Moderate hyperhomocysteinemia induced by short-term dietary methionine overload alters bone microarchitecture and collagen features during growth

Accepted Manuscript Moderate hyperhomocysteinemia induced by short-term dietary methionine overload alters bone microarchitecture and collagen features during growth

Petar Milovanovic, Dragan Hrncic, Ksenija Radotic, Mira Stankovic, Dragosav Mutavdzic, Danijela Djonic, Aleksandra Rasic-Markovic, Dragan Djuric, Olivera Stanojlovic, Marija Djuric PII: DOI: Reference:

S0024-3205(17)30512-X doi:10.1016/j.lfs.2017.10.008 LFS 15377

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Please cite this article as: Petar Milovanovic, Dragan Hrncic, Ksenija Radotic, Mira Stankovic, Dragosav Mutavdzic, Danijela Djonic, Aleksandra Rasic-Markovic, Dragan Djuric, Olivera Stanojlovic, Marija Djuric , Moderate hyperhomocysteinemia induced by short-term dietary methionine overload alters bone microarchitecture and collagen features during growth. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Lfs(2017), doi:10.1016/j.lfs.2017.10.008

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Moderate hyperhomocysteinemia induced by short-term dietary methionine overload alters bone microarchitecture and collagen features during growth

Petar Milovanovic1, Dragan Hrncic2, Ksenija Radotic3, Mira Stankovic3, Dragosav

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Mutavdzic3, Danijela Djonic1, Aleksandra Rasic - Markovic2, Dragan Djuric2, Olivera

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Stanojlovic2, Marija Djuric1,*

Laboratory for Anthropology, Institute of Anatomy, Faculty of Medicine, University

Laboratory for Neurophysiology, Institute of Medical Physiology "Richard Burian",

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of Belgrade, Belgrade, Serbia

Faculty of Medicine, University of Belgrade, Belgrade, Serbia Department of Life Sciences, Institute for Multidisciplinary Research, University of

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Belgrade, Belgrade, Serbia

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* Corresponding author:

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Prof. Marija Djuric, MD, PhD

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Laboratory for Anthropology, Institute of Anatomy, Faculty of Medicine, University of Belgrade, Dr Subotica 4/2, 11 000 Belgrade, Serbia Tel: +381 11 2686 172, e-mail: [email protected]

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Abstract Aims: In general, hyperhomocysteinemia is increasingly appreciated as a risk factor for various diseases, including osteoporosis. However, its effects in non-adults remain largely unknown. Our aim was to determine whether dietary-caused increased

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homocysteine levels have deleterious effects on bone structure during growth. Main methods: We developed a model of moderate hyperhomocysteinemia caused by

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short-term methionine nutritional overload in growing rats. 30-days-old male Wistar

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albino rats were randomly assigned to either experimental group subject to a 30-days hypermethionine diet or control group. High-resolution 3D assessment of bone

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geometry and microarchitecture, as well as fluorescence spectroscopic analysis of bone

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matrix were performed.

Key findings: Short-term moderate hyperhomocysteinemia (~30 µmol/L) achieved in

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the study notably affected bone and cartilage characteristics. Parameters of the cortical

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bone geometry in the experimental group indicated peculiar reorganization of the bone cross-section. Trabecular bone microarchitecture was especially sensitive to hyperhomocysteinemia showing clearly negative bone balance in the experimental

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group (almost 30% reduced bone volume, mainly due to ~25% decrease in trabecular

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number as well as markedly reduced trabecular connections). Fluorescent spectroscopy of bone matrix revealed multiple alterations to collagen spectra due to homocysteine accumulation in bone, indicative of broken collagenous cross-links. Significance: Given that appropriate accrual of bone mass during growth has important effects on the risk of osteoporosis in adulthood, understanding the skeletal effects of dietary-induced hyperhomocysteinemia in non-adults is essential for interpreting its

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importance as a modifiable risk factor for osteoporosis and improving programs to preserve/re-establish bone health.

