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1.
Braz Oral Res ; 34: e014, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32074214

RESUMO

Although dental implants and bone regenerative procedures are important approaches for the reestablishment of esthetics and function in young patients with a history of generalized aggressive periodontitis (GAP), no predictable outcomes have been reported, and the host osteo-immunoinflammatory response may play a relevant role in this context. In view of the lack of molecular investigations into the bone tissue condition of young patients with periodontitis, the aim of this study was to evaluate the gene expression of bone-related factors in this population. Bone biopsies were obtained from the posterior mandible in 16 individuals previously diagnosed with GAP and on periodontal support therapy and from 17 periodontally healthy (PH) patients. The gene expression of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß, receptor activator of the NF-κB ligand (RANKL), osteoprotegerin (OPG), osteocalcin (OC), bone sialoprotein (BSP), and type I collagen (COL-I), important biomarkers of bone turnover, was evaluated by qRT-PCR. Lower TGF-ß and OPG mRNA levels were observed in GAP patients compared to PH individuals (p ≤ 0.05). There were no between-group differences in levels of TNF-α, BSP, RANKL, OC, or COL-I mRNA (p>0.05). In young adults, a history of periodontal disease can negatively modulate the gene expression of important bone-related factors in alveolar bone tissue. These molecular outcomes may contribute to the future development of therapeutic approaches to benefit bone healing in young patients with history of periodontitis via modulation of osteo-immuno-inflammatory biomarkers.


Assuntos
Periodontite Agressiva/genética , Expressão Gênica , Adulto , Periodontite Agressiva/metabolismo , Processo Alveolar/química , Biomarcadores , Colágeno Tipo I/análise , Colágeno Tipo I/genética , Estudos Transversais , Feminino , Humanos , Sialoproteína de Ligação à Integrina/análise , Sialoproteína de Ligação à Integrina/genética , Masculino , Osteocalcina/análise , Osteocalcina/genética , Osteoprotegerina/análise , Osteoprotegerina/genética , Ligante RANK/análise , Ligante RANK/genética , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Método Simples-Cego , Estatísticas não Paramétricas , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta/genética , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/genética , Adulto Jovem
2.
PLoS One ; 15(1): e0225589, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923243

RESUMO

Carbon nanotubes (CNTs) have desirable mechanical properties for use as biomaterials in orthopedic and dental area such as bone- and tooth- substitutes. Here, we demonstrate that a glass surface densely coated with single-walled carbon nanotubes (SWNTs) stimulate the osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs). MSCs incubated on SWNT- and multi-walled carbon nanotube (MWNT)-coated glass showed high activities of alkaline phosphatase that are markers for early stage osteogenic differentiation. Expression of Bmp2, Runx2, and Alpl of MSCs showed high level in the early stage for MSC incubation on SWNT- and MWNT-coated surfaces, but only the cells on the SWNT-coated glass showed high expression levels of Bglap (Osteocalcin). The cells on the SWNT-coated glass also contained the most calcium, and their calcium deposits had long needle-shaped crystals. SWNT coating at high density could be part of a new scaffold for bone regeneration.


Assuntos
Diferenciação Celular , Nanotubos de Carbono/química , Osteogênese , Tecidos Suporte/química , Fosfatase Alcalina/metabolismo , Animais , Células da Medula Óssea/classificação , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Cálcio/química , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Vidro/química , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Ratos , Ratos Endogâmicos F344
3.
Mol Cell Biochem ; 463(1-2): 91-100, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31606864

RESUMO

Baicalin (BAI), a sort of flavonoid monomer, acquires from Scutellaria baicalensis Georgi, which was forcefully reported in diversified ailments due to the pleiotropic properties. But, the functions of BAI in osteoblast differentiation have not been addressed. The intentions of this study are to attest the influences of BAI in the differentiation of osteoblasts. MC3T3-E1 cells or rat primary osteoblasts were exposed to BAI, and then cell viability, ALP activity, mineralization process, and Runx2 and Ocn expression were appraised through implementing CCK-8, p-nitrophenyl phosphate (pNPP), Alizarin red staining, western blot, and RT-qPCR assays. The microRNA-217 (miR-217) expression was evaluated in MC3T3-E1 cells or rat primary osteoblasts after BAI disposition; meanwhile, the functions of miR-217 in BAI-administrated MC3T3-E1 cells were estimated after miR-217 inhibitor transfection. The impacts of BAI and miR-217 inhibition on Wnt/ß-catenin and MEK/ERK pathways were probed to verify the involvements in BAI-regulated the differentiation of osteoblasts. BAI accelerated cell viability, osteoblast activity, and Runx2 and Ocn expression in MC3T3-E1 cells or rat primary osteoblasts, and the phenomena were mediated via activations of Wnt/ß-catenin and MEK/ERK pathways. Elevation of miR-217 was observed in BAI-disposed MC3T3-E1 cells or rat primary osteoblasts, and miR-217 repression annulled the functions of BAI in MC3T3-E1 cell viability and differentiation. Additionally, the activations of Wnt/ß-catenin and MEK/ERK pathways evoked by BAI were both restrained by repression of miR-217. These explorations uncovered that BAI augmented the differentiation of osteoblasts via activations of Wnt/ß-catenin and MEK/ERK pathways by ascending miR-217 expression.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Flavonoides/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , MicroRNAs/biossíntese , Osteoblastos/metabolismo , Animais , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Camundongos , MicroRNAs/genética , Osteoblastos/citologia , Osteocalcina/genética , Osteocalcina/metabolismo
4.
Arch Oral Biol ; 109: 104582, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31605918

