Osteoporosis Samples Research Articles

RNA sequencing-based approaches to identifying disulfidptosis-related diagnostic clusters and immune landscapes in osteoporosis.

Disulfidptosis, a newly recognized cell death triggered by disulfide stress, has garnered attention for its potential role in osteoporosis (OP) pathogenesis. Although sulfide-related proteins are reported to regulate the balance of bone metabolism in OP, the precise involvement of disulfidptosis regulators remains elusive. Herein, leveraging the GSE56815 dataset, we conducted an analysis to delineate disulfidptosis-associated diagnostic clusters and immune landscapes in OP. Subsequently, vertebral bone tissues obtained from OP patients and controls were subjected to RNA sequencing (RNA-seq) for the validation of key disulfidptosis gene expression. Our analysis unveiled seven significant disulfidptosis regulators, including FLNA, ACTB, PRDX1, SLC7A11, NUBPL, OXSM, and RAC1, distinguishing OP samples from controls. Furthermore, employing a random forest model, we identified four diagnostic disulfidptosis regulators including FLNA, SLC7A11, NUBPL, and RAC1 potentially predictive of OP risk. A nomogram model integrating these four regulators was constructed and validated using the GSE35956 dataset, demonstrating promising utility in clinical decision-making, as affirmed by decision curve analysis. Subsequent consensus clustering analysis stratified OP samples into two different disulfidptosis subgroups (clusters A and B) using significant disulfidptosis regulators, with cluster B exhibiting higher disulfidptosis scores and implicating monocyte immunity, closely linked to osteoclastogenesis. Notably, RNA-seq analysis corroborated the expression patterns of two disulfidptosis modulators, PRDX1 and OXSM, consistent with bioinformatics predictions. Collectively, our study sheds light on disulfidptosis patterns, offering potential markers and immunotherapeutic avenues for future OP management.

SIRT1 and ZNF350 as novel biomarkers for osteoporosis: a bioinformatics analysis and experimental validation.

Osteoporosis (OP) is characterized by bone mass decrease and bone tissue microarchitectural deterioration in bone tissue. This study identified potential biomarkers for early diagnosis of OP and elucidated the mechanism of OP. Gene expression profiles were downloaded from Gene Expression Omnibus (GEO) for the GSE56814 dataset. A gene co-expression network was constructed using weighted gene co-expression network analysis (WGCNA) to identify key modules associated with healthy and OP samples. Functional enrichment analysis was conducted using the R clusterProfiler package for modules to construct the transcriptional regulatory factor networks. We used the "ggpubr" package in R to screen for differentially expressed genes between the two samples. Gene set variation analysis (GSVA) was employed to further validate hub gene expression levels between normal and OP samples using RT-PCR and immunofluorescence to evaluate the potential biological changes in various samples. There was a distinction between the normal and OP conditions based on the preserved significant module. A total of 100 genes with the highest MM scores were considered key genes. Functional enrichment analysis suggested that the top 10 biological processes, cellular component and molecular functions were enriched. The Toll-like receptor signaling pathway, TNF signaling pathway, PI3K-Akt signaling pathway, osteoclast differentiation, JAK-STAT signaling pathway, and chemokine signaling pathway were identified by Kyoto Encyclopedia of Genes and Genomes pathway analysis. SIRT1 and ZNF350 were identified by Wilcoxon algorithm as hub differentially expressed transcriptional regulatory factors that promote OP progression by affecting oxidative phosphorylation, apoptosis, PI3K-Akt-mTOR signaling, and p53 pathway. According to RT-PCR and immunostaining results, SIRT1 and ZNF350 levels were significantly higher in OP samples than in normal samples. SIRT1 and ZNF350 are important transcriptional regulatory factors for the pathogenesis of OP and may be novel biomarkers for OP treatment.

