Bone Tissue Of Rats Research Articles

Вплив препаратів кріоконсервованої плаценти на перебіг неспецифічного гонартриту в експерименті

Актуальність. Лікування патології суглобів як дегенеративного, так і травматичного генезу — процес тривалий, багатокомпонентний, завжди вимагає спільних зусиль лікаря та пацієнта. Вибір оптимальних засобів терапії складний та неоднозначний. Біологічно активні речовини — одні з потужних засобів боротьби із запальним процесом, що є постійним супутником пошкодження. Завдяки своїм антиоксидантним, імуномодулюючим та регенеративним властивостям фрагменти плаценти визнані перспективним засобом лікування артропатій різної етіології. Мета дослідження — визначення морфологічних характеристик складових структур колінних суглобів щурів у нормі в умовах експериментального артриту та після введення кріоконсервованої плаценти. Матеріали та методи. Експериментальне дослідження проводилось на 115 білих щурах. З інтактною групою (10 тварин) порівнювались 3 контрольні групи по 35 тварин кожна, яким виконувались: 1) моделювання каргінаніндукованого асептичного запалення колінного суглоба; 2) підшкірна імплантація фрагмента плаценти; 3) підшкірна імплантація фрагмента плаценти на фоні асептичного запалення колінного суглоба. Гістологічні препарати готували за загальними методиками із забарвленням гематоксиліном та еозином та за Ван Гізоном. Результати. При створенні запального вогнища зміни в хрящовій тканині визначались із третьої доби у вигляді лакунарності, каріопікнозу як етап некробіозу хондроцитів, що був особливо виражений із 10-ї до 14-ї доби. Морфологічні зміни кісткової тканини щурів, яким вводився ƛ-карагінан, відмічались на 7-му добу дослідження у вигляді резорбції та деструкції кісткових балок. У подальшому (10-та доба) запальний процес призводив до картини фрагментованої архітектоніки кісткової тканини, що спостерігалась до 21-ї доби. Із боку синовіальної оболонки спостерігаються гіперплазія ворсин, лімфоїдна інфільтрація, поява додаткових шарів синовіїцитів як результат проліферативної реакції. Найбільш виражені зміни спостерігались у покривному шарі синовіальної оболонки. При однократному введенні кріоконсервованої плаценти ознаки запалення вірогідно зменшувались: обмежувалась площа деструкції хряща та кістки, у синовіальній оболонці знижувались ознаки мукоїдного набухання. Процеси функціонального кровонаповнення мікроциркуляторного русла переважали над ішемічними. Стимуляція регенеративних процесів забезпечувала структурне відновлення на 21-шу добу спостереження. Висновки. Препарати кріоконсервованої плаценти впливають на перебіг запального процесу в умовах експериментального остеоартриту. Найбільш вірогідний терапевтичний ефект визначається з боку синовіальної оболонки.

Effect of Eight Weeks Aerobic Exercise and Vitamin-D Supplementation on Osteocalcin and Alkaline Phosphatase Gene Expression in Male Rats Poisoned with Hydrogen Peroxide

Introduction: Free radicals increase with age and disease, so the aim of this study was to investigate the effect of exercise and vitamin D on the expression of alkaline phosphatase and osteocalcin genes in bone tissue of rats poisoned with hydrogen peroxide. Methods: In this experimental trial, 36 adult male Wistar rats were randomized into six groups of six rats, 1) control; 2) hydrogen peroxide; 3) hydrogen peroxide + vitamin D; 4) hydrogen peroxide + exercise; 5) hydrogen peroxide + exercise and vitamin D and 6) Sham. For eight weeks, groups 2, 3, 4, and 5 were given daily dose of 1 mmol/kg hydrogen peroxide on even days, groups 3 and 5 received 0.5 mg / kg of Vitamin-D daily, and sham group received only vitamin D solvent intraperitoneally. Groups 4 and 5 performed aerobic exercise 3 day/week. Osteocalcin and alkaline phosphatase gene expression were measured by PCR and were analyzed using independent t-test, two-way analysis of variance and Boferroni’s post hoc test with SPSS 16 (p≤0.05). Results: The interactive effect of exercise and vitamin D on increasing alkaline phosphatase and osteocalcin was significant. (p≤0.05); exercise increased alkaline phosphatase and osteocalcin (p ≤ 0.05); vitamin D was also associated with increased alkaline phosphatase and osteocalcin (p=0.0001). The greatest effect on increasing osteocalcin and alkaline phosphatase showed in groups 5 and 3, respectively (p=0.001). Conclusion: Exercise and vitamin D had a positive effect on bone tissue, so that even the systemic effect of hydrogen peroxide could not change the results of this constructive effect.