RESUMO

OBJECTIVE: The aim of this study was to evaluate the proliferation and odontogenic differentiation of human dental pulp cells (hDPCs) and human umbilical cord mesenchymal stem cells (hUCMSCs) in three-dimensional co-culture system which was established with the help of bone morphogenetic protein-2 (BMP-2) and hydrogel. METHODS: hDPCs and hUCMSCs were cultured in different concentrations of hydrogel to explore the more suitable concentrations for subsequent experiments. hUCMSCs and hDPCs induced by BMP-2 were co-cultured in the hydrogel. MTT assay was used to measure the cell viability. The differentiation into odontoblast-like cells were measured by the mRNA expression of dentin salivary phosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), alkaline phosphatase and osteocalcin. Alizarin red staining was performed for the formation of mineralized nodules. RESULTS: hUCMSCs and hDPCs could grow and proliferate in hydrogel scaffold. The growth rate of cells in lower concentrations hydrogels were higher than that of high concentrations hydrogels (P < 0.05). The study showed that 0.25% hydrogel scaffold was more suitable for subsequent experiments than other groups. Compared with hUCMSCs-monoculture and hDPCs-monoculture, the co-culture groups exhibited more proliferative potential, alkaline phosphatase activity and mineralization nodule formation (P < 0.05). The mRNA expression in co-culture groups were higher than that of hUCMSCs-monoculture, closed to or even higher than that of hDPCs-monoculture. CONCLUSION: 0.25% hydrogel was the suitable concentration in co-culture system for subsequent experiments. The co-culture groups had stronger abilities of odontoblastic differentiation and mineralization than cells-monoculture groups, indicated that the co-culture conditions could regulate cell proliferation and differentiation within a certain range.


Assuntos
Diferenciação Celular , Polpa Dentária/citologia , Hidrogéis , Células-Tronco Mesenquimais/citologia , Fosfatase Alcalina/genética , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Proteínas da Matriz Extracelular/genética , Humanos , Odontoblastos/citologia , Osteocalcina/genética , Fosfoproteínas/genética , Sialoglicoproteínas/genética , Cordão Umbilical/citologia
5.
Mater Sci Eng C Mater Biol Appl ; 106: 110289, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31753386

RESUMO

This paper systematically investigates the biomedical performance of selective laser melted (SLM) porous Ti6Al4V ELI scaffolds for bone implantation through in vitro and in vivo experiments. Scaffolds with pore sizes of 500 µm, 600 µm and 700 µm and porosities of 60% and 70% were manufactured in order to explore the optimum pore size and porosity. Rat bone marrow mesenchymal stem cells (rBMMSCs) were used in the in vitro experiments. Cell Counting Kit-8, live/dead staining and scanning electron microscope were used to assess the cytotoxicity of the porous scaffolds. DNA content quantification was performed to investigate cell proliferation on the porous scaffolds. The osteogenic differentiation of cells was measured by alkaline phosphatase (ALP) activity and osteogenic gene expressions, including bone morphogenetic protein-2 (BMP-2), collagen type 1α1 (COL-1), osteocalcin (OCN), osteopontin (OPN) and runt-related transcription factor-2 (RUNX-2). The Sprague-Dawley (SD) rat models with distal femoral condyles defect were used in the in vivo experiments. Micro-CT analysis and histological analysis were performed after implantation surgery to reveal the bone ingrowth into the porous scaffolds. All in vitro data were analyzed by one-way ANOVA followed by Tukey post hoc tests, in vivo data were analyzed using Kruskall-Wallis ANOVA and Conover-Inman post-hoc test. Based on the in vitro and in vivo experiments, it is found that the porous scaffolds manufactured by SLM did not induce a cytotoxic effect. Among all the porous scaffolds, the scaffold with a pore size of 500 µm and porosity of 60% showed the best cell proliferation and osteogenic differentiation (in vitro experiments) and bone ingrowth (in vivo experiments).


