Evaluation of circulating microRNA signature in patients with erosive hand osteoarthritis: The HOAmiR study.

OBJECTIVES: To identify circulating micro-RNAs differentially expressed in patients with erosive hand osteoarthritis (HOA) compared to patients with non-erosive HOA and patients without HOA. METHODS: In the screening phase, 768 well-characterized micro-RNAs using Taqman low-density array cards were measured in 30 sera from 10 patients with erosive HOA, 10 patients with non-erosive HOA, and 10 controls without HOA, matched for age and body mass index (BMI). In a second step, we validated the micro-RNAs identified at the screening phase (adjusted p value < 0.05 after false discovery rate correction using Benjamini-Hochberg method and literature review) in larger samples (60 patients with erosive HOA and 60 patients without HOA matched for age and BMI). RESULTS: In the screening phase, we identified 21 down-regulated and 4 up-regulated micro-RNAs of interest between erosive HOA and control groups. Among these, 9 micro-RNAs (miR-373-3p, miR-558, miR-607, miR-653-5p, miR-137 and miR448 were down-regulated, and miR-142-3p, miR-144-3p and miR-34a-5p were up-regulated) were previously described in chondrocytes homeostasis or OA. We found only one significantly down-regulated micro-RNA between erosive and non-erosive HOA. In the validation phase, we showed replication of a single micro-RNA the significant downregulation of miR-196-5p, that had been previously identified in the screening phase among patients with erosive HOA compared to those without HOA. After reviewing the literature and the miRNA-gene interaction prediction model, we found that this microRNA could interact with bone homeostasis and HOXC8, which could explain its role in osteoarthritis. CONCLUSIONS: We found that miR-196-5p was down-regulated in patients with erosive HOA and some of its targets could explain a role in OA.

A new serum biochemical marker of synovium turnover predicts radiographic progression in patients with early arthritis.

OBJECTIVE: To investigate whether serum Col 3-4, a new biochemical marker of synovial tissue turnover, was associated with progression of joint damage in patients with early arthritis. METHODS: A total of 788 early arthritis patients (<6 months of symptoms, 82% diagnosis of RA, 18% undifferentiated arthritis) from the prospective ESPOIR study were investigated. Progression was defined as an increase of 1 or 5 unit(s) in radiographic van der Heijde modified Sharp score between baseline and 1 or 5 years, respectively. Associations between baseline Col 3-4 and progression were assessed by logistic regression. RESULTS: Each standard deviation increase of baseline Col 3-4 levels was associated with an increased 5-yr total damage progression with an odds ratio (OR, 95% CI) of 1.51 (1.21, 1.88), which remained significant when DAS28, C-reactive protein and anti-citrullinated protein antibodies positivity were included in the model [OR (95% CI): 1.34 (1.01, 1.76)]. Further adjustment for bone erosion did not modify the association. Patients with both Col 3-4 in the highest quintile and bone erosion had a >2-fold higher risk of progression [OR (95% CI): 7.16 (2.31, 22)] than patients with either high Col 3-4 [2.91 (1.79, 4.73)] or bone erosion [2.36 (2.38, 3.70)] alone. Similar associations were observed for prediction of 12 months progression. CONCLUSIONS: Increased serum Col 3-4 is associated with a higher risk of structural progression, independently of major risk factors. Col 3-4 may be useful in association with bone erosion to identify patients with early arthritis at higher risk.

OPN, BSP, and Bone Quality-Structural, Biochemical, and Biomechanical Assessment in OPN-/-, BSP-/-, and DKO Mice.