Корекція дефіциту вітаміну D високими дозами холекальциферолу

На сьогодні у зв’язку зі значною поширеністю дефіциту вітаміну D все більше застосовуються болюсні схеми призначення високих доз вітаміну D у різних режимах. У статті наведено обґрунтування застосування високих доз вітаміну D, висвітлено питання гіпервітамінозу D й гіперчутливості до вітаміну D, подані аналіз літературних даних ефективності застосування препаратів вітаміну D у дозі 50 000 МО при деяких соматичних захворюваннях, а також результати власних експериментальних досліджень впливу високих доз вітаміну D на показники кісткової тканини в щурів старечого віку із системним остеопорозом та його ускладненнями.

Nanomaterial-induced mesenchymal stem cell differentiation into osteoblast for counteracting bone resorption in the osteoporotic rats

The current approach was designed to unearth the therapeutic potential of osteoblasts infusion, yielded from cultivating rat mesenchymal stem cells of bone marrow source in osteogenic differentiation media supplied with either hydroxyapatite nanoparticles (HA-NPs), chitosan/hydroxyapatite nanomaterials (C/HA-NPs), or chitosan nanoparticles, in the osteoporotic rats. The successful migration of the osteoblasts to the diseased bones of rats in C/HA-NPs and HA-NPs groups was evidenced by PCR screening of the Y-linked sex-determining gene (SRY) in the femoral bone tissue. Serum bone biomarker levels and gene expression patterns of cathepsin K, receptor activator of nuclear factor kappa B ligand (RANKL), and osteoprotegerin (OPG) were assessed. Additionally, histological examination of the femoral bone tissues of rats was performed. The current outcomes revealed that osteoblast implantation, resulted from C/HA-NPs or HA-NPs group, significantly lessened bone sialoprotein level. In Addition, it yielded a significant decline in the gene expression patterns of cathepsin K, RANKL, and RANKL/OPG proportion as well as up-regulation in BMP-2 and Runx-2 gene expression levels as opposed to the untreated ovariectomized animals. Moreover, it could restrain bone resorption and refine bone histoarchitecture. Conclusively, this study sheds light on the therapeutic significance of osteoblasts transplantation in alleviating the intensity of the bone remodeling cycle, consequently representing a hopeful therapeutic approach for primary osteoporosis.

Biodegradable dual-crosslinked adhesive glue for fixation and promotion of osteogenesis

The effective fixation of fracture fragments is the foundation for the smooth healing process of fracture. Due to the presence of small fracture fragments, the comminuted fracture cannot be fixed under high mechanical strength leading to the bone nonunion and delayed healing. In this study, the primary crosslinked injectable bone glue was fabricated by the Schiff base reaction between low substitution degree methacrylate gelatin (GelMa) and Oxidized dextran (oDex) covalently crosslinked with Aminated mesoporous bioactive glass nanoparticles (AMBGN), then the second crosslinked bone glue was formed among small fracture fragments after the irradiation. By this method, the osteogenic dual-crosslinking biodegradable bone glue was synthesized with enhanced mechanical capability. This bone glue was biocompatible and biodegradable, in addition, after the second crosslinking, the mechanical strength was 2.1 times higher than that in primary cross-linking. Moreover, the dual-crosslinked bone glue also exhibited excellent adhesion ability on the isolated rat bone tissues. Additionally, the bone glue can increase the proliferation of bone marrow mesenchymal stem cells (BMSCs) and enhance the expression of Alkaline phosphatase (ALP) and the deposition of calcium nodules with the up-regulating expression of the osteogenic protein (Runx2) and adhesin (Vinculin) in vitro. Furthermore, the bone glue also presented enhanced osteogenesis in the rat calvarial critical-size defect models in vivo. Overall, this osteogenic dual-crosslinking biodegradable bone glue with high mechanical strength provided a new approach in treating the comminuted fracture fragments.

Efficacy of using a complex of minerals and vitamins for prevention of complications in bone tissue and the digestive tract in rats with hypothyroidism

