Calcif Tissue Int DOI 10.1007/s00223-015-9996-2

ORIGINAL RESEARCH

The Role of Collagen Organization on the Properties of Bone Patrick Garnero1

Received: 12 January 2015 / Accepted: 7 April 2015 Ó Springer Science+Business Media New York 2015

Abstract Bone is a complex tissue constituted by a collagen matrix filled in with crystal of hydroxyapatite (HAP). Bone mechanical properties are influenced by the collagen matrix which is organized into hierarchical structures from the individual type I collagen heterotrimer flanked by linear telopeptides at each end to the collagen fibrils that are interconnected by enzymatic and non-enzymatic crosslinks. Although most studies focused on the role of collagen cross-links in bone strength, other organizational features may also play a role. At the molecular level it has been shown that homotrimer of type I collagen found in bone tissue of some patients with osteogenesis imperfecta (OI) is characterized by decreased mechanical competence compared to the regular heterotrimer. The state of C-telopeptide isomerization—which can be estimated by the measurement in body fluids of the native and isomerized isoforms—has also been shown to be associated with bone strength, particularly the post-yield properties independent of bone size and bone mineral density. Other higher hierarchical features of collagen organization have shown to be associated with changes in bone mechanical behavior in ex vivo models and may also be relevant to explain bone fragility in diseases characterized by collagen abnormalities e.g., OI and Paget’s disease. These include the orientation of collagen fibrils in a regular longitudinal direction, the D-spacing period between collagen fibrils and the collagen-HAP interfacial bonding. Preliminary data indicate that some of these organizational features can change during treatment with bisphosphonate, raloxifene,

& Patrick Garnero [email protected] 1

INSERM UMR 1033, Lyon, France

and PTH suggesting that they may contribute to their antifracture efficacy. It remains however to be determined which of these parameters play a specific and independent role in bone matrix properties, what is the magnitude of mechanical strength explained by collagen organization, whether they are relevant to explain osteoporosis-induced bone fragility, and how they could be monitored non-invasively to develop efficient bone quality biomarkers. Keywords Bone matrix
Bone strength
Collagen organization
Type I collagen homotrimer
Osteogenesis imperfecta
Paget’s disease
Osteoporosis

Introduction Although clinical assessment of bone mineral density (BMD) by dual X-ray absorptiometry, which largely reflects the mineral phase of bone tissue, is the current gold standard for the diagnosis of osteoporosis, about 50 % of postmenopausal women with incident fracture have BMD levels above the WHO criteria for osteoporosis [1, 2], indicating that factors not reflected in BMD measurement contribute to bone fracture resistance. Indeed, bone fragility also depends on the morphology and architecture of bone as well as on the material properties of bone matrix that cannot be readily assessed. Bone is an intricate material which is characterized by a complex hierarchical structure. From a biochemical point of view, it is composed of collagen fibers filled with stiff, hard mineral crystal of hydroxyapatite (HAP). Type I collagen makes up 90 % of the organic matrix. Mineral plays a major role in determining bone stiffness and yield strength, whereas post-yield properties such as ultimate strength and toughness are believed to be dependent on an

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P. Garnero: The Role of Collagen Organization on the Properties of Bone

intact collagen network [3, 4]. The importance of collagen integrity in bone properties is demonstrated by manipulations to the collagen, such as thermal-induced collagen denaturation [5], formalin fixation [6], and high-energy gamma or X-ray irradiation [7–9], which all reduce the toughness of bone. The molecular bases that underlie the effects of collagen on bone mechanical properties involve the structure of the triple helix, the stabilizing hydrogen bounding, and the intermolecular cross-linking and other enzymatic and nonenzymatic post-translational modifications. The objectives of this review is first to describe the organization of collagen molecules in bone tissue, then discuss its influence on bone strength and the changes occurring in diseases characterized by an altered collagen organization. It has to be mentioned that the non-collagenous proteins which account for 10 % of the organic matrix are also involved in the mechanical properties of bone tissue as recently reviewed [10] but their analysis is beyond the scope of this paper.