Osteopontin (OPN) and Bone Sialoprotein (BSP), abundantly expressed by osteoblasts and osteoclasts, appear to have important, partly overlapping functions in bone. In gene-knockout (KO, -/-) models of either protein and their double (D)KO in the same CD1/129sv genetic background, we analyzed the morphology, matrix characteristics, and biomechanical properties of femur bone in 2 and 4 month old, male and female mice. OPN-/- mice display inconsistent, perhaps localized hypermineralization, while the BSP-/- are hypomineralized throughout ages and sexes, and the low mineralization of young DKO mice recovers with age. The higher contribution of primary bone remnants in OPN-/- shafts suggests a slow turnover, while their lower percentage in BSP-/- indicates rapid remodeling, despite FTIR-based evidence in this genotype of a high maturity of the mineralized matrix. In 3-point bending assays, OPN-/- bones consistently display higher Maximal Load, Work to Max. Load and in young mice Ultimate Stress, an intrinsic characteristic of the matrix. Young male and old female BSP-/- also display high Work to Max. Load along with low Ultimate Stress. Principal Component Analysis confirms the major role of morphological traits in mechanical competence, and evidences a grouping of the WT phenotype with the OPN-/- and of BSP-/- with DKO, driven by both structural and matrix parameters, suggesting that the presence or absence of BSP has the most profound effects on skeletal properties. Single or double gene KO of OPN and BSP thus have multiple distinct effects on skeletal phenotypes, confirming their importance in bone biology and their interplay in its regulation.

Plasma Cartilage Acidic Protein 1 Measured by ELISA Is Associated With the Progression to Total Joint Replacement in Postmenopausal Women.

OBJECTIVE: To investigate the association of plasma cartilage acidic protein 1 (CRTAC1), a novel biochemical marker of osteoarthritis (OA), and total joint replacement (TJR) in postmenopausal women. METHODS: The association of plasma CRTAC1 with the incidence of TJR was investigated in a prospective cohort including 478 postmenopausal women. A total of 38 women underwent a TJR for OA during a median follow-up of 18 years. Every one of the TJR cases were age- and BMI (kg/m2)-matched with 2 controls with no TJR from the same cohort. Plasma CRTAC1 was measured before TJR. The association between CRTAC1 and TJR incidence was investigated by conditional logistic regression. RESULTS: Increased CRTAC1 was associated with a higher risk of TJR with an odds ratio (OR) of 1.80 (95% CI 1.11-2.92) for 1 SD increase, which remained significant after adjusting for Western Ontario and McMaster Universities Osteoarthritis Index, knee OA baseline severity (Kellgren-Lawrence grade), hip OA, and hip bone mineral density. Urinary crosslinked C-telopeptide of type II collagen (CTX-II) was also associated with a higher risk of TJR with an adjusted OR of 1.83 (95% CI 1.11-3.00). When CRTAC1 and CTX-II were included in the same model, both markers were significantly associated with TJR with similar ORs. CONCLUSION: CRTAC1 is a new risk indicator of TJR for OA in postmenopausal women. Combined with knee and hip OA and CTX-II, it may help to identify subjects at risk for TJR.

Spatial Distribution of Microcracks in Osteoarthritic Femoral Neck: Influence of Osteophytes on Microcrack Formation.

Osteophytes have been suggested to influence the bone mechanical properties. The aim of this study was to compare the microcrack density in osteophytes with that in the other parts of the osteoarthritic femoral neck (FN). The presence of microcracks was investigated in the ultra-distal FN and in the osteophytes in samples obtained during hip arthroplasty in 24 postmenopausal women aged 67 ± 10 years. Furthermore, the 3D microarchitecture and the collagen crosslinks contents were assessed by high-resolution peripheral quantitative computed tomography and high-performance liquid chromatography, respectively. Osteophytes were present in the 24 FN, mainly at the level of the inferior quadrant. Microcracks were present in all FN with an average of 2.8 per sample. All observed microcracks were linear. The microcrack density (Cr.N/BV; #/mm2) was significantly higher in cancellous than in cortical bone (p = 0.004), whereas the microcrack length (Cr.Le, µm) was significantly greater in cortical bone (p = 0.04). The collagen crosslinks ratio pyridinoline/deoxypyridinoline was significantly and negatively correlated with Cr.N/BV in the posterior (r' = - 0.68, p = 0.01) and inferior (r' = - 0.53, p = 0.05) quadrants. Microcracks were observed in seven osteophytes in seven patients. When microcracks were present in the osteophyte area, Cr.N/BV was also significantly higher in the whole FN and in the quadrant of the osteophyte compared to the cases without microcrack in the osteophyte (p < 0.03). In conclusion, in FN from hip osteoarthritis microcracks are present in all FNs but in only 23% of the osteophytes. The microcrack formation was greater and their progression was smaller in the cancellous bone than in the cortex. The spatial distribution of microcracks varied according to the proximity of the osteophyte, and suggests that osteophyte may influence microcrack formation related to changes in local bone quality.