Significant consequences of hypothyroidism are impairments in all types of metabolism, including bone metabolism, pathological changes in the digestive tract and the development of deficiency of vitamins, macro-and micronutrients.The relevance of the study is due to the widespread occurrence of hypothyroidism and the feasibility of developing effective methods for the prevention of the pathology and its complications. The study was aimed at the condition of bone tissue and digestive tract in rats with hypothyroidism, as well as the effectiveness of preventive addition of a complex of vitamins and minerals. The studies were performed on rats with hypothyroidism that was caused using thyrostatic mercazolyl for 50 days, administered orally. Prophylaxis was performed by adding a prophylactic complex of vitamins P, C, D and minerals Ca, Mg, Cu, Se, Mn to the daily diet. In the bone tissue of rats, the indicators of destruction (elastase and acid phosphatase activity) and mineralization (alkaline phosphatase activity, calcium content) as well as the antioxidant state (activities of catalase, glutathione reductase, malonic dialdehyde content) were studied. In the gums and mucous membranes of the digestive tract of rats, we determined indicators of antioxidant status (catalase activity, malonic dialdehydecontent), inflammation (activities of elastase, acid phosphatase) and dysbiosis (activities of urease activity, lysozyme). In the bone tissue of rats with hypothyroidism, activation of destruction processes, reduction of calcium levels, activation of lipid peroxidation and antioxidant enzymes were found. Hypothyroidism also led to pathological disorders in the gums of rats and gastric mucosa, small and large intestine, namely – the development of inflammation, increased membrane permeability, activation of lipid peroxidation, increased contamination with opportunistic bacteria against the background of reduced antioxidant protection. In general, preventive use of the complex contributed to the normalization of the studied parameters in the gums, mucous membranes of the stomach, intestines, as well as remodeling processes and antioxidant-prooxidative state in the bone tissue of animals with hypothyroidism. In the conditions of hypothyroidism, the proposed complex of vitamins and minerals provided notable antioxidant, anti-inflammatory and antidysbiotic effects in the tissues of the gastrointestinal tract, thus overcoming the calcium deficiency in the blood and stopping the destructive and oxidative processes in the bone tissue of animals with hypothyroidism.

Морфометричні та метаболічні порушення у кістковій тканині лабораторних щурів з хронічним токсичним гепатитом

The development of osteodystrophy in diseases of the hepatobiliary system is a known fact, but the mechanism of the formation of metabolic disorders in bone tissue in liver pathology is not fully understood. The purpose of the study was to study morphological and biochemical changes in the bone tissue of female and male rats during the modeling of chronic toxic hepatitis. Materials and methods. Modeling of toxic hepatitis was carried out on 1 month old male and female Wistar rats by intraperitoneal injection of hydrazine sulfate 50 mg / kg twice a week for 3 months. The experiment was conducted in accordance with the norms and principles of the EU Council Directive on the protection of vertebrates used for scientific purposes. Morphometric measurements (density, content of mineral-organic, mineral and organic components) were carried out on the femurs and the last lumbar vertebra. Elastase activity, acidic phosphatase (indicators of bone resorption); alkaline phosphatase activity and calcium content (indicators of the synthesis of new bone tissue) were determined in homogenates of the bone tissue of the jaws and femur. The mineralizing index was calculated from the ratio of the activities of acidic phosphatase and alkaline phosphatase. Results and discussion. A decrease in the quantities organic matrix with a preserved intensity savings of bone tissue mineral parts in females and a decrease intensity in bone tissue mineralization in males under the influence of toxic hepatitis were found. Biochemical studies of bone tissue showed the activation of the destruction of the protein matrix of the bone, the intensification of mineralization in females, as well as the activation of hydrolysis of hydroxyapatite, along with a decrease in the level of calcium in the bone tissue of animals of both sexes against the background of modeling of chronic toxic hepatitis. Conclusion. The studies carried out have established multidirectional disorders of the metabolism and structure of bone tissue in males and females, induced by prolonged administration of hydrazine sulfate: in females, suppression of the formation of an organic matrix with a preserved intensity of bone mineralization, and in males, a decrease in bone mineralization against the background of a slightly increased intensity of synthesis of the organic component. The established patterns made it possible to reduce the negative effect of Hydrazine Sulfate on the protein metabolism of rat bone tissue, indirectly through the metabolism of estrogens. The results of our study provided experimental justification for the development of a scheme for the Prevention of osteodystrophic conditions in conditions of chronic exposure to toxins or impaired liver function

The Mechanism of the Yigutang-Mediated P13K/AKT/GSK-3β Signal Pathway to Regulate Osteogenic Differentiation of Bone Marrow Stromal Stem Cells to Treat Osteoporosis.

