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1.
Actual. osteol ; 19(1): 18-29, ago. 2023. tab
Article in English | LILACS, UNISALUD, BINACIS | ID: biblio-1511400

ABSTRACT

MicroRNAs (miRNAs) are small non-coding RNA molecules that play critical roles in post-transcriptional gene regulation. They function by binding to target messenger RNA (mRNA) molecules, leading to their degradation or inhibiting their translation into proteins. In the context of skeletal diseases, such as osteoporosis, osteoarthritis, and bone metastasis, there is growing evidence osteoblastic miRNAs, are involved in the regulation of bone formation and maintenance.Osteoblasts are bone-forming cells responsible for synthesizing and depositing the extracellular matrix, which ultimately mineralizes to form bone tissue. Osteoblastic miRNAs modulate various aspects of osteoblast function, including proliferation, differentiation, mineralization, and apoptosis. Dysregulation of these miRNAs can disrupt the balance between bone formation and resorption, leading to skeletal diseases.The therapeutic implications of targeting osteoblastic miRNAs in skeletal diseases are significant. Modulating the expression levels of specific miRNAs holds promise for developing novel therapeutic strategies to enhance bone formation, prevent bone loss, and promote bone regeneration. Potential therapeutic approaches include the use of synthetic miRNA mimics to restore miRNA expression in diseases associated with miRNA downregulation or the use of anti-miRNA oligonucleotides to inhibit miRNA function in diseases associated with miRNA upregulation.miRNA-based therapies are still in the early stages of development, and further research is needed to fully understand the complexity of miRNA networks. Additionally, the delivery of miRNAs to specific target tissues and cells remains a challenge that needs to be addressed for effective clinical translation. Nonetheless, targeting osteoblastic miRNAs represents a promising avenue for future therapeutic interventions in skeletal diseases. (AU)


Los micro-ARNs (miARNss) son pequeños ARN no codificantes que desempeñan un papel fundamental en la regulación génica postranscripcional. Ejercen su función al unir-se a moléculas de ARN mensajero (ARNm), promoviendo su degradación e inhibiendo su traducción en proteínas. En el contexto de las enfermedades esqueléticas, como la osteoporosis, la osteoartritis y la metástasis ósea existe evidencia de que los miARNs osteoblásticos están involucrados en la regulación de la formación y del mantenimiento óseo. Los osteoblastos son células formadoras de hueso responsables de sintetizar y depositar la matriz extracelular, que finalmente se mineraliza para formar el hueso. Los miARNs derivados de osteoblastos modulan varios aspectos de la función de estas células, incluida la proliferación, diferenciación, mineralización y la apoptosis. La desregulación de estos miARNs puede alterar el equilibrio entre la formación y la resorción ósea, lo que lleva a enfermedades óseas. Las implicaciones terapéuticas de los miARNs osteoblásticos en enfermedades esqueléticas son significativas. La modulación de los niveles de expresión de miARNs específicos es prometedora para desarrollar nuevas estrate-gias terapéuticas a fin de mejorar la formación, prevenir la pérdida y promover la regeneración ósea. Los enfoques terapéuticos potenciales incluyen el uso de miméticos de miARNs para restaurar la expresión de miARNs o el uso de oligonucleótidos anti-miARNs para inhibir su función. Las terapias basadas en miARNs aún se encuentran en las primeras etapas de desarrollo. La administración de miARNs a las células y los tejidos específicos sigue siendo un desafío para lograr una aplicación clínica eficaz. (AU)


Subject(s)
Humans , Osteoblasts/cytology , Osteogenesis/genetics , MicroRNAs/genetics , Osteoclasts/cytology , Bone Diseases/prevention & control , Signal Transduction , Gene Expression Regulation , MicroRNAs/biosynthesis , MicroRNAs/physiology , MicroRNAs/therapeutic use
2.
Biol. Res ; 56: 17-17, 2023. ilus, tab, graf
Article in English | LILACS | ID: biblio-1439484

ABSTRACT

BACKGROUND: Spontaneous spheroid culture is a novel three-dimensional (3D) culture strategy for the rapid and efficient selection of progenitor cells. The objectives of this study are to investigate the pluripotency and differentiation capability of spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells (AB-MSCs); compare the advantages of spontaneous spheroids to those of mechanical spheroids; and explore the mechanisms of stemness enhancement during spheroid formation from two-dimensional (2D) cultured cells. METHODS: AB-MSCs were isolated from the alveolar bones of C57BL/6 J mice. Spontaneous spheroids formed in low-adherence specific culture plates. The stemness, proliferation, and multi-differentiation capacities of spheroids and monolayer cultures were investigated by reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, alkaline phosphatase (ALP) activity, and oil-red O staining. The pluripotency difference between the spontaneous and mechanical spheroids was analyzed using RT-qPCR. Hypoxia-inducible factor (HIFs) inhibition experiments were performed to explore the mechanisms of stemness maintenance in AB-MSC spheroids. RESULTS: AB-MSCs successfully formed spontaneous spheroids after 24 h. AB-MSC spheroids were positive for MSC markers and pluripotency markers (Oct4, KLF4, Sox2, and cMyc). Spheroids showed higher Ki67 expression and lower Caspase3 expression at 24 h. Under the corresponding conditions, the spheroids were successfully differentiated into osteogenic and adipogenic lineages. AB-MSC spheroids can induce neural-like cells after neurogenic differentiation. Higher expression of osteogenic markers, adipogenic markers, and neurogenic markers (NF-M, NeuN, and GFAP) was found in spheroids than in the monolayer. Spontaneous spheroids exhibited higher stemness than mechanical spheroids did. HIF-1α and HIF-2α were remarkably upregulated in spheroids. After HIF-1/2α-specific inhibition, spheroid formation was significantly reduced. Moreover, the expression of the pluripotency genes was suppressed. CONCLUSIONS: Spontaneous spheroids from AB-MSCs enhance stemness and pluripotency. HIF-1/2α plays an important role in the stemness regulation of spheroids. AB-MSC spheroids exhibit excellent multi-differentiation capability, which may be a potent therapy for craniomaxillofacial tissue regeneration.