Keywords: Bone; Homocysteine; Fluorescent spectroscopy; Development; Tissue

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microarchitecture; Collagen

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INTRODUCTION

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Homocysteine is an amino acid that is an endogenous product of methionine metabolism [1]. Increased blood level of homocysteine is nowadays considered as a risk

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factor for the diseases of different organ systems [2-6]. The reported prevalence of

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hyperhomocysteinemia (homocysteine level >15μmol/L) is high and varies between investigated populations. It is more frequent in men and generally increases with age,

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also depending on geographical location and ethnicity [7]. The overall prevalence of

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hyperhomocysteinemia ranges from 15.4% in Greek children [8] to 27.5% in general Chinese population [7], 29.3% in Framingham population or even 44.5% in elderly patients in Buenos Aires [9].

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Recently, homocysteine effects on the skeleton also started attracting attention of

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researchers [10-13]. In this regard, homocysteine has been identified as a potential risk factor for osteoporosis and bone fragility [12, 14]. Epidemiological studies investigated the relationship between homocysteine levels and bone mineral density (BMD) – a surrogate marker of bone strength, showing a decreased BMD [15] and subsequently an increased fracture risk in the patients with hyperhomocysteinemia [16]. Moreover, negative effects of hyperhomocysteinemia on bone were found to be partly independent of the decrease in BMD [14]. However, as the human studies are limited in 3

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documenting and understanding the origins of adverse homocysteine effects on bone strength, various animal models with distinct advantages and disadvantages have been developed recently [13, 17-20]. In particular, regarding bone pathology, the animal models predominantly focused on severe [13, 21] and long-term hyperhomocysteinemia

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[13, 19-21] that can be especially relevant for genetic types of hyperhomocysteinemia due to enzymes defects or those occurring in chronic renal disease in humans [1, 22].

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However, nutrition is a factor that can cause rather moderate hyperhomocysteinemia,

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that may be more frequent than genetic mutation in enzymes of homocysteine metabolism [23-25]. Hence, homocysteine as a modifiable risk factor is an important

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potential target for prevention and treatment programs related to wide range of diseases,

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including bone fragility/osteoporosis, beside cardiovascular and brain disorders. Since animal studies dealing with hyperhomocysteinemia focused only on adult

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female rats, it is still unknown whether and how hyperhomocysteinemia affects bone

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structure during growth and development. In general, accrual of bone mass during growth and development has important effects on risk of osteoporosis in adulthood [26], where one standard deviation increase in peak bone mass may reduce the lifetime

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fracture risk by 50% [27]. Therefore, individuals still in period of growth should already

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be a target for preventing later osteoporosis with all its complications. Clinical data in a pediatric population with increased homocysteine levels showed higher biochemical markers of bone turnover and a decline in BMD [28]; yet, clarifying the structural basis of increased bone fragility requires high-resolution imaging on a suitable animal model. Recently, Hrncic et al. [29] have developed a specific model of mild hyperhomocysteinemia induced by methionine nutritional overload in young male rats

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and showed particular brain vulnerability in this model even during a short-term treatment (30 days). Therefore, in this study we have used an animal model of short-term dietaryinduced moderate hyperhomocysteinemia to reveal its effects on bone structural

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integrity during growth and development. Specifically, an approach combining the methods based on X-ray absorption (high resolution micro-CT imaging of both cortical

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and trabecular bone compartments’ micro-architecture and bone geometry) and

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fluorescence-emission (fluorescence spectroscopic analysis of bone matrix) may unravel various structural imprints of hyperhomocysteinemia and contribute to understanding of

MATERIALS AND METHODS

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Animals

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the alterations in bone development characteristics.

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Young male Wistar albino rats (Military Medical Academy Breeding Laboratories, Belgrade, Serbia, local certified supplier) were used in the study. All rats were kept in a sound-attenuated room under a constant laboratory environment (temperature 22±2 ºC,

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relative humidity 50-60%, 12/12 h light/dark cycle starting at 8 AM) in groups of 2 in

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standard transparent, plastic cages. Water was provided ad libitum during the entire experiment.

All experiment procedures were carried out in accordance with the Directive of the European Parliament and of the Council (2010/63/EU) and approved by the Ethical Committee of the Republic of Serbia (Permission No 298/5-2).

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Experimental groups and protocol for inducing hyperhomocysteinemia Animals were randomly assigned to either control group (n=8) or experimental group (Hyperhomocysteinemia

group:

n=8).