Objectives To explore the mechanism of Yigutang mediating the P13K/AKT/GSK-3β signaling pathway to regulate the osteogenic differentiation of bone marrow stromal stem cells to treat osteoporosis (OP). Methods Sixty 12-week-old female SD rats were randomly divided into the normal group, model group, Yigutang group, and estrogen group, with 15 cases in each group. In the model group, Yigutang group, and estrogen group, the ovaries on both sides were removed to construct the model, and the bone mineral density (BMD) of the upper metaphysis of the right femur of the rats in each group was detected. The left femur of each group of rats was removed, and the load-deformation curve of the left femur of each group of rats was calculated. The number of osteoblasts was observed by H&E staining. After extracting the right femurs of rats in each group, real-time fluorescent quantitative PCR and Western blot were used to detect the expression levels of genes and proteins related to the P13K/AKT/GSK-3β signaling pathway. Results The BMD of the upper metaphysis of the right femur in the Yigutang group and the estrogen group was significantly higher than that of the model group (P < 0.05), while both Yigutang and estrogen groups had no significant difference compared with the normal group (P > 0.05). In addition, there was no significant difference between the Yigutang group and the estrogen group (P > 0.05). The elastic load and maximum load of the Yigutang group and the estrogen group were significantly higher than the model group (P < 0.05), but both were lower than the normal group (P < 0.05). There was no significant difference between the Yigutang group and the estrogen group (P > 0.05). The number of osteoblasts in the Yigutang group and the estrogen group was significantly higher than the model group (P < 0.05), but both were lower than the normal group (P < 0.05). There was no significant difference between the Yigutang group and the estrogen group (P > 0.05). The P13K gene expression in the right femoral bone tissue of rats in the Yigutang group and the estrogen group was significantly higher than that in the model group (P < 0.05), and the AKT gene expression did not change significantly (P > 0.05). The gene expression of GSK-3β was significantly lower than that of the model group (P < 0.05). Compared with the normal group, the gene expression of P13K, AKT, and GSK-3β in the right femur bone tissue of the Yigutang group and the estrogen group did not change significantly (P > 0.05). The protein expression of P13K and P-AKt in the right femoral bone tissue of rats in the Yigutang group and the estrogen group was significantly higher than that of the model group (P < 0.05), and the protein expression of AKT did not change significantly (P > 0.05). The protein expression of GSK-3β was significantly lower than the model group (P < 0.05). Compared with the normal group, the protein expression of P13K, AKT, P-AKt, and GSK-3β in the right femoral bone tissue of the Yigutang group and the estrogen group did not change significantly (P > 0.05). Conclusions Yigutang can regulate the differentiation of bone marrow stromal stem cells into osteoblasts, which may be achieved by regulating the P13K/AKT/GSK-3β signaling pathway.

Bisphenol A exposure prenatally delays bone development and bone mass accumulation in female rat offspring via the ERβ/HDAC5/TGFβ signaling pathway

Previous studies have suggested that bisphenol A (BPA) has a toxic effect on bone development; however, its pathological mechanism has not been fully elucidated. In the present study, pregnant Wistar rats were intragastrically administered BPA (10 μg/kg per day) during gestational days 14–21. Then, bone tissues were obtained from neonatal rats on postnatal day 1 for histological analysis, and the bone mass of adult rat offspring was analyzed by micro-CT at postnatal week 10. Furthermore, osteoprogenitors from neonatal rats were obtained and treated with various concentrations of BPA in vitro to clarify the associated mechanism. In vivo, we found that prenatal BPA exposure reduced body weight and body length in female neonatal rats but not in male neonatal rats. Meanwhile, BPA exposure during pregnancy delayed bone development and reduced bone mass only in female rat offspring. Moreover, BPA exposure during pregnancy inhibited osteogenic function and downregulated the transforming growth factor β (TGF β) signaling pathway in the bone tissue of female neonatal rats. Our in vitro findings further indicated that various concentrations of BPA suppressed the osteogenic function of osteoprogenitors by downregulating the TGFβ signaling pathway. Meanwhile, BPA downregulated H3K9ac and expression levels of TGFβ via the ERβ/HDAC5 signaling pathway. Collectively, this research revealed that prenatal BPA exposure impairs bone development and bone mass accumulation in female rat offspring, which was attributed to inhibitory osteogenic function via the ERβ/HDAC5/TGFβ signaling pathway.

Rapamycin activates autophagy by inhibiting mTOR pathway to alleviate early osteoporosis in rats with skeletal fluorosis