Subject(s)
Animals , Mice , Spheroids, Cellular , Mesenchymal Stem Cells , Osteogenesis/genetics , Stem Cells , Cell Differentiation , Cells, Cultured , Cell Culture Techniques/methods , Hypoxia/metabolism , Mice, Inbred C57BL
3.
Biol. Res ; 56: 9-9, 2023. ilus, tab, graf
Article in English | LILACS | ID: biblio-1429910

ABSTRACT

BACKGROUND: Knowledge about regulating transcription factors (TFs) for osteoblastogenesis from mesenchymal stem cells (MSCs) is limited. Therefore, we investigated the relationship between genomic regions subject to DNA-methylation changes during osteoblastogenesis and the TFs known to directly interact with these regulatory regions. RESULTS: The genome-wide DNA-methylation signature of MSCs differentiated to osteoblasts and adipocytes was determined using the Illumina HumanMethylation450 BeadChip array. During adipogenesis no CpGs passed our test for significant methylation changes. Oppositely, during osteoblastogenesis we identified 2462 differently significantly methylated CpGs (adj. p < 0.05). These resided outside of CpGs islands and were significantly enriched in enhancer regions. We confirmed the correlation between DNA-methylation and gene expression. Accordingly, we developed a bioinformatic tool to analyse differentially methylated regions and the TFs interacting with them. By overlaying our osteoblastogenesis differentially methylated regions with ENCODE TF ChIP-seq data we obtained a set of candidate TFs associated to DNA-methylation changes. Among them, ZEB1 TF was highly related with DNA-methylation. Using RNA interference, we confirmed that ZEB1, and ZEB2, played a key role in adipogenesis and osteoblastogenesis processes. For clinical relevance, ZEB1 mRNA expression in human bone samples was evaluated. This expression positively correlated with weight, body mass index, and PPARγ expression. CONCLUSIONS: In this work we describe an osteoblastogenesis-associated DNA-methylation profile and, using these data, validate a novel computational tool to identify key TFs associated to age-related disease processes. By means of this tool we identified and confirmed ZEB TFs as mediators involved in the MSCs differentiation to osteoblasts and adipocytes, and obesity-related bone adiposity.


Subject(s)
Humans , Osteogenesis/genetics , Mesenchymal Stem Cells , Transcription Factors/genetics , Transcription Factors/metabolism , Cell Differentiation/genetics , DNA Methylation
4.
Acta Physiologica Sinica ; (6): 429-438, 2023.
Article in Chinese | WPRIM | ID: wpr-981018

ABSTRACT

It has been well documented that exercise can improve bone metabolism, promote bone growth and development, and alleviate bone loss. MicroRNAs (miRNAs) are widely involved in the proliferation and differentiation of bone marrow mesenchymal stem cells, osteoblasts, osteoclasts and other bone tissue cells, and regulation of balance between bone formation and bone resorption by targeting osteogenic factors or bone resorption factors. Thus miRNAs play an important role in the regulation of bone metabolism. Recently, regulation of miRNAs are shown to be one of the ways by which exercise or mechanical stress promotes the positive balance of bone metabolism. Exercise induces changes of miRNAs expression in bone tissue and regulates the expression of related osteogenic factors or bone resorption factors, to further strengthen the osteogenic effect of exercise. This review summarizes relevant studies on the mechanism whereby exercise regulates bone metabolism via miRNAs, providing a theoretical basis for osteoporosis prevention and treatment with exercise.


Subject(s)
Humans , MicroRNAs/metabolism , Osteogenesis/genetics , Cell Differentiation , Osteoblasts , Bone Resorption/metabolism
5.
Journal of Experimental Hematology ; (6): 170-178, 2023.
Article in Chinese | WPRIM | ID: wpr-971120

ABSTRACT

OBJECTIVE@#To investigate the expression of pyruvate kinase M2 (PKM2) in bone marrow mesenchymal stem cells (BMSCs) in myeloma bone disease (MBD) and its effect on osteogenic and adipogenic differentiation of BMSCs.@*METHODS@#BMSCs were isolated from bone marrow of five patients with multiple myeloma (MM) (MM group) and five with iron deficiency anemia (control group) for culture and identification. The expression of PKM2 protein were compared between the two groups. The differences between osteogenic and adipogenic differentiation of BMSCs were assessed by using alkaline phosphatase (ALP) and oil red O staining, and detecting marker genes of osteogenesis and adipogenesis. The effect of MM cell line (RPMI-8226) and BMSCs co-culture on the expression of PKM2 was explored. Functional analysis was performed to investigate the correlations of PKM2 expression of MM-derived BMSCs with osteogenic and adipogenic differentiation by employing PKM2 activator and inhibitor. The role of orlistat was explored in regulating PKM2 expression, osteogenic and adipogenic differentiation of MM-derived BMSCs.@*RESULTS@#Compared with control, MM-originated BMSCs possessed the ability of increased adipogenic and decreased osteogenic differentiation, and higher level of PKM2 protein. Co-culture of MM cells with BMSCs markedly up-regulated the expression of PKM2 of BMSCs. Up-regulation of PKM2 expression could promote adipogenic differentiation and inhibit osteogenic differentiation of MM-derived BMSCs, while down-regulation of PKM2 showed opposite effect. Orlistat significantly promoted osteogenic differentiation in MM-derived BMSCs via inhibiting the expression of PKM2.@*CONCLUSION@#The overexpression of PKM2 can induce the inhibition of osteogenic differentiation of BMSCs in MBD. Orlistat can promote the osteogenic differentiation of BMSCs via inhibiting the expression of PKM2, indicating a potential novel agent of anti-MBD therapy.