Collagen Synthesis, Structure, and Post-translational Modifications Type I collagen is the most abundant type of collagen and is widely distributed in almost all connective tissues with the exception of hyaline cartilage. It is the major protein in bone, skin, tendon, and ligament sclera, cornea, and blood vessels. Type I collagen comprises approximately 95 % of the entire collagen content of bone and about 80–90 % of the total proteins present in bone. Other types of collagen, such as types III and V, are present at low levels in bone [11] and appear to modulate the type I collagen fibril diameter. Collagen is synthesized as a procollagen precursor which is a helical rod of three intertwining polypeptide chains in a heterodimer consisting of two identical a1 helices and one different a2 helix. Each chain contains approximately 1000 amino acids in which every third residue is glycine and is positioned toward the center of the supercoil [12]. Proline typically occupies the next position, and there is an abundance of hydroxyproline in the third position. Hydroxyproline residue facilitates thermal stability through intramolecular hydrogen bonding [13]. Hydroxylysine is also a characteristic residue of bone collagen and gives rise to cross-linking. Two specific enzymes, called galactosyltransferase and galactosylhydroxylysyl-glucosyltransferase, act on the e-hydroxyl group of a hydroxylysine during the biosynthesis of procollagen [14]. They result in the formation of the a-1,2-glucosyl-galactosylhydroxylysine (Glc-Gal-Hyl) and the b-1-galactosyl-hydroxylysine (Gal-Hyl). As hydroxylases, these enzymes act

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as long as the collagen triple helix is not completely formed. The degree of glycosylation changes according to the collagen type and the tissue. Bone contains essentially Gal-Hyl, whereas soft tissues such as synovium [15] and skin contain mainly Glc-Gal-Hyl [16]. Collagen molecules in bone tissue are organized into hierarchical structures, where the lowest hierarchical level consists of triple helical collagen molecule (Fig. 1). Collagen fibrils consist of high-aspect-ratio polypeptides, tropocollagen molecules, with a length of about 300 nm and a diameter of about 1.5 nm flanked by small terminal globular domains known as the N- and C-propeptides. Proteolytic cleavage of the propeptides results in triple helical collagen molecules that have short N- and C-telopeptides at each end. They can assemble into fibrils which are arranged in a staggered configuration. This structure creates an observable periodicity known as the D-band where D = 67 nm (Fig. 1). Molecules in a fibril are deposited side by side and parallel but staggered with respect to each other, where the molecular axes are parallel to fibril direction [17]. A gap between two consecutive collagen molecules is known as ‘‘gap region’’ and measures 0.54 D or about 36 nm. Collagen fibers have a diameter of 80–100 nm and a length up to the millimeter range and are formed through the bundling of several microfibrils that each contains clusters of five collagen molecules. At the next hierarchical level, multiple fibrils make up the collagen fiber, formed with the participation cross-linking molecules (Fig. 2). In bone, the organic collagen protein is stiffened via the inclusion of the mineral HAP crystals that emerge from the gap regions. Collagen enzymatic and non-enzymatic cross-links play a major role in collagen organization and bone strength. The process of formation of cross-linking molecules along with their relationships with bone biochemical properties are described in detail in another paper in this issue (see Saito and Marumo in this issue). Racemization and Isomerization Besides enzymatic and non-enzymatic cross-links, collagen also undergoes racemization and isomerisation. Racemization is due to the spontaneous conversion of the native Lenantiomeric form of amino acids or sugars to the rare D form. Accumulation of D isomer is common in tissues with low turnover such as dentin, dermis, and cartilage. b isomerization is due to the transfer of the peptide bound between aspartic acid residues and the adjacent amino acid from the a carboxyl group to the b or c carboxyl group (Fig. 3). In bone, racemization and isomerization involve the aspartic acid residue within the C-telopeptide of type I collagen in the so called CTX sequence [18, 19] (Fig. 3). Isomerisation in the type I collagen C-telopeptides causes a kink in the peptide

P. Garnero: The Role of Collagen Organization on the Properties of Bone Fig. 1 The different hierarchical levels of type I collagen fibrils organization

N

Asp C

Lys pentosidine Arg

C PYD/DPD Advanced Glycation End Products (AGE) - non-crosslink type AGE (carboxymethylysine) - Intermolecular crosslink (eg pentosidine, glucosepane)