Assuntos
Diferenciação Celular , Proliferação de Células , Osteogênese , Tecidos Suporte/química , Titânio/química , Animais , Células da Medula Óssea/citologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fraturas Ósseas/terapia , Lasers , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Porosidade , Ratos , Ratos Sprague-Dawley , Engenharia Tecidual , Titânio/toxicidade , Microtomografia por Raio-X
6.
J Ethnopharmacol ; 248: 112329, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-31672526

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Mesenchymal stem cells (MSCs) are multipotent stem cells possessing regenerative potential. Symphytum officinale (SO) is a medicinal plant and in homoeopathic literature, believed to accelerate bone healing. AIM OF THE STUDY: This study aimed to determine if homoeopathic doses of SO could augment osteogenesis in MSCs as they differentiate into osteoblasts in vitro. MATERIALS AND METHODS: Bone marrow samples were obtained from patients who underwent bone grafting procedures (n = 15). MSCs were isolated, expanded and characterized by flow cytometry (CD90, CD105). Cytotoxicity of SO was evaluated by MTT assay. Osteogenic differentiation was induced in MSCs with ß-glycerophosphate, ascorbic acid and dexamethasone over 2 weeks. Different homoeopathic doses of SO (MT, 3C, 6C, 12C and 30C) were added to the basic differentiation medium (BDM) and efficiency of MSCs differentiating into osteoblasts were measured by evaluating expression of Osteocalcin using flow cytometry, and alkaline phosphatase activity using ELISA. Gene expression analyses for osteoblast markers (Runx-2, Osteopontin and Osteocalcin) were evaluated in differentiated osteoblasts using qPCR. RESULTS: Flow cytometry (CD90, CD105) detected MSCs isolated from bone marrow (93-98%). MTT assay showed that the selected doses of SO did not induce any cytotoxicity in MSCs (24 hours). The efficiency of osteogenic differentiation (2 weeks) for different doses of Symphytum officinale was determined by flow cytometry (n = 10) for osteoblast marker, Osteocalcin, and most doses of Symphytum officinale enhanced osteogenesis. Interestingly, gene expression analysis for Runx-2 (n = 10), Osteopontin (n = 10), Osteocalcin (n = 10) and alkaline phosphatase activity (n = 8) also showed increased osteogenesis with the addition of Symphytum officinale to BDM, specially mother tincture. CONCLUSIONS: Our findings suggest that homoeopathic dose (specially mother tincture) of Symphytum officinale has the potential to enhance osteogenesis.


Assuntos
Conservadores da Densidade Óssea/farmacologia , Diferenciação Celular/efeitos dos fármacos , Confrei , Homeopatia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Extratos Vegetais/farmacologia , Fosfatase Alcalina/metabolismo , Conservadores da Densidade Óssea/isolamento & purificação , Diferenciação Celular/genética , Linhagem Celular , Confrei/química , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/genética , Osteopontina/genética , Osteopontina/metabolismo , Fenótipo , Extratos Vegetais/isolamento & purificação
7.
Ann Anat ; 227: 151427, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31614180

RESUMO

Mandibular/alveolar (m/a) bone, as a component of the periodontal apparatus, allows for the proper tooth anchorage and function of dentition. Bone formation around the tooth germs starts prenatally and, in the mouse model, the mesenchymal condensation turns into a complex vascularized bone (containing osteo-blasts, -cytes, -clasts) within only two days. This very short but critical period is characterized by synchronized cellular and molecular events. The m/a bone, as others, is subjected to endocrine regulations. This not only requires vasculature to allow the circulation of active molecules (ligands), but also the expression of corresponding cell receptors to define target tissues. This contribution aimed at following the dynamics of calciotropic receptors´ expression during morphological transformation of a mesenchymal condensation into the initial m/a bone structure. Receptors for all three calciotropic systemic regulators: parathormone, calcitonin and activated vitamin D (calcitriol), were localized on serial histological sections using immunochemistry and their relative expression was quantified by q-PCR. The onset of calciotropic receptors was followed along with bone cell differentiation (as checked using osteocalcin, sclerostin, RANK and TRAP) and vascularization (CD31) during mouse prenatal/embryonic (E) days 13-15 and 18. Additionally, the timing of calciotropic receptor appearance was compared with that of estrogen receptors (ESR1, ESR2). PTH receptor (PTH1r) appeared in the bone already at E13, when the first osteocalcin-positive cells were detected within the mesenchymal condensation forming the bone anlage. At this stage, blood vessels were only lining the condensation. At E14, the osteoblasts started to express the receptor for activated vitamin D (VDR). At this stage, the vasculature just penetrated the forming bone. On the same day, the first TRAP-positive (but not yet multinucleated) osteoclastic cells were identified. However, calcitonin receptor was detected only one day later. The first Sost-positive osteocytes, present at E15, were PTH1r and VDR positive. ESR1 almost copied the expression pattern of PTH1r, and ESR2 appearance was similar with VDR with a significant increase between E15 and E18. This report focuses on the in vivo situation and links morphological transformation of the mesenchymal cell condensation into a bone structure with dynamics of cell differentiation/maturation, vascularization and onset of receptors for calciotropic endocrine signalling in developing m/a bone.