Cortical Fractal Analysis and Collagen Crosslinks Content in Femoral Neck After Osteoporotic Fracture in Postmenopausal Women: Comparison with Osteoarthritis.

The femoral neck (FN) has been previously characterized by thinner cortices in osteoporotic fracture (HF) when compared to hip osteoarthritis (HOA). The purposes of this study were to complete the previous investigations on FNs from HF and HOA by analyzing the complexity of the cortical structure and to approach the intrinsic properties of cortical bone by assessing the collagen crosslink contents. FN samples were obtained during arthroplasty in 35 postmenopausal women (HF; n = 17; mean age 79 ± 2 years; HOA; n = 18; mean age 66 ± 2 years). The cortical fractal dimension (Ct.FD) and lacunarity (Ct.Lac) derived from high-resolution peripheral quantitative tomography (isotropic voxel size: 82 µm) images of FN by using Ctan software and Fraclac running in ImageJ were analyzed. The collagen crosslinks content [pyridinoline, deoxypyridinoline, pentosidine (PEN)] were assessed in cortical bone. Ct.FD was significantly lower (p < 0.0001) in HF than HOA reflecting a decreased complexity and was correlated to the age and BMD. In two sub-groups, BMD- and age-matched, respectively, Ct.FD remained significantly lower in HF than HOA (p < 0.001). Ct.Lac was not different between HF and HOA. PEN content was two times higher in HF than HOA (p < 0.0001) independently of age. In conclusion, FN with HF was characterized by a less complex cortical texture and higher PEN content than HOA. In addition to the decreased bone mass and BMD previously reported, these modifications contribute to the lower bone quality in HF than HOA in postmenopausal women.

The C-Terminal Intact Forms of Periostin (iPTN) Are Surrogate Markers for Osteolytic Lesions in Experimental Breast Cancer Bone Metastasis.

Periostin is an extracellular matrix protein that actively contributes to tumor progression and metastasis. Here, we hypothesized that it could be a marker of bone metastasis formation. To address this question, we used two polyclonal antibodies directed against the whole molecule or its C-terminal domain to explore the expression of intact and truncated forms of periostin in the serum and tissues (lung, heart, bone) of wild-type and periostin-deficient mice. In normal bones, periostin was expressed in the periosteum and specific periostin proteolytic fragments were found in bones, but not in soft tissues. In animals bearing osteolytic lesions caused by 4T1 cells, C-terminal intact periostin (iPTN) expression disappeared at the invasive front of skeletal tumors where bone-resorbing osteoclasts were present. In vitro, we found that periostin was a substrate for osteoclast-derived cathepsin K, generating proteolytic fragments that were not recognized by anti-periostin antibodies directed against iPTN. In vivo, using an in-house sandwich immunoassay aimed at detecting iPTN only, we observed a noticeable reduction of serum periostin levels (- 26%; P < 0.002) in animals bearing osteolytic lesions caused by 4T1 cells. On the contrary, this decrease was not observed in women with breast cancer and bone metastases when periostin was measured with a human assay detecting total periostin. Collectively, these data showed that mouse periostin was degraded at the bone metastatic sites, potentially by cathepsin K, and that the specific measurement of iPTN in serum should assist in detecting bone metastasis formation in breast cancer.

Serum Col3-4: A new type III and IV collagen biochemical marker of synovial tissue turnover in patients with rheumatoid arthritis.