Subject(s)
Humans , Adipogenesis , Bone Diseases/metabolism , Bone Marrow Cells , Cell Differentiation , Cells, Cultured , Mesenchymal Stem Cells/physiology , Multiple Myeloma/metabolism , Orlistat/pharmacology , Osteogenesis/genetics
6.
Journal of Experimental Hematology ; (6): 154-161, 2023.
Article in Chinese | WPRIM | ID: wpr-971118

ABSTRACT

OBJECTIVE@#To investigate the effect of adipocytes in the bone marrow microenvironment of patients with multiple myeloma (MM) on the pathogenesis of MM.@*METHODS@#Bone marrow adipocytes (BMA) in bone marrow smears of health donors (HD) and newly diagnosed MM (ND-MM) patients were evaluated with oil red O staining. The mesenchymal stem cells (MSC) from HD and ND-MM patients were isolated, and in vitro co-culture assay was used to explore the effects of MM cells on the adipogenic differentiation of MSC and the role of BMA in the survival and drug resistance of MM cells. The expression of adipogenic/osteogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4, FASN and ALP both in MSC and MSC-derived adipocytes was determined with real-time quantitative PCR. The Western blot was employed to detect the expression levels of IL-6, IL-10, SDF-1α, TNF-α and IGF-1 in the supernatant with or without PPAR-γ inhibitor.@*RESULTS@#The results of oil red O staining of bone marrow smears showed that BMA increased significantly in patients of ND-MM compared with the normal control group, and the BMA content was related to the disease status. The content of BMA decreased in the patients with effective chemotherapy. MM cells up-regulated the expression of MSC adipogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4 and FASN, but the expression of osteogenic differentiation-related gene ALP was significantly down-regulated. This means that the direct consequence of the interaction between MM cells and MSC in the bone marrow microenvironment is to promote the differentiation of MSC into adipocytes at the expense of osteoblasts, and the cytokines detected in supernatant changed. PPAR-γ inhibitor G3335 could partially reverse the release of cytokines by BMA. Those results confirmed that BMA regulated the release of cytokines via PPAR-γ signal, and PPAR-γ inhibitor G3335 could distort PPAR-γ mediated BMA maturation and cytokines release. The increased BMA and related cytokines effectively promoted the proliferation, migration and drug resistance of MM cells.@*CONCLUSION@#The BMA and its associated cytokines are the promoting factors in the survival, proliferation and migration of MM cells. BMA can protect MM cells from drug-induced apoptosis and plays an important role in MM treatment failure and disease progression.


Subject(s)
Humans , Osteogenesis/genetics , Bone Marrow/metabolism , Multiple Myeloma/metabolism , Drug Resistance, Neoplasm , Peroxisome Proliferator-Activated Receptors/pharmacology , Cell Differentiation , Adipogenesis , Cytokines/metabolism , Adipocytes/metabolism , Bone Marrow Cells/metabolism , Cells, Cultured , PPAR gamma/pharmacology , Tumor Microenvironment
7.
Chinese Journal of Reparative and Reconstructive Surgery ; (12): 615-621, 2023.
Article in Chinese | WPRIM | ID: wpr-981641

ABSTRACT

OBJECTIVE@#To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1).@*METHODS@#The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture.@*RESULTS@#The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( P>0.05).@*CONCLUSION@#Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.


Subject(s)
Animals , Female , Mice , Cell Differentiation , Cells, Cultured , Core Binding Factor Alpha 1 Subunit/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Mesenchymal Stem Cells , Mice, Inbred C57BL , MicroRNAs/metabolism , Osteocalcin/metabolism , Osteogenesis/genetics , RNA, Messenger/genetics
8.
International Journal of Oral Science ; (4): 19-19, 2022.
Article in English | WPRIM | ID: wpr-929147

ABSTRACT

Parental imprinting is an epigenetic process leading to monoallelic expression of certain genes depending on their parental origin. Imprinting diseases are characterized by growth and metabolic issues starting from birth to adulthood. They are mainly due to methylation defects in imprinting control region that drive the abnormal expression of imprinted genes. We currently lack relevant animal or cellular models to unravel the pathophysiology of growth failure in these diseases. We aimed to characterize the methylation of imprinting regions in dental pulp stem cells and during their differentiation in osteogenic cells (involved in growth regulation) to assess the interest of this cells in modeling imprinting diseases. We collected dental pulp stem cells from five controls and four patients (three with Silver-Russell syndrome and one with Beckwith-Wiedemann syndrome). Methylation analysis of imprinting control regions involved in these syndromes showed a normal profile in controls and the imprinting defect in patients. These results were maintained in dental pulp stem cells cultured under osteogenic conditions. Furthermore, we confirmed the same pattern in six other loci involved in imprinting diseases in humans. We also confirmed monoallelic expression of H19 (an imprinted gene) in controls and its biallelic expression in one patient. Extensive imprinting control regions methylation analysis shows the strong potential of dental pulp stem cells in modeling imprinting diseases, in which imprinting regions are preserved in culture and during osteogenic differentiation. This will allow to perform in vitro functional and therapeutic tests in cells derived from dental pulp stem cells and generate other cell-types.