N+

Lox

Telopeptide Hyl or Lys

+ Helical Hyl or Amadori Lys rearrangement

Hydroxyallysine Allysine

DHLNL HLNL

+ Hydroxyallysine or Allysine

HLKLN LKLN

Immature divalent enzymatic crosslinks

- PYD/DPD - Pyrrole

Mature trivalent enzymatic crosslinks

Amadori Product + Lys or Arg Schiff Base Allysine

Glycation Glycoxylation Oxydative stress

+ Glucose/pentose Lys or Hyl Helical domain

Fig. 2 Pathways of enzymatic and non-enzymatic crosslink formation in bone collagen. deH-DHLNL dehydro-dihydroxylysinonorleucine, deH-HLNL dehydro-hydroxylysinonorleucine, deH-LNL

dehydro-lysinonorleucine, HLKNL hydroxylysino-5-k-ketonorleucine, LKNL lysine-5-ketonorleucine, PYD pyridinoline, DPD deoxypyridinoline

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P. Garnero: The Role of Collagen Organization on the Properties of Bone

N-telopepde

K K

C-telopepde Xlink

K O

N H

OH N H

O

O

EKAHD (asp) G (gly)GR (CTX sequence)

Isomerizaon OH

Native CTX newly synthesized

N H

OH

O H N OH O

Isomerized

Bone area with increased bone turnover

O

CTX

age-relatedpredominant in “healthy adults”

(Paget’s disease, bone metastasis)

Fig. 3 Pathways of isomerisation of the Asp-Gly motif within the CTX sequence in the C-telopeptide of type I collagen a1 chain. When collagen molecules are newly synthesized the Asp-Gly motif is in a a form, i.e., the peptide bound between Asp and Gly involves the carboxyl group in position a. During maturation of collagen in bone matrix there is a spontaneous isomerisation which consists in the transfer of the peptide bound to the carboxyl group in position b. The

two isoforms a and b are in equilibrium which can be altered in case of high bone remodeling found for example in patients with Paget’s disease or bone metastases. The relative abundance of a and b isoforms can be measured in urine by immunoassays using antibodies recognizing specifically the a and b isomers of the eight amino acid CTX sequence which is a breakdown product of collagen generated by collagenolytic enzymes particularly cathepsin K

backbone and may thus alter the properties of the collagen molecule. For example, it has been shown that the degree of bone collagen C-telopeptide isomerisation influences the collagenolytic activity of cathepsin K which cleaves type I collagen at multiple sites including the C-telopeptide close to the isomerisation motif [20]. It is also possible that isomerisation interferes with the formation of enzymatic crosslinks and consequently has indirectly an impact on bone strength [21]. It remains unclear whether isomerisation has beneficial or detrimental effects on bone strength. In vitro and clinical studies suggest, however, that the relation between isomerisation and bone strength is likely to follow a U-shaped pattern, with maturation that is too high or too low compromising tissue strength. Aspartic acid isomerization at 37 °C has been studied in vitro using synthetic CTX peptides and immature fetal bovine bone collagen extract that consists mainly of native a CTX isomer [18]. At the steady state, there is equilibrium between the native and isomerized peptide, with about 20 % of CTX peptide remaining in its original a form and 80 % is b isomerized [18]. Thus, the b isoform concentration cannot exceed 80 % of the total amount of a and b type I collagen isoforms. In healthy adults, because on average the rate of bone remodeling is slower than isomerization, equilibrium is achieved, resulting in a fairly constant a/b CTX ratio from the age of 20 years and

upward [19]. Conversely, in disease states characterized by increased bone remodeling, such as in Pagetic bone [22] or metastatic tissue [23], the steady state is not achieved leading to an excess of native versus isomerized isoform. To sum up, isomerization is a slow process that induces conformation modifications of proteins, disrupting protein regulation, or function. In bone tissue, C-telopeptide isomerisation could serve as a biomarker of collagen matrix maturation which can be measured by the detection in body fluids of native and isomerized CTX breakdown products.

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Collagen Organization and Bone Strength As previously mentioned, collagen molecules in bone matrix are organized at different hierarchical levels, i.e., molecular level, collagen fibrils, and collagen fibers (Fig. 1). Below is described the role of these different hierarchical structures in bone properties. Structure of the Collagen Molecule and Mechanical Properties The type I collagen molecule is a heterotrimer consisting of two a-1 chains and one a-2 chain. Homotrimeric collagen, consisting of three a-1 chains, has been studied to