The objective of this study was to develop a serum biochemical marker of the degradation of type III and IV collagens, as an index of synovium turnover, and evaluate its performance in patients with rheumatoid arthritis (RA). An enzyme-linked immunosorbent assay for serum synovial collagen fragments (Col3-4) was developed using an antibody recognizing a specific sequence from human type III collagen, which shares 70% homology with type IV collagen. Immunohistochemistry was performed to localize Col3-4 and the matrix metalloprotease MMP-9 which is upregulated in RA synovial fibroblasts in the synovial tissue from a RA patient. Serum Col3-4 was measured in patients with RA (n = 66, 73% women, mean age 62 years, median disease activity score 28 with erythrocyte sedimentation rate (DAS28-ESR) 2.6) and in sex and age matched healthy controls (n = 70, 76% women, mean age 59 years). Col3-4 immunoassay demonstrated adequate analytical performances and recognized a circulating neoepitope resulting from the cleavage of type III and IV collagens. In RA synovium tissue, Col3-4 fragments were localized in the lining layer where destructive fibroblasts are present and around blood vessels rich in type IV collagen. MMP-9 colocalized with Col3-4 staining and efficiently released Col3-4 fragments from type III and type IV collagen digestion. Serum Col3-4 was markedly increased in patients with RA (+240% vs controls, p < 0.0001) and correlated with DAS28-ESR (r = 0.53, p < 0.0001). Patients with RA and active disease (DAS28-ESR > 3.2, n = 20) had 896% (p < 0.0001) higher levels than subjects with low activity (n = 46). Serum Col3-4 is a specific and sensitive biochemical marker reflecting MMP- mediated type III and IV collagen degradation from synovial tissue. Serum Col3-4 levels are markedly increased in patients with RA, particularly in those with active disease, suggesting that it may be useful for the clinical investigation of RA.

Cathepsin K preferentially solubilizes matured bone matrix.

Bone collagen undergoes a series of enzymatic and nonenzymatic posttranslational modifications with maturation. The aim of this study was to analyze the collagenolytic efficiency of cathepsin K in relation to the extent of bone collagen age. Bone collagen posttranslational maturation was induced in vitro by preincubating bovine fetal cortical bone specimens at 37 °C for different times. The collagen enzymatic cross-links pyridinoline (PYD) and deoxypyridinoline (DPD), the advanced glycation end product pentosidine (PEN), and the native (α) and ß-isomerized C-telopeptide (CTX) isomers were measured in each bone specimen. After extraction, bone collagen was incubated with human recombinant cathepsin K at different concentrations and its collagenolytic activity was measured by the release of hydroxyproline. To assess the affinity of cathepsin K for isomerized and nonisomerized CTX isomers, incubation with cathepsin K was also performed in the presence of various concentrations of a specific inhibitor. We showed that preincubation of bone collagen at 37 °C induces a marked increase in the bone concentration of PYD, DPD, and PEN and of CTX isomerization as reflected by the ratio of α-/ßCTX. This increase was associated with a parallel increase in the efficiency of cathepsin K to solubilize bone collagen. When cathepsin K was incubated in the presence of an inhibitor, the ß-isomerized form of collagen from 3-month- and 8-year-old bovine bone was more susceptible to degradation than the native α form. These results suggest that the collagenolytic activity of cathepsin K may be increased toward more matured bone collagen.

Ultrasounds could be considered as a future tool for probing growing bone properties.