Subject(s)
Adult , Animals , Humans , DNA Methylation , Dental Pulp , Genomic Imprinting , Osteogenesis/genetics , Stem Cells
9.
China Journal of Orthopaedics and Traumatology ; (12): 379-386, 2022.
Article in Chinese | WPRIM | ID: wpr-928328

ABSTRACT

OBJECTIVE@#To investigate the effect of RUNX2 gene overexpression vector modified exosomes derived from bone marrow mesenchymal stem cells (BMSCs) combined with calcium carbonate scaffold system in bone defect.@*METHODS@#Rabbit BMSCs were used as the research object, and BMSCs were identified by flow cytometry. Construct RUNX2 gene overexpression vector, transfect BMSCs with lentivirus, and collect exosomes by ultracentrifugation. The morphology of exosomes was observed by transmission electron microscope, the expression of exosome marker CD63 was detected by Western blot, and the calcium carbonate scaffold was constructed by three chamber parallel automatic temperature control reaction system. According to whether the RUNX2 gene overexpression vector was transfected or not, the complex of BMSCs and calcium carbonate scaffold was divided into three groups, namely BMSCs group, RUNX2 overexpression group and exosome group. The osteogenic differentiation of BMSCs was detected by oil red O staining and RT-PCR. There were 9 clean adult healthy male New Zealand white rabbits, aged (12.97±1.21) months, with a body weight of (19.3±3.6) kg, with 3 rabbits in each group. The animal model of skull defect was constructed by surgical method, and the repair of bone defect was evaluated by imaging, he staining and Masson staining.@*RESULTS@#The results of flow cytometry showed that the expression of CD29 protein, CD44 protein, CD11b protein and CD45 protein on the surface of BMSCs were 99.5%, 100%, 0.1% and 0.1%, respectively. Transmission electron microscopy showed that the exosomes were bilayer vesicles with a diameter of 50 to 150 nm. Western blot showed that the molecular marker CD63 of exosomes was positive. Oil red O staining showed that the osteogenic differentiation of BMSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group (P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group (P<0.05). MSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group(P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group(P<0.05).@*CONCLUSION@#Compared with RUNX2 gene overexpression vector transfection, extraction of exosomes directly can promote the differentiation of BMSCs into osteoblasts more efficiently, and the combination with calcium carbonate scaffold can better promote the healing of bone defects. So as to provide new ideas and methods for the clinical treatment of bone defects.


Subject(s)
Animals , Humans , Male , Rabbits , Calcium Carbonate/metabolism , Core Binding Factor Alpha 1 Subunit/metabolism , Exosomes/metabolism , Osteogenesis/genetics , RNA, Messenger/metabolism
10.
Chinese Journal of Biotechnology ; (12): 1159-1172, 2022.
Article in Chinese | WPRIM | ID: wpr-927771

ABSTRACT

It is known that low-frequency pulsed electromagnetic fields (PEMFs) can promote the differentiation and maturation of rat calvarial osteoblasts (ROBs) cultured in vitro. However, the mechanism that how ROBs perceive the physical signals of PEMFs and initiate osteogenic differentiation remains unknown. In this study, we investigated the relationship between the promotion of osteogenic differentiation of ROBs by 0.6 mT 50 Hz PEMFs and the presence of polycystin2 (PC2) located on the primary cilia on the surface of ROBs. First, immunofluorescence staining was used to study whether PC2 is located in the primary cilia of ROBs, and then the changes of PC2 protein expression in ROBs upon treatment with PEMFs for different time were detected by Western blotting. Subsequently, we detected the expression of PC2 protein by Western blotting and the effect of PEMFs on the activity of alkaline phosphatase (ALP), as well as the expression of Runx-2, Bmp-2, Col-1 and Osx proteins and genes related to bone formation after pretreating ROBs with amiloride HCl (AMI), a PC2 blocker. Moreover, we detected the expression of genes related to bone formation after inhibiting the expression of PC2 in ROBs using RNA interference. The results showed that PC2 was localized on the primary cilia of ROBs, and PEMFs treatment increased the expression of PC2 protein. When PC2 was blocked by AMI, PEMFs could no longer increase PC2 protein expression and ALP activity, and the promotion effect of PEMFs on osteogenic related protein and gene expression was also offset. After inhibiting the expression of PC2 using RNA interference, PEMFs can no longer increase the expression of genes related to bone formation. The results showed that PC2, located on the surface of primary cilia of osteoblasts, plays an indispensable role in perceiving and transmitting the physical signals from PEMFs, and the promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2. This study may help to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.


Subject(s)
Animals , Rats , Alkaline Phosphatase/metabolism , Electromagnetic Fields , Osteoblasts/metabolism , Osteogenesis/genetics , TRPP Cation Channels/physiology
11.
Journal of Southern Medical University ; (12): 988-996, 2022.
Article in Chinese | WPRIM | ID: wpr-941031

ABSTRACT

OBJECTIVE@#To explore whether the effect of low-frequency pulsed electromagnetic fields (PEMFs) in promoting osteoblast mineralization and maturation is related to the primary cilia, polycystin2 (PC2) and sAC/PKA/CREB signaling pathway.@*METHODS@#We detected the expression levels of PC2, sAC, PKA, CREB and their phosphorylated proteins in primary rat calvarial osteoblasts exposed to 50 Hz 0.6 mT PEMFs for 0, 5, 15, 30, 60, 90, and 120 min. We blocked PC2 function with amiloride hydrochloride and detected the changes in the activity of sAC/PKA/CREB signal pathway and the mineralization and maturation of the osteoblasts. These examinations were repeated in the osteoblasts after specific knockdown of PC2 via RNA interference and were the co-localization of PC2, sAC, PKA, CREB and their phosphorylated proteins with the primary cilia were using immunofluorescence staining. The expressions of PC2 and the signaling proteins of sAC/PKA/CREB pathway were detected after inhibition of primary ciliation by RNA interference.@*RESULTS@#The expression levels of PC2, sAC, p-PKA and p- CREB were significantly increased in the osteoblasts after exposure to PEMFs for different time lengths (P < 0.01). Blocking PC2 function or PC2 knockdown in the osteoblasts resulted in failure of sAC/PKA/CREB signaling pathway activation and arrest of osteoblast mineralization and maturation. PC2, sAC, p-PKA and p-CREB were localized to the entire primary cilia or its roots, but PKA and CREB were not detected in the primary cilia. After interference of the primary cilia, PEMFs exposure no longer caused increase of PC2 expression and failed to activate the sAC/PKA/CREB signaling pathway or promote osteoblast mineralization and maturation.@*CONCLUSION@#PC2, located on the surface of the primary cilia of osteoblasts, can perceive and transmit the physical signals from PEMFs and promote the mineralization and maturation of osteoblasts by activating the PC2/ sAC/PKA/CREB signaling pathway.


