Background: Oxidative stress is a known to be associated with in the pathogenesis of many inflammatory diseases, including periodontitis. Ursolic acid is a pentacyclic triterpenoid with has antimicrobial, antioxidative, and anticancer properties. However, the role of ursolic acid in the regulating of osteogenesis remains undetermined. This study was aimed to elucidate the crucial osteogenic effects of ursolic acid and its ability to inhibit oxidative stress by targeting the immediate early response 3 (IER3)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Methods: Cell proliferation was determined using water-soluble tetrazolium salt assay, cell differentiation was evaluated by alkaline phosphatase (ALP) activity, and formation of calcium nodules was detected using alizarin red S stain. Generation of reactive oxygen species (ROS) was determined using by DCFH-DA fluorescence dye in hydrogen peroxide ($H_2O_2$)-treated MG-63 cells. Expression levels of IER3, Nrf2, and heme oxygenase-1 (HO-1) were analyzed using western blot analysis. Results: Our results showed that ursolic acid up-regulated the proliferation of osteoblasts without any cytotoxic effects, and promoted ALP activity and mineralization. $H_2O_2$-induced ROS generation was found to be significantly inhibited on treatment with ursolic acid. Furthermore, in $H_2O_2$-treated cells, the expression of the early response genes: IER3, Nrf2, and Nrf2-related phase II enzyme (HO-1) was enhanced in the presence of ursolic acid. Conclusion: The key findings of the present study elucidate the protective effects of ursolic acid against oxidative stress conditions in osteoblasts via the IER3/Nrf2 pathway. Thus, ursolic acid may be developed as a preventative and therapeutic agent for mineral homeostasis and inflammatory diseases caused due to oxidative injury.
Postsurgical infections are caused by implant-related pathogenic microorganisms that lead to graft rejection. Hence, an intrinsically antibacterial material is required to produce a biocompatible biomaterial with osteogenic properties that could address this major issue. Hence, this current research aims to make strontium-doped hydroxyapatite nanorods (SrHANRs) via an ethylene diamine tetraacetic acid (EDTA)-enable microwave mediated method using Anodontia alba seashells for biomedical applications. This investigation also perceives that EDTA acts as a soft template to accomplish Sr-doping and mesoporous structures in pure hydroxyapatite nanorods (HANRs). The X-ray diffraction (XRD) and transmission electron microscopy (TEM) analysis reveals the crystalline and mesoporous structures, and Brunauer-Emmett-Teller (BET) indicates the surface area of all the samples, including pure HANRs and doped HANRs. In addition, the biocidal ability was tested using various implant-related infectious bacteria pathogens, and it was discovered that Sr-doped HANRs have excellent biocidal properties. Furthermore, toxicity evaluation using zebrafish reports the non-toxic nature of the produced HANRs. Incorporating Sr2+ ions into the HAp lattice would enhance biocompatibility, biocidal activity, and osteoconductive properties. As a result, the biocompatible HANRs materials synthesized with Sr-dopants may be effective in bone regeneration and antibacterial in-built implant applications.