Objective: To investigate the effects of rapamycin target protein (mTOR) pathway and autophagy on bone formation and bone resorption in fluorosis osteoporosis in rats. Methods: In September 2018, a rat model of skeletal fluorosis was established by intragastric administration of fluorine. The experimental animals were divided into control group, 10 mgF(-)/kg group, 20 mgF(-)/kg group, 2 mg/kg rapamycin (RAPA) +10 mgF(-)/kg group and 2 mg/kg RAPA+20 mgF(-)/kg group, 20 per group. The experiment lasted for 3 months. The changes of bone tissue in rats were observed by hematoxylin-eosin (HE) staining. Bone mineral density (BMD) and biomechanical indexes, such as Modulus of elasticity, Stiffness, Maximum stress and Maximum load, were measured by BMD and biomechanical biometer. Serum levels of alkaline phosphatase (ALP) , osteocalcin (BGP) , osteoprotectin (OPG) , type I procollagen amino-terminal peptide (PINP) , tartrate-resistant acid phosphatase (TRACP) and nuclear factor kappa B receptor activator ligand (RANKL) were determined by enzymatic linked immunosorbent assay (ELISA) . Bone tissue phosphorylated mTOR (p-mTOR) , autophagy-related index selective autophagy adaptor protein p62, microtubule associated protein II (LC3-II) , ALP, osteoblastic transcription factor (Osterix) , and RNT Expression of related transcription factor 2 (Runx2) and bone resorption indicator RANKL were detected by Western blotting. Results: Compared with the control group, dental fluorosis in the 10 mgF(-)/kg and 20 mgF(-)/kg groups was significantly increased, periosteum thickness and absorption lacunae appeared, and BGP, OPG, PINP, TRACP and RANKL in serum contents were increased (P<0.05) , BMD, Modulus of elasticity, Stiffness, Maximum stress and Maximum load of bone tissue decreased significantly (P<0.05) , and the expressions of p-mTOR and p62 were decreased (P<0.05) , also the expressions of ALP, Osterix, Runx2 and RANKL were increased (P<0.05) . Compared with 10 mgF(-)/kg and 20 mgF(-)/kg groups, there were no obvious dental fluorosis symptoms in 2 mg/kg RAPA+10 mgF(-)/kg group and 2 mg/kg RAPA+20 mgF(-)/kg group, and serum ALP, BGP and OPG levels were significantly increased (P<0.05) . TRACP and RANKL contents were significantly decreased (P<0.05) . BMD, Modulus of elasticity, Stiffness, Maximum stress and Maximum load were significantly increased (P<0.05) . The levels of p-mTOR, p62 and RANKL in bone tissues were decreased (P<0.05) , and the expressions of LC3-II, LC3-II/LC3-I, ALP, Osterix and Runx2 were increased (P<0.05) . Conclusion: RAPA may activate autophagy by inhibiting mTOR phosphorylation, and inhibit bone resorption while promoting bone formation, thus alleviating early osteoporosis in skeletal fluorosis rats.

Simvastatin intervention mitigates hypercholesterolemia-induced alveolar bone resorption in rats.

Simvastatin promotes bone formation and increases bone mineral density in patients with hyperlipidemia and ameliorates hypercholesterolemia-induced microstructure changes in the jaw bone of animals. However, whether and how treatment with simvastatin can modulate the hypercholesterolemia-induced alveolar bone resorption is unclear. The present study aimed to examine the therapeutic efficacy and potential mechanisms of simvastatin application in hypercholesterolemia-induced alveolar bone resorption. The association between hyperlipidemia and alveolar bone resorption in 100 patients with periodontitis was examined. Additionally, male Sprague-Dawley rats were fed a standard rodent chow (NC) for 32 weeks or a high cholesterol diet (HCD) for 24 weeks. The HCD-fed rats were randomized, continually fed with HCD and treated with vehicle saline (HC) or simvastatin by gavage (5 mg/kg; SIM, n=10/group) for 8 weeks. The morphological changes to alveolar bone resorption in rats were analyzed by linear measurements. The relative levels of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand RANKL, nuclear factor-κB (NF-κB), microtubule-associated protein 1 light chain 3 (LC3) and p62 in the alveolar bone tissues were determined by reverse transcription-quantitative PCR and/or immunohistochemistry. Sulcus bleeding index (SBI), clinical attachment loss (CAL), probing depth (PD) and the distance of cemantoenamel junction-alveolar bone crest (CEJ-ABC) in patients with hyperlipidemia were significantly greater than that in the controls (P<0.001). The levels of hyperlipidemia were positively correlated with the values of SBI, CAL, PD and CEJ-ABC in this population. Compared with the NC rats, higher levels of alveolar bone resorption, NF-κB expression, higher ratios of RANKL/OPG mRNA transcripts and LC3 to p62 expression were detected in the alveolar bone tissues of HC group. Simvastatin intervention significantly mitigated hypercholesterolemia-induced alveolar bone loss and RANKL mRNA transcription, but increased the ratios of LC3/p62 protein expression in the alveolar bone tissues of rats. Hyperlipidemia is associated with alveolar bone resorption and simvastatin treatment alleviated the hypercholesterolemia-related alveolar bone loss by down-regulating the NF-κB expression.

The effect of high concentration of zoledronic acid on tooth induced movement and its repercussion on root, periodontal ligament and alveolar bone tissues in rats

Zoledronic acid (ZA) is often prescribed for osteoporosis or resorptive metabolic bone disease. This study aims to evaluate the effect of ZA on orthodontic tooth movement (OTM) and root and bone resorption and its repercussion on root, periodontal ligament and alveolar bone tissues. The experimental group consisted of 72 Wistar rats divided in four subgroups: Naive, Saline and Zoledronic Acid groups at the concentration of 0.2 mg/kg [ZA (0.2)] or 1.0 mg/kg [ZA (1.0)]. The animals were subjected to i.v (dorsal penile vein) administrations of ZA or saline solution, on days 0, 7, 14 and 42. Under anesthesia, NiTi springs were installed in the first left maxillary molar with 50gf allowing the OTM, except for the negative control group (N) for mesial movement of the left first maxillary teeth. The animals were sacrificed and maxillae were removed for macroscopic and histopathological analyzes, scanning electron microscopy, computerized microtomography and confocal microscopy. Treatment with ZA decreased the OTM and the number of osteoclasts and loss of alveolar bone when compared to the naive and saline groups. Reduction of radicular resorption, increased necrotic areas and reduced vascularization in the periodontal ligament were observed in the ZA groups. ZA interferes with OTM and presents anti-resorptive effects on bone and dental tissues associated with a decreased vascularization, without osteonecrosis.