Integration analysis of lncRNA and mRNA expression data identifies DOCK4 as a potential biomarker for elderly osteoporosis

BackgroundWe aimed to identify some potential biomarkers for elderly osteoporosis (OP) by integral analysis of lncRNA and mRNA expression data.MethodsA total of 8 OP cases and 5 healthy participants were included in the study. Fasting peripheral venous blood samples were collected from individuals, and total RNA was extracted. RNA-seq library was prepared and sequenced on the Illumina HiSeq platform. Differential gene expression analysis was performed using “DESeq2” package in R language. Functional enrichment analysis was conducted using the “clusterProfiler” package, and the cis- and trans-regulatory relationships between lncRNA and target mRNA were analyzed by the lncTar software. A protein-protein interaction (PPI) network was constructed using the STRING database, and hub genes were identified through the MCODE plugin in Cytoscape.ResultsWe identified 897 differentially expressed lncRNAs (DELs) and 1366 differentially expressed genes (DEGs) between normal and OP samples. After co-expression network analysis and cis-trans regulatory genes analysis, we identified 69 candidate genes regulated by lncRNAs. Then we further screened 7 genes after PPI analysis. The target gene DOCK4, trans-regulated by two lncRNAs, was found to be significantly upregulated in OP samples. Additionally, 4 miRNAs were identified as potential regulators of DOCK4. The potential diagnostic value of DOCK4 and its two trans-regulatory lncRNAs was supported by ROC analysis, indicating their potential as biomarkers for OP.ConclusionDOCK4 is a potential biomarker for elderly osteoporosis diagnostic. It is identified to be regulated by two lncRNAs and four miRNAs.

Unveiling the unexplored novel signatures for osteoporosis via a detailed bioinformatics and molecular experiments based approach.

Osteoporosis (OP) stands as a prevalent bone ailment affecting the elderly, globally. The identification of reliable diagnostic markers crucially aids OP clinical management. Utilizing the GEO database (GSE35959), we acquired expression profiles for OP and normal samples. Differential expression genes (DEGs) and hub genes were pinpointed through STRING, GEO2R, and Cytoscape. The competing endogenous RNA (ceRNA) network was constructed using miRTarBase, miRDB, and MiRcode databases. Gene Ontology (GO) and KEGG pathway enrichment analyses were performed via DAVID. Validation involved clinical OP samples from the Pakistani population, with Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) assessing hub gene expression. A total of 2124 differentially expressed genes (DEGs) were identified between OP and normal samples in GSE35959. The selected hub genes among these DEGs were Splicing Factor 3a Subunit 1 (SF3A1), Ataxin 2 Like (ATXN2L), Heat Shock Protein 90 Beta Family Member 1 (HSP90B1), Cluster of Differentiation 74 (CD74), DExH-Box Helicase 29 (DHX29), ALG5 Dolichyl-Phosphate Beta-Glucosyltransferase (ALG5), NudC Domain Containing 2 (NUDCD2), and Ras-related protein Rab-2A (RAB2A). Expression validation of these genes on the Pakistani OP patients revealed significant up-regulation of SF3A1, ATXN2L, and CD74 and significant (P < 0.05) down-regulation of HSP90B1, DHX29, ALG5, NUDCD2, and RAB2A in OP patients. Receiver operating characteristic (ROC) analysis demonstrated that these hub genes displayed considerable diagnostic accuracy for detecting OP. The ceRNA network analysis of the hub genes revealed some important hub genes' regulatory miRNAs and lncRNAs. Via KEGG analysis, hub genes were found to be enriched in N-Glycan biosynthesis, Thyroid hormone synthesis, IL-17 signaling pathway, Prostate cancer, AMPK signaling pathway, Spliceosome, Estrogen signaling pathway, and Fluid shear stress and atherosclerosis, etc., pathways. The identified eight hub genes in the present study could reliably distinguish OP patients from normal individuals, which may provide novel insight into the diagnostic research of OP.

A novel prognostic 6-gene signature for osteoporosis.

IntroductionThe incidence of osteoporosis (OP) keeps increasing due to global aging of the population. Therefore, identifying the diagnostic and prognostic biomarkers of OP is of great significance.MethodsmRNA data from OP and non-OP samples were obtained from GEO database, which were divided into training set (GSE35959) and testing sets (GSE7158, GSE62402, GSE7429 and GSE56815). Gene modules most significantly related to OP were revealed using weighted gene co-expression network analysis (WGCNA) and differentially expressed genes (DEGs) between OP and normal samples in training set were identified using limma R package. Thereafter, above two gene sets were intersected to obtain the genes potentially related to OP. Protein-protein interaction (PPI) pairs were screened by STRING database and visualized using Cytoscape, while the plug-in cytoHubba was used to screen hub genes by determining their topological parameters. Afterwards, a diagnostic model was constructed using those hub genes, whose creditability was further evaluated by testing sets.ResultsThe results of WGCNA analysis found the Black module was most significantly related to OP, which included altogether 1286 genes. Meanwhile, 2771 DEGs were discovered between OP patients and the normal controls. After taking the intersection, 479 genes were identified potentially correlated with the development of OP. Subsequently, six hub genes were discovered through PPI network construction and node topological analysis. Finally, we constructed a support vector machine model based on these six genes, which can accurately classified training and testing set samples into OP and normal groups.ConclusionOur current study constructed a six hub genes-based diagnostic model for OP. Our findings may shed some light on the research of the early diagnosis for OP and had certain practical significance.