Jie Ni;Aili Ye;Liya Gong;Xiafei Zhao;Sisi Fu;Jieya Guo
Nutrition Research and Practice
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v.18
no.4
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pp.479-497
/
2024
BACKGROUND/OBJECTIVES: Activating brown adipose tissue (BAT) and browning of white adipose tissue (WAT) can protect against obesity and obesity-related metabolic conditions. Cryptotanshinone (CT) regulates lipid metabolism and significantly ameliorates insulin resistance. Adenosine-5'-monophosphate (AMP)-activated protein kinase (AMPK), a receptor for cellular energy metabolism, is believed to regulate brown fat activity in humans. MATERIALS/METHODS: The in vivo study included high-fat-fed obese mice administered orally 200/400 mg/kg/d CT. They were evaluated through weight measurement, the intraperitoneal glucose tolerance test (IPGTT), intraperitoneal insulin tolerance test (IPITT), cold stimulation test, serum lipid (total cholesterol, triglycerides, and low-density lipoprotein) measurement, hematoxylin and eosin staining, and immunohistochemistry. Furthermore, the in vitro study investigated primary adipose mesenchymal stem cells (MSCs) with incubation of CT and AMPK agonists (acadesine)/inhibitor (Compound C). Cells were evaluated using Oil Red O staining, Alizarin red staining, flow cytometry, and immunofluorescence staining to identify and observe the osteogenic versus adipogenic differentiation. Quantitative real-time polymerase chain reaction and the Western blot were used to observe related gene expression. RESULTS: In the diet-induced obesity mouse model mice CT suppressed body weight, food intake, glucose levels in the IPGTT and IPTT, serum lipids, the volume of adipose tissue, and increased thermogenesis, uncoupling protein 1, and the AMPK pathway expression. In the in vitro study, CT prevented the formation of lipid droplets from MSCs while activating brown genes and the AMPK pathway. AMPK activator enhanced CT's effects, while the AMPK inhibitor reversed the effects of CT. CONCLUSION: CT promotes adipose tissue browning to increase body thermogenesis and reduce obesity by activating the AMPK pathway. This study provides an experimental foundation for the use of CT in obesity treatment.
This study has been performed to evaluate the relationship between the remained mineral components in a decalcified bone matrix and an ectopic bone formation efficiency. The freezed rat diaphyseal cortical bones measuring 0.5cm in length were demineralized in heated 0.6N HCl at $60^{\circ}C$ for 5, 10, 15, 20, 25, 30, 35, 40 minutes, respectively, using a controlled heat ultrasonic cleaner. Each 1cc of decalcifying solution taken during decalcification procedure was used to calculate calcium content using calcium dignostics kit under 600nm of spectrophotomer. After decalcification, each specimen was also weighed. Then each prepared specimen was implanted into the dorsal pouch of 24 Sprague-Dawley rats divided into 8 groups by time course. The implants were harvested at 1, 2, and 3 weeks and prepared for routine H-E stain specimens to evaluate osteogenic activity. The results are as follows: 1. There was statistical significant difference in change of calcium concentration up to demineralization of 30 minutes and each allogenic bones decalcifed up to 20 minutes revealed 99.65% of decalcification in average. 2. There was statistical significant difference in change of weight in demineralized allogenic bone up to 20 minutes treatment but, no significant change was noted after that time. 3. The histologic analysis revealed active ectopic bone formation in the implanted allografts demineralized for 20, 25, 30 minutes, respectively. However, the other groups of allografts showed relatively poor osteoinductive activity. These findings suggest that complete decalcification with a minimized degeneration of collagen matrix is necessary to induce maximal osteogenesis by decalcified bone allograft.
Purpose: Gene therapy (ex vivo) has recently been used as a means of delivering bone morphogenetic proteins (BMPs) to sites of tissue regeneration. In the present study, we investigated the effect of co-transduction of adenoviruses expressing BMP-2 and BMP-7 on osteogenesisof C2C12 cells in vitro. Methods: A replication-defective human adenovirus 5 (Ad5) containing a cDNA for BMPs in the E1 region of the virus (Ad5BMP-2 and Ad5BMP-7) was constructed by in vivo homologous recombination. Functional activity of Ad5BMP-2 and Ad5BMP-7 were evaluated in mouse stromal cells (W20-17cells). C2C12 cells are transduced with various MOI (multiplicity of infection) of Ad5BMP-2 and Ad5BMP-7 to assess most effective and stable titer. Based on this result, C2C12 cells were transduced with Ad5BMP-2 and Ad5BMP-7 alone or by combination. BMPs expression, alkaline phosphatase (ALPase) activity, cell proliferation, and mineralization were assessed. Results: Ad5BMP-2 and Ad5BMP-7 are successfully transduced to W20-17 cells, and secreted BMPs stimulated cell differentiation. Also, C2C12 cells transduced with Ad5BMPs showed expression of BMPs and increased ALPaseactivity. In all groups, cell proliferation was observed over times. At 7days, cells co-transduced with Ad5BMP-2 and Ad5BMP-7 showed lower proliferation than the others. C2C12 cells co-transduced with Ad5BMP-2 and Ad5BMP-7 had greater ALPaseactivity than that would be predicted if effect of individual Ad5BMPs were additive. Little mineralized nodule formation was detected in cells transduced with individual Ad5BMPs. In contrast, Ad5BMP-2 and Ad5BMP-7 combination stimulated mineralization after culturing for 10 days in mineralizing medium. Conclusions: Present study demonstrated that adenoviruses expressing BMPs gene successfully produced BMPs protein and these BMPs stimulated cells to be differentiated into osteoblastic cells. In addition, the osteogenic activity of Ad5BMPs can be synergistically increased by co-transduction of cells with Ad5BMP-2 and Ad5BMP-7.