P. Garnero: The Role of Collagen Organization on the Properties of Bone

investigate the role of the a-2 chain on collagen stability and mechanical integrity [24]. In humans, homotrimeric collagen is mainly found in fetal tissues [25], fibrosis [26], and cancer [27, 28]. Significant amounts of type I collagen homotrimer have, however, also been reported in the subchondral bone of patients with osteoarthritis [29]. Cultured human osteoblasts from patients carrying the Sp1 COL1A1 polymorphism ‘‘ss’’ were found to secrete up to 10–15 % of a1(I) homotrimers [30]. Several studies have reported an association between this COL1A1 polymorphism and increased bone fragility in the general population, independent of BMD [31–33]. It is thus possible that synthesis of type I collagen homotrimers could be relevant in osteoporosis-related bone fragility, although it remains unknown how this collagen form could alter mechanical properties at the tissue level. Some insights may however be gained from the investigation of a mouse model of the genetic brittle bone disease due to the osteogenesis imperfecta murine (oim) mutation of type I collagen. This model is characterized by a complete replacement of the a-2 chain by an a-1 chain, resulting in the synthesis of homotrimeric collagen. The oim phenotype has skeletal fragility, reduced stature, and bone deformities, mimicking moderate to severe osteogenesis imperfect (OI) in humans. Experimental studies have shown that the mechanical strength of oim bone and tail tendon is significantly less than that of the normal mice [34–37]. At the molecular level it has been proposed that the a-2 chain has a critical role in the integrity of the triple helix of collagen and organization of collagen fibers [38] as shown by a loss of lateral packing in the collagen fibrils in the oim mouse. Using differential scanning calorimetry (DSC) study, it was estimated that oim collagen has *5 % greater volume fraction of water, which leads to an increased lateral distance between collagen molecules and consequently reduced cross-linking at the fibril hierarchical level [39]. More recently, the differential behavior of hetero and homo dimer at the molecular level was analyzed by full atomistic simulations using the NAMD [40] and the CHARMM force field [41]. Such analysis is used to explain the mechanisms that affect the overall behavior of molecules, including mechanical characteristics. These studies showed that homotrimer are more flexible and less stiff than heterotrimer, because it has larger kink angle leading to a larger distance between homotrimer molecules in the fibril [42]. There is also some evidence suggesting that changes in the isomerisation of the C-telopeptide of collagen are associated with fracture resistance. In an ex vivo model of fetal bovine cortical bone allowing control for bone size and BMD, it was shown that a higher degree of isomerisation (i.e., a lower a/b CTX ratio) was positively associated with bending and compressive mechanical properties particularly the yield stress and the post-yield energy

absorption. In contrast, no significant association was observed with the elastic modulus [21]. An increased level of a/b CTX ratio in bone tissue from human vertebral, indicating a deficit of isomerisation, has also been shown to be associated with decreased compressive bone strength, after accounting for the contribution of BMD [43]. It is, however, unlikely that the minor conformational changes induced by isomerisation, would directly modify the mechanical behavior of collagen molecules. More likely isomerisation is an index of other yet to be identified alterations that are impacting the mechanical properties of the collagen matrix. Collagen Fibril Orientation and Mechanical Properties Bone strength is also dependent on the orientation of collagen fibrils that can change with age and the direction of the load. Circularly polarized light microscopy (PLM) of cross sections from the midshaft of cadaveric femurs revealed age and gender differences in collagen fiber orientation [44] suggesting that age-related changes in bone mechanical properties could be related in part to the changes in fiber orientation. Strength of bone is higher in the direction of physiological loading that corresponds to the orientation of osteons in the cortical bone [45, 46]. In equine cortical bone, the lateral cortex is monotonically stronger, but less fatigue resistant, than tissue from the medial and dorsal regions. Plane and circularly PLM showed that longitudinally disposed collagen correlated with greater modulus and monotonic strength [47]. Similar findings were more recently reported in mouse cortical bone in which PLM studies showed that the anterior specimens contain relatively more longitudinal fibers than posterior sections and this orientation was correlated with improved tensile mechanical properties [48]. Supportive findings have also been generated from the study of the senescence-accelerated mouse strain P6 (SAMP6), which is regarded as a murine model of senile osteoporosis [49]. In this model post-yield displacement, fracture displacement, and fracture energy is decreased compared to control SAMR1 mice, whereas the rigidity is similar in both strains [50]. The main difference between the two strains was that collagen fibers of SAMP6 bone were significantly less oriented compared with SAMR1 mice both in longitudinal and transverse section. Analysis of demineralised bone showed that collagen from SAMP6 is characterized by lower ultimate energy. Other collagen alterations were a modest decrease of collagen content (-13 %) and a slight increase in hydroxyle residues whereas there was no difference between mice strains in the enzymatic lysylpyridinoline and non-enzymatic pentosidine cross-links. Thus, intact bones from SAMP6 mice are weak and brittle

