The Ratio 1660/1690 cm−1 Measured by Infrared Microspectroscopy Is Not Specific of Enzymatic Collagen Cross-Links in Bone Tissue

In postmenopausal osteoporosis, an impairment in enzymatic cross-links (ECL) occurs, leading in part to a decline in bone biomechanical properties. Biochemical methods by high performance liquid chromatography (HPLC) are currently used to measure ECL. Another method has been proposed, by Fourier Transform InfraRed Imaging (FTIRI), to measure a mature PYD/immature DHLNL cross-links ratio, using the 1660/1690 cm−1 area ratio in the amide I band. However, in bone, the amide I band composition is complex (collagens, non-collagenous proteins, water vibrations) and the 1660/1690 cm−1 by FTIRI has never been directly correlated with the PYD/DHLNL by HPLC. A study design using lathyritic rats, characterized by a decrease in the formation of ECL due to the inhibition of lysyl oxidase, was used in order to determine the evolution of 1660/1690 cm−1 by FTIR Microspectroscopy in bone tissue and compare to the ECL quantified by HPLC. The actual amount of ECL was quantified by HPLC on cortical bone from control and lathyritic rats. The lathyritic group exhibited a decrease of 78% of pyridinoline content compared to the control group. The 1660/1690 cm−1 area ratio was increased within center bone compared to inner bone, and this was also correlated with an increase in both mineral maturity and mineralization index. However, no difference in the 1660/1690 cm−1 ratio was found between control and lathyritic rats. Those results were confirmed by principal component analysis performed on multispectral infrared images. In bovine bone, in which PYD was physically destructed by UV-photolysis, the PYD/DHLNL (measured by HPLC) was strongly decreased, whereas the 1660/1690 cm−1 was unmodified. In conclusion, the 1660/1690 cm−1 is not related to the PYD/DHLNL ratio, but increased with age of bone mineral, suggesting that a modification of this ratio could be mainly due to a modification of the collagen secondary structure related to the mineralization process

Étude des modifications et maturations du collagène de type I dans la résistance mécanique osseuse

L influence des maturations du collagène, de types enzymatique (désoxypyridinoline et pyridinoline) et non-enzymatique (produits de glycation avancée (AGEs) telle que la pentosidine (PEN) et isomérisation des C-télopeptides du collagène) a été étudiée sur le comportement biomécanique osseux. Dans notre modèle in vitro de glycation d os bovin nous avons démontré l action inhibitrice de laminoguanidine sur la formation de la PEN et de son inertie vis-à-vis de la phase minérale. De plus, une étude sur des vertèbres humaines a permis de quantifier la PEN et le ratio de collagène isomérisé dans l os cortical et trabéculaire. Ainsi, la PEN et l isomérisation du collagène apporteraient une contribution supplémentaire dans l explication des propriétés biomécaniques en plus de la densité minérale osseuse. Nos travaux suggèrent que les modifications des propriétés intrinsèques de la matrice osseuse, liées au vieillissement, contribueraient à la résistance mécanique osseuseThe influence of maturations of collagen, enzymatic types (deoxypyridinoline and pyridinoline) and non-enzymatic (advanced glycation end products (AGEs) such as pentosidine (PEN) and isomerization of C-telopeptides of collagen) was studied on bone biomechanical behavior. In our in vitro bovine bone model of glycation, we showed the inhibiting action of the aminoguanidine on the formation of the PEN and its inertia with respect to the mineral phase. Moreover, another study on human vertebrae allowed us to quantify PEN content and the ratio of isomerized collagen in cortical and trabecular bone. Thus, PEN and isomerization of collagen would bring an additional explanation of biomechanical properties in addition, to bone mineral density. Our results suggest that the modifications of the intrinsic properties of bone matrix, related to ageing, would contribute to bone strengthLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Association between collagen cross-links and trabecular microarchitecture properties of human vertebral bone.