Juvenile bone growth is well described (physiological and anatomical) but there are still lacks of knowledge on intrinsic material properties. Our group has already published, on different samples, several studies on the assessment of intrinsic material properties of juvenile bone compared to material properties of adult bone. The purpose of this study was finally to combine different experimental modalities available (ultrasonic measurement, micro-Computed Tomography analysis, mechanical compression tests and biochemical measurements) applied on small cubic bone samples in order to gain insight into the multiparametric evaluation of bone quality. Differences were found between juvenile and adult groups in term of architectural parameters (Porosity Separation), Tissue Mineral Density (TMD), diagonal stiffness coefficients (C33, C44, C55, C66) and ratio between immature and mature cross-links (CX). Diagonal stiffness coefficients are more representative of the microstructural and biochemical parameters of child bone than of adult bone. We also found that compression modulus E was highly correlated with several microstructure parameters and CX in children group while it was not at all correlated in the adult group. Similar results were found for the CX which was linked to several microstructure parameters (TMD and E) only in the juvenile group. To our knowledge, this is the first time that, on a same sample, ultrasonic measurements have been combined with the assessment of mechanical and biochemical properties. It appears that ultrasonic measurements can provide relevant indicators of child bone quality (microstructural and biochemical parameters) which is promising for clinical application since, B-mode ultrasound is the preferred first-line modality over other more constraining imaging modalities (radiation, parent-child accessibility and access to the patient's bed) for pediatric patients.

A Signature of Circulating miRNAs Associated With Fibrous Dysplasia of Bone: the mirDys Study.

Fibrous dysplasia (FD) is a rare bone disease caused by activating mutations of GNAS encoding the Gsα protein, enhancing cyclic adenosine monophosphate (cAMP) production by overstimulation of adenylyl cyclase and impairing osteoblastic differentiation. The clinical presentation ranges from asymptomatic to polyostotic forms with severe disability, explained by the mosaic distribution of the GNAS mutation. Physicians have to deal with the gap of knowledge in FD pathogenesis, the absence of prognostic markers and the lack of specific treatment. The identification of specific biomarkers for FD is an important step to improve the clinical and therapeutic approaches. An epigenetic regulation driven by microRNAs (miRNAs), known as promising biomarkers in bone disease, could be involved in FD. We have sought circulating miRNAs that are differentially expressed in FD patients compared to controls and would reflect dysregulations of osteogenesis-related genes and bone disorder. The global miRNA profiling was performed using Next Generation Sequencing in patient serum collected from a discovery cohort of 20 patients (10 polyostotic and 10 monostotic) and 10 controls. From these, we selected 19 miRNAs for a miRNA validation phase from serum of 82 patients and 82 controls, using real-time qPCR. Discovery screening identified 111 miRNAs differentially expressed in patient serum, after adjusting for the false discovery rate (FDR). Among the 82 patients, 55% were polyostotic, and 73% were women with a mean age of 42 years. Six miRNAs (miR-25-3p, miR-93-5p, miR-182-5p, miR-324-5p, miR-363-3p, and miR-451a) were significantly overexpressed in serum, with FDR <0.05. The expression level of these six miRNAs was not associated with the FD severity. In conclusion, we identified a signature of circulating miRNAs associated with FD. These miRNAs are potential negative regulators of gene expression in bone cell progenitors, suggesting their activity in FD by interfering with osteoblastic and osteoclastic differentiation to impair bone mineralization and remodeling processes. © 2020 American Society for Bone and Mineral Research.

Anisotropic elastic properties of human femoral cortical bone and relationships with composition and microstructure in elderly.

The strong dependence between cortical bone elasticity at the millimetre-scale (mesoscale) and cortical porosity has been evidenced by previous studies. However, bone is an anisotropic composite material made by mineral, proteins and water assembled in a hierarchical structure. Whether the variations of structural and compositional properties of bone affect the different elastic coefficients at the mesoscale is not clear. Aiming to understand the relationships between bone elastic properties and compositions and microstructure, we applied state-of-the-art experimental modalities to assess these aspects of bone characteristics. All elastic coefficients (stiffness tensor of the transverse isotropic bone material), structure of the vascular pore network, collagen and mineral properties were measured in 52 specimens from the femoral diaphysis of 26 elderly donors. Statistical analyses and micromechanical modeling showed that vascular pore volume fraction and the degree of mineralization of bone are the most important determinants of cortical bone anisotropic mesoscopic elasticity. Though significant correlations were observed between collagen properties and elasticity, their effects in bone mesoscopic elasticity were minor in our data. This work also provides a unique set of data exhibiting a range of variations of compositional and microstructural cortical bone properties in the elderly and gives strong experimental evidence and basis for further development of biomechanical models for human cortical bone. STATEMENT OF SIGNIFICANCE: This study reports the relationships between microstructure, composition and the mesoscale anisotropic elastic properties of human femoral cortical bone in elderly. For the first time, we provide data covering the complete anisotropic elastic tensor, the microstructure of cortical vascular porosity, mineral and collagen characteristics obtained from the same or adjacent samples in each donor. The results revealed that cortical vascular porosity and degree of mineralization of bone are the most important determinants of bone anisotropic stiffness at the mesoscale. The presented data gives strong experimental evidence and basis for further development of biomechanical models for human cortical bone.