CircRNA_0001795 sponges miRNA-339-5p to regulate yes-associated protein 1 expression and attenuate osteoporosis progression

ABSTRACT Osteoporosis (OP) is one of the most common bone diseases, especially in women after menopause. Increasing evidence shows that non-coding RNAs are implicated in the pathogenesis of OP. In this study, based on the published circular RNA profiling data between OP patients and healthy controls, we found that circRNA_0001795 (circ_0001795) is downregulated in OP samples, which was further validated in the OP samples collected in this study. We therefore investigated the functional role and molecular mechanism of circ_0001795 in the osteogenic differentiation of human bone marrow stromal cells (hBMSCs) hBMSCs by alkaline phosphatase (ALP) activity assay, ALP and Alizarin Red S (ALS) Staining, luciferase reporter assay. Our data revealed that the overexpression of circ_0001795 could significantly promote the osteogenic differentiation of hBMSCs. MiRNA-339-5p (miR-339-5p) was identified as a target of circ_0001795, and miR-339-5p mimic attenuated the effect of circ_0001795 overexpression. MiR-339-5p downregulated yes-associated protein 1 (YAP1), which mediates the effect of circ_0001795 overexpression. Overall, this study uncovered the role of circ_0001795/miR-339-5p/YAP1 axis in regulating osteogenic differentiation, indicating that targeting Circ_0001795 could serve as a novel therapeutic target for OP.

Targeting Filamin A alleviates ovariectomy-induced bone loss in mice via the WNT/β-catenin signaling pathway

Osteoporosis (OP) is a worldwide prevalent chronic metabolic bone disease, causing by a disruption of the balance between bone resorption and formation. Estrogen deficiency and aging are the main causes for disturbances in bone remodeling activity and bone loss, however, the mechanisms underlying bone remodeling regulation require clarification if novel targets for OP treatment are to be identified. In this investigation, we showed that filamin A (FLNA) accumulated in osteoblasts (OBs) and osteoclasts (OC) in bone from human OP samples, and mice with age-related and postmenopausal OP. FLNA negatively modulated in vitro osteogenic differentiation and positively promoted RANKL-induced osteoclastic differentiation. Mechanistically, FLNA interacted with low-density lipoprotein receptor-related proteins 6 (LRP6) to inhibit β-catenin expression, and enhanced nuclear factor of activated T cell c1 (NFATc1)-dependent osteoclastogenic gene expression to inhibit osteogenesis, and promote osteoclastogenesis. Inhibiting FLNA with calpeptin activated WNT/β-catenin signaling, resulting in prominent protective effects of bone loss in ovariectomy (OVX)-induced postmenopausal OP mice. Our findings revealed that FLNA not only participated in OP pathogenesis, but could be a new target to stimulate bone formation and inhibit bone resorption. Targeting FLNA with calpeptin may be a promising therapeutic approach for postmenopausal OP in the future.

MiRNA Let-7a-5p targets RNA KCNQ1OT1 and Participates in Osteoblast Differentiation to Improve the Development of Osteoporosis.

It is known that miRNA mediates the formation of osteogenesis, but the mechanism by which miRNA let-7a-5p regulates osteogenesis in osteoporosis (OP) is not yet understood. This paper aims to probe into the regulatory mechanism of miRNA let-7a-5p in the development of OP. Fresh femoral trabecular bones of patients with osteoporotic fracture (OP group, n = 25) and non-OP osteoarthritis (Non-OP group, n = 23) who underwent hip replacement in our hospital from December 2016 to December 2019 were collected. The expression and protein levels of miRNA let-7a-5p and V-AKT murine thymoma viral oncogene homolog 3 (RNA KCNQ1OT1) were detected. C2C12 cells were purchased and osteogenic differentiation model was constructed by BMP2 induction. After miRNA let-7a-5p up-regulation or down-regulation by transfection of corresponding mimics and inhibitors, the impacts of miRNA let-7a-5p and RNA KCNQ1OT1 on osteogenic differentiation-related factors (OC, ALP, COL1A1) in C2C12 cells were analyzed. The determination of targeting correlation of miRNA let-7a-5p with RNA KCNQ1OT1 was performed by dual-luciferase reporter (DLR). In OP samples, miRNA let-7a-5p was notably declined while RNA KCNQ1OT1 were remarkably up-regulated. MiRNA let-7a-5p reduced in C2C12 cells as BMP2 treatment proceeded. MiRNA let-7a-5p up-regulation or RNA KCNQ1OT1 down-regulation increased OC, ALP, COL1A1 levels and ALP activity. RNA KCNQ1OT1 was directly targeted to miR-497-5p. RNA KCNQ1OT1 up-regulation weakened the promoting effect of miRNA let-7a-5p up-regulation on osteoblast differentiation. MiRNA let-7a-5p up-regulation can target to reduce RNA KCNQ1OT1 and promote osteoblast differentiation, thereby improving the development of osteoporosis.