International audienceIt has been suggested that age-related deterioration in trabecular microarchitecture and changes in collagen cross-link concentrations may contribute to skeletal fragility. To further explore this hypothesis, we determined the relationships among trabecular bone volume fraction (BV/TV), microarchitecture, collagen cross-link content, and bone turnover in human vertebral trabecular bone. Trabecular bone specimens from L2 vertebrae were collected from 51 recently deceased donors (54-95 years of age; 20 men and 30 women). Trabecular bone volume and microarchitecture was assessed by microCT and bone formation, reflected by osteoid surface (OS/BS, %), was measured by 2D histomorphometry. Pyridinoline (PYD), deoxypyridinoline (DPD), pentosidine (PEN) and collagen content in the cancellous bone were analysed by high-performance liquid chromatography. Associations between variables were investigated by Pearson correlations and multiple regression models, which were constructed with BV/TV and collagen cross-links as explanatory variables and microarchitecture parameters as the dependent variables. RESULTS: Microarchitecture parameters were modestly to strongly correlated with BV/TV (r(2)=0.10-0.71). The amount of mature enzymatic PYD and DPD cross-links were not associated with the microarchitecture, either before or after adjustment for BV/TV. However, there was a positive correlation between PEN content and trabecular number (r=0.45, p=0.001) and connectivity density (r=0.40, p=0.004), and a negative correlation between PEN content and trabecular separation (r=-0.29, p=0.04). In the multiple regression models including BV/TV, age and PEN content was still significantly associated with several of the microarchitecture variables. In summary, this study suggests a link between trabecular microarchitecture and the collagen cross-link profile. As PEN reflects non-enzymatic glycation of collagen and generally increases with bone age, the association between PEN and trabecular architecture suggests that the preserved trabeculae may contain mainly old bone and have undergone little remodeling. Thus, vertebral fragility may not only be due to alterations in bone architecture but also to modification of collagen cross-link patterns thereby influencing bone's mechanical behavior

Determinants of microdamage in elderly human vertebral trabecular bone.

Previous studies have shown that microdamage accumulates in bone as a result of physiological loading and occurs naturally in human trabecular bone. The purpose of this study was to determine the factors associated with pre-existing microdamage in human vertebral trabecular bone, namely age, architecture, hardness, mineral and organic matrix. Trabecular bone cores were collected from human L2 vertebrae (n = 53) from donors 54-95 years of age (22 men and 30 women, 1 unknown) and previous cited parameters were evaluated. Collagen cross-link content (PYD, DPD, PEN and % of collagen) was measured on surrounding trabecular bone. We found that determinants of microdamage were mostly the age of donors, architecture, mineral characteristics and mature enzymatic cross-links. Moreover, linear microcracks were mostly associated with the bone matrix characteristics whereas diffuse damage was associated with architecture. We conclude that linear and diffuse types of microdamage seemed to have different determinants, with age being critical for both types

Effects of preexisting microdamage, collagen cross-links, degree of mineralization, age, and architecture on compressive mechanical properties of elderly human vertebral trabecular bone.

International audiencePrevious studies have shown that the mechanical properties of trabecular bone are determined by bone volume fraction (BV/TV) and microarchitecture. The purpose of this study was to explore other possible determinants of the mechanical properties of vertebral trabecular bone, namely collagen cross-link content, microdamage, and mineralization. Trabecular bone cores were collected from human L2 vertebrae (n = 49) from recently deceased donors 54-95 years of age (21 men and 27 women). Two trabecular cores were obtained from each vertebra, one for preexisting microdamage and mineralization measurements, and one for BV/TV and quasi-static compression tests. Collagen cross-link content (PYD, DPD, and PEN) was measured on surrounding trabecular bone. Advancing age was associated with impaired mechanical properties, and with increased microdamage, even after adjustment by BV/TV. BV/TV was the strongest determinant of elastic modulus and ultimate strength (r²  = 0.44 and 0.55, respectively). Microdamage, mineralization parameters, and collagen cross-link content were not associated with mechanical properties. These data indicate that the compressive strength of human vertebral trabecular bone is primarily determined by the amount of trabecular bone, and notably unaffected by normal variation in other factors, such as cross-link profile, microdamage and mineralization

Pictures obtained under fluorescent light of microdamage in orange: A) Linear microcrack, B) Diffuse damage, C) Few cross-hatching microdamage were found.

<p>Pictures obtained under fluorescent light of microdamage in orange: A) Linear microcrack, B) Diffuse damage, C) Few cross-hatching microdamage were found.</p

Bivariate Scattergrams.

<p>A) Age of donor vs Crack density – r = 0.36, p = 0.010, B) BV/TV vs Diffuse damage density, r = −0.42, p = 0.003, C) Degree of mineralization vs crack density, r = −0.31, p = 0.02, D) Mineralization index vs Crack length, r = 0.36, p = 0.008, E) Mineral maturity vs Crack length, r = 0.30, p = 0.03, F) Mature cross-links vs Crack length, r = −0.30, p = 0.03.</p

Descriptive statistics, mean ± SD, range, median and interquartile range (IQR).

<p>Descriptive statistics, mean ± SD, range, median and interquartile range (IQR).</p

Spearman partial coefficient after adjustment for age; dependent variables shown along the top and independent variables along the side of the table.

*<p>p<0.05,</p>**<p>p<0.01.</p

Bivariate spearman coefficient correlation (r_sp) between microdamage, age, architecture, mineralization degree, microhardness, mineral at crystal level, organic matrix and collagen cross-links.

*<p>p<0.05,</p>**<p>p<0.01.</p