To

induce

hyperhomocysteinemia,

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experimental protocol using hypermethionine diet described previously was followed

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[29]. In brief, the animals were fed from 30th to 60th postnatal day ad libitum with standard or methionine-enriched diet, respectively. In comparison to the standard diet

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(#4RF21GLP, Mucedola SLR, Milano, Italy), methionine-enriched diet (#4RF21-

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CUSTOM-Met2x, Mucedola SLR, Milano, Italy) contained double content of methionine (7.70 vs. 3.85 g/kg), and the same amount of folic acid (2.3 mg/kg), vitamin

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B6 (10.7 mg/kg), vitamin B12 (0.027 mg/kg), choline (2256 mg/kg) and other amino

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acids and microelements. The food consumption and body weight were monitored on a daily basis, which allowed us to confirm that both groups had comparable food intake

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between the groups (p=0.620).

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and regular mass increments, so that final body weight did not differ significantly

Specimen preparation

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On the 61st postnatal day rats were euthanized by decapitation and samples were

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collected for further analyses. Blood samples were collected in heparin coated tubes, centrifuged at 2500 × g for 10 min, and the isolated samples stored at −80°C until use. Total blood homocysteine concentration was determined by commercially available kit using method of immunonephelometry according to manufacturer’s instructions (Homocysteine Assay Kit, #605 1-A, BN II, Dade Berhing, Marburg, Germany) as previously referred in details [29]. The right femur of each animal was dissected and cleaned of adherent soft tissues. 6

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Micro-computed tomography The right femur of each animal was covered with thin layer of parafilm to avoid specimen’s desiccation and fixed on a sample holder with consistent proximal-distal

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orientation. The distal femur’s portion was scanned using Skyscan 1172 microcomputed tomography system (Bruker mikroCT, Skyscan, Belgium). The scanning

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conditions were 60 kV, 167 µA, 10 W, 640 ms exposure time, 0.5 mm Aluminum filter,

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2K camera binning and 6 µm isotropic resolution, rotation step of 0.4° and frame averaging of 3. The reconstruction of the projection images was accomplished using

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NRecon software (Bruker microCT, Belgium) on InstaRecon platform (InstaRecon,

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USA) with appropriate thermal drift correction and misalignment compensation, Gaussian smoothing of 1, and appropriate ring artifact and beam hardening corrections.

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Ct.An program version 1.14 (Skyscan, Belgium) was used for quantitative analysis of

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the data. With regard to the peculiarities of rat femur anatomy, trabecular bone was marked manually on slides to define the trabecular volume of interest, while cortical bone was automatically selected using custom processing routine. The global threshold

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of 68/255 was chosen to distinguish between the mineralized bone (gray levels above

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68) and marrow spaces (gray values below 68). The same threshold was applied for all specimens to allow inter-individual comparisons of microarchitectural parameters. The following microarchitectural parameters of the cortical bone were evaluated in automatic 3D analysis: percent bone volume (BV/TV, %), bone surface to volume ratio (BS/BV, 1/mm), fractal dimension (FD, dimensionless), total porosity (Po_tot, %), pore diameter (Po.Dm, mm), and pore spacing (Po.Sp, mm).

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Trabecular microarchitectural parameters analyzed were percent bone volume (BV/TV, %), bone surface to volume ratio (BS/BV, 1/mm), trabecular bone pattern factor that represents inverse of connectivity (Tb.Pf, 1/mm), fractal dimension (FD, dimensionless), structure model index (SMI, dimensionless), degree of anisotropy (DA,

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dimensionless), trabecular thickness (Tb.Th, mm), trabecular number (Tb.N, 1/mm), and trabecular separation (Tb.Sp, mm).

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Mean gray scale index value was calculated in Ct.An program to estimate the

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mineralization level of the trabecular and cortical compartments in both groups of cases. Parameters of bone geometry were also assessed: average object equivalent

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circle diameter (Av.Obj.ECD, mm) denoting a diameter of a circle with the same area as

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given bone cross-sectional area (i.e., surrogate for diaphyseal cross-section area), bone perimeter (B.Pm, mm) that represents total perimeter of the diaphyseal bone cross-

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section, cortical thickness (Ct.Th, mm), and eccentricity (Ecc, dimensionless) reflecting

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the shape of the diaphyseal cross-section. To rule out that the potential inter-group differences stem from differences in bone length, bone geometry parameters were further adjusted for bone length.

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Given that growth plate cartilage of the distal femur has a very complex

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morphology, for the sake of consistency the epiphyseal growth plate mean thickness was measured on micro-CT reconstructions on a sagittal section passing half-way between two condyles.