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P. Garnero: The Role of Collagen Organization on the Properties of Bone

compared with SAMR1 controls which is attributed primarily to poorer organization of collagen fibers and, to a lesser extent, reduced collagen content. Collagen orientation is likely to also be important to explain the mechanical competence of trabecular bone as suggested from studies performed in vertebrae. Puustjarvi et al. [51] reported in a dog model that although long-term running significantly reduces BMD in the vertebrae, there was no change in the bone mechanical properties. By using PLM, they showed a reorganization of collagen fibrils with a preferred longitudinal orientation after running. Conversely, there was no change of the collagen content or maturation as monitored by enzymatic cross-links. Thus, collagen reorganization during exercise may contribute to the maintenance of vertebral bone strength despite decreased mineral density.

Lessons Taken from Diseases Due to Abnormalities in Collagen Organization Valuable information on the influence of collagen organization on bone properties can also been obtained from the study of diseases characterized by altered collagen matrix. The typical example is OI, but defects in collagen structure are also observed in other metabolic bone diseases such as Paget’s disease of bone and metastatic bone diseases. There are also marked alterations in collagen structure and bone strength in Lathyrism which is a rare disease that occurs in humans and in some animals after ingesting the seeds of sweet peas, Lathyrus odoratus. This disease is characterized by severe abnormalities of bones, joints, and blood vessels due to increased fragility of collagen fibrils. These defects are due to the inhibition of enzymatic collagen cross-links by the aminopropionitrile contained in the seeds, which irreversibly inhibits lysyl oxidase, an enzyme which is required for the formation of cross-links [52, 53]. These defects will be described in the chapter reviewing the contribution of collagen cross-links in bone properties. Osteogenesis Imperfecta (OI) OI is a heritable syndrome that affects both mineralized and non-mineralized tissues, and clinically, it causes increased bone fragility [54]. OI is characterized by a low bone mass, a reduced trabecular thickness and number, and a decreased bone formation at the cellular level [55]. Animal studies and investigations in human subjects suggest that skeletal fragility in OI is primarily due to the defect in collagen synthesis. The collagen abnormalities are the result of a mutation primarily of the two type I collagen genes, COLIA1 and COLIA2. More recently, however, other mutations causing recessive cases have been described. They include CRTAP, LEPRE1, and PPIB,

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which encode three proteins forming the prolyl 3-hydroxylase complex; PLOD2 and FKBP10, which encode, respectively, lysyl hydroxylase 2 and a foldase required for its activity in forming mature cross-links in bone collagen; SERPINH1, which encodes the collagen chaperone HSP47; SERPINF1, which encodes pigment epithelium-derived factor required for osteoid mineralization; and BMP1, which encodes the type I procollagen C-propeptidase [56]. Over 200 different mutations have been identified, but the most frequent is a point mutation that affects the conserved glycine residue adjacent to a bulky side chain amino acid in either COL1A1 or COL1A2. The severity of the disease depends on which amino acid is substituted for the glycine, which of the two a-chains is affected, and in which position the mutation takes in the triple helix. One type of mutation affects the proa1(I) or proa2(I) alleles that impair transcription and mRNA stability and produce low amounts of the secreted heterotrimer which is however normal in structure. The other group of mutations results in the secretion of structurally abnormal proa1(I) chains. The abnormal heterotrimers are incorporated into the matrix, resulting in qualitative alterations of bone tissue. An increase in the degree of hydroxylation of lysine in type I collagen has been reported, leading to slower triple helix formation in severe cases. This may lead to the formation of thin collagen molecules—although not observed in all studies—resulting in increased brittleness of bone [57–60]. In contrast, no change in the concentration of mature enzymatic cross-links has been reported. There is also some indirect evidence that the pattern of type I collagen C-telopeptide isomerisation may be altered in adult patients with mild OI. It was shown that compared to healthy agematched controls the urinary ratio a/b CTX was increased by 49 % in OI subjects. There was no correlation between urinary a/b CTX and the conventional biochemical markers of bone formation and bone resorption, indicating that this index of bone collagen matrix abnormalities could provide additional information on bone status in OI compared to conventional markers of bone turnover [61]. Detailed information on the impact of alterations of collagen organization on bone properties can also been obtained from the well defined mouse models of OI. Existing mouse models for OI include the oim mutation [62], Mov-13 mouse [63], Brittle IV mouse [64], and Crtap mouse [65], but the oim and brittle IV mouse have been the most used. As previously mentioned, the oim mouse is characterized by genetic mutations on the COLIA2 gene and produces a1(I) collagen homotrimers and non-functional proa2(I) chains. Besides the direct influence of the intrinsic behavior of homotrimer described above, other abnormalities in collagen fibrils organization have been reported in this model. These include a decrease of collagen content, reduced collagen fiber diameter, and a reduction in