Assuntos
Mandíbula/crescimento & desenvolvimento , Osteogênese/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Diferenciação Celular , Imuno-Histoquímica , Camundongos , Osteoblastos/fisiologia , Osteocalcina/análise , Osteocalcina/genética , Osteoclastos/fisiologia , Osteócitos/fisiologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Receptores da Calcitonina/metabolismo
8.
J Recept Signal Transduct Res ; 39(5-6): 407-414, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31847659

RESUMO

Activating transcription factor 4 (ATF4) promotes bone formation in human bone marrow mesenchymal stem cells. However, the underlying mechanisms of ATF4 in high glucose-induced injury of osteoblast still remain unclear. Small interfering RNA and plasmid targeting ATF4 were used to transfect MC3T3-E1 cells to knock down and overexpress ATF4 using Lipofectamin 3000. Cell viability, alkaline phosphatase (ALP) activity and levels were determined by MTT, ALP kit assay, quantitative real-time (qRT)-PCR and Western blot. Osteocalcin (OCN) expression was determined by ELISA, PCR and Western blot. The mRNA and protein levels of ATF4, glucose regulated protein 78 kDa (GRP78) and C/EBP homologous protein (CHOP) were detected by PCR and Western blot. In the current study, viabilities of MC3T3-E1 cells were inhibited by high glucose. Meanwhile, the mRNA and protein levels of ATF4 were effectively up-regulated in high glucose-incubated MC3T3-E1 cells. By conducting functional experiments, silencing ATF4 induced by small interfering RNA partially reversed the inhibitory effects of high glucose on viabilities of MC3T3-E1 cells. We also found that the expressions of ER stress-related proteins (ATF4, GRP78 and CHOP) were higher in high glucose-treated MC3T3-E1 cells but were inhibited by siATF4. However, overexpression of AFT4 had opposite results, and high glucose attenuated the protein levels of osteogenic marker genes ALP and OCN, which were further inhibited by ATF4 knockout gene. Thus, ATF4 was a necessary gene for high glucose to inhibit the proliferation and differentiation of MC3T3-E1 cells.


Assuntos
Fator 4 Ativador da Transcrição/genética , Diferenciação Celular/genética , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fosfatase Alcalina/genética , Animais , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Inativação de Genes , Inativação Gênica , Glucose/toxicidade , Proteínas de Choque Térmico/genética , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Osteoblastos/efeitos dos fármacos , Osteocalcina/genética , RNA Mensageiro/genética , Fator de Transcrição CHOP/genética
9.
J Diabetes Res ; 2019: 9302636, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31886290

RESUMO

Type 2 diabetes mellitus (T2DM) belongs to the diseases with hereditary predisposition, so both environmental and genetic factors contribute to its development. Recent studies have demonstrated that the skeleton realizes systemic regulation of energy metabolism through the secretion of osteocalcin (OCN). Thus, the association analysis between HindIII single nucleotide polymorphism of OCN gene (BGLAP) promoter region and T2DM development in Ukrainian population was carried out. 153 individuals diagnosed with T2DM and 311 control individuals were enrolled in the study. The genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The lack of association between BGLAP HindIII single nucleotide polymorphism (SNP) and T2DM development among Ukrainians was found. Further studies with extended groups of comparison are needed to confirm the obtained results.


Assuntos
Diabetes Mellitus Tipo 2/genética , Osteocalcina/genética , Polimorfismo de Nucleotídeo Único , Idoso , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/epidemiologia , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Regiões Promotoras Genéticas , Fatores de Risco , Ucrânia/epidemiologia
10.
Pathol Res Pract ; 215(12): 152722, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31718857

RESUMO

BACKGROUND: Periodontal ligament stem cells (PDLSCs) could differentiate into osteoblasts and have a great prospect in treating bone diseases. microRNAs (miRs) and nuclear factor kappa-B (NF-κB) signaling pathway have proved pivotal in regulating osteogenic differentiation. This study intended to discuss the mechanism of miR-132 and NF-κB in PDLSC osteogenesis. METHODS: PDLSCs were firstly cultured, induced, and identified by detecting the surface markers and observing cell morphology. Levels of osteogenic markers alkaline phosphatase (ALP), bone morphogenetic proteins 2 (BMP2), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN), along with miR-132 expression were measured. The osteoblast activity and mineral deposition were detected by ALP and alizarin red S (ARS) stainings. The targeting relationship between miR-132 and growth differentiation factor 5 (GDF5) was verified. The gain-and loss-of-function was performed to discuss roles of miR-132 and GDF5 in osteogenic differentiation of PDLSCs. Besides, levels of NF-κB signaling pathway-related proteins were measured. RESULTS: In osteogenic differentiation of PDLSCs, levels of ALP, BMP2, Runx2 and OCN were upregulated while miR-132 was downregulated. Overexpressing miR-132 reduced levels of osteogenic markers, osteoblast activity, ALP and ARS intensity and the activation of NF-κB axis. GDF5 is a target of miR-132 and GDF5 overexpression reversed the inhibitory effects of overexpressed miR-132 on PDLSC osteogenesis. CONCLUSION: Together, miR-132 could inhibit PDLSC osteogenesis via targeting GDF5 and activating NF-κB axis. These data provide useful information for PDLSC application in periodontal therapy.