Fluorescence spectroscopy Fluorescence steady state emission spectrum of macromolecules may be a sum of two or more individual components corresponding to various fluorophores – emitting 8

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structural entities. Determining the number and emission profiles of components in an integral spectrum is a prerequisite to get a specific insight into the structure, complementary to the other analytical techniques. This can be achieved by measurement of a series of emission spectra at different excitation wavelengths in a wavelength range,

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thus obtaining excitation-emission matrices (EEMs) that are subsequently analyzed by using advanced statistical methods [30-32]. Such an approach provides fine information

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about molecular structure and its changes. Measurement of EEMs for tissue samples in

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vitro is a fast and noninvasive technique relying on the endogenous fluorophores to obtain diagnostic information for clinical studies and diagnostic analysis [33-34].

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Collagen fluorescence emission has a broad band in range 400-440 nm and it has been

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exploited as an intrinsic indicator of the pathological changes in different tissues. For instance, changes in collagen emission properties were observed in precancerous colon

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tissue sections [35-36], malignant bladder mucosa in vitro [37] and dysplastic cervical

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tissue in comparison to healthy control cases [38]. The fluorescent spectroscopy method has also proved itself useful for assessment of different solid materials [39-40]. Diaphyseal specimens were cut and slightly polished under constant water

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irrigation (Unipol 810, MTI Corporation, USA) using the finest carbide papers

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(smoothness of 4000) to remove very thin superficial bone layer and better expose collagen. Fluorescence spectra of diaphyseal specimens from control and treated animals were collected using a Fluorolog FL3-221 spectrofluorimeter (JobinYvon Horiba, Paris, France) equipped with a 450W Xe lamp and a photomultiplier tube. The spectra were measured in the front-face configuration of the measuring cavity, which is particularly important for solid specimens, such as bone. The slits on the excitation and emission beams were fixed at 2 nm. The integration time was 0.1 s. The spectra were 9

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corrected for dark counts and Rayleigh masking was applied. In each measurement three scans were averaged. For each sample a series of 11 emission spectra were collected, by excitation at different wavelengths, in the range 270-300 nm, with 3 nm step. The emission spectra were measured in the 310-525 nm range, with 1 nm

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increment. The bone excitation-emission landscapes were compared with the collagen

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spectra from the literature [41].

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Statistical analysis

According to the ethical approvals, the groups were limited to the size acceptable for

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most experimental studies on rats, where the RRR principle of animal research is

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considered. To ensure the statistical power of at least 80%, the groups sizes were verified based on preliminary data on the trabecular BV/TV (considered as the primary

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outcome variable), expected differences to be detected and probability of type-I error

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(alpha) of 0.05.

Kolmogorov-Smirnov test confirmed Gaussian distribution of all measured parameters. We used Student’s t-test for independent samples to compare various

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quantitative parameters between the experimental and control groups. Linear regression

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analysis was performed to determine the dependence of microarchitectural features on homocysteine concentrations. The statistical analyses were performed in SPSS statistical software (ver. 15) at the two-sided significance level of 0.05. In the mathematical-statistical analysis of the fluorescence spectra we used two matrices, corresponding to the bone samples of control and hyperhomocysteinemic animals. Each matrix was analysed by using Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) method [32], which extracted the number of components, as 10

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well as their emission profiles. All analyses were performed using The Unscrambler software package (Camo ASA).

RESULTS

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Hypermethionine diet in growing rats caused moderate hyperhomocysteinemia (32.83 ± 15.62 µmol/L), significantly higher from the values in the control group (8.67 ± 1.21

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µmol/L, p=0.006).

(342.5 ± 30.8 g vs. 336.3 ± 15.8 g, p=0.620).

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Experimental and control groups did not differ significantly in body weight

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Femur length was not significantly changed between the groups, although

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showing a tendency to increased length in the experimental group (3.45 ± 0.05 cm vs. 3.52 ± 0.08 cm, p=0.07). However, epiphyseal growth plate thickness was higher in the

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experimental group (0.749±0.262 mm vs. 0.309±0.173 mm, p=0.002) (Supplementary

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Fig. 1). Regression analysis showed that both the femur length and epiphyseal growth plate thickness correlated positively with the measured concentrations of homocysteine

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(r=0.567, p=0.027; r=0.792, p=0.000).