An emerging potential therapeutic target for osteoporosis: LncRNA H19/miR-29a-3p axis.

Osteoporosis (OP) is a complex systemic disease characterized by a loss of bone density, leading to bone fragility and an increase risk of fractures of the hip, spine and wrist. The clinical therapeutic effect is still far from satisfactory. Thus, further studies are urgently needed to explore the pathogenesis of OP. In this study, our aim is to explore the underlying molecular mechanism of lncRNA H19/miR-29a-3p axis for regulating of inflammation, proliferation and apoptosis in OP. The expression of lncRNA H19 was significantly upregulated in OP samples compared with the health control. Subsequently, we found that miR-29a-3p is the target of lncRNA H19 in OP. Furthermore, the knockdown of lncRNA H19 was validated to promote the expression of pro-inflammatory mediators, repress cell proliferation and inhibit cell apoptosis in vitro. Moreover, the modulating effects of lncRNA-H19 on the expressions of pro-inflammatory mediators, cell proliferation and apoptosis in vitro were diminished after co-transfecting with miR-29a-3p inhibitor and siRNA-H19. Thus, we concluded that lncRNA H19/miR-29a-3p axis was involved in the development of OP. This study might provide a better understanding of OP development and a potential therapeutic target for OP intervention.

Osteoporosis-related variations of trabecular bone properties of proximal human humeral heads at different scale lengths

Osteoporosis (OP) is a skeletal disorder responsible for the weakening of the bone structure and, consequently, for an increased fracture risk in the elderly population. In the past, bone mineral density (BMD) variation was considered the best OP indicator, but recently the focus has shifted toward the variation of microstructural bone parameters. This work is based on the characterisation of 8-mm cylindrical biopsies harvested from proximal humeral heads belonging to healthy and osteoporotic patients, in order to assess the OP-related variations of bone properties at different scale lengths. In particular, bone biopsies underwent micro-computed tomography analysis to study the most relevant features of bone architecture and extrapolate the tissue mineral density (TMD) value of bone trabeculae. Compression tests and nanoindentations were performed to investigate the macro- and micromechanical properties of bone biopsies, respectively. In addition, XRD analyses were performed to obtain the mean hydroxyapatite (HA) crystallite size, while Raman spectroscopy investigated the collagen secondary structure. Thermogravimetric analysis was performed to evaluate the ratio between organic and inorganic phases.From the obtained results, OP samples showed a more anisotropic and less interconnected structure responsible for reduced compression strength. From this, it can be supposed that OP caused an alteration of bone structure that led to inferior macroscopic mechanical properties. Furthermore, OP samples possessed higher TMD and bigger HA crystals that are correlated to an increase of the hardness value obtained by means of nanoindentation. This less controlled HA crystal growth is probably due to an alteration of the organic matrix structure, as revealed by the increase of the random coil contribution in the Raman spectra of the OP bone. This higher crystal content led to an increase in trabecular density and hardness. In conclusion, the obtained data showed that OP affects bone properties at different scale lengths causing an alteration of its morphological, structural and mechanical features.