Assuntos
Diferenciação Celular , Fator 5 de Diferenciação de Crescimento/metabolismo , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Osteogênese , Ligamento Periodontal/metabolismo , Células-Tronco/metabolismo , Adolescente , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fator 5 de Diferenciação de Crescimento/genética , Humanos , MicroRNAs/genética , Osteocalcina/genética , Osteocalcina/metabolismo , Ligamento Periodontal/citologia , Transdução de Sinais , Adulto Jovem
11.
Chin J Nat Med ; 17(10): 756-767, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31703756

RESUMO

Peptides from Pilose antler aqueous extract (PAAE) have been shown to stimulate the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the underlying molecular mechanisms are not well understood. Here, PAAE was isolated and purified to explore the molecular mechanisms underlying PAAE's effects on BMSCs as well as its osteoprotective effects in ovariectomized rats. Our results showed that PAAE promoted proliferation and differentiation of BMSCs to become osteoblasts by enhancing ALP activity and increasing extracellular matrix mineralization. The trabecular microarchitecture of ovariectomized rats was also found to be protected by PAAE. Quantitative reverse transcription-polymerase chain reaction (Quantitative RT-PCR) results suggest that PAAE also increased the expression of osteogenic markers including, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), bone morphogenetic protein-2 (BMP-2), and collagen I (COL-I). Immunoblotting results indicated that PAAE upregulated the levels of BMP-2 and Runx2 and was associated with Smad1/5 phosphorylation. PAAE A at the concentration of 200 µg·mL-1 showed the strongest effect on proliferation and osteogenic differentiation of BMSCs after 48 h. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), we identified the molecular weight of PAAE A and found that it is less than 3000 Da and showed several significant peaks. In conclusion, PAAE activates the BMP-2/Smad1, 5/Runx2 pathway to induce osteoblastic differentiation and mineralization in BMSCs and can inhibit OVX-induced bone loss. These mechanisms are likely responsible for its therapeutic effect on postmenopausal osteoporosis.


Assuntos
Chifres de Veado/química , Proteína Morfogenética Óssea 2/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteogênese/efeitos dos fármacos , Osteoporose Pós-Menopausa/tratamento farmacológico , Peptídeos/administração & dosagem , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Proteína Morfogenética Óssea 2/genética , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Cervos , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteoporose Pós-Menopausa/genética , Osteoporose Pós-Menopausa/metabolismo , Osteoporose Pós-Menopausa/fisiopatologia , Peptídeos/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Proteína Smad1/genética , Proteína Smad5/genética
12.
Int J Med Sci ; 16(11): 1466-1472, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31673238

RESUMO

Non-steroidal anti-inflammatory drugs (NSAIDs), including cyclooxygenase-2 (COX-2)-selective NSAIDs, are associated with adverse effects on bone tissue. These drugs are frequently the treatment of choice but are the least studied with respect to their repercussion on bone. The objective of this study was to determine the effects of celecoxib on cultured human osteoblasts. Human osteoblasts obtained by primary culture from bone samples were treated with celecoxib at doses of 0.75, 2, or 5µM for 24 h. The MTT technique was used to determine the effect on proliferation; flow cytometry to establish the effect on cell cycle, cell viability, and antigenic profile; and real-time polymerase chain reaction to measure the effect on gene expressions of the differentiation markers RUNX2, alkaline phosphatase (ALP), osteocalcin (OSC), and osterix (OSX). Therapeutic doses of celecoxib had no effect on osteoblast cell growth or antigen expression but had a negative impact on the gene expression of RUNX2 and OSC, although there was no significant change in the expression of ALP and OSX. Celecoxib at therapeutic doses has no apparent adverse effects on cultured human osteoblasts and only inhibits the expression of some differentiation markers. These characteristics may place this drug in a preferential position among NSAIDs used for analgesic and anti-inflammatory therapy during bone tissue repair.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Celecoxib/farmacologia , Proliferação de Células/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Ciclo-Oxigenase 2/genética , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteocalcina/genética , Osteogênese/efeitos dos fármacos , Cultura Primária de Células , Fator de Transcrição Sp7/genética
13.
Int J Mol Sci ; 20(20)2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658685

RESUMO

The bone regeneration efficiency of bone marrow mesenchymal stem cells (BMSCs) and dental pulp mesenchymal stem cells (DPSCs) combined with xenografts in the craniofacial region remains unclear. Accordingly, this study commenced by comparing the cell morphology, cell proliferation, trilineage differentiation, mineral synthesis, and osteogenic gene expression of BMSCs and DPSCs in vitro. Four experimental groups (empty control, Bio-Oss only, Bio-Oss+BMSCs, and Bio-Oss+DPSCs) were then designed and implanted in rabbit calvarial defects. The BMSCs and DPSCs showed a similar morphology, proliferative ability, surface marker profile, and trilineage-differentiation potential in vitro. However, the BMSCs exhibited a higher mineral deposition and expression levels of osteogenic marker genes, including alkaline phosphatase (ALP), runt related transcription factor 2 (RUNX2), and osteocalcin (OCN). In the in vivo studies, the bone volume density in both MSC groups was significantly greater than that in the empty control or Bio-Oss only group. Moreover, the new bone formation and Collagen I / osteoprotegerin protein expressions of the scaffold+MSC groups were higher than those of the Bio-Oss only group. Finally, the Bio-Oss+BMSC and Bio-Oss+DPSC groups had a similar bone mineral density, new bone formation, and osteogenesis-related protein expression. Overall, the DPSCs seeded on Bio-Oss matched the bone regeneration efficacy of BMSCs in vivo and hence appear to be a promising strategy for craniofacial defect repair in future clinical applications.


