Bone Histomorphology Research Articles

Morin Improves the Bone Histomorphology and Biochemical Markers in an Animal Model of Ovariectomy‐Induced Osteoporosis by Suppressing Autophagy and Apoptosis

ABSTRACTOsteoporosis (OP) is the most prevalent metabolic bone disease and an important postmenopausal consequence. This study aimed to investigate the effects of morin, a flavonoid with beneficial properties, on ovariectomy‐induced OP. Animals were ovariectomized (OVX) and treated with different doses of morin (15, 30, and 45 mg/kg/day) or estradiol (10 μg/kg/day) for 10 weeks by gavage. Then bone histo‐stereology, bone‐related biochemical indicators, and gene and protein levels of autophagy and apoptosis‐related markers were analyzed. In comparison to controls, OVX significantly decreased the number of osteoblasts (5.78 × 106 vs. 1.66 × 106) and osteocytes (32.55 × 106 vs. 11.92 × 106), whereas increasing the number of osteoclasts (83.38 × 103 vs. 392.1 × 103). Moreover, OVX caused a remarkable decrease in bone structures and Ca, P, and estradiol levels while increasing ALP and OC (p < 0.001). The administration of 45 mg/kg/day morin restored the effects of OP on bone histomorphology and biochemical markers (p < 0.05). Further studies revealed that morin caused a 7.1% and 36.6% decrease in the bone level of LC3 and BECN1 proteins, respectively, compared to the OVX group. Also, morin caused a significant decrease of 47.4% in the CASP3 level and a significant increase of 23.6% in the BCL‐2 level compared to OVX animals (p < 0.001). The present findings showed that morin is potentially able to improve the bone‐related histomorphological and biochemical changes caused by osteoporosis, which is probably attributed to the suppression of apoptosis‐ and autophagy‐caused cell death.

Geranylgeranyl diphosphate synthase inhibition impairs osteoclast differentiation, morphology and resorptive activity

Abstract Nitrogen bisphosphonates (NBPs), such as zoledronic acid, target the enzyme farnesyl diphosphate synthase (FDPS) in the isoprenoid biosynthetic pathway (IBP), and are the frontline treatment for osteolytic bone diseases. The strong affinity of these agents for bone limits their distribution out of the skeleton. Geranylgeranyl diphosphate synthase (GGDPS) is directly downstream to FDPS in the IBP and novel GGDPS inhibitors such as RAM2061 have been shown to have key drug-like features including prolonged half-life, metabolic stability, and systemic distribution. Furthermore, RAM2061 exerts anti-neoplastic benefits in mouse models of multiple myeloma and Ewing sarcoma. Therefore, we are interested in determining the potential impact of RAM2061 on osteoclast biology and bone remodeling. Studies utilizing undifferentiated RAW264.7 cells demonstrated that treatment with RAM2061 depletes cells of GGPP, impairs protein geranylgeranylation and induces markers of the unfolded protein response pathway and apoptosis. Differentiation of RAW264.7 cells to mature osteoclasts is disrupted by RAM2061, resulting in decreased numbers of mature osteoclasts, altered morphology and decreased tartrate resistant acid phosphatase activity. Treatment of fully differentiated RAW264.7 cells with RAM2061 led to decreased resorptive activity. Confocal microscopy studies revealed that RAM2061 disrupts Cdc42 localization, inhibiting proper actin ring formation in osteoclasts. No significant impact on bone turnover markers or bone histomorphology was observed following a 3-week treatment of CD-1 mice with RAM2061, although decreased numbers of osteoclasts were observed. Liquid chromatography–tandem mass spectrometry studies confirmed accumulation of RAM2061 in bone from the in vivo studies as well as hydroxyapatite binding in vitro. In conclusion, these studies are the first to demonstrate the anti-osteoclastic activity of GGDPS inhibitor treatment and support future studies exploring the therapeutic benefit of this novel therapy in the setting of pathological bone remodeling.

Chromium Picolinate Regulates Bone Metabolism and Prevents Bone Loss in Diabetic Rats.

Diabetic osteoporosis (DOP) is an abnormal metabolic disease caused by long-term hyperglycemia. In this study, a model rat of streptozotocin (STZ)-induced diabetes was established, and chromium picolinate (5 mg·kg-1) was given; the changes in blood glucose and body weight were detected before and after administration; and bone mineral density (BMD), bone morphology, bone turnover markers, inflammatory cytokines, and oxidative stress indicators were observed in each group. We found that after chromium picolinate (CP) intervention for 8 weeks, the blood glucose level was decreased; the BMD, the bone histomorphology parameters, and the pathological structure were improved; the expression of bone resorption-related proteins was downregulated; and the expression of bone formation-related proteins was upregulated. Meanwhile, serum antioxidant activity was increased, and inflammatory cytokine levels were decreased. In conclusion, CP could alleviate DOP by anti-oxidation, inhibition of bone turnover, anti-inflammation, and regulation of the OPG/RANKL/RANK signaling pathway. Therefore, CP has important application values for further development as a functional food or active medicine in DOP treatment.

YTHDC1 inhibits osteoclast differentiation to alleviate osteoporosis by enhancing PTPN6 messenger RNA stability in an m6A-hUR-dependent manner.

YTHDC1 has been confirmed to mediate osteoporosis (OP) progression by regulating osteogenic differentiation. However, whether YTHDC1 mediates osteoclast differentiation and its molecular mechanism remains unclear. Quantitative real-time polymerase chain reaction and Western blot analysis were performed to detect the levels of YTHDC1, PTPN6, NFATc1, TRAP, RUNX2, alkaline phosphatase, and HUR. YTHDC1 knockout mice was constructed by CRISPR/Cas9 system, and the OP mice model was established by ovariectomy. Hematoxylin and eosin staining and micro-computed tomography were used to evaluate bone formation and bone mass. Mouse primary bone marrow macrophage cells were isolated and induced into osteoclasts. TRAP-positive cells were detected using TRAP staining. MeRIP-qPCR, RIP-qPCR assay, RNA affinity isolation assay, and co-immunoprecipitation assay were used to confirm the interactions among YTHDC1, PTPN6, and HUR. YTHDC1 expression was reduced and positively correlated with lumbar bone mineral density in OP patients. In the ovariectomy model of YTHDC1 knockout mice, bone formation was reduced, bone histomorphology was changed, and osteoclastic-related factor (NFATc1 and TRAP) levels were enhanced. Overexpression YTHDC1 inhibited osteoclast differentiation. YTHDC1 increased PTPN6 messenger RNA stability in an m6A-dependent manner. Moreover, YTHDC1 interacted with HUR to positively regulate PTPN6 expression. PTPN6 knockdown promoted osteoclast differentiation, and this effect was reversed by overexpressing HUR or YTHDC1. YTHDC1 was involved in regulating OP progression through inhibiting osteoclast differentiation by enhancing PTPN6 messenger RNA stability in an m6A-HUR-dependent manner.

Biochanin A abrogates osteoclastogenesis in type 2 diabetic osteoporosis via regulating ROS/MAPK signaling pathway based on integrating molecular docking and experimental validation

BackgroundThere are accumulating type 2 diabetes patients who have osteoporosis simultaneously. More effective therapeutic strategies should be discovered. Biochanin A (BCA) has been indicated that can play a role in improving metabolic disorders of type 2 diabetes and preventing osteoporosis. But whether BCA can treat type 2 diabetic osteoporosis has not been studied.PurposeTo investigate if the BCA can protect against type 2 diabetic osteoporosis and clarify the mechanism.MethodsMicro-CT and histology assays were performed to detect the trabecular bone and analyze the bone histomorphology effect of BCA. CCK-8 assay was performed to detect the toxicity of BCA. TRAcP staining, immunofluorescence and hydroxyapatite resorption assay were used to observe osteoclasts differentiation and resorptive activity. Molecular docking provided evidence about BCA regulating the MAPK axis via prediction by the algorithm. QRT-PCR and Western Blotting were utilized to detect the expression of osteoclastogenesis-related markers and MAPK signaling pathway.ResultsAccumulation of bone volume after BCA treatment could be found based on the 3D reconstruction. Besides, there were fewer osteoclasts in db/db mice treated with BCA than db/db mice treated with saline. In vitro, we found that BCA hadn’t toxicity in osteoclasts precursor, but also inhibited differentiation of osteoclasts. Further, we found that BCA suppresses osteoclastogenesis via ROS/MAPK signaling pathway.ConclusionBCA can prevent type 2 diabetic osteoporosis by restricting osteoclast differentiation via ROS/MAPK signaling pathway.

Enhancing cranial defect repair in rats: investigating the effect of combining Total Flavonoids from Rhizoma Drynariae with calcium phosphate/collagen scaffolds

ObjectiveTo investigate the therapeutic efficacy of total flavonoids of Rhizoma Drynariae (TFRD) in conjunction with a calcium phosphate/collagen scaffold for the repair of cranial defects in rats.MethodsThe subjects, rats, were segregated into four groups: Control, TFRD, Scaffold, and TFRD + Scaffold. Cranial critical bone defects, 5 mm in diameter, were artificially induced through precise drilling. Post-surgery, at intervals of 2, 4, and 8 weeks, micro-CT scans were conducted to evaluate the progress of skull repair. Hematoxylin–eosin and Masson staining techniques were applied to discern morphological disparities, and immunohistochemical staining was utilized to ascertain the expression levels of local osteogenic active factors, such as bone morphogenetic protein 2 (BMP-2) and osteocalcin (OCN).ResultsUpon examination at the 8-week mark, cranial defects in the Scaffold and TFRD + Scaffold cohorts manifested significant repair, with the latter group displaying only negligible foramina. Micro-CT examination unveiled relative to its counterparts, and the TFRD + Scaffold groups exhibited marked bone regeneration at the 4- and 8-week intervals. Notably, the TFRD + Scaffold group exhibited substantial bone defect repair compared to the TFRD and Scaffold groups throughout the entire observation period, while histomorphological assessment demonstrated a significantly higher collagen fiber content than the other groups after 2 weeks. Immunohistochemical analysis further substantiated that the TFRD + Scaffold had augmented expression of BMP-2 at 2, 4 weeks and OCN at 2 weeks relative to other groups.ConclusionsThe synergistic application of TFRD and calcium phosphate/collagen scaffold has been shown to enhance bone mineralization, bone plasticity, and bone histomorphology especially during initial osteogenesis phases.

Functionalized Virus Nanoparticles Alleviates Osteoporosis via Targeting the Function of RANK-Specific Motifs.

Osteoporosis is a common skeletal disease characterized by excessive osteoclast-induced bone loss. RANKL/RANK signaling pathway is essential for osteoclastogenesis and is a key target for osteoporosis. However, regarding the fact that RANKL/RANK also functions beyond bone, the total block of RANKL/RANK will have unwanted impact on other organs. Our previous study revealed that mutation of RANK-specific motifs inhibited osteoclastogenesis without effects on other organs in mice. However, the instability and low cellular uptake efficiency limited the application of the therapeutic peptide originating from the amino acid sequence of RANK-specific motifs (RM). To this end, in this study, the peptide RM (SRPVQEQGGA (C to N terminal)) was chemically modified onto the surface of the plant virus-based nanoparticles cowpea chlorotic mottle virus (CCMV). Subsequent experiments showed that the novel virus nanoparticles RM-CCMV had excellent biocompatibility and stability, which ultimately facilitated its cellular uptake efficiency and improved its inhibitive effects on osteoclastogenesis. Moreover, RM-CCMV achieved bone enrichment and suppressed bone resorption by inhibiting osteoclastogenesis and improving the parameters of bone histomorphology in murine femurs. To be mentioned, the effective dose of CCMV conjugated RM was only 6.25% of free RM. In summary, these results have provided a promising therapeutic strategy for osteoporosis.

METTL14 represses osteoclast formation to ameliorate osteoporosis via enhancing GPX4 mRNA stability.

Excessive bone resorption by osteoclasts results in the development of multiple bone disorders including osteoporosis. This study aimed to explore the biological function of methyltransferase-like14 (METTL14) in osteoclast formation, as well as its related mechanisms. Expression levels of METTL14, GPX4 and osteoclast-related proteins TRAP, NFATc1, c-Fos were detected by qRT-PCR and Western blotting. The osteoporosis model was established in mice by bilateral ovariectomy (OVX). Bone histomorphology was determined by micro-CT and H&E staining. NFATc1 expression in bone tissues was determined by immunohistochemical staining. Proliferation of primary bone marrow macrophages cells (BMMs) was assessed by MTT assay. Osteoclast formation was observed by TRAP staining. The regulatory mechanism was evaluated by RNA methylation quantification assay, MeRIP-qPCR, dual luciferase reporter assay, and RIP, respectively. METTL14 was down-regulated in the serum samples of postmenopausal osteoporotic women, which was positively associated with bone mineral density (BMD). Osteoclast formation was promoted in OVX-treated METTL14+/- mice as compared with wild-type littermates. Conversely, METTL14 overexpression repressed RANKL-induced osteoclast differentiation of BMMs. Mechanistically, METTL14-mediated m6A modification post-transcriptionally stabilized glutathione peroxidase 4 (GPX4), with the assistance of Hu-Antigen R (HuR). Finally, GPX4 depletion-mediated osteoclast formation in BMMs could be counteracted by METTL14 or HuR overexpression. Collectively, METTL14 inhibits osteoclastogenesis and bone resorption via enhancing GPX4 stability through an m6A-HuR dependent mechanism. Therefore, targeting METTL14 might be a novel promising treatment strategy for osteoporosis.

A new thin sectioning method for observation of higher resolution images in bone histomorphology

A new thin sectioning method for observation of higher resolution images in bone histomorphology

Peiminine regulates bone-fat balance by canonical Wnt/β-catenin pathway in an ovariectomized rat model.

Peiminine is a major biologically active component of Fritillaria thunbergii Miq that exhibits good anticancer, antiinflammatory, and anti-osteoclast effects. However, its effects on osteoporosis (OP) remain unknown. This study aimed to explore whether Peiminine was able to regulate osteogenesis and adipogenesis in ovariectomized (OVX) rat. The effects on the differentiation of bone marrow stem cells (BMSCs), function of Wnt/β-catenin pathway, ALP activity, calcium nodule deposition, as well as adipocyte formation in vitro by Peiminine at different concentrations, were detected. The curative effects of Peiminine on the ovariectomy-induced osteoporosis model by micro-CT and bone histomorphology assays were analyzed. The promotion of osteogenic differentiation and inhibition of adipogenic differentiation by Peiminine (5-40 μg/mL) was detected and the optimum concentration was 20 μg/mL. Mechanistically, Peiminine regulated the fate of BMSCs in vitro, and activated Wnt/β-catenin signaling pathway by restraining phosphorylation of β-catenin and promoting the nuclear translocation of β-catenin. Moreover, Peiminine prevented ovariectomy-induced osteoporosis by alleviating trabecular bone loss and inhibiting adipose formation. Our data suggested that Peiminine could attenuate ovariectomy-induced osteoporosis by alleviating trabecular bone loss and inhibiting adipose formation. These encouraging discoveries could lay the foundation for Peiminine to be a promising preventive treatment strategy for skeletal diseases, such as osteoporosis.

Isoorientin ameliorates osteoporosis and oxidative stress in postmenopausal rats

Context Isoorientin has many biological activities, including antioxidant, anti-inflammatory, antitumor. However, the effect of isoorientin on postmenopausal osteoporosis remains unclear. Objective To evaluate the effect of isoorientin on postmenopausal osteoporosis. Materials and methods Sprague-Dawley rats were divided into five groups (n = 5): sham, model, 17-β-oestradiol (E2, 10 μg/kg/day), low-dose isoorientin (L-Iso, 50 mg/kg), and high-dose isoorientin (H-Iso, 100 mg/kg). The rats were ovariectomized, treated by gavage daily for 12 weeks, and serum and femur samples were collected. Bone mineral density, bone metabolism, and oxidative stress were assessed. H&E staining, immunohistochemistry, and western blotting were employed. Results Isoorientin improved the bone mineral density of the lumbar vertebrae (2.01 ± 0.05 g/cm3 in H-Iso group vs. 1.74 ± 0.07 g/cm3 in model group) and femur (1.46 ± 0.06 g/cm3 vs. 1.19 ± 0.03 g/cm3), increased the trabecular bone number (1.97 ± 0.03 vs. 1.18 ± 0.13) and thickness (0.27 ± 0.02 vs. 0.16 ± 0.03 mm). Isoorientin decreased the separation degree of trabecular bone, ameliorated bone histomorphology changes, and significantly improved the mechanical properties. Isoorientin diminished MDA (by 60%) and increased SOD (by 49.2%), and GSH-Px (by 159%) activity. Furthermore, osteoprotegerin (OPG), nuclear factor erythroid 2-like 2 (Nrf2), haem oxygenase (HO-1), NAD(P)H quinone dehydrogenase 1(NQO1), and oestrogen receptor 1(ESR1) protein expression increased, while receptor activator of nuclear factor-κB ligand (RANKL) protein expression decreased after treatment. Conclusions Isoorientin ameliorates osteoporosis via upregulating OPG and Nrf2/ARE signalling, suggesting isoorientin maybe a potential therapeutic drug for PMOP.

Alveolar bone and tibia responses to hormonal and mineral abnormalities in rats with chronic kidney disease: A pilot study.

As the impact of chronic kidney disease (CKD) severity on different bone types remains unclear, we induced increasing levels of CKD severity in a rat model and investigated hormone and mineral levels as well as alveolar and tibia bone histomorphology. Rats were divided into sham operation (sham), 4/6 nephrectomy (4/6Nx), 5/6Nx, and 4/6Nx with hyperphosphorous (HP) diet (4/6NxHP). At week 20, BUN, FGF23, PTH, and P were estimated in plasma. Bone parameters were evaluated by microCT, and osteoclasts and osteoid areas were evaluated by TRAP and H&E stains, respectively. The 4/6NxHP and 5/6Nx groups had elevated PTH, and the 4/6NxHP group alone had elevated P. Compared to the 4/6Nx group, the 4/6NxHP group demonstrated increased FGF23 and P. In the alveolar bone, the 4/6NxHP group had reduced bone volume and BMD compared to the sham and 4/6Nx groups. In the tibia cortical bone, bone surface density was higher in the 4/6NxHP group compared to the sham group. Tibia cortical bone volume was negatively correlated with FGF23 and P. Moreover, alveolar bone volume was negatively correlated with FGF23, PTH, and P. Our results demonstrate that hormone and mineral levels vary with CKD severity, and alveolar bone loss strongly correlates with these hormone and mineral alterations.

Efficiency of Nanohydroxyapatite on Repairing Type II Diabetes Dental Implant-Bone Defect.

The purpose of this study was to see how a nanohydroxyapatite (n-HA) composite polyamide 66 (PA66) affected the repair of bone defects in diabetics with titanium implants, as well as to develop experimental materials for the creation of the interface between bone tissue and titanium implants. Rabbit bone marrow mesenchymal stem cells (MSCs) were isolated using n-HA/PA66 composite material, and the effect of coculture with the material on cell proliferation was analyzed after induction of mineralization. Bone defect models of diabetic experimental rabbits and titanium implants were prepared. Normal rabbits with bone defects were used as control (NC group, N = 8). After the diabetic bone defect (DM group, N = 8) and the implantation of n-HA/PA66 composite material (n-HA/PA66 group, N = 8), the differences in body weight, blood glucose, scanning electron microscopy of the implant-bone interface, bone mineral density, new bone trabecular parameters, histomorphology, and biomechanical properties of the implant-bone interface were compared and analyzed. In vitro test results showed that MSC cell growth could be promoted by mineralization induction, the cell growth condition was good after coculture with n-HA/PA66, and the proliferation activity of MSCs was not affected by the material. In vivo test results showed that the body weight of the DM group and n-HA/PA66 group was considerably inferior to that of the NC group, and the blood glucose was dramatically superior to that of the NC group (P < 0.05). However, the body weight of the n-HA/PA66 group was dramatically superior to that of the DM group (P < 0.05). The bone mineral density, bone volume fraction (BV/TV), bone surface area fraction (BS/BV), bone trabecular thickness (Tb.Th), bone trabecular number (Tb.N), bone trabecular area, and biomechanical properties in the DM group were considerably inferior to those in the NC group and n-HA/PA66 group (P < 0.05). The trabecular space (Tb.Sp) in the NC group and n-HA/PA66 group was dramatically superior to that in the NC group (P < 0.05). The bone mineral density, BV/TV, BS/BV, Tb.Th, Tb.N, trabecular area, and biomechanical properties of the n-HA/PA66 group were dramatically superior to those of the NC group (P < 0.05), while Tb.Sp was considerably inferior to that of the NC group (P < 0.05). These findings showed that the n-HA/PA66 material had good biocompatibility and minimal cytotoxicity, and that filling the space between the surrounding bone and the titanium implant can enhance bone repair. This research paved the way for future research into the tissue-engineered bone in the field of oral surgery.

Dynamics of hematological parameters and acute phase reactions for osteosubstitution by autofibrin and hydroxyapatite ceramics with β-tricalcium phosphate fragmentary bone fractures in dogs

Osteo-replacement materials are considered to be the most promising direction in solving the problem of restoring the regenerative potential of bone tissue, especially fragmentary fractures. However, the mechanism of their influence on the histomorphology of bone regenerates and the pathochemical phase of reparative osteogenesis require further and comprehensive justification. The aim of the study was to establish the dynamics of hematological parameters and acute phase response to osteosubstitution by hydroxyapatite ceramics with β-tricalcium phosphate and platelet-enriched autofibrin in fragmentary bone fractures in dogs. Control and research groups of animals were formed, each of which included dogs with fractures of both shoulder and forearm bones, which came in 2019- 2021 to the clinic of small pets of the Faculty of Veterinary Medicine of Bila Tserkva NAU. After general and local anesthesia, extracortical osteosynthesis and replacement of bone defects were performed: in the first experimental group (n = 10) autofibrin enriched with platelets (PRF), and in the second (n = 10) - its combination with hydroxyapatite ceramics (PRF + HA/β -TCP-700); in the control group (n = 10) the defects were left to heal under the blood clot. Blood samples for hematological and biochemical studies were taken after injury no later than 24 hours and on the 3rd, 7th, 14th, 21st and 42nd day after osteosynthesis. In peripheral blood, the number of erythrocytes, platelets and leukocytes was determined by conventional methods, and hemoglobin - hemoglobin cyanide. The content of nitric oxide (NO) in the blood serum was determined by the method of Green in the modification of Golikov, haptoglobin - by reaction with rivanol kits PJSC "Reagent" (Ukraine) and α2-macroglobulin - by KM Veremeenko. Statistical processing of the results was performed using the program Statistica 10 (StatSoft Inc, USA, 2011). It was found that bone trauma in dogs is accompanied by a decrease in peripheral blood of erythrocytes by 1.3 times (p &lt;0.001) and an increase of 1.1 times (p &lt;0.05) the number of leukocytes with a tendency to increase hemoglobin and platelets, compared with indicators of clinically healthy animals. After osteosynthesis in all groups leukocytosis developed. During osteosubstitution, the intensity and duration of the leukocyte reaction decreased significantly. In the first experimental group, the peak of NO concentration occurred on the 21st day, and in the case of combined osteosubstitution, the phase of its changes was noted, which had two peaks: on the 7th in 1.5 and on the 21st in 1.8 (p &lt;0.001) is several times higher than in the control group. The concentration of haptoglobin after bone injury in dogs increased 1.1 times (p &lt;0.001) compared with clinically healthy animals and reached its peak in all groups on the 3rd day (p &lt;0.001). No changes in α2-macroglobulin concentration were observed in the groups during the entire study period. Combined osteosubstitution by calcium-phosphate ceramics with platelet-enriched fibrin induces early osteogenic processes at a lower intensity of the acute phase reaction at the phase peaks of nitric oxide - an inducer of angiogenesis, which indicates the viability of animals. Key words: nitric oxide, acute phase proteins, reparative potential, platelets.

Aptamer-immobilized bone-targeting nanoparticles in situ reduce sclerostin for osteoporosis treatment

Bone-targeted drug delivery to reduce systemic complications and maintain sufficient doses in bone tissues is the main challenge for osteoporosis treatment. Here, we develop a novel, and simply synthesized bone-targeting nanomedicine (DNA-MSN, DNAM) containing a PEGylated dendritic mesoporous silica nanoparticle (MSN) core (~ 65 nm) and an anti-sclerostin aptamer (Aptscl56) layer, to treat osteoporosis. The nanoparticle core protects the immobilized Aptscl56 from rapid nuclease degradation and renal filtration, prolonging in vivo half-lives. The DNAM-immobilized Aptscl56 layer exhibits dual functions to direct bone-attachment of ovariectomized mice, due to the interaction between phosphate groups in DNA aptamer Aptscl56 and bone calcium in hydroxyapatites, and to in situ capture sclerostin with picomolar affinities. Moreover, we show DNAM significantly reverses the serum level of sclerostin and osteoporotic bone loss to a normal level, improving bone histomorphology parameters and mechanical properties in the femur, and recovering serum levels of bone turnover markers, without systemic toxicity. Notably, the therapeutic effect of DNAM is superior to the "gold standard" drug alendronate, and the systemic dose of DNAM-immobilized Aptscl56 is only 25% of free Aptscl56. The present study provides insights into the regulation of unwanted circulating biohazards and represents a promising approach to deliver nanomedicines to treat osteoporosis.

Serum β-catenin changes vary among different stages of osteonecrosis of the femoral head: an exploratory biomarker study

BackgroundWnt/β-catenin signaling pathway is closely related to the pathogenesis Osteonecrosis of the femoral head (ONFH). β-catenin, as a major component of Wnt signaling pathway, plays a vital role in the proliferation of osteoblasts. But the effect of altering β-catenin level on the early diagnosis and staging of ONFH has not been studied. Our purpose is to investigate the role of β-catenin level in the progress of ONFH.MethodOne hundred and one patients with three stages of ONFH and fifty healthy controls were recruited between May 2016 and November 2016. We divided the patients into 32 cases of stage II, 41 cases of stage III and 28 cases of stage IV according to the Association Research Circulation Osseous (ARCO) classification. We evaluated the clinical bone histomorphology, expression position and level of β-catenin as well as the plasma β-catenin level. We investigated the level of β-catenin from the serum and tissue samples using ELISA and Western Blot assay. We also evaluated the expression of β-catenin in bone tissue by immunohistochemistry. Data were analyzed by independent t-test and ANOVA.ResultsWe found that the mean (± SD) serum level of β-catenin was 66.99 ± 3.032 ng/ml in the ONFH patients, which was higher than 20.14 ± 1.715 ng/ml observed in the control group (P < 0.001). Moreover, the β-catenin levels were 49.30 ± 4.649 ng/ml, 72.54 ± 4.864 ng/ml and 79.10 ± 4.773 ng/ml in the ONFH patients with ARCO stage II, stage III and stage IV respectively, showing significant difference among them (P < 0.001). We also found that the area under the curve (AUC) calculated by ROC curve analysis to determine the values for β-catenin levels in ONFH compared with those in the control group was 0.9358 (P < 0.001), where the sensitivity was 77.23% and specificity was 98.00%.ConclusionOur results indicate that the increased β-catenin may play a vital role in the progress of ONFH and the level of β-catenin is correlated with ARCO stages. The cut-off concentration may be used as one of the sensitive marks to assess the disease process of ONFH.

A comparison of three decalcification agents for assessments of cranial fracture histomorphology.

The extraction of mineral calcium from bone by decalcification is a critical step in the preparation of histological samples for light microscopy. This study assessed the time required for complete decalcification and the resultant histomorphological preservation of bone histomorphology by three decalcification agents: 7% hydrochloric acid (HCl), 5% nitric acid, and 10% ethylenediaminetetraacetic acid (EDTA). The goal of this study was to identify which decalcification agent provides the optimal combination of expedient processing and quality histological outcomes of cranial fracture samples. HCl provided the most rapid decalcification ( =3.57 days), nitric acid followed closely ( =10.35 days), while EDTA took significantly longer on average ( =78.97 days) but encompassed a broader range of times. Decalcification agent, sample thickness, sample width, and decedent age are significant predictors of decalcification time. Sample visualization quality, measured for tissues, cells, and nuclei on a five-point Likert scale, was highest for samples decalcified in 10% EDTA, second highest using 5% nitric acid, and lowest for 7% HCl. The quality difference between EDTA and nitric acid was not highly significant for any of the three features. For basic assessments of bone histomorphology, the study results indicate 5% nitric acid is suitable for the decalcification of adult specimens and samples thicker than 3 mm. EDTA is a suitable agent for thin samples of the cranial vault (<3mm) from infants and young children less than three years old, decalcifying samples in a timeframe comparable to nitric acid while providing the best quality and clarity of samples.

Exercise improves bone formation by upregulating the Wnt3a/\u03b2-catenin signalling pathway in type 2 diabetic mice

BackgroundThe bone formation ability of type 2 diabetes is inhibited, and exercise can effectively improve the bone formation of T2DM. However, whether exercise can mediate the Wnt3a/β-catenin pathway to improve the mechanism of bone formation and metabolism still needs further research.MethodsA T2DM mouse model was established by a high-fat diet and STZ injection, and the mice were trained with swimming and downhill running exercise. Alizarin red staining is used to observe the changes of the left femoral trabecular bone; micro-CT is used to analyze the trabecular and cortical BMD, BV/TV, BS/BV, BS/TV, Tb.Th, Tb.Sp; the ALP staining of skull was used to observe the changes in ALP activity of bone tissues at the skull herringbone sutures; ALP staining was performed to observe the changes in the number of OBs and ALP activity produced by differentiation; Quantitative PCR was used to detect mRNA expression; Western blot was used to detect protein expression levels.ResultsWhen the Wnt3a/β-catenin pathway in the bones of T2DM mice was inhibited, the bone formation ability of the mice was significantly reduced, resulting in the degradation of the bone tissue morphology and structure. Swimming caused the significant increase in body weight and Runx2 mRNA expression, while downhill running could significantly decrease the body weight of the mice, while the tibia length, wet weight, and the trabecular morphological structure of the distal femur and the indexes of bone histomorphology were significantly improved by activating the Wnt3a/β-catenin pathway.ConclusionsBone formation is inhibited in T2DM mice, leading to osteoporosis. Downhill running activates the Wnt3a/β-catenin pathway in the bones of T2DM mice, promotes OB differentiation and osteogenic capacity, enhances bone formation metabolism, and improves the bone morphological structure.

Effects and Mechanism of Zishen Jiangtang Pill on Diabetic Osteoporosis Rats Based on Proteomic Analysis.

Objective To explore the effect and mechanism of ZJP on DOP rats by proteomic analysis. Materials and Methods After the establishment of diabetes model by Streptozocin (STZ, 60 mg/kg), 40 Wistar rats were equally divided into normal group, model group (diabetic rats), high-dose group (3.0 g/kg/d ZJP), and low-dose group (1.5 g/kg/d ZJP) and received treatment for 3 months. Histological changes in bone and pancreas tissues were observed by hematoxylin and eosin staining, electron microscopy, and immunofluorescence. Proteomic and bioinformatic analyses were performed to identify the differentially expressed proteins. The fingerprint and active ingredients of ZJP were identified via high-performance liquid chromatography (HPLC). Results Compared with the model group, ZJP could rescue the weight, fasting blood glucose, and fasting insulin of rats in both high-dose and low-dose group. ZJP could also improve the microstructures of pancreatic islet cells, bone mass, and trabecular and marrow cavities in DOP rats. Bioinformatic analysis suggested that ZJP might influence DOP via multiple pathways, mainly including ribosomes, vitamin digestion and absorption, and fat digestion and absorption. The primary active ingredients, including notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, icariin, and ginsenoside Rb1, were detected. Conclusion ZJP could significantly improve the histomorphology and ultrastructure of bone and islets tissues and might serve as an effective alternative medicine for the treatment of DOP.

MicroRNA-151a-3p Functions in the Regulation of Osteoclast Differentiation: Significance to Postmenopausal Osteoporosis.

BackgroundStudies have found the pivotal role of miRNAs in the progression of postmenopausal osteoporosis (OP). However, the function of miRNAs in OP is unclear. This study aimed to explore the biological functions of microRNA-151a-3p in OP.MethodsRT-qPCR was employed to assess the expression of microRNA-151a-3p in serum isolated from OP patients and healthy controls. Dual-energy X-ray absorptiometry (DXA) was used to measure the bone mineral density (BMD) of the lumbar spine. The expression levels of c-Fos, NFATc1, and TRAP were tested by Western blot. Ovariectomized (OVX) rats were treated with antago microRNA-151a-3p or antago NC, and then serum and lumbar vertebrae were collected for ELISA and bone histomorphology analysis.ResultsThe expression of microRNA-151a-3p in postmenopausal women with osteoporosis was significantly up-regulated, and microRNA-151a-3p level was negatively correlated with BMD. During osteoclastogenesis, microRNA-151a-3p level was obviously increased. Overexpression of microRNA-151a-3p promoted the differentiation of RANKL-induced THP-1 and RAW264.7 cells into osteoclasts, whereas silencing of microRNA-151a-3p resulted in the opposite results. Silencing of microRNA-151a-3p in OVX rats altered osteoclastogenesis-related factors and raised BMD.ConclusionMicroRNA-151a-3p could partly regulate osteoporosis by promoting osteoclast differentiation, and miRNA-151a-3p could be a potential therapeutic target for postmenopausal osteoporosis.