Relationships between human cortical bone toughness and collagen cross-links on paired anatomical locations.

Human cortical bone fracture processes depend on the internal porosity network down to the lacunar length scale. Recent results show that at the collagen scale, the maturation of collagen cross-links may have a negative influence on bone mechanical behavior. While the effect of pentosidine on human cortical bone toughness has been studied, the influence of mature and immature enzymatic cross-links has only been studied in relation to strength and work of fracture. Moreover, these relationships have not been studied on different paired anatomical locations. Thus, the aim of the current study was to assess the relationships between both enzymatic and non-enzymatic collagen cross-links and human cortical bone toughness, on four human paired anatomical locations. Single Edge Notched Bending toughness tests were performed for two loading conditions: a quasi-static standard condition, and a condition representative of a fall. These tests were done with 32 paired femoral diaphyses, femoral necks and radial diaphyses (18 women, age 81 ±â€¯12 y.o.; 14 men, age 79 ±â€¯8 y.o.). Collagen enzymatic and non-enzymatic crosslinks were measured on the same bones. Maturation of collagen was defined as the ratio between immature and mature cross-links (CX). The results show that there was a significant correlation between collagen cross-link maturation and bone toughness when gathering femoral and radial diaphyses, but not when considering each anatomical location individually. These results show that the influence of collagen enzymatic and non-enzymatic cross-links is minor when considering human cortical bone crack propagation mechanisms.

Extracellular post-translational modifications of collagen are major determinants of biomechanical properties of fetal bovine cortical bone.

Mechanical behavior of bone depends on its mass and architecture, and on the material properties of the matrix, which is composed of a mineral phase and an organic component mainly constituted of type I collagen. Mineral accounts largely for the stiffness of bone, whereas type I collagen provides bone its ductility and toughness, i.e., its ability to undergo deformation and absorb energy after it begins to yield. The molecular mechanisms underlying the effect of alterations in type I collagen on bone mechanical properties are unclear. We used an in vitro model of fetal bovine cortical bone specimens (n = 44), where the extent of type I collagen cross-linking was modified by incubation at 37 degrees C for 0, 60, 90 and 120 days, keeping constant the architecture and the mineral content. At each incubation time, the following parameters were determined: (1) the bone concentration of enzymatic (pyridinoline; PYD and deoxypyridinoline, DPD) and non-enzymatic (pentosidine) crosslinks by HPLC, (2) the extent of aspartic acid isomerization of the type I collagen C-telopeptide (CTX) by ELISA of native (alpha CTX) and isomerized (beta CTX) forms, (3) the mineral density by DXA, (4) the porosity by micro-computed tomography and (5) the bending and compressive mechanical properties. Incubation of bone specimens at 37 degrees C for 60 days increased the level (per molecule of collagen) of PYD (+98%, P = 0.005), DPD (+42%, P = 0.013), pentosidine (+55-fold, P = 0.005), and the degree of type I collagen C-telopeptide isomerization (+4.9-fold, P = 0.005). These biochemical changes of collagen were associated with a 30% decrease in bending and compressive yield stress and a 2.5-fold increase in compressive post-yield energy absorption (P < 0.02 for all), with no significant change of bone stiffness. In multivariate analyses, the level of collagen cross-linking was associated with yield stress and post-yield energy absorption independently of bone mineral density, explaining up to 25% of their variance. We conclude that the extent and nature of collagen cross-linking contribute to the mechanical properties of fetal bovine cortical bone independently of bone mineral density.

Nonenzymatic Glycation and Degree of Mineralization Are Higher in Bone From Fractured Patients With Type 1 Diabetes Mellitus.

Low-energy fractures are frequent complications in type 1 diabetes mellitus patients (T1DM). Modifications of bone intrinsic composition might be a potential cause of fragility observed in diabetic subjects. Advanced glycation end products (AGEs) were found in numerous connective tissues from T1DM patients. However, whether AGEs are present at high levels in bone matrix from diabetic subjects is unknown. Moreover, whether elevated AGEs in the bone matrix impair mineralization has not been addressed in humans. The purposes of this study were 1) to determine whether bone matrix from fracturing and nonfracturing T1DM contained more AGEs than bone from healthy patients (CTL), and 2) to compare the degree of mineralization of bone and hardness between fracturing and nonfracturing T1DM versus CTL. We analyzed iliac crest bone biopsies from 5 fracturing T1DM patients, 5 nonfracturing T1DM patients, and 5 healthy subjects, all age- and sex-matched. AGEs (pentosidine) in bone matrix was measured by high-performance liquid chromatography separately in trabecular and cortical bone. The degree of mineralization of bone (DMB) was assessed by digitized microradiography, and mechanical properties by micro- and nanohardness tests. Trabecular bone from fracturing T1DM exhibited significantly higher levels of pentosidine than CTL (p = 0.04) and was more mineralized than nonfracturing T1DM (p = 0.04) and CTL (p = 0.04). Trabecular bone was not significantly different in pentosidine between nonfracturing T1DM and CTL. Cortical bone from nonfracturing T1DM was not significantly different from CTL. Positive correlations were found between HbA1c and pentosidine (r' = 0.79, p < 0.003) and between HbA1c and DMB (r' = 0.64, p < 0.02). Both modifications could lead to less flexible bone (reduced modulus of elasticity) and a tendency toward low-energy fractures in T1DM patients.

Low-intensity continuous ultrasound triggers effective bisphosphonate anticancer activity in breast cancer.

Ultrasound (US) is a non-ionizing pressure wave that can produce mechanical and thermal effects. Bisphosphonates have demonstrated clinical utility in bone metastases treatment. Preclinical studies suggest that bisphosphonates have anticancer activity. However, bisphosphonates exhibit a high affinity for bone mineral, which reduces their bioavailability for tumor cells. Ultrasound has been shown to be effective for drug delivery but in interaction with gas bubbles or encapsulated drugs. We examined the effects of a clinically relevant dose of bisphosphonate zoledronate (ZOL) in combination with US. In a bone metastasis model, mice treated with ZOL+US had osteolytic lesions that were 58% smaller than those of ZOL-treated animals as well as a reduced skeletal tumor burden. In a model of primary tumors, ZOL+US treatment reduced by 42% the tumor volume, compared with ZOL-treated animals. Using a fluorescent bisphosphonate, we demonstrated that US forced the release of bisphosphonate from the bone surface, enabling a continuous impregnation of the bone marrow. Additionally, US forced the penetration of ZOL within tumors, as demonstrated by the intratumoral accumulation of unprenylated Rap1A, a surrogate marker of ZOL antitumor activity. Our findings made US a promising modality to trigger bisphosphonate anticancer activity in bone metastases and in primary tumors.

Low-Intensity Ultrasound Promotes Clathrin-Dependent Endocytosis for Drug Penetration into Tumor Cells.

Ultrasound-mediated drug delivery is a field of research with promising results, although the exact mechanisms underlying intracellular delivery of therapeutic compounds remain to be elucidated. Many studies use drug carriers and cavitation to enhance drug uptake into tumor cells. However, cavitation could induce cell lysis and remain difficult to control and predict in vivo. In this study, low-intensity ultrasound was delivered using two transducers working at 2.9 and 1.3 MHz. The maximal peak negative pressure was 0.29 MPa to avoid cavitation. Low-intensity ultrasound induced clathrin-mediated endocytosis and forced the penetration of a bisphosphonate (zoledronic acid) into MCF-7 human breast cancer cells potentially as a result of mechanical stresses. When sonication parameters were adjusted to create mild hyperthermia in addition to the mechanical stress, further significant accumulation of ZOL was observed. These results provide better insight into the role of acoustic parameters in drug uptake.

Ratio between mature and immature enzymatic cross-links correlates with post-yield cortical bone behavior: An insight into greenstick fractures of the child fibula.

As a determinant of skeletal fragility, the organic matrix is responsible for the post-yield and creep behavior of bone and for its toughness, while the mineral apatite acts on stiffness. Specific to the fibula and ulna in children, greenstick fractures show a plastic in vivo mechanical behavior before bone fracture. During growth, the immature form of collagen enzymatic cross-links gradually decreases, to be replaced by the mature form until adolescence, subsequently remaining constant throughout adult life. However, the link between the cortical bone organic matrix and greenstick fractures in children remains to be explored. Here, we sought to determine: 1) whether plastic bending fractures can occur in vitro, by testing cortical bone samples from children's fibula and 2) whether the post-yield behavior (ωp plastic energy) of cortical bone before fracture is related to total quantity of the collagen matrix, or to the quantity of mature and immature enzymatic cross-links and the quantity of non-enzymatic cross-links. We used a two-step approach; first, a 3-point microbending device tested 22 fibula machined bone samples from 7 children and 3 elderly adults until fracture. Second, biochemical analysis by HPLC was performed on the sample fragments. When pooling two groups of donors, children and elderly adults, results show a rank correlation between total energy dissipated before fracture and age and a linear correlation between plastic energy dissipated before fracture and ratio of immature/mature cross-links. A collagen matrix with more immature cross-links (i.e. a higher immature/mature cross-link ratio) is more likely to plastically deform before fracture. We conclude that this ratio in the sub-nanostructure of the organic matrix in cortical bone from the fibula may go some way towards explaining the variance in post-yield behavior. From a clinical point of view, therefore, our results provide a potential explanation of the presence of greenstick fractures in children.

Periostin deficiency increases bone damage and impairs injury response to fatigue loading in adult mice.

Bone damage removal and callus formation in response to fatigue loading are essential to prevent fractures. Periostin (Postn) is a matricellular protein that mediates adaptive response of cortical bone to loading. Whether and how periostin influences damage and the injury response to fatigue remains unknown. We investigated the skeletal response of Postn(-/-) and Postn(+/+) mice after fatigue stimulus by axial compression of their tibia. In Postn(+/+) mice, cracks number and surface (CsNb, CsS) increased 1h after fatigue, with a decrease in strength compared to non-fatigued tibia. At 15 days, CsNb had started to decline, while CtTV and CtBV increased in fatigued vs non-fatigued tibia, reflecting a woven bone response that was present in 75% of the fatigued bones. Cortical porosity and remodelling also prominently increased in the fatigued tibia of Postn(+/+) mice. At 30 days, paralleling a continuous removal of cortical damage, strength of the fatigued tibia was similar to the non-fatigue tibia. In Postn(-/-) mice, cracks were detectable even in the absence of fatigue, while the amount of collagen crosslinks and tissue hardness was decreased compared to Postn(+/+). Fatigue significantly increased CsNb and CsS in Postn(-/-), but was not associated with changes in CtTV and CtBV, as only 16% of the fatigued bones formed some woven bone. Cortical porosity and remodelling did not increase either after fatigue in Postn(-/-), and the level of damage remained high even after 30 days. As a result, strength remained compromised in Postn(-/-) mice. Contrary to Postn(+/+), which osteocytic lacunae showed a change in the degree of anisotropy (DA) after fatigue, Postn(-/-) showed no DA change. Hence periostin appears to influence bone materials properties, damage accumulation and repair, including local modeling/remodeling processes in response to fatigue. These observations suggest that the level of periostin expression could influence the propensity to fatigue fractures.

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.