Subject(s)
Animals , Rats , Cell Differentiation , Electromagnetic Fields , Osteoblasts , Osteogenesis/genetics , Signal Transduction
12.
International Journal of Oral Science ; (4): 30-30, 2022.
Article in English | WPRIM | ID: wpr-939849

ABSTRACT

Human adipose-derived stem cells (hASCs) are a promising cell type for bone tissue regeneration. Circular RNAs (circRNAs) have been shown to play a critical role in regulating various cell differentiation and involve in mesenchymal stem cell osteogenesis. However, how circRNAs regulate hASCs in osteogenesis is still unclear. Herein, we found circ_0003204 was significantly downregulated during osteogenic differentiation of hASCs. Knockdown of circ_0003204 by siRNA or overexpression by lentivirus confirmed circ_0003204 could negatively regulate the osteogenic differentiation of hASCs. We performed dual-luciferase reporting assay and rescue experiments to verify circ_0003204 regulated osteogenic differentiation via sponging miR-370-3p. We predicted and confirmed that miR-370-3p had targets in the 3'-UTR of HDAC4 mRNA. The following rescue experiments indicated that circ_0003204 regulated the osteogenic differentiation of hASCs via miR-370-3p/HDAC4 axis. Subsequent in vivo experiments showed the silencing of circ_0003204 increased the bone formation and promoted the expression of osteogenic-related proteins in a mouse bone defect model, while overexpression of circ_0003204 inhibited bone defect repair. Our findings indicated that circ_0003204 might be a promising target to promote the efficacy of hASCs in repairing bone defects.


Subject(s)
Animals , Humans , Mice , Adipose Tissue/metabolism , Cell Differentiation/genetics , Cells, Cultured , Histone Deacetylases/metabolism , MicroRNAs/metabolism , Osteogenesis/genetics , RNA, Circular/metabolism , Repressor Proteins/metabolism , Signal Transduction , Stem Cells/metabolism
13.
Journal of Zhejiang University. Science. B ; (12): 529-546, 2022.
Article in English | WPRIM | ID: wpr-939825

ABSTRACT

Mammalian bone is constantly metabolized from the embryonic stage, and the maintenance of bone health depends on the dynamic balance between bone resorption and bone formation, mediated by osteoclasts and osteoblasts. It is widely recognized that circadian clock genes can regulate bone metabolism. In recent years, the regulation of bone metabolism by non-coding RNAs has become a hotspot of research. MicroRNAs can participate in bone catabolism and anabolism by targeting key factors related to bone metabolism, including circadian clock genes. However, research in this field has been conducted only in recent years and the mechanisms involved are not yet well established. Recent studies have focused on how to target circadian clock genes to treat some diseases, such as autoimmune diseases, but few have focused on the co-regulation of circadian clock genes and microRNAs in bone metabolic diseases. Therefore, in this paper we review the progress of research on the co-regulation of bone metabolism by circadian clock genes and microRNAs, aiming to provide new ideas for the prevention and treatment of bone metabolic diseases such as osteoporosis.


Subject(s)
Animals , Circadian Clocks/genetics , Circadian Rhythm/genetics , Mammals/genetics , MicroRNAs/genetics , Osteogenesis/genetics , Osteoporosis/genetics
14.
China Journal of Orthopaedics and Traumatology ; (12): 978-984, 2021.
Article in Chinese | WPRIM | ID: wpr-921928

ABSTRACT

OBJECTIVE@#To explore the effects of siRNA hsa-circ-0000885 modified bone marrow mesenchymal stem cells (BMSCs) on osteogenic differentiation, cell proliferation and apoptosis in order to provide new ideas and methods for the clinical treatment of osteoporosis (OP).@*METHODS@#From September 2018 to February 2020, 13 patients with osteoporosis admitted to our hospital were selected as the research objects, including 11 females and 2 males, with an age of (65.45±10.77) years old. After obtaining the informed consent of patients, peripheral blood tissues were extracted. Then the expression level of cir-cRNA in peripheral blood mononuclear cells(PBMC) was detected by circ RNA chip. The expression of circ RNA was silenced by siRNA technology. The BMSCs were transfected with lentivirus. According to the siRNA interference plasmid hsa-circ-0000885, the cells were divided into the blank group, the empty vector group and the siRNA interference group. After 72 hours of treatment, the cell cycle was detected by flow cytometry, the apoptosis level was detected by AV-PI kit, and the osteogenic differentiation ability of BMSCs was detected by ALP staining.@*RESULTS@#The expression of hsa-circ-0000885 in PBMC of patients with osteoporosis was significantly higher than that of healthy controls (@*CONCLUSION@#The lentivirus mediated siRNA hsa-circ-0000885 plasmid transfected into BMSCs and osteoclast co culture system can promote cell proliferation, inhibit apoptosis and promote osteogenic differentiation of BMSCs, which can be used as a potential therapeutic target for OP patients.