Changes in Rat Bone Tissue at the Site of the Defect In Vivo under the Effect of a Cryogenically Structured Albumin Sponge Containing a Bioregulator.

We performed a morphological study of the bone tissue after implantation of a cryogenically structured albumin sponge containing a bioregulator isolated from blood serum into an extensive experimental defect of the femur. By day 90, no complete reparation of the bone tissue was achieved in the control group (without implantation of 3D carrier), a loose spongy bone is formed at the site of the defect. After implantation of the 3D carrier without serum bioregulator, the defect was closed, but the formed bone was loose and contained no inflammation foci. After the defect was filed with the albumin sponge with the bioregulator, the repair pattern corresponded to the processes of epimorphic tissue regeneration. The results suggest that cryogenically structured protein material in combination with a serum bioregulator ensured complete restoration of the bone tissue.

MicroRNA-182 inhibits osteogenic differentiation of bone marrow mesenchymal stem cells by targeting Smad1.

The present study aimed to screen abnormally expressed microRNAs (miRs/miRNAs) in patients with postmenopausal osteoporosis (POP) and explore their mechanisms via functional verification. Bone marrow mesenchymal stem cells (BMSCs) were extracted from healthy controls and patients with POP. Differences in osteogenic differentiation and proliferation of human BMSCs were compared between the two groups using Cell Counting Kit-8 (CCK-8) assay and alizarin red staining. A rat model of POP was established. Compared with patients with POP, human BMSCs in healthy controls had significantly enhanced viability at 24, 36, 48 and 72 h. The results of alizarin red staining revealed that the deposition of calcium minerals in human BMSCs were significantly lower in patients with POP. Based on miRNA microarray and reverse transcription-quantitative polymerase chain reaction (PCR) results, the expression levels of miR-7010 and miR-467c decreased, while miR-132 and miR-182 expression increased in the human BMSCs of patients with POP. Alizarin red staining showed that miR-182 markedly suppressed the osteogenic differentiation of primary rat BMSCs in rats. Western blotting and immunofluorescence assay revealed that miR-182 inhibited the expression of osteogenesis markers runt-related transcription factor 2, osterix and actinin-associated LIM protein. The results of the luciferase reporter assay showed that Smad1 is the direct target of miR-182. In rat primary BMSCs, Smad1 overexpression abolished the inhibitory effect of miR-182 on osteogenesis, indicating that miR-182 inhibits osteogenic differentiation of primary rat BMSCs in rats by targeting Smad1. Finally, in vivo experimental results showed that the biomechanical characteristics of bone tissues in POP rats were significantly enhanced by miR-182 inhibition, while they were significantly weakened by miR-182 overexpression. MiR-182 inhibits osteogenic differentiation of rat BMSCs, thus aggravating POP in rats.

The value of the hedgehog signal in osteoblasts in fluoride-induced bone-tissue injury

ObjectiveThis study was designed to observe the expression of important hedgehog (Hh) signal factors in the bone tissue of rats with chronic fluorosis and cultured osteoblasts in order to investigate the role and significance of the Hh signal in fluoride-induced bone injury.MethodsHealthy Sprague-Dawley (SD) rats were randomly divided into four groups: the control group, the fluorosis group (F Group), the fluoride + blocker group (F + Cycl group: rats were treated with fluoride + cyclopamine), and the fluoride + blocker control group (F + DMSO group). After 6 months of intervention, the urinary fluoride content of rats in each group was detected. The primary osteoblasts of rats were selected for cell experiment, and the experiment was carried out after the cells were passaged from the second to the fourth generation.ResultsThe proliferation rate of primary rat osteoblasts presented time-affected and dose-affected relationships in a short time under treatment with a low dose of sodium fluoride (NaF), but the proliferation of osteoblasts was inhibited by long-term and high-dose NaF exposure. In the F group, the alkaline phosphatase (ALP) activity of osteoblasts increased gradually. The ALP activity was lower in the F + Cycl group than in the F group, and there was no significant difference between the F + DMSO group and F group. With the increase in fluoride exposure, the expression of Hh signal factors and osteogenic-related factor proteins increased gradually. The expressions of Indian hedgehog (Ihh), smoothened (Smo), Glioma-associated oncogene homolog (Gli) 2, and Runt-related transcription factor 2 (Runx2)in the F + Cycl group increased with the dose of fluoride but they were significantly inhibited compared with the F group. Compared with the control group, the content of urinary fluoride in the F group was significantly higher (P < 0.05), but there was no significant change in urinary fluoride content in the F + Cycl group and the F + DMSO group. Compared with the control group, the serum bone alkaline phosphatase (BALP) contents of rats in the other groups increased after 6 months’ intake of fluoride water (P < 0.05). After drug blocking, the serum BALP content in the F + Cycl group was lower than that in the F + DMSO group (P < 0.05). The BALP content in the F + DMSO group was similar to that in the F group: it did not decrease. The mRNA expressions of Ihh, Smo, Gli2, and Runx2 in bone tissue of the F group were significantly higher than those in the control group (P < 0.05). After cyclopamine blocking, the expressions decreased (P < 0.05), but the differences between the F + DMSO group and F group were not statistically significant.ConclusionHh signal plays an important role in fluoride-induced bone injury. The effective inhibition of cyclopamine is expected to be a new target for the treatment of skeletal damage caused by fluorosis.