Assuntos
Medula Óssea/metabolismo , Regeneração Óssea/fisiologia , Polpa Dentária/citologia , Células-Tronco Mesenquimais/citologia , Osteogênese/fisiologia , Fosfatase Alcalina/genética , Animais , Osso e Ossos/anormalidades , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Cálcio/análise , Diferenciação Celular , Proliferação de Células , Colágeno , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Xenoenxertos , Minerais , Osteoblastos/citologia , Osteocalcina/genética , Osteogênese/genética , Osteoprotegerina , Coelhos
14.
Mol Cells ; 42(11): 763-772, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31659886

RESUMO

Periodontitis is characterized by the loss of periodontal tissues, especially alveolar bone. Common therapies cannot satisfactorily recover lost alveolar bone. Periodontal ligament stem cells (PDLSCs) possess the capacity of self-renewal and multilineage differentiation and are likely to recover lost alveolar bone. In addition, periodontitis is accompanied by hypoxia, and hypoxia-inducible factor-1α (HIF-1α) is a master transcription factor in the response to hypoxia. Thus, we aimed to ascertain how hypoxia affects runt-related transcription factor 2 (RUNX2), a key osteogenic marker, in the osteogenesis of PDLSCs. In this study, we found that hypoxia enhanced the protein expression of HIF-1α, vascular endothelial growth factor (VEGF), and RUNX2 ex vivo and in situ. VEGF is a target gene of HIF-1α, and the increased expression of VEGF and RUNX2 proteins was enhanced by cobalt chloride (CoCl2, 100 µmol/L), an agonist of HIF-1α, and suppressed by 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1, 10 µmol/L), an antagonist of HIF-1α. In addition, VEGF could regulate the expression of RUNX2, as RUNX2 expression was enhanced by human VEGF (hVEGF165) and suppressed by VEGF siRNA. In addition, knocking down VEGF could decrease the expression of osteogenesis-related genes, i.e., RUNX2, alkaline phosphatase (ALP), and type I collagen (COL1), and hypoxia could enhance the expression of ALP, COL1, and osteocalcin (OCN) in the early stage of osteogenesis of PDLSCs. Taken together, our results showed that hypoxia could mediate the expression of RUNX2 in PDLSCs via HIF-1α-induced VEGF and play a positive role in the early stage of osteogenesis of PDLSCs.


Assuntos
Diferenciação Celular/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica , Ligamento Periodontal/citologia , Células-Tronco/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Hipóxia Celular , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/genética , Interferência de RNA , Células-Tronco/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
15.
Bull Exp Biol Med ; 167(5): 681-684, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31630302

RESUMO

The effects of bone graft materials on the inflammatory response and biochemical markers of bone remodeling were studied on a rabbit model of fracture augmentation with the following grafts: ß-tricalcium phosphate, demineralized bone matrix, nanostructured carbon implant, and porous titanium implant made by additive 3D printing. The markers of bone remodeling and the blood system response in the postoperative period were studied. It was found that porous titanium implant and ß-tricalcium phosphate induced osteogenesis and minimized osteoclastic resorption. Augmentation with nanostructured carbon implant and demineralized bone matrix stimulated the processes of osteoclastic resorption.


Assuntos
Materiais Biocompatíveis/farmacologia , Transplante Ósseo/métodos , Fosfatos de Cálcio/farmacologia , Cementoplastia/métodos , Fraturas Intra-Articulares/terapia , Osseointegração/efeitos dos fármacos , Titânio/farmacologia , Fosfatase Alcalina/sangue , Fosfatase Alcalina/genética , Animais , Biomarcadores/metabolismo , Técnica de Desmineralização Óssea , Matriz Óssea/química , Remodelação Óssea , Reabsorção Óssea/metabolismo , Carbono/metabolismo , Carbono/farmacologia , Colágeno Tipo I/sangue , Colágeno Tipo I/genética , Feminino , Fraturas Intra-Articulares/metabolismo , Fraturas Intra-Articulares/cirurgia , Nanoestruturas/química , Osseointegração/fisiologia , Osteocalcina/sangue , Osteocalcina/genética , Peptídeos/sangue , Peptídeos/genética , Porosidade , Coelhos , Tíbia/efeitos dos fármacos , Tíbia/lesões , Tíbia/metabolismo , Tíbia/cirurgia
16.
J Mol Histol ; 50(6): 493-502, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31522346