P. Garnero: The Role of Collagen Organization on the Properties of Bone

immature enzymatic cross-links which all contribute to the alteration of the tensile properties of collagen fibers and mineral crystallinity of bone [66]. More recent data comparing the mechanical properties of mineralized and demineralised bone from oim mouse suggest, however, that the decrease in bone strength is more likely to be dependent on an altered interaction between the collagen and mineral than a direct effect of collagen defects [67]. The brittleness of the Mov13 bones is attributed largely to the loss of energy-dissipating features in the microstructure [68]. The lamellar bone present in the Mov13 bone does not dissipate energy with the same efficiency through interlamellar cleavage as does the non-mutant bone during fracture. In addition, the number of lamellar interfaces is decreased in the Mov13 mice compared with controls due to an 80 % increase in woven bone tissue. Finally, the Brtl mouse, is a model of human Type IV OI which has a classical glycine substitution (a cysteine is substituted for a glycine at the 349 position of the triple helix in one Col1a1 allele) [64]. In this model, there is no significant change in the degree of post-translational modifications and cross-linking but the mutation leads to smaller diameter of collagen fibers in vitro and disrupts crystalline organization [69]. Recent data indicate that the distribution of the D-period spacing in collagen fibrils is also altered in this model [70]. The D-periodic spacing is a key metric of fibril morphology. Indeed, this measure captures different aspects of fibril structure which may be related to the state of the individual molecular triple helix, post-translational modifications, and cross-linking. Changes in the D-period spacing are likely driven by alterations in the end-to-end spacing of collagen molecules within the fibril due to changes in intracellular tropocollagen processing and assembly, by a change in the tightness of the twist of the fibril, or even by differences in mineralization within the fibrils themselves. Tensile stretching of an individual collagen fibril indicated an association between fibril D-spacing and fibril mechanical properties [71, 72]. Interestingly, data in sheep have shown that long-term estrogen deficiency induced by ovariectomy resulted in a larger proportion of bone collagen fibers with a D-spacing lower than 64 nm, whereas in sham animals only 7 % had decreased values [73]. Thus, changes in D-spacing distribution could also be relevant in the context of postmenopausal osteoporosis, although currently there is no direct evidence of an association between this measure and bone strength at the tissue level. Paget’s Disease of Bone and Metastatic Bone Disease Paget’s disease of bone is a localized disease characterized by increased bone remodeling, bone hypertrophy, and abnormal bone structure. In patients with Paget’s disease,

increased bone fragility occurs despite an increase in bone density and bone size at most skeletal sites. In Pagetic bone sites, both bone resorption and bone formation are markedly increased. This high bone turnover does not allow proper organization of collagen fibers and leads to the formation of abnormal woven bone with an irregular arrangement of collagen fibers that are not deposited in a lamellar fashion with decreased mechanical properties [74]. Using immunohistochemistry analysis with antibodies recognizing specifically and distinctively the native and isomerized form of type I collagen C-telopeptide, it was shown that the immature woven bone was characterized by a lower degree of b isomerisation of type I collagen C-telopeptide which could be detected non-invasively by the measurement in urine of the ratio between a and b CTX [22]. A single injection of the bisphosphonate zoledronic acid was able to normalize urinary CTX ratio in patients with active Paget’s disease. This may be indicative—although this was not formally proven—of a progressive replacement of woven bone by a lamellar bone with a normal degree of isomerisation [75], as suggested by histological studies of patients following efficient bisphosphonate treatment. The woven bone and the associated alterations in type I collagen C-telopeptide isomerisation are not specific for Paget’s disease but reflect an extremely high rate of bone turnover which can be encountered in other clinical situations such as the metastatic bone found in patients with solid tumors [23].