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Geometry and cortical thickness There was a tendency to an increased average object equivalent circle diameter of the femoral diaphysis in the experimental group (p=0.09), denoting a wider cross section due to hyperhomocysteinemia. The trend was maintained even after the adjustment for bone length (p=0.1), revealing that the result was not an artifact of different bone length. Total perimeter of the diaphyseal cross-section was higher in the experimental group (32.09±3.02 mm vs. 29.65±1.32 mm, p=0.064). Adjusting the diaphyseal perimeter for 11

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the variations in bone length even increased the difference (0.93 vs. 0.86, p=0.05), emphasizing the consequence of hyperhomocysteinemia. Yet, cortical thickness did not show any difference between the groups (Ct.Th=0.35 ± 0.02 mm in both groups, p=0.82), even after the adjustment for bone length. The eccentricity parameter was

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significantly lower in the control group (Ecc=0.64±0.03 vs. 0.69±0.04, p=0.011), suggesting also a change in the overall bone shape (less circular, more elongated cross-

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section) in the experimental group.

Cortical microarchitecture

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Micro-CT assessment of cortical microarchitectural parameters (Table 1) did not show

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statistically significant inter-group differences (p>0.05), although a slight tendency to a 5% higher total porosity was noticed in the experimental group. There was no

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significant difference in mineralization level, as reflected in mean gray scale index

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(p>0.05) (Table 1).

Trabecular microarchitecture

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Unlike cortical bone, the trabecular microarchitecture was significantly deteriorated due

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to hyperhomocysteinemia (Table 2, Fig. 1). Specifically, trabecular bone volume fraction was almost 30% lower than in the control group, mainly due to nearly a 25% decrease in trabecular number. Increased trabecular pattern factor revealed markedly reduced trabecular connections in the experimental group. There was no significant difference in mineralization level, as reflected in mean gray scale index (p>0.05) (Table 2).

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Furthermore, regression analysis on homocysteine concentration and trabecular microarchitectural parameters revealed that most trabecular microarchitectural properties were linearly dependant on homocysteine concentration. This was evident for the parameters related to bone quantity showing a significant decrease in trabecular

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bone volume fraction (r=-0.770, p=0.001) and trabecular number (r=-0.763, p=0.001), as well as an increase in trabecular separation (r=0.739, p=0.002). Likewise, the

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parameters related to bone internal organization were also linearly dependent on

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homocysteine concentrations, such as bone surface to volume ratio (r=0.589, p=0.021), trabecular bone pattern factor (r=0.807, p=0.000), structure model index (r=0.782,

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p=0.001), and fractal dimension (r=-0.729, p=0.002).

Bone matrix composition through fluorescence spectroscopy

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The excitation-emission landscapes for the bones from the control group and

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hyperhomocysteinemia group revealed collagen spectra containing two peaks, high wavelength peak being higher in relation to low wavelength peak (Fig. 2 A,B). In the experimental group, there was an increase in magnitude of the low wavelength peak

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relative to the high wavelength peak. The results of MCR analysis of the emission

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profiles in the control group showed the first maximum around 400 – 405 nm and another one at 435 nm (Fig. 2 C). Hyperhomocysteinemia-affected bone showed shifts of peaks to lower wavelengths: 390 – 395 nm and 425 – 435 nm. An additional peak (visible as a shoulder in the raw spectra) also appeared at a lower wavelength in the bone from the experimental group (350 – 365 nm) (Fig. 2 D).

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DISCUSSION In comparison with other studies performing a prolonged diet (2-5 months) with moderately [20] or more severely increased blood levels of homocysteine in adult female rats (more than 50 μmol/L) [13, 19, 21], here we show that in growing male rats

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even a short-term hypermethionine diet (30 days) and subsequently moderate hyperhomocysteinemia were strong enough to affect bone structural integrity. This is

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important especially for understanding the cases of nutritional hyperhomocysteinemia

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that express a less severe increase in homocysteine concentrations [24] and do not necessarily persist for long periods of time; even so, our findings showed that bone

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structure and development could be affected to a significant extent, mainly through

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changes in tissue microarchitectural organization and collagen characteristics. Hyperhomocysteinemia is frequently classified as mild (homocysteine level

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range: 15 - 30 µmol/L), moderate (30 - 100 µmol/L) and severe (> 100 µmol/L). Severe