RESUMO

Bone regeneration is impaired in patients with osteoporosis. Previous studies have shown that periostin (Postn) shows great potential in bone regeneration treatments. However, the role of Postn in bone marrow mesenchymal stem cells (BMMSCs) remains to be elucidated. In this study, we isolated BMMSCs from ovariectomized rats (OVX-BMMSCs) and normal rats. Then, the expression levels of Postn and osteogenesis in OVX-BMMSCs were detected by alizarin red and alkaline phosphatase substrate staining, qPCR, and western blotting. We found that the levels of Postn in OVX-BMMSCs were significantly reduced. Furthermore, Postn overexpression in OVX-BMMSCs using recombinant lentivirus could improve the expression of alkaline phosphatase, runt-related transcription factor 2, and osteocalcin and reduce the expression of sclerostin. Besides, micro-computed tomography analysis, hematoxylin-eosin, and Masson's staining showed that the healing of the alveolar bone defect in osteoporotic rats could be promoted using Postn-modified OVX-BMMSC sheets. In conclusion, Postn-modified OVX-BMMSCs might restore the osteogenic capacity and promote alveolar bone regeneration, which may serve as a new therapeutic approach for bone regeneration in osteoporosis.


Assuntos
Processo Alveolar/metabolismo , Células da Medula Óssea/metabolismo , Regeneração Óssea/fisiologia , Moléculas de Adesão Celular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteoporose/metabolismo , Processo Alveolar/diagnóstico por imagem , Animais , Regeneração Óssea/genética , Moléculas de Adesão Celular/genética , Células Cultivadas , Feminino , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/genética , Osteogênese/fisiologia , Osteoporose/genética , Ovariectomia , Ratos Sprague-Dawley , Microtomografia por Raio-X
17.
Int J Biochem Cell Biol ; 116: 105614, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31550547

RESUMO

Osteopontin (OPN) is an osteoblast-derived secretory protein that plays a role in bone remodeling, osteoblast responsiveness, and inflammation. We recently found that osteoblast differentiation is type-specific, with conditions of JNK inactivation inducing osteoblasts that preferentially express OPN (OPN-type). Since OPN-type osteoblasts highly express osteogenesis-inhibiting proteins and Rankl, an important inducer of osteoclastogenesis, an increased appearance of OPN-type osteoblasts may be associated with inefficient and poor-quality bone regeneration. However, whether specific osteogenic inducers can modulate OPN-type osteoblast differentiation is completely unknown. Here, we demonstrate that bone morphogenic protein 9 (BMP9) prevents induction of OPN-type osteoblast differentiation under conditions of JNK inhibition. Although JNK inactivation suppressed both BMP2- and BMP9-induced matrix mineralization and osteocalcin expression, the expression of Rankl and specific cytokines such as Gpha2, Esm1, and Sfrp1 under similar conditions was increased in all cells except those treated with BMP9. Increased expression of Id4, a critical transcriptional regulator of OPN-type osteoblast differentiation, was similarly prevented only in BMP9-treated cells. We also found that BMP9 specifically induces the expression of Hey1, a bHLH transcriptional repressor, and that Id4 inhibits the suppressive effects of Hey1 on Opn promoter activity by forming Id4-Hey1 complexes in osteoblasts. Using site-direct mutagenesis, ChIP, and immunoprecipitation, we elucidated that BMP9-induced overexpression of Hey1 can overcome the effects of Id4 and suppress OPN expression. We further found that p38 activation and JNK inactivation are involved in BMP9-induced Hey1 expression. Collectively, these data suggest that BMP9 is a unique osteogenic inducer that regulates OPN-type osteoblast differentiation.


Assuntos
Proteínas de Ciclo Celular/genética , Fator 2 de Diferenciação de Crescimento/farmacologia , Proteínas Inibidoras de Diferenciação/genética , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteopontina/genética , Animais , Animais Recém-Nascidos , Proteína Morfogenética Óssea 2/farmacologia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Glicerofosfatos/farmacologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas Inibidoras de Diferenciação/metabolismo , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/genética , Osteopontina/metabolismo , Cultura Primária de Células , Proteoglicanas/genética , Proteoglicanas/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
18.
Int J Mol Sci ; 20(17)2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31461878

RESUMO

Previously, we detected a higher degree of mineralization in fetal calf serum (FCS) compared to serum-free cultured jaw periosteum derived osteoprogenitor cells (JPCs). By Raman spectroscopy, we detected an earlier formation of mineralized extracellular matrix (ECM) of higher quality under serum-free media conditions. However, mineralization potential remained too low. In the present study, we aimed to investigate the biochemical composition and subsequent biomechanical properties of the JPC-formed ECM and minerals under human platelet lysate (hPL) and FCS supplementation. JPCs were isolated (n = 4 donors) and expanded under FCS conditions and used in passage five for osteogenic induction under both, FCS and hPL media supplementation. Raman spectroscopy and Alizarin Red/von Kossa staining were employed for biochemical composition analyses and for visualization and quantification of mineralization. Osteocalcin gene expression was analyzed by quantitative PCR. Biomechanical properties were assessed by using atomic force microscopy (AFM). Raman spectroscopic measurements showed significantly higher (p < 0.001) phosphate to protein ratios and in the tendency, lower carbonate to phosphate ratios in osteogenically induced JPCs under hPL in comparison to FCS culturing. Furthermore, higher crystal sizes were detected under hPL culturing of the cells. With respect to the ECM, significantly higher ratios of the precursor protein proline to hydroxyproline were detected in hPL-cultured JPC monolayers (p < 0.001). Additionally, significantly higher levels (p < 0.001) of collagen cross-linking were calculated, indicating a higher degree of collagen maturation in hPL-cultured JPCs. By atomic force microscopy, a significant increase in ECM stiffness (p < 0.001) of FCS cultured JPC monolayers was observed. The reverse effect was measured for the JPC formed precipitates/minerals. Under hPL supplementation, JPCs formed minerals of significantly higher stiffness (p < 0.001) when compared to the FCS setting. This study demonstrates that hPL culturing of JPCs leads to the formation of an anorganic material of superior quality in terms of biochemical composition and mechanical properties.