During bone remodeling, there is requirement of differentiation of osteoblastic cells. Previously, we identified proteins differentially expressed in soft tissue during bone healing. Of these proteins, we focused the effect of LTF on differentiation of osteoblast. In order to analyze the osteogenic ability of LTF, we treated conditioned media collected from human LTF-stably transfected HEK293T cells into osteoblastic MC3T3-E1. The results showed that the activity and expression of alkaline phosphatase were increased in MC3T3-E1 cells treated with conditioned media containing LTF in dose- and time-dependent manner. At the same time, we observed the significant increase of the expression of osteoblastic genes, such as ALP, BSP, COL1A1, and OCN, and along with matrix mineralization genes, such as DMP1 and DMP2, in LTF conditioned media-treated groups. Moreover, the result of treating recombinant human LTF directly into osteoblastic MC3T3-E1 showed the same pattern of treating conditioned media containing LTF. Our study demonstrated that LTF constitutively enhances osteoblastic differentiation via induction of osteoblastic genes and activation of matrix mineralization in MC3T3-E1 cells.
Kim, Eun-Cheol;Park, Jaesuh;Kwon, Il Keun;Lee, Suk-Won;Park, Su-Jung;Ahn, Su-Jin
Journal of Periodontal and Implant Science
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v.47
no.5
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pp.273-291
/
2017
Purpose: Although static magnetic fields (SMFs) have been used in dental prostheses and osseointegrated implants, their biological effects on osteoblastic and cementoblastic differentiation in cells involved in periodontal regeneration remain unknown. This study was undertaken to investigate the effects of SMFs (15 mT) on the osteoblastic and cementoblastic differentiation of human osteoblasts, periodontal ligament cells (PDLCs), and cementoblasts, and to explore the possible mechanisms underlying these effects. Methods: Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, mineralized nodule formation based on Alizarin red staining, calcium content, and the expression of marker mRNAs assessed by reverse transcription polymerase chain reaction (RT-PCR). Signaling pathways were analyzed by western blotting and immunocytochemistry. Results: The activities of the early marker ALP and the late markers matrix mineralization and calcium content, as well as osteoblast- and cementoblast-specific gene expression in osteoblasts, PDLCs, and cementoblasts were enhanced. SMFs upregulated the expression of Wnt proteins, and increased the phosphorylation of glycogen synthase $kinase-3{\beta}$ ($GSK-3{\beta}$) and total ${\beta}-catenin$ protein expression. Furthermore, p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK), and nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) pathways were activated. Conclusions: SMF treatment enhanced osteoblastic and/or cementoblastic differentiation in osteoblasts, cementoblasts, and PDLCs. These findings provide a molecular basis for the beneficial osteogenic and/or cementogenic effect of SMFs, which could have potential in stimulating bone or cementum formation during bone regeneration and in patients with periodontal disease.