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hyperhomocysteinemia is relatively rare and is caused by genetic defects, while mild and moderate hyperhomocysteinemia are considerably more frequent in general population [42]. In the current study, we used a rodent model of moderate

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hyperhomocysteinemia induced by methionine-enriched diet during 30 days (from 30th

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to 60th postnatal day). Taking into account lifespan of rodents and humans and temporal characteristics of their development, it has been calculated that ~10-daysperiod in growing rats corresponds to one human year [43]. Thus, duration of high methionine intake in this model relates to approximately three years of altered dietary habits in humans, and here developed hyperhomocysteinemia corresponds to chronic moderate hyperhomocysteinemia in humans. On the other hand, in most rodent models

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there were long-term nutritional interventions lasting up to 3-5 months (corresponding to duration of 9-15 years in humans). Bone structure is built and rebuilt based on balanced bone formation and bone resorption processes [44], where relative predominance of bone resorption leads to

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osteoporotic bones [45-47]. It is accepted that bone microarchitecture can reflect imbalances between bone formation and bone resorption processes at the

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microstructural level [45]. Previous experimental studies already reported an increase in

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biochemical markers of bone resorption following long-term hypermethionine diet [13, 20], whereas clinical data in pediatric population showed that hyperhomocysteinemia

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was associated with an increase both in bone resorption and formation markers [28].

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The current study showed deteriorated trabecular bone microarchitecture in the experimental group (Fig. 1), where decreased trabecular number, thinning of the

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remaining trabeculae, losing intertrabecular connections, as well as a shift from rather

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plate-like to more rod-like trabecular shapes may all suggest an overall predomination of bone resorption processes. All changes followed a dose-dependent pattern of homocysteine concentrations. It has to be noted, however, that although these

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microarchitectural parameters suggest predominance of bone resorption over bone

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formation, further studies will require use of cellular analyses and dynamic histomorphometry to get definite mechanistic explanations. As cortical microarchitecture did not show significant deterioration in the rats with hyperhomocysteinemia, it may suggest that cortical bone is less susceptible to deleterious effects of homocysteine in comparison to the trabecular bone. This could be a consequence of a much higher bone surface to volume ratio measured in the trabecular bone in either group (Tables 1 and 2). Nevertheless, it is also possible that the time 15

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frame of hypermethionine diet and the level of homocysteine achieved in our study were just not high enough to induce thorough intra-cortical bone loss that would be evident in deteriorated cortical microarchitecture. In contrast to slight changes in microarchitecture, overall geometry of the cortical bone cross-section showed

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remarkable changes due to hypermethionine diet, confirming that cortical bone is not “immune” to the effects of increased homocysteine concentrations. Namely, the

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experimental group demonstrated an increase in cortical perimeter (with unchanged

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cortical thickness) as well as a wider and more elongated cross-section. Considering the way how morphology of cortical cross-sections can change in different life periods [47],

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the changes found in hyperhomocysteinemia group are compatible with remarkable

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processes of bone resorption and bone formation occurring simultaneously at the opposite surfaces. Namely, while bone is lost from the endosteal surface, equal

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we found in this study.

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thickness is added to the periosteal surface, which preserves mean cortical thickness as

While most studies agree that homocysteine stimulates osteoclastic activity [4849], it is less clear how it affects osteoblasts and apparently effects of homocysteine on

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osteoblasts are contradictory. Namely, it was reported that homocysteine disturbs

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osteoblasts’ function as evidenced in a decreased osteocalcin secretion [50], but also that it has the ability to stimulate the differentiation of preosteoblastic cells in culture [51]. Although the present study did not directly evaluate the cellular characteristics, the observed changes in cortical bone geometry might indicate that osteoblasts at the periosteal surface (unlike other surfaces) are relatively uninhibited by increased homocysteine levels. From the mechanistic point of view, it is possible that increased periosteal expansion is a compensatory mechanism to offset the negative effects of 16

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decreased bone architecture (endosteal cortical resorption and significant trabecular bone loss) and preserve bone strength, as suggested in aging human femora [44]. This explanation rises from the fact that in tubular structures even a slight increase in the outer diameter notably increases bending strength [44, 52]. However, as bone cross-

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sections are reorganized to a more elongated shape in the experimental group, one could speculate that altered ossification process may be a factor here. Regression analysis

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showed that both the femur’s length and epiphyseal growth plate thickness correlated

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positively with the measured concentrations of homocysteine. Hence, it is likely that there is a delayed conversion of cartilage to bone, with preserved or even increased

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cartilage growth, both of which led to a significantly thicker growth plate cartilage and

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also a tendency to longer femora in the experimental group.