MicroRNA-935-modified bone marrow mesenchymal stem cells-derived exosomes enhance osteoblast proliferation and differentiation in osteoporotic rats

AimsThe involvement of several microRNAs (miRNAs) in osteogenic differentiation has been indicated recently. Also, exosomes, derived from different cells, could shuttle specific miRNAs to other cell systems. Nevertheless, the effect and mechanism of microRNA-935 (miR-935)-containing exosomes in osteoblasts remain basically unclear. The current work was set to inspect the relevance of bone marrow mesenchymal stem cells (BMSCs)-derived exosomes (BMSC-exo) carrying miR-935 to osteoporotic rats. MethodsThe extracted BMSCs and purchased osteoblasts were cultured, followed by exosome isolation and identification. After cell grouping, osteoblasts were co-cultured with BMSCs. CCK-8, alizarin red staining as well as ALP staining were performed to detect osteoblast proliferation and activity. The binding connection between miR-935 and signal transducer and activator of transcription 1 (STAT1) was measured by dual-luciferase reporter gene assays. The expression profiles of miR-935, STAT1 and osteoblast-related proteins were assessed by RT-qPCR and Western blot. A rat model with osteoporosis was induced, and the BMD, BV/TV, Tb.N, Tb.Th and Tb.Sp values in rat bone tissues were observed by Micro-CT. ResultsBMSC-exo inhibited STAT1 levels by the delivery of miR-935 into osteoblasts, while STAT1 silencing promoted ALP activity in osteoblasts and mineralized nodules. STAT1 was identified as a target gene of miR-935. Moreover, in vivo experiments showed that in ovariectomized rats, silencing of miR-935 significantly reduced BMD, BV/TV, Tb.N, Tb.Th and increased Tb.Sp. ConclusionBMSC-exo carry miR-935 to promote osteoblast proliferation and differentiation through targeting STAT1.

Netrin-1 regulates ERK1/2 signaling pathway and autophagy activation in wear particle-induced osteoclastogenesis.

BackgroundArtificial joint replacement surgery is often accompanied by osteolysis induced aseptic loosening around the prosthesis. Wear particles from joint replacement are thought to be one of the main factors leading to local inflammation and osteolysis at the prosthesis site. The aim of this study was to investigate the molecular mechanism of osteoclast formation and dissolution induced by wear particles and the potential roles of Netrin‐1, the ERK1/2 pathway and autophagy activation in this process.MethodsThe messenger RNA levels in cells and tissues were detected with real‐time quantitative PCR. The western blotting was used to detect the expression of proteins. A CCK‐8 kit was used to detect the viability of RAW 264.7 cells. Moreover, an air pouch model of bone resorption was established. Immunohistochemistry was used to detect the expression of TRAP and Netrin‐1 in rat bone tissue. Cell culture supernatants were collected in the rat air pouch model of bone resorption, and the levels of RANKL and OPG were detected with enzyme‐linked immunosorbent assay. The protein levels of TRAP and Netrin‐1 in bone tissue were examined by immunohistochemistry.ResultsTitanium wear particles induced osteoclast formation and autophagy activation. Moreover, blocking autophagy suppressed the osteoclastogenesis after exposure to wear particles in vitro. The activation of the ERK1/2 pathway and the overexpression of Netrin‐1 were both found to play important roles in osteoclastogenesis mediated by autophagy. Moreover, 3‐MA effectively decreased the secretion of proinflammatory cytokines mediated by wear particles.ConclusionBlockade of autophagy inhibits the osteoclastogenesis and inflammation induced by wear particles, thus potentially providing novel treatment strategies for abnormal osteoclastogenesis and aseptic prosthesis loosening induced by wear particles.

Silencing long noncoding RNA colon cancer-associated transcript-1 upregulates microRNA-34a-5p to promote proliferation and differentiation of osteoblasts in osteoporosis.