Subject(s)
Aged , Female , Humans , Middle Aged , Apoptosis/genetics , Cell Differentiation , Cell Proliferation/genetics , Cells, Cultured , Coculture Techniques , Lentivirus , Leukocytes, Mononuclear , Mesenchymal Stem Cells , Osteoclasts , Osteogenesis/genetics , RNA, Small Interfering/genetics , Transfection
15.
Acta odontol. latinoam ; 33(2): 125-134, Sept. 2020. graf
Article in English | LILACS | ID: biblio-1130743

ABSTRACT

ABSTRACT Melatonin (MLT) is a potential signaling molecule in the homeostasis of bone metabolism and may be an important mediator of bone formation and stimulation. The aim of this in vitro study was to evaluate the effect of MLT on the viability, mRNA/protein expression and mineralization of pre-osteoblastic cells. The concentrations 5, 2.5, 1, 0.1 and 0.01 mM MLT were tested on pre-osteoblastic cells (MC3T3) compared to control (no MLT), evaluating proliferation and cell viability (C50), gene expression (RT-PCR) and secretion (ELISA) of COL-I and OPN at 24h, 48h and 72h, and the formation of mineral nodules (alizarin red and fast red) after 10 days of treatment. MLT at 5 and 2.5 mM proved to be cytotoxic (C50), so only 0.01, 0.1 and 1 mM were used for the subsequent analyses. OPN mRNA expression increased with MLT at 0.1 mM - 1 mM, which was followed by increased secretion of OPN both at 24h and 72h compared to the remaining groups (p <0.05). COL-I mRNA and COL-1 secretion followed the same pattern as OPN at 0.1 mM MLT at 72h of treatment (p <0.05). Regarding mineralization, all MLT doses (except 1mM) caused an increase (p <0.05) in the formation of mineral nodules compared to the control. Melatonin at 0.01mM - 1mM had a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/ secretion and mineralization, thereby fostering osteogenesis.


RESUMO A melatonina (MLT) é uma molécula potencial de sinalização na homeostase do metabolismo ósseo e pode ser um importante mediador da formação e estimulação óssea. O objetivo deste estudo in vitro foi avaliar o efeito da MLT na viabilidade, na expressão do mRNA da proteína e mineralização de células préosteoblásticas. As concentrações de MLT 5, 2,5, 1, 0,1 e 0,01 mM foram testadas em células pré-osteoblásticas da linhagem MC3T3 em comparação ao controle (sem MLT), avaliando a proliferação e a viabilidade celular (C50), expressão gênica (rtPCR) e secreção (Elisa) de Colágeno tipo 1 (COL-I) e osteopontina (OPN) às 24, 48 e 72 horas, além da formação de nódulos minerais por meio do teste vermelho de Alizarina fast red após 10 dias de tratamento. MLT a 5 e 2,5 mM provou ser tóxico (C50). Portanto, as concentrações de 0,01, 0,1 e 1 mM foram utilizadas para as análises subsequentes. A expressão do mRNA da OPN aumentou com MLT a 0,1 mM-1mM, seguida pela secreção aumentada de OPN às 24 e 72 horas em comparação aos demais grupos (p<0,05). O mRNA de COL-I e a secreção de COL-I seguiram o mesmo padrão do OPN a 0,1 mM de MLT em 72 horas de tratamento (p<0,05). Em relação à mineralização, todas as doses de MLT (exceto 1mM) causaram aumento (p<0,05) na formação de nódulos minerais em comparação ao controle. A MLT na concentração entre 0,01mM a 1 mM teve um efeito estimulador sobre os osteoblastos, ao regular positivamente a expressão e secreção de COL-I e OPN, além da mineralização, favorecendo a osteogênese.


Subject(s)
Humans , Osteoblasts/drug effects , Osteogenesis/drug effects , Osteogenesis/genetics , Peptide Fragments/metabolism , Cell Differentiation/drug effects , Cell Differentiation/genetics , Matrix Metalloproteinase 2/metabolism , Osteopontin/metabolism , Melatonin/pharmacology , Osteoblasts/metabolism , Peptide Fragments/genetics , RNA, Messenger/genetics , Enzyme-Linked Immunosorbent Assay , Gene Expression , Gene Expression Regulation, Developmental/drug effects , Matrix Metalloproteinase 2/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Osteopontin/genetics , Real-Time Polymerase Chain Reaction
16.
Neotrop. ichthyol ; 16(2): [e170164], jun. 2018. tab, ilus, graf
Article in English | LILACS, VETINDEX | ID: biblio-948591

ABSTRACT

Comparisons of the external morphology and analysis of osteological features of the postcranial and appendicular skeletons of three southwestern Atlantic flatfish species of the genus Paralichthys (P. isosceles, P. orbignyanus and P. patagonicus) were carried out. Bones are described, and detailed morphological, morphometric and meristic characteristics of these flounders are given in order to provide information about the external and internal morphology of three species of Paralichthys occurring in the south-west Atlantic waters that add new information and will help regarding within the framework of a phylogenetic study of the group. Interspecific differences were found in the number of vertebrae and intermuscular bones, as well as in the morphology and morphometry of vertebrae, caudal skeletons, pectoral and pelvic girdle bones. Relationships between bones are discussed and bone characteristics compared with those found in other species of Paralichthys and in other pleuronectiform species. The position of Paralichthys isosceles within Paralichthys is discussed, along with other congeners such as P. triocellatus and P. oblongus.(AU)


Se llevaron a cabo comparaciones de la morfología externa y el análisis de las características osteológicas de los esqueletos postcraneal y apendicular de tres especies de peces planos del Atlántico sudoccidental del género Paralichthys (P. isosceles, P. orbignyanus y P. patagonicus). Se describen los huesos, y se proporcionan características morfológicas, morfométricas y merísticas detalladas de estos lenguados con el fin de aportar información sobre la morfología externa e interna de tres especies de Paralichthys presentes en el Atlántico sudoccidental. Esta nueva información contribuirá al marco de un estudio filogenético del grupo. Se encontraron diferencias interespecíficas en el número de vértebras y huesos intermusculares, así como en la morfología y morfometría de las vértebras, los esqueletos caudales, los huesos de las cinturas pectoral y pélvica. Se discuten las relaciones entre los huesos y las características óseas en comparación con las encontradas en otras especies de Paralichthys y de otros Pleuronectiformes. Se discute la posición de Paralichthys isosceles dentro del género Paralichthys, junto con otros congéneres como P. oblongus y P. triocellatus.(AU)