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Homocysteine effects on bone matrix

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It is likely that microarchitectural and geometry changes after hypermethione diet largely originate from the methionine and homocysteine’s effects on bone re/modeling. However, bone quality is also affected by homocysteine’s direct effects on bone matrix.

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It was suggested by Herrmann et al. that in severe hyperhomocysteinemia the

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collagenous part of the bone matrix is a predilection site for accumulation of homocysteine [13]. It is important that collagen solutions with added homocysteine cannot form insoluble fibrils in vitro [53], and recent cell culture studies demonstrated that homocysteine reduces the amount of enzymatic cross-links of collagen due to a decreased activity and expression of lysyl oxidase [4]. In the current study we analyzed the fluorescence of the matrix, with focus on collagen fluorescence spectra [37-38, 41, 54], considering that in load-bearing tissues collagen fluorescence is based on 17

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intramolecular cross-links (hydroxylysyl pyridoline and lysyl pyridinoline) [55]. Hence, two peaks in the collagen spectra obtained in the present study may correspond to two different forms of collagen. In accordance to the literature data [41], a peak with a higher wavelength would indicate protein composed of extremely long chains of amino

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acids with preserved inter-chain cross links, while the lower wavelength peak points to the collagen with more hydrolyzed structure composed of short amino acid chains with

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reduced amount of cross-links. In the experimental group, there was an increase in

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magnitude of the low wavelength peak (390 – 395 nm) relative to the high wavelength peak (425 – 435 nm), suggesting the state of collagen in which many of the cross-links

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are broken. In addition, both peaks shifted to lower wavelengths (peaks’ positions: 390

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– 395 nm and 425 – 435 nm, instead of 400 – 405 nm and 435 nm, respectively), and an additional third peak at low wavelengths (350 – 365 nm) appeared, also supporting

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changed organization in the collagen due to hyperhomocysteinemia. Additional studies

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are necessary to explain the exact chemical mechanisms of reduced cross-linking, one of which could be as reported on a mice model of cystathionine β-synthase deficiency where homocysteine-tiolactone interacted with lysine residues that are required for

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establishing collagen cross-links [56].

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The characteristics of collagen are nowadays considered as an important determinant of bone quality [57] and alterations in collagen configuration and crosslinks are increasingly linked to impaired bone toughness [58-61]. Therefore, our spectroscopic

findings

suggested

that

moderate

hyperhomocysteinemia

was

accompanied by breaking of collagen cross-links which may be harmful for bone quality at the matrix level. However, despite general agreement about the importance of spectroscopic methods for assessment of collagen properties [62], further studies are 18

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necessary to obtain morphological insights into collagen alterations in various metabolic conditions. Bone strength stems from all levels of bone hierarchical structure, from nanolevel [59, 63-66] to micro- [45, 67-71] and macro-levels [72-74]. The study is limited

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by the lack of whole bone mechanical testing that could directly show the mechanical effects of the experimental protocol. Yet, there is a sound pool of studies relating the

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values of micro-CT parameters to bone mechanical properties, so that microarchitecture

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can be considered as an indirect measure of bone strength [68, 75-77]. However, the focus of this study was not on direct assessment of bone mechanical properties, but

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rather on evaluating changes in bone structure due to hypermethionine diet, providing

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additional insights into the deleterious effects of homocysteine. Further studies are warranted to investigate whether the observed homocysteine effects are uniform

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throughout the skeleton or some bones are more affected.

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Due to the study design (in both the current and majority of other studies) in which termination of dietary intervention corresponds to rodent life termination due to tissue sampling, it is not yet known if homocysteine-induced alterations in bone are

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reversible. Although additional intake of B-vitamins can reduce homocysteine blood

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levels, a few studies showed that it could not reduce the risk of osteoporotic fracture during five years of follow-up in adult patients with cardiovascular or cerebrovascular diseases [78-80]. It might be expected that, in non-adult population, reducing homocysteine concentration before the peak bone mass has been achieved is essential for recovering bone quality.

