chem Lab Med. 2013;51:333–8. Recent clinical evidence that oxidative stress and bone mass/volume loss are associated in PMOP. 2. Sendur OF, Turan Y, Tastaban E, Serter M. Antioxidant status in patients with osteoporosis: a controlled study. Joint Bone Spine. 2009;76:514–8. 3. Maggio D, Barabani M, Pierandrei M, Polidori MC, Catani M, Mecocci P, et al. Marked decrease in plasma antioxidants in aged osteoporotic women: results of a cross-sectional study. J Clin Endocrinol Metab. 2003;88:1523–7. 4. Muthusami S, Ramachandran I, Muthusamy B, Vasudevan G, Prabhu V, Subramaniam V, et al. Ovariectomy induces oxidative stress and impairs bone antioxidant system in adult rats. Clin Chim Acta. 2005;360:81–6. 5. Hamada Y, Fujii H, Fukagawa M. Role of oxidative stress in diabetic bone disorder. Bone. 2009;45 Suppl 1:S35–8. 6.• Grune T, Catalgol B, Jung T. Oxidative stress and protein oxidation. In: Protein Oxidation and Aging: John Wiley and Sons; 2013, p. 1–214. A good review of oxidative stress and protein oxidation with sections on various diseases including osteoporosis. 7. Avery NC, Bailey AJ. The effects of the Maillard reaction on the physical properties and cell interactions of collagen. Pathol Biol. 2006;54:387–95. 8. Biemel KM, Reihl O, Conrad J, Lederer MO. Formation pathways for lysine-arginine cross-links derived from hexoses and pentoses by Maillard processes: unraveling the structure of a pentosidine precursor. J Biol Chem. 2001;276:23405–12. 9. Sell DR, Nagaraj RH, Grandhee SK, Odetti P, Lapolla A, Fogarty J, et al. Pentosidine: a molecular marker for the cumulative damage to proteins in diabetes, aging, and uremia. Diabetes Metab Rev. 1991;7:239–51. 10. Hein G, Weiss C, Lehmann G, Niwa T, Stein G, Franke S. Advanced glycation end product modification of bone proteins and bone remodelling: hypothesis and preliminary immunohistochemical findings. Ann Rheum Dis. 2006;65:101–4. 11. Bucciarelli LG, Wendt T, Rong L, Lalla E, Hofmann MA, Goova MT, et al. RAGE is a multi-ligand receptor of the immunoglobulin superfamily: implications for homeostasis and chronic disease. Cell Mol Life Sci. 2002;59:1117–28. 12. Allen MR, Gineyts E, Leeming DJ, Burr DB, Delmas PD. Bisphosphonates alter trabecular bone collagen cross-linking and