Long noncoding RNAs (lncRNAs) have been revealed to be related to multiple physiological and pathology processes such as development, carcinogenesis, and osteogenesis. It is reported that lncRNAs might exert function in osteoblast differentiation and bone formation. Here, we determined this study to clarify whether lncRNA CCAT1 could regulate osteoblast proliferation and differentiation in ovariectomized rats with osteoporosis. The osteoporosis models were established by bilateral ovariectomy and treated with CCAT1 siRNAs to discuss the effect of CCAT1 on pathological changes and osteocyte apoptosis in ovariectomized rats with osteoporosis. The osteoblasts from ovariectomized rats were cultured in vitro, which were then treated with CCAT1 siRNAs to explore the role of CCAT1 in osteoblast proliferation and differentiation. Moreover, the relationships among CCAT1, miR-34a-5p, and SMURF2 were confirmed. CCAT1 and SMURF2 were amplified while miR-34a-5p expression was inhibited in bone tissues and osteoblasts of ovariectomized rats with osteoporosis. Inhibited CCAT1 improved pathology and restricted osteocyte apoptosis of bone tissues in ovariectomized rats with osteoporosis in vivo, and also enhanced differentiation, mineralization abilities, and proliferation, and suppressed apoptosis of osteoblasts from ovariectomized rats in vitro through upregulating miR-34a-5p expression. LncRNA CCAT1 could competitively bind with miR-34a-5p to prevent the degradation of its target gene SMURF2. Results of this research suggested that the CCAT1 inhibits the proliferation and differentiation of osteoblasts in rats with osteoporosis by binding to miR-34a-5p, providing novel biomarkers for osteoporosis treatment.

MiR-29c-3p reduces bone loss in rats with diabetic osteoporosis via targeted regulation of Dvl2 expression.

The aim of this study was to investigate the influence of micro ribonucleic acid (miR)-29c-3p on rats with diabetic osteoporosis (DOP) and its underlying mechanism. A total of 30 specific pathogen-free (SPF)-grade male Wistar rats aged 6-week-old were randomly selected and divided into three groups according to different intervention means, including: NC group (control rats only injected with normal saline), DOP group (rats with DOP induced by injection of streptozotocin), and ME group (DOP rats injected with miR-29c-3p agonist for 4 consecutive weeks). The changes in blood glucose and body weight were recorded in the rats of each group every week. Enzyme-linked immunosorbent assay (ELISA) was applied to detect the content of bone turnover markers (BTMs) in serum, such as alkaline phosphatase (ALP), osteocalcin (OC), and procollagen type I N-terminal propeptide (PINP). The variations in serum calcium (Ca) and phosphorus (P) levels in the abdominal aorta were determined using an atomic absorption spectrometer in the three groups. Meanwhile, bone mineral density (BMD) of femur and lumbar vertebra (L1-L4) were examined. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the changes in messenger RNA (mRNA) expressions of miR-29c-3p and Disheveled 2 (Dvl2) in the bone tissues of intervened rats. In addition, the staining and expression changes of Dvl2 protein in bone tissues were determined via immunohistochemistry. The rats in NC group had normal behavioral activities, normally increased body weight, sensitive responses, as well as normal and stable blood glucose. In DOP group, the rats manifested clinical symptoms of diabetes mellitus (DM) (i.e., polydipsia, polyphagia, polyuria, and weight loss), lackluster hairs, decreased behavioral activities, slow responses, and blood glucose at a concentration higher than 16.7 mmol/L. However, the blood glucose rose first, and then, declined and it was maintained at a level higher than normal concentration in ME group. Meanwhile, the rate of weight loss significantly decreased. The results of qRT-PCR indicated that the relative expression level of miR-29c-3p in bone tissues of DOP group was remarkably lower than that in NC group (p<0.01). However, it was significantly higher in ME group than that in DOP group (p<0.05). DOP group exhibited significantly upregulated serum BTMs (ALP, CTX-1, OC, TRACP-5b, and PIPN) when compared with NC group (p<0.05) and ME group (p<0.05). Furthermore, femoral BMD decreased in DOP group (p<0.05) while increased in ME group, showing statistically significant difference between the two groups (p<0.05). Immunohistochemistry results indicated that the bone tissues of DOP rats were deeply stained, and protein expression of Dvl2 protein was significantly higher in comparison with NC group. The bone tissues were lightly stained in ME group, and the protein expression of Dvl2 was lower than that in DOP group. Besides, qRT-PCR results demonstrated that the mRNA expression changes of Dvl2 were consistent with its protein expression trends. MiR-29c-3p reduces bone loss in rats with DOP via targeted regulation of Dvl2 expression.

Vibratory Platform in the Tibial Bone Tissue of Wistar Rats Subjected to Immobilization

Vibratory Platform in the Tibial Bone Tissue of Wistar Rats Subjected to Immobilization