Subject(s)
Animals , Osteogenesis/genetics , Flatfishes/genetics , Flatfishes/anatomy & histology
17.
Actual. osteol ; 13(3): 207-213, Sept - DIc. 2017. ilus, graf
Article in English | LILACS | ID: biblio-1117111

ABSTRACT

Osteocytes are the most abundant bone cell and are formed when osteoblasts become embedded in the bone matrix. Through changes in gene expression and paracrine effects, osteocytes regulate the number of osteoblasts, bone forming cells, and osteoclasts, bone resorbing cells, which are needed to maintain bone mass. MLO-Y4 is the better characterized osteocytic cell line; however, lacks expression of sclerostin, the product of the SOST gene, which is fundamental for osteocyte function and blocks bone formation. With the objective to isolate MLO-Y4 clones with different gene expression profiles, we performed cultures at very low density of MLO-Y4 cells stably transfected with nuclear green fluorescent protein (MLOnGFP). Cell morphology was visualized under a fluorescence microscope. Once the cells reached 80% confluency, RNA was extracted and quantitative real time PCR was performed. Clones exhibit different sizes and morphology, with some cells showing a spindle-like shape and others with abundant projections and a star-like shape. Gene expression also differed among clones. However, none of the clones examined expressed SOST. We conclude that the MLO-nGFP clones constitute a useful tool to study osteocyte differentiation and the role of osteocytes in the control of bone formation and resorption in vitro. (AU)


Los osteocitos son las células más abundantes del hueso y se forman cuando los osteoblastos se encuentran rodeados de matriz ósea. A través de cambios en la expresión génica y efectos paracrinos, los osteocitos controlan el número de osteoblastos que forman el hueso, y osteoclastos que resorben el hueso, células necesarias para mantener la masa ósea. Las células MLO-Y4 son la línea celular osteocítica más investigada; sin embargo, no expresan esclerostina, el pro esclerostina, el producto del gen SOST que bloquea la formación ósea y es indispensable para la función de los osteocitos. Con el objetivo de aislar clones de las células MLO-Y4 con diferentes perfiles de expresión génica, realizamos cultivos a muy baja densidad de las células transfectadas en forma estable con proteína verde fluorescente nuclear (MLO-nGFP). La morfología celular fue evaluada utilizando un microscopio de fluorescencia. Una vez que las células alcanzaron el 80% de confluencia, el ARN fue extraído y analizado por PCR cuantitativa en tiempo real. Las células de los diferentes clones tienen diferentes tamaños y morfología, algunas células son fusiformes y otras con proyecciones citoplasmáticas abundantes y en forma de estrella. La expresión de los genes también varió en los distintos clones. Sin embargo, ninguno de ellos expresó SOST. En conclusión, los clones de las células MLO-nGFP constituyen una herramienta útil para estudiar la diferenciación de los osteocitos y el rol de estas células en el control de la formación y resorción ósea in vitro. (AU)


Subject(s)
Humans , Male , Female , Osteoblasts/cytology , Osteoclasts/cytology , Osteocytes/cytology , Cell Line , Clone Cells/cytology , Osteoblasts/metabolism , Osteoclasts/metabolism , Osteocytes/metabolism , Osteogenesis/genetics , Bone Resorption/genetics , In Vitro Techniques , RNA/analysis , Gene Expression , Polymerase Chain Reaction , Collagen/genetics , Alkaline Phosphatase/metabolism , Fluorescence , Anti-Bacterial Agents/administration & dosage
18.
Actual. osteol ; 13(3): 233-242, Sept - DIc. 2017. ilus, tab
Article in Spanish | LILACS | ID: biblio-1117496

ABSTRACT

La displasia fibrosa ósea es un trastorno no hereditario del desarrollo esquelético caracterizado por una proliferación anormal de fibroblastos y diferenciación deficiente de osteoblastos que conduce a un reemplazo del tejido óseo esponjoso por tejido conectivo fibroso. Es producida por una mutación somática activadora del gen GNAS1 que induce una activación y proliferación de células mesenquimales indiferenciadas con formación de tejido fibroso y trabéculas óseas anómalas. Existen formas monostóticas, poliostóticas y craneofaciales con diversos grados de dolor, deformidades y fracturas óseas, aunque muchos casos son asintomáticos. En ocasiones se producen quistes óseos aneurismáticos, hemorragias, compromisos neurológicos y raramente osteosarcomas. Algunos casos se asocian a síndrome de McCune-Albright, síndrome de Mazabraud y a osteomalacia por hipofosfatemia por pérdida tubular renal inducida por el FGF23 producido por el tejido displásico. Los hallazgos en las radiografías convencionales son característicos, aunque variables y de carácter evolutivo. La gammagrafía ósea es la técnica de imagen con mayor sensibilidad para determinar la extensión de la enfermedad. El diagnóstico diferencial incluye múltiples lesiones óseas de características similares y en raras ocasiones se requiere biopsia ósea o estudio genético para confirmarlo. No existe un consenso unánime acerca del abordaje terapéutico de estos pacientes, razón por la cual es necesario un enfoque multidisciplinario. La conducta puede ser expectante o quirúrgica según el tipo de lesiones y es importante el manejo del dolor y de las endocrinopatías asociadas. La mayor experiencia publicada se refiere al uso de bifosfonatos y, más recientemente, denosumab. Los tratamientos actuales son insuficientes para modificar el curso de la enfermedad y es necesario el desarrollo de nuevas moléculas que actúen específicamente en el gen GNAS1 o sobre las células mesenquimales afectadas. (AU)