Heme oxygenase (HO-1) catalyzes heme to carbon monoxide (CO), biliverdin/bilirubin, and iron and is known to prevent the pathogenesis of several human diseases. We assessed the beneficial effect of heme degradation products on osteoclastogenesis induced by receptor activator of NF-${\kappa}B$ ligand (RANKL). Treatment of RAW264.7 cells with CORM-2 (a CO donor) and bilirubin, but not with iron, decreased RANKL-induced osteoclastogenesis, with CORM-2 having a more potent anti-osteogenic effect. CORM-2 also inhibited RANKL-induced osteoclastogenesis and osteoclastic resorption activity in marrow-derived macrophages. Treatment with hemin, a HO-1 inducer, strongly inhibited RANKL-induced osteoclastogenesis in wild-type macrophages, but was ineffective in $HO-1^{+/-}$ cells. CORM-2 reduced RANKL-induced NFATc1 expression by inhibiting IKK-dependent NF-${\kappa}B$ activation and reactive oxygen species production. These results suggest that CO potently inhibits RANKL-induced osteoclastogenesis by inhibiting redox-sensitive NF-${\kappa}B$-mediated NFATc1 expression. Our findings indicate that HO-1/CO can act as an anti-resorption agent and reduce bone loss by blocking osteoclast differentiation.
Journal of the Korean Association of Oral and Maxillofacial Surgeons
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v.35
no.5
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pp.304-309
/
2009
Purpose: Fibrous dysplasia (FD) is a fibro-osseous disease associated with activating missense mutations of the gene encoding the $\alpha$-subunit of stimulatory G protein. FD may affect a single bone (called monostotic form) or multiple bones (called polyostotic form). The extent of lesions reflects the onset time of mutation. In this study, cells from monostotic FD in maxilla of a patient were isolated and cultured in vitro for characterization. Materials and Methods: The single cells were released from FD lesion which was surgical specimen from 15 years-old boy. These isolated cells were cultured in vitro and tested their proliferation activity with MTT assay. In osteogenic media, these cells underwent differentiation process comparing with its normal counterpart i.e. bone marrow stromal cells. The proliferated FD cells were detached and transplanted into the dordsal pocket of nude mouse and harvested in 6 weeks and 12 weeks. Results and Summary: FD cells have an increased proliferation rate and poor differentiation. As a result, cells isolated from FD lesion decreased differentiation into osteoblast and increased proliferation capacity. MTT assay presented that proliferation rate of FD cells were higher than control. However, the mineral induction capacity of FD was lesser than that of control. Monostotic FD cells make fewer amounts of bone ossicles and most of them are woven bone rather than lamellar bone in vivo transplantation. In transplanted FD cells, hematopoietic marrow were not seen in the marrow space and filled with the organized fibrous tissue. Therefore, they were recapitulated to the original histological features of FD lesion. Collectively, these results indicated that the FD cells were shown that the increased proliferation and decreased differentiation potential. These in vitro and in vivo system can be useful to test FD cell's fate and possible.
Kim, Dong-Jin;Yun, Jeong-Ho;Jung, Ui-Won;Yoo, Yun-Jung;Kim, Yun-Chul;You, Hyung-Keun;Kim, Chong-Kwan;Choi, Sung-Ho
Journal of Periodontal and Implant Science
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v.34
no.4
/
pp.791-805
/
2004
The purpose of this study was to evaluate the effects of DFPR on differentiation of mouse calvarial cell in vitro, to examine the possibility for periodontal regeneration. $10{\mu}g/ml$ of DFPR was used as experimental concentration. osteogenic medium only was assigned as control, Experimental 1 was supplemented with 10nM dexamethasone, Experimental 2 with $10{\mu}g/ml$ DFPR and Experimental 3 with l0nM dexamethasone + $10{\mu}g/ml$ DFPR. cellular activity was evaluated by MTT method at 8, 12, 16 days, expression of mRNA of ALP, osteopontin, osteocalcin, collagen type-l was detected by RT-PCR method at 4, 8, 12, 16 days of culture. extent of mineralization was observed by Von Kossa staining at 16 day of culture. The results are as follows 1)Any acceleration of differentiation was not observed at expression of differentiation marker, 2) Decrease in expression of extracelluar matrix and in bone nodule formation was observed The results suggested that DFPR have negative effect on the rate of differentiation on rat calvarial cell, decrease extracelluar matrix formation ,decrease bone nodule formation. Ongoing studies are necessary in order to determine effect of DFPR on periodontal regeneration.
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