Investigation of transcriptome mechanism associated with osteoporosis explored by microarray analysis

Microarray data of osteoporosis (OP) were analyzed based on prediction of transcription factors (TFs) or their targets as well as influences of TFs or TF network to uncover key TFs in OP. The microarray data E-GEOD-35956 was downloaded from the GPL570 platform. Differentially expressed genes (DEGs) with logarithm of fold change (|logFC|) >2 and P-value <0.05 were identified between OP samples and normal controls. TF genes were screened from the DEGs based on ITFP, Marbach 2016, TRRUST databases. TF targets were enriched from DEGs using Fisher's exact test. TF targets were selected based on their impact factors. TF targets were chosen from TF network analysis. Finally, key TFs were identified by based on TFs coverage. A total of 300 DEGs were obtained. There were no TF genes screened from the DEGs. In total 165, 87 and 178 TF targets were screened from DEGs respectively based on Fisher's exact test, influence of TFs or TF network analysis. According to the optimal TF set with TFs having maximum coverage of DEGs, 178 TF targets was the most. Thus, the optimal sets of TFs were FOXO1, KLF16, RXRA, RARA, HNF4A, CEBPB, ESR1, SOX8, ZNF219, and SP1. Altogether, these results suggested identified crucial TFs in OP might play a significant role in OP development, showing these key TFs probably would aid in unveiling the underlying molecular mechanisms and may be therapeutic targets, diagnostic or prognostic biomarkers for OP.

Screening of differentially expressed genes in male idiopathic osteoporosis via RNA sequencing.

As a type of osteoporosis (OP), male idiopathic OP (MIO) is a bone disorder that occurs in young males and is a public health problem worldwide. However, the detailed pathogenesis of MIO remains to be elucidated. In the present study, blood samples of patients with MIO, senile OP, postmenopausal OP and normal controls (NCs) were obtained for RNA sequencing. Compared with the NC group, differentially expressed genes (DEGs) in the three types of OP were identified. DEGs that were common among the three types of OP and the DEGs that were unique to patients with MIO were determined. Gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted. MIO-specific and OP-specific protein-protein interaction (PPI) networks were constructed. Compared with NCs, a total of 519, 368 and 1,472 DEGs were identified in samples from MIO, senile OP and postmenopausal OP, respectively. Tetraspanin 5 (TSPAN5) and α-synuclein (SNCA) were unique DEGs in MIO that were not identified in the other two types of OP compared with NCs. Furthermore, the expression of carbonic anhydrase 1 (CA1) and S100 calcium-binding protein P (S100P) in MIO was significantly different compared with senile OP, postmenopausal OP and NC samples. ‘MAPK signaling pathway’, ‘type I diabetes mellitus’ and ‘hematopoietic cell lineage’ were among significantly enriched pathways of DEGs in MIO. SNCA and CDC-like kinase 1 were the hub genes in the MIO-specific PPI network. In conclusion, the mitogen-activated protein kinase signaling and type I diabetes mellitus pathways may be involved in bone formation; SNCA and TSPAN5 may be associated with bone resorption. These two pathways and two genes may serve a role in MIO. CA1 and S100P may regulate the process of MIO by modulation of calcification and dysregulation of calcium binding. These findings may have provided an experimental basis for elucidating the underlying mechanisms and developing potential diagnostic biomarkers of MIO.

Diversity analysis of gut microbiota in osteoporosis and osteopenia patients.

Some evidence suggests that bone health can be regulated by gut microbiota. To better understand this, we performed 16S ribosomal RNA sequencing to analyze the intestinal microbial diversity in primary osteoporosis (OP) patients, osteopenia (ON) patients and normal controls (NC). We observed an inverse correlation between the number of bacterial taxa and the value of bone mineral density. The diversity estimators in the OP and ON groups were increased compared with those in the NC group. Beta diversity analyses based on hierarchical clustering and principal coordinate analysis (PCoA) could discriminate the NC samples from OP and ON samples. Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria constituted the four dominant phyla in all samples. Proportion of Firmicutes was significantly higher and Bacteroidetes was significantly lower in OP samples than that in NC samples (p < 0.05), Gemmatimonadetes and Chloroflexi were significantly different between OP and NC group as well as between ON and NC group (p < 0.01). A total of 21 genera with proportions above 1% were detected and Bacteroides accounted for the largest proportion in all samples. The Blautia, Parabacteroides and Ruminococcaceae genera differed significantly between the OP and NC group (p < 0.05). Linear discriminant analysis (LDA) results showed one phylum community and seven phylum communities were enriched in ON and OP, respectively. Thirty-five genus communities, five genus communities and two genus communities were enriched in OP, ON and NC, respectively. The results of this study indicate that gut microbiota may be a critical factor in osteoporosis development, which can further help us search for novel biomarkers of gut microbiota in OP and understand the interaction between gut microbiota and bone health.