isomerization in beagle dog vertebra. Osteoporos Int. 2008;19:329– 37. 13. Verzijl N, Degroot J, Oldehinkel E, Bank RA, Thorpe SR, Baynes JW, et al. Age-related accumulation of Maillard reaction products in human articular cartilage collagen. Biochem J. 2000;350(Pt 2):381–7. 14. Verzijl N, Degroot J, Thorpe SR, Bank RA, Shaw JN, Lyons TJ, et al. Effect of collagen turnover on the accumulation of advanced glycation end products. J Biol Chem. 2000;275:39027–31. 15.•• Saito M, Marumo K. Collagen cross-links as a determinant of bone quality: a possible explanation for bone fragility in aging, osteoporosis, and diabetes mellitus. Osteoporos Int. 2010;21:195–214. An excellent and comprehensive review of bone collagen crosslinking and the changes associated with loss of bone quality in various diseases including osteoporosis and diabetes mellitus. 16. Odetti P, Rossi S, Monacelli F, Poggi A, Cirnigliaro M, Federici M, et al. Advanced glycation end products and bone loss during aging. Ann N Y Acad Sci. 2005;1043:710–7. 17. Hein GE. Glycation endproducts in osteoporosis—is there a pathophysiologic importance? Clin Chim Acta. 2006;371:32–6. 18.•• Saito M, Marumo K, Kida Y, Ushiku C, Kato S, Takao-Kawabata R, et al. Changes in the contents of enzymatic immature, mature, and non-enzymatic senescent cross-links of collagen after onceweekly treatment with human parathyroid hormone (1–34) for 18 months contribute to improvement of bone strength in ovariectomized monkeys. Osteoporos Int. 2011;22:2373–83. An important study of bone collagen crosslinking changes in a primate OVX model of PM-OP. 19.•• Saito M, Marumo K, Soshi S, Kida Y, Ushiku C, Shinohara A. Raloxifene ameliorates detrimental enzymatic and nonenzymatic collagen cross-links and bone strength in rabbits with hyperhomocysteinemia. Osteoporos Int. 2010;21:655–66. This important study gives evidence for the important role of oxidative stress in altering bone collagen crosslinking (decreased enzymatic and increased pentosidine crosslinking). Furthermore, it demonstrates one of the ways raloxifene may imparts its beneficial effects and the role of hyperhomocysteinemia in increasing oxidative stress and increasing pentosidine content. 20. Saito M, Fujii K, Soshi S, Tanaka T. Reductions in degree of mineralization and enzymatic collagen cross-links and increases in glycation-induced pentosidine in the femoral neck cortex in cases of femoral neck fracture. Osteoporos Int. 2006;17:986–95. 21. McCarthy AD, Etcheverry SB, Bruzzone L, Lettieri G, Barrio DA, Cortizo AM. Non-enzymatic glycosylation of a type I collagen matrix: effects on osteoblastic development and oxidative stress. BMC Cell Biol. 2001;2:16. 22. Almeida M, Han L, Martin-Millan M, Plotkin LI, Stewart SA, Roberson PK, et al. Skeletal involution by age-associated oxidative stress and its acceleration by loss of sex steroids. J Biol Chem. 2007;282:27285–97. 23.• Goettsch C, Babelova A, Trummer O, Erben RG, Rauner M, Rammelt S, et al. NADPH oxidase 4 limits bone mass by promoting osteoclastogenesis. J Clin Invest. 2013;123:4731–8. This study highlights the role of NOX4 in osteoclastogenesis. This may link increased oxidative stress and increase bone resorption in PM-OP. 24. Almeida M, Han L, Ambrogini E, Bartell SM, Manolagas SC. Oxidative stress stimulates apoptosis and activates NF-kappaB in osteoblastic cells via a PKCbeta/p66shc signaling cascade: counter regulation by estrogens or androgens. Mol Endocrinol. 2010;24: 2030–7. 25. Baynes JW, Thorpe SR. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes. 1999;48:1–9. 26. Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes. 1991;40:405–12. 27.• Fleming TH, Humpert PM, Nawroth PP, Bierhaus A. Reactive metabolites and AGE/RAGE-mediated cellular dysfunction affect the