Assuntos
Cálcio/metabolismo , Arcada Osseodentária/citologia , Osteoblastos/metabolismo , Periósteo/metabolismo , Fosfatos/metabolismo , Calcificação Fisiológica , Carbonatos/metabolismo , Células Cultivadas , Colágeno/metabolismo , Meios de Cultura/farmacologia , Matriz Extracelular/metabolismo , Humanos , Osteoblastos/efeitos dos fármacos , Osteoblastos/ultraestrutura , Osteocalcina/genética , Osteocalcina/metabolismo , Periósteo/citologia , Prolina/metabolismo
19.
Am J Physiol Cell Physiol ; 317(5): C932-C941, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31411920

RESUMO

Exosome secretion is an important paracrine way of endothelial progenitor cells (EPCs) to modulate resident endothelial cells. The osteocalcin (OCN)-expressing EPCs have been found to be increased in cardiovascular disease patients and are considered to be involved in the process of coronary atherosclerosis. Since OCN has been proven to prevent endothelial dysfunction, this study aimed to evaluate the effect of exosomes derived from OCN-overexpressed EPCs on endothelial cells. Exosomes derived from EPCs (Exos) and OCN-overexpressed EPCs (OCN-Exos) were isolated and incubated with rat aorta endothelial cells (RAOECs) with or without the inhibition of OCN receptor G protein-coupled receptor family C group 6 member A (GPRC6A). The effects of exosomes on the proliferation activity of endothelial cells were evaluated by CCK-8 assay, and the migration of endothelial cells was detected by wound healing assay. A tube formation assay was used to test the influence of exosomes on the angiogenesis performance of endothelial cells. Here, we presented that OCN was packed into Exos and was able to be transferred to the RAOECs via exosome incorporation, which was increased in OCN-Exos groups. Compared with Exos, OCN-Exos had better efficiency in promoting RAOEC proliferation and migration and tube formation. The promoting effects were impeded after the inhibition of GPRC6A expression in RAOECs. These data suggest that exosomes from OCN-overexpressed EPCs have a beneficial regulating effect on endothelial cells, which involved enhanced OCN-GPRC6A signaling.


Assuntos
Proliferação de Células/fisiologia , Células Progenitoras Endoteliais/metabolismo , Exossomos/metabolismo , Neovascularização Fisiológica/fisiologia , Osteocalcina/biossíntese , Animais , Movimento Celular/fisiologia , Expressão Gênica , Osteocalcina/genética , Ratos
20.
Colloids Surf B Biointerfaces ; 182: 110332, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31325776

RESUMO

Micro/nano-topography (MNT) is an important variable affecting osseointegration of bone biomaterials, but the underlying mechanisms are not fully understood. We probed the role of a AMOT130/YAP pathway in osteoblastic differentiation of bone marrow mesenchymal stems cultured on titanium (Ti) carrying MNTs. Ti surfaces with two well-defined MNTs (TiO2 nanotubes of different diameters and wall thicknesses) were prepared by anodization. Rat BMSCs were cultured on flat Ti and Ti surfaces carrying MNTs, and cell behaviors (i.e., morphology, F-actin development, osteoblastic differentiation, YAP localization) were studied. Ti surfaces carrying MNTs increased F-actin formation, osteoblastic gene expression, and protein AMOT130 production in BMSCs (all vs. flat Ti), and the surface carrying larger nantubes was more effective, confirming osteoblastic differentiation induced by MNTs. Elevation of the AMOT130 level (by inhibiting its degradation) increased the osteoblastic gene expression, F-actin formation, and nuclear localization of YAP. These show that, AMOT130/YAP is an important pathway mediating the translation of MNT signals to BMSC osteoblastic commitment, likely via the cascade: AMOT130 promotion of F-actin formation, increased YAP nuclear import, and activation of osteoblastic gene expression.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mecanotransdução Celular/genética , Proteínas de Membrana/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Titânio/farmacologia , Actinas/genética , Actinas/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Nanotubos/química , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/genética , Osteopontina/genética , Osteopontina/metabolismo , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Propriedades de Superfície , Titânio/química
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