Fibrous dysplasia of bone is a noninherited developmental anomaly of bone characterized by abnormal proliferation of fibroblasts and differentiation of osteoblasts that cause a replacement of trabeculous bone by fibrous connective tissue. It is caused by a somatic mutation in the GNAS1 gene, which induces an undifferentiated mesenquimal cells activation and proliferation with formation of fibrous tissue and abnormal osseous trabeculae. There are monostotic, polyostotic and craniofacial variants with different grades of bone pain, deformities and fractures, although many cases remain asymptomatic. Aneurysmal bone cysts, bleeding, neurological compromise and infrequently osteosarcoma are possible complications. Some cases are associated to McCune-Albright syndrome, Mazabraud syndrome or hypophosphatemia and osteomalacia due to to renal tubular loss induced by FGF23 produced by dysplastic tissue. The findings on conventional radiography are characteristic although variable and evlolve with time. Bone scintigraphy is the most sensitive technique to evaluate the extent of disease. Differential diagnosis include several osseous lesions of similar appearance and, in some cases, bone biopsy or genetic testing may be necessary. Today, there is no consensus regarding the therapeutic approach for these patients and it is necessary a multidisciplinary medical team. Watchful waiting or surgical interventions can be indicated, depending on the type of bone lesions. Bone pain and associated endocrinopathies management are very important. Most published experience refers to the use of bisphosphonates and, more recently, denosumab. Current treatments are insufficient to modify the natural curse of the disease and therefore, new molecules with specific action on GNAS1 gene or affected mesenchymal cells are necessary. (AU)


Subject(s)
Humans , Male , Female , Child, Preschool , Child , Adolescent , Adult , Young Adult , Fibrous Dysplasia of Bone/etiology , Fibrous Dysplasia of Bone/drug therapy , Osteogenesis/genetics , Osteomalacia/complications , Congenital Abnormalities , Vitamin D/therapeutic use , Osteosarcoma/etiology , Calcium/therapeutic use , Hypophosphatemia/blood , Bone Cysts, Aneurysmal/etiology , Diagnosis, Differential , Diphosphonates/administration & dosage , Diphosphonates/adverse effects , Fractures, Bone/pathology , Mesenchymal Stem Cells/pathology , Pain Management , Fibrous Dysplasia, Monostotic/etiology , Fibrous Dysplasia of Bone/genetics , Fibrous Dysplasia of Bone/blood , Fibrous Dysplasia of Bone/diagnostic imaging , Fibrous Dysplasia, Polyostotic/etiology , Fibrous Dysplasia, Polyostotic/diagnostic imaging , Craniofacial Fibrous Dysplasia/etiology , Mutation/genetics
19.
Biol. Res ; 50: 43, 2017. tab, graf
Article in English | LILACS | ID: biblio-950890

ABSTRACT

BACKGROUND: Understanding the molecular basis underlying the formation of bone-forming osteocytes and lipid-storing adipocytes will help provide insights into the cause of disorders originating in stem/progenitor cells and develop therapeutic treatments for bone- or adipose-related diseases. In this study, the role of RGS2 and RGS4, two members of the regulators of G protein signaling (RGS) family, was investigated during adipogenenic and osteogenenic differentiation of human mesenchymal stem cells (hMSCs). RESULTS: Expression of RGS2 and RGS4 were found to be inversely regulated during adipogenesis induced by dexamethasone (DEX) and 3-isobutyl-methylxanthine, regardless if insulin was present, with RGS2 up-regulated and RGS4 down-regulated in response to adipogenic induction. RGS2 expression was also up-regulated during osteogenesis at a level similar to that induced by treatment of DEX alone, a shared component of adipogenic and osteogenic differentiation inducing media, but significantly lower than the level induced by adipogenic inducing media. RGS4 expression was down-regulated during the first 48 h of osteogenesis but up-regulated afterwards, in both cases at levels similar to that induced by DEX alone. Expression knock-down using small interfering RNA against RGS2 resulted in decreased differentiation efficiency during both adipogenesis and osteogenesis. On the other hand, expression knock-down of RGS4 also resulted in decreased adipogenic differentiation but increased osteogenic differentiation. CONCLUSIONS: RGS2 and RGS4 are differentially regulated during adipogenic and osteogenic differentiation of hMSCs. In addition, both RGS2 and RGS4 play positive roles during adipogenesis but opposing roles during osteogenesis, with RGS2 as a positive regulator and RGS4 as a negative regulator. These results imply that members of RGS proteins may play multifaceted roles during human adipogenesis and osteogenesis to balance or counterbalance each other's function during those processes.


Subject(s)
Humans , Osteocytes/cytology , Osteogenesis/physiology , Gene Expression Regulation/physiology , RGS Proteins/metabolism , Adipogenesis/physiology , Mesenchymal Stem Cells/cytology , Osteogenesis/genetics , Time Factors , Gene Expression Regulation/genetics , RGS Proteins/genetics , Adipogenesis/genetics
20.
Belo Horizonte; s.n; 2016. 82 p. ilus.
Thesis in Portuguese | LILACS, BBO | ID: biblio-916177

ABSTRACT

Os enxertos de origem autógena são osteogênicos e possuem como vantagens a produção de tecido ósseo originário do próprio organismo. A sua limitação é a grande morbidade cirúrgica. O biovidro é uma cerâmica bioativa com disponibilidade ilimitada que leva a uma cirurgia com menor morbidade. O presente estudo teve como objetivo avaliar o biovidro teste durante o reparo ósseo e compará-lo com outros substitutos ósseos, em defeitos cirúrgicos experimentais em tíbia de ratos. O biovidro teste (CEELBIO, Belo Horizonte, Brasil), previamente caracterizado foi comparado com o Biogran® (Biomet 3i Inovattions Inc., Palm Beach Gardens, USA), através de espectroscopia na região do infravermelho, microscopia eletrônica de varredura (MEV), espectroscopia de dispersão em energia (EDS) e fluorescência de Rx. Após as análises estruturais, deu-se início ao estudo in vivo. Foram utilizados 