Curr Osteoporos Rep

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

aging process – a mini-review. Gerontology. 2010;57(5):435–43. The paper presents a model of the negative effects of AGE/RAGE mediated cellular dysfunction that can be applied to better understand PM-OP. Zhou Z, Immel D, Xi CX, Bierhaus A, Feng X, Mei L, et al. Regulation of osteoclast function and bone mass by RAGE. J Exp Med. 2006;203:1067–80. McCarthy AD, Etcheverry SB, Cortizo AM. Advanced glycation endproduct-specific receptors in rat and mouse osteoblast-like cells: regulation with stages of differentiation. Acta Diabetol. 1999;36:45–52. Ding KH, Wang ZZ, Hamrick MW, Deng ZB, Zhou L, Kang B, et al. Disordered osteoclast formation in RAGE-deficient mouse establishes an essential role for RAGE in diabetes related bone loss. Biochem Biophys Res Commun. 2006;340:1091–7. Takagi M, Kasayama S, Yamamoto T, Motomura T, Hashimoto K, Yamamoto H, et al. Advanced glycation endproducts stimulate interleukin-6 production by human bone-derived cells. J Bone Miner Res. 1997;12:439–46. Miyata T, Notoya K, Yoshida K, Horie K, Maeda K, Kurokawa K, et al. Advanced glycation end products enhance osteoclast-induced bone resorption in cultured mouse unfractionated bone cells and in rats implanted subcutaneously with devitalized bone particles. J Am Soc Nephrol. 1997;8:260–70. Saito M, Fujii K, Mori Y, Marumo K. Role of collagen enzymatic and glycation induced cross-links as a determinant of bone quality in spontaneously diabetic WBN/Kob rats. Osteoporos Int. 2006;17: 1514–23. Franke S, Siggelkow H, Wolf G, Hein G. Advanced glycation end products influence the mRNA expression of RAGE, RANKL and various osteoblastic genes in human osteoblasts. Arch Physiol Biochem. 2007;113:154–61. McCarthy AD, Etcheverry SB, Bruzzone L, Cortizo AM. Effects of advanced glycation end-products on the proliferation and differentiation of osteoblast-like cells. Mol Cell Biochem. 1997;170:43–51. Alikhani M, Alikhani Z, Boyd C, MacLellan CM, Raptis M, Liu R, et al. Advanced glycation end products stimulate osteoblast apoptosis via the MAP kinase and cytosolic apoptotic pathways. Bone. 2007;40:345–53. Yan SD, Schmidt AM, Anderson GM, Zhang J, Brett J, Zou YS, et al. Enhanced cellular oxidant stress by the interaction of advanced glycation end products with their receptors/binding proteins. J Biol Chem. 1994;269:9889–97. Kislinger T, Fu C, Huber B, Qu W, Taguchi A, Du Yan S, et al. N(epsilon)-(carboxymethyl)lysine adducts of proteins are ligands for receptor for advanced glycation end products that activate cell signaling pathways and modulate gene expression. J Biol Chem. 1999;274:31740–9. Wautier MP, Chappey O, Corda S, Stern DM, Schmidt AM, Wautier JL. Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE. Am J Physiol Endocrinol Metab. 2001;280:E685–94. Oxlund H, Mosekilde L, Ortoft G. Reduced concentration of collagen reducible cross links in human trabecular bone with respect to age and osteoporosis. Bone. 1996;19:479–84. Saito M, Fujii K, Marumo K. Degree of mineralization-related collagen crosslinking in the femoral neck cancellous bone in cases of hip fracture and controls. Calcif Tissue Int. 2006;79:160–8. Opsahl W, Zeronian H, Ellison M, Lewis D, Rucker RB, Riggins RS. Role of copper in collagen cross-linking and its influence on selected mechanical properties of chick bone and tendon. J Nutr. 1982;112:708–16. Masse PG, Rimnac CM, Yamauchi M, Coburn SP, Rucker RB, Howell DS, et al. Pyridoxine deficiency affects biomechanical properties of chick tibial bone. Bone. 1996;18:567–74. Oxlund H, Barckman M, Ortoft G, Andreassen TT. Reduced concentrations of collagen cross-links are associated with reduced strength of bone. Bone. 1995;17:365S–71.

45.