Effect of Osteoporosis Treatments on Collagen Organization Recent studies reviewed in details in a separate paper of this issue (see Saito and Marumo) performed in animals and humans indicated that some treatments including bisphosphonate, PTH, and analogs of vitamin D3 [76, 77] induced significant changes in type I collagen enzymatic or non-enzymatic AGE cross-links and collagen maturation as measured by the ratio of mature versus immature collagen cross-links. Conversely, the effects of treatment on the other features of collagen organization are scarce, probably due to the methodological limitations to detect and quantify them. A recent study investigated the effect of raloxifene on the interface between collagen fibrils and HAP crystals. The water which is bound to collagen and HAP can induce changes in that interface and alter toughness. Bound water in human bone decreases with age and is strongly correlated with bone toughness [78, 79]. The ex vivo treatment of canine cortical bone with raloxifene (but also 17 b estradiol) increases bone toughness without affecting preyield properties which were correlated with the increase of water content in bone as determined by 3D ultrashort echo

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P. Garnero: The Role of Collagen Organization on the Properties of Bone Table 1 Decreased type I collagen isomerisation (increased urinary a/b CTX ratio) as a BMD-independent risk factor for fracture in postmenopausal women (OFELY study) and elderly men (MrOs study)

Unadjusted BMD adjusted a

OFELY studya 660 women; mean age 62 year, all fractures (n = 97), 10 year follow-up Relative risk(95 % CI) for a/b CTX levels above the upper limit of the premenopausal range

MrOS studyb Clinical vertebral fractures (n = 93), controls (n = 730); 7.3 year follow-up Relative risk (95 % CI) for a/b CTX in quartile 3 versus 3 lower quartiles

2.01 (1.22–3.31)

7.3 (4.5–11.8)

1.83 (1.10–3.05)

9.5 (4.5–12.8)

Adapted from Garnero et al. [82] and

b

Bauer DC et al. [84]

time magnetic resonance imaging [80]. This effect appears to be dependent on the hydroxyl groups present on raloxifene. Interestingly, using atomic force microscopy this study also showed that in samples treated by raloxifene the D-period spacing of collagen fibrils was slightly increased along with its distribution. It is thus possible that raloxifene weakens the collagen-HAP interfacial bonding allowing slipping in that plane and consequently increasing the period of post-yield deformation as suggested by modeling experiments [81]. These bone material property changes, if they are translated to the clinical situation, may explain part of the efficacy of raloxifene to reduce fracture risk despite only modest effects on BMD and bone turnover. As indicated above, treatment with bisphosphonate induces changes in the degree of type I collagen isomerisation in patients with Paget’s disease of bone, due to the potent inhibition of bone turnover induced by this class of drug.

Collagen Organization Features as Non-invasive Clinical Biomarkers of Bone Quality Few clinical studies have investigated the association between serum and urinary levels of collagen post-translational modifications and bone strength. One of the main limitations of the measurements of enzymatic cross-links in blood and in urine is that systemic levels reflect mainly the levels of bone turnover and not directly the alterations of their content per amount of collagen matrix. In addition, because most of the collagen cross-links including AGEs are not exclusively distributed in bone, there is also an issue of tissue specificity which is likely to impair the sensitivity of detecting significant associations. Nevertheless, a series of clinical investigations reviewed elsewhere in this issue of the journal (see Saito and Marumo) have reported an association between non-enzymatic AGEs (such as serum and urine pentosidine levels and serum carboxymethyllysine) and fracture risk, independent of BMD in subjects with osteoporosis or type 2 diabetes. Among the collagen post-translational modifications which can be monitored non-invasively, the pattern of type I