Microstructural alterations of femoral head articular cartilage and subchondral bone in osteoarthritis and osteoporosis

Explore whether osteoporosis (OP) in humans influences the morphological status of the articular cartilage and the subchondral bone. Explore the relationship between the macroscopic aspect of the articular surface and the rate of microscopic changes of both the cartilage and the subchondral bone in OP and osteoarthritis (OA). Femoral heads after total hip replacement were obtained from patients with OP or hip OA (OP, n=56; OA, n=12). Cartilage degeneration was assessed using the Mankin grading system whereas subchondral bone was evaluated using histomorphometry and Micro-computed Tomography (μCT) scanning system. Thickness of the cartilage layers and subchondral cortical bone (SCB) was measured. Samples with higher total Mankin score have significantly reduced cartilage thickness. Mankin score differed between all OP specimens. In OP samples with lower Mankin scores the thickness of SCBshows a trend of an increase caused by increased levels of bone remodeling. In OP samples with higher Mankin scores we observed thinning of SCB. Structural indices of subchondral trabecular bone (STB) were significantly lower in OP than in OA samples. Thinning of SCB, found in OP samples with higher Mankin scores could be related with the progression of the cartilage degeneration indicating an early-stage OA. Increased levels of bone remodeling and evidently changed morphology of subchondral bone found in OP samples with lower Mankin score indicated that bony bed level must have a role in the progression of the cartilage degeneration.

Osteoarthritic versus osteoporotic bone and intra‐skeletal variations in normal bone: Evaluation with µCT and bone histomorphometry

Several studies have shown that in contrast to osteoporosis (OP), osteoarthritis (OA) is characterized by high bone mineral density (BMD). Bone strength not only depends on mineral content as determined by dual X-ray absorptiometry (DXA), but also on bone microarchitecture. We studied intertrochanteric bone from normal controls and OA and OP patients by bone histomorphometry (BHM) and microcomputed tomography (µCT) as well as DXA in order to first, test the differences between OA and OP comparing both groups to healthy controls, second, to assess variations between three different skeletal sites in controls and third, to determine the level of agreement between µCT, BHM, and DXA. Analysis was performed on 115 samples from OA and OP patients, and controls. We found significant differences between OA and OP samples in structural parameters and in the osteoid fraction (p < 0.05). The majority of the intra-skeletal differences were shown between lumbar spine and femoral head samples (p < 0.05). Significant agreements were found between µCT and BHM and DXA (r = 0.32-0.45, p < 0.05). Our findings suggest differences in intertrochanteric bone between OA and OP, the age-related intra-skeletal variations and a correlation between microscopic and macroscopic bone evaluation methods.

Reproducibility and agreement of micro-CT and histomorphometry in human trabecular bone with different metabolic status

The use of micro-computed tomography (micro-CT) to study bone microstructure is continuously increasing. Thus, it is important to ensure that micro-CT can differentiate healthy and pathological bone. This study aimed to determine whether the reproducibility of bone histomorphometry and micro-CT, and agreement between the techniques, vary in bone samples with different metabolic status. Iliac crest biopsies (n=36) were obtained from healthy subjects (n=10) and from patients with osteoporosis (OP) (n=15) or renal osteodystrophy (ROD) (n=11). Micro-CT and histomorphometry analyses were repeated twice. Results were analyzed in separate groups and after pooling the data. Bone histomorphometry detected generally known differences between the diseases, whereas micro-CT did not detect differences between normal and ROD samples as effectively. Repeated measurements for BV/TV, Tb.Th, Tb.N, and Tb.Sp exhibited linear correlation coefficients (ρ) of 0.87-0.92 [coefficients of variations (CV), 8.3-27.2%] for histomorphometry and of 0.66-0.94 (CV, 4.4-23.4%) for micro-CT. There were no significant differences in reproducibility among samples from different study groups. Correlations between BV/TV (micro-CT) and mineralized bone volume (Md.V/TV, histomorphometry) were weaker than between BV/TV (micro-CT) and BV/TV (histomorphometry). When comparing the techniques, BV/TV, Tb.Th, and Tb.N displayed moderate correlations (ρ=0.39-0.62, P<0.05), and the agreement for BV/TV was highest in OP samples. The agreement between the techniques using clinical bone samples was moderate. Especially, micro-CT was less effective than bone histomorphometry in differentiating ROD from normal samples. The reproducibility was not affected by the health status of bone. Histomorphometry is still needed in clinical practice to study the remodeling balance in bone, and the methods are complementary.