Kafantari H, Kounadi E, Fatouros M, Milonakis M, Tzaphlidou M. Structural alterations in rat skin and bone collagen fibrils induced by ovariectomy. Bone. 2000;26:349–53. 46. Roschger P, Paschalis EP, Fratzl P, Klaushofer K. Bone mineralization density distribution in health and disease. Bone. 2008;42:456–66. 47. Landis WJ, Jacquet R. Association of calcium and phosphate ions with collagen in the mineralization of vertebrate tissues. Calcif Tissue Int. 2013;93:329–37. 48. Eyre DR, Weis MA. Bone collagen: new clues to its mineralization mechanism from recessive osteogenesis imperfecta. Calcif Tissue Int. 2013;93:338–47. 49. Gupta HS, Zioupos P. Fracture of bone tissue: the 'hows' and the 'whys'. Med Eng Phys. 2008;30:1209–26. 50. Nyman JS, Roy A, Tyler JH, Acuna RL, Gayle HJ, Wang X. Agerelated factors affecting the postyield energy dissipation of human cortical bone. J Orthop Res. 2007;25:646–55. 51. Nyman JS, Roy A, Acuna RL, Gayle HJ, Reyes MJ, Tyler JH, et al. Age-related effect on the concentration of collagen crosslinks in human osteonal and interstitial bone tissue. Bone. 2006;39:1210–7. 52. Wang X, Li X, Shen X, Agrawal CM. Age-related changes of noncalcified collagen in human cortical bone. Ann Biomed Eng. 2003;31:1365–71. 53. Wang X, Shen X, Li X, Agrawal CM. Age-related changes in the collagen network and toughness of bone. Bone. 2002;31:1–7. 54. Wang X, Bank RA, Tekoppele JM, Hubbard GB, Athanasiou KA, Agrawal CM. Effect of collagen denaturation on the toughness of bone. Clin Orthop Relat Res. 2000;371:228–39. 55. Zioupos P, Currey JD, Hamer AJ. The role of collagen in the declining mechanical properties of aging human cortical bone. J Biomed Mater Res. 1999;45:108–16. 56. Vashishth D, Gibson GJ, Khoury JI, Schaffler MB, Kimura J, Fyhrie DP. Influence of nonenzymatic glycation on biomechanical properties of cortical bone. Bone. 2001;28:195–201. 57. Tang SY, Vashishth D. The relative contributions of non-enzymatic glycation and cortical porosity on the fracture toughness of aging bone. J Biomech. 2011;44:330–6. 58. Tang SY, Vashishth D. Non-enzymatic glycation alters microdamage formation in human cancellous bone. Bone. 2010;46:148–54. 59.• Willett TL, Sutty S, Gaspar A, Avery N, Grynpas M. In vitro nonenzymatic ribation reduces postyield strain accommodation in cortical bone. Bone. 2013;52:611–22. This paper draws attention to the short comings of the ribation model. 60. Biemel KM, Friedl DA, Lederer MO. Identification and quantification of major Maillard cross-links in human serum albumin and lens protein. Evidence for glucosepane as the dominant compound. J Biol Chem. 2002;277:24907–15. 61. Sell DR, Biemel KM, Reihl O, Lederer MO, Strauch CM, Monnier VM. Glucosepane is a major protein cross-link of the senescent human extracellular matrix. Relationship with diabetes. J Biol Chem. 2005;280:12310–5. 62.•• Gallant MA, Brown DM, Hammond M, Wallace JM, Du J, Deymier-Black AC, et al. Bone cell-independent benefits of raloxifene on the skeleton: a novel mechanism for improving bone material properties. Bone. 2014;61:191–200. A very interesting study suggesting that the positive effects of raloxifene on bone quality and in treating osteoporosis may be partly cellindependent. In vitro soaking of bone specimens in a raloxifene solution improved ductility. 63. Allen MR, Hogan HA, Hobbs WA, Koivuniemi AS, Koivuniemi MC, Burr DB. Raloxifene enhances material-level mechanical properties of femoral cortical and trabecular bone. Endocrinology. 2007;148:3908–13. 64. Allen MR, Iwata K, Sato M, Burr DB. Raloxifene enhances vertebral mechanical properties independent of bone density. Bone. 2006;39:1130–5.

Curr Osteoporos Rep 65.

Voziyan PA, Hudson BG. Pyridoxamine: the many virtues of a maillard reaction inhibitor. Ann N Y Acad Sci. 2005;1043: 807–16.

66.

Voziyan PA, Hudson BG. Pyridoxamine as a multifunctional pharmaceutical: targeting pathogenic glycation and oxidative damage. Cell Mol Life Sci. 2005;62:1671–81.