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collagen C-telopeptide isomerisation is a potential candidate. Indeed, the measurement of the ratio between native and isomerised isoforms of CTX in urine provides a good indication of the extent of collagen isomerisation in bone. In the OFELY prospective study, we found that women with a urinary a/b CTX ratio in the highest quartile had an increased risk of incident fracture independent of both the level of hip BMD and of bone turnover rate measured by serum bonespecific alkaline phosphatase [82]. An increase in the a/b CTX ratio was also reported in elderly postmenopausal Japanese women with hip fracture [83]. Finally, a strong BMDindependent association between high urinary a/b CTX ratio and the incidence of vertebral fracture was also reported in older men participating in the MrOs study [84], whereas conventional bone markers were not predictive (Table 1). This suggests that in aged men, the skeletal fragility may be more dependent on the alterations of the collagen matrix organization than quantitative abnormalities in bone turnover. The fracture efficacy of anti-resorptive and anabolic therapies is only partly explained by changes in BMD and bone turnover, suggesting that other factors, including modifications of bone matrix properties may play a role. A decrease in the urinary a/b CTX ratio has been reported in postmenopausal women receiving alendronate 10 or 20 mg/day and oral daily (2.5 mg) or intermittent ibandronate, but not in those receiving raloxifene or estradiol [85] consistent with animal studies. Conversely, in women receiving PTH 1–84 for 2 years [86], we reported a marked increase of urinary a/b CTX suggesting a decrease of collagen maturation as expected from the effect of PTH to stimulate the production of new bone matrix. Whether these changes in systemic markers of collagen organization observed with bisphosphonate and PTH therapy will translate into alterations of fracture risk independently of BMD remains to be investigated.

Conclusions and Research Questions Altogether, the data reviewed above indicate that the organization of the collagen matrix at all hierarchical levels including the structure of individual collagen molecules,

P. Garnero: The Role of Collagen Organization on the Properties of Bone

the orientation of collagen fibers, their interaction with mineral and water, and the pattern of post-translational modifications, plays an independent role in determining the bone mechanical competence, more specifically the postyield properties. However, important questions remain to be addressed to better characterize the contribution of collagen organization in bone mechanical properties and ultimately use some of these alterations as biological markers of bone quality. Most of the existing studies have described the collagen organization in bone biospecimens from humans or in defined animal models of bone fragility. However, there are few studies that have investigated the direct relationship between the alterations detected and the potential resulting mechanical effects both at the collagen matrix and the tissue levels. Because the different hierarchal levels of collagen organization are intimately interconnected and are interacting with HAP, it remains challenging to dissect out the specific effect of a given alteration on the bone properties. The identification of the most important collagen traits is, however, critical to concentrate the research efforts on the methods to quantify them at the tissue levels. Most of the studies were performed in animal models or in human diseases characterized by dramatic changes in bone structure and collagen matrix, such as OI and Paget’s disease of bone. It remains unknown whether and which of the collagen defects are relevant to explain the subtle abnormalities in bone properties observed in osteoporosis. The direct non-invasive assessment of the different hierarchical levels of collagen organization, including for example the D-period spacing, remains challenging, although this is critical to develop collagen matrix quality biomarkers to be used in large clinical studies. It is, however, possible that some of these features are determined by genetic polymorphisms that would represent a convenient way to predict them. An example of this connection is the association of the Sp1 polymorphism in the COL1A1 gene with an increase of type I collagen homotrimer synthesis. A field that needs to be explored in more details is the impact of therapies on collagen organization, which would be very useful in order to identify new mechanisms that predict anti-fracture efficacy, in addition to other factors such as BMD, bone dimensions, microstructure, and mineralization. One aspect which would also increase our understanding of the contribution of the organic matrix is the analysis of the influence of the non-collagenous proteins of bone tissue on collagen organization. From a translational point of view, at the present time one of the only noninvasive biomarker of collagen quality/maturation seems to be the isomerisation of the C-telopeptide. However, to be qualified as a valid surrogate biomarker, we need to better characterize the relationship between this subtle conformational change and alterations in the mechanical behavior

of the collagen molecule and ultimately the bone strength. As previously indicated, it is likely that isomerization is just an indirect index of more dramatic changes occurring elsewhere in collagen organization. Another candidate biomarker is collagen homotrimer. It remains, however, unclear what is the prevalence of this collagen form in the general population and at which ratio of homo versus heterotrimer, the mechanical impacts of bone can be observed. It seems, however, feasible to develop a non-invasive biological test to quantify the synthesis of type I collagen homotrimers based on the detection in body fluids of the homotrimer-specific N-terminal propeptide [87].

Conflict of interest interest to disclose.

Patrick Garnero does not have any conflict of

Human and Animal Rights and Informed Consent All studies reviewed in this paper have been performed following regulatory requirements for human and animal rights and informed consent statements have been obtained from each patient.

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