19

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CONCLUSIONS In summary, the experimental study in young male rats demonstrated that short-term moderate hyperhomocysteinemia affected bone structural integrity during growth and development. Clearly, hyperhomocysteinemia affected both trabecular and cortical

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compartments, but trabecular bone microarchitecture was especially deteriorated. Moreover, at the bone matrix level, hyperhomocysteinemia was associated with signs

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indicative of collagen cross-links breakage. Obviously, through both bone architecture

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and matrix effects, moderately increased homocysteine concentration affects bone development and impairs bone quality (Fig. 3). Understanding of skeletal effects of

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dietary-induced hyperhomocysteinemia is essential for understanding its importance as

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a modifiable risk factor for osteoporosis and improving preventive and treatment programs to preserve or re-establish bone health. In particular, given that the impact of

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bone loss in old age is predetermined by peak bone mass acquired during the skeletal

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growth, pediatric age may already be a time to prevent adult osteoporosis.

Acknowledgments: The study was supported by the Ministry of Education and Science

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of the Republic of Serbia (grant numbers: III 45005, 175032, 173017). The funding

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agency had no role in the study design, analyses or writing of the manuscript.

Conflict of interest: All authors declare that they have no conflicts of interest.

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Figure legends Fig. 1 Micro-CT scans of the distal femoral trabecular bone in control and experimental rats. 2D maximum intensity projection images in (A) control and (B) experimental group (scale bars: 1 mm). White circular outlines denote the sites of 3D reconstructed

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images. Representative 3D reconstruction of trabecular bone in control group (C) and

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experimental group (D) (scale bars 0.5 mm). (E, F) 3D reconstruction of the micro-CT

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scans of the cortical bone in control (E) and experimental group (F) (scale bars 1 mm).

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Fig. 2 Fluorescence spectroscopy in control and experimental rats. (A, B) Excitationemission landscape for the raw spectra of control and experimental group, respectively.

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(C, D) Estimated emission profiles obtained by MCR-ALS method for control and experimental group, respectively. Blue and red colors denote 400-405 nm and 435 nm

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components, respectively (C). Red, green and blue colors denote 350-365 nm, 390-395

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nm and 425-435 nm components, respectively (D).

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Fig. 3 Summary figure

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Tables Table 1. Cortical microarchitecture in experimental and control groups (Experimental group – fed 30 days with double content of methionine comparing to

Group

Mean

Percent bone volume

control

81.69

experimental

80.73

Bone surface / volume

control

28.31

experimental

Fractal dimension

2.65 0.211 2.53

2.443

0.032 0.865

2.446

0.021

control

18.31

3.01

19.27

2.46

0.098

0.006

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experimental control

0.512

Pore separation [mm]

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experimental control

0.124 0.093

0.006

0.041

0.004

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Pore diameter [mm]

Mineralization

0.220

experimental

0.039

0.003

control

115.48

2.66

114.69

2.92

(mean gray scale index) experimental

0.512

2.46

experimental

Total porosity [%]

P-value

3.01

30.08

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control

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ratio [1/mm]

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[%]

SD

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Parameter

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standard diet, Control group – fed with standard diet)

0.582

Table 2. Trabecular micro-architecture in experimental and control groups 37

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Parameter

Group

Mean

SD

Percent bone volume

control

31.14

2.02

P-value

[%]

experimental

22.40

3.16

Bone surface / volume

control

60.83

3.84

ratio [1/mm]

experimental

67.33

4.04

Trabecular pattern

control

-1.88

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0.000

0.007

6.06

control

0.527

Structure model index

control Degree of anisotropy

control

[mm]

0.000 0.159 0.132

2.104

0.085

2.613

0.631

0.035 0.000

experimental

2.495

0.049

0.054

0.003

control

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Trabecular thickness

0.132

D

Fractal dimension

3.00

2.132

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experimental

1.082

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experimental

0.048

experimental

0.052

0.003

control

5.722

0.458

experimental

4.331

0.435

Trabecular separation

control

0.138

0.013

[mm]

experimental

0.175

0.015

control

103.72

3.28

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[1/mm]

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Trabecular number

Mineralization

0.000

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experimental

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factor [1/mm]

1.95

0.000

0.000

0.876 (mean gray scale index) experimental

103.95

2.32

38

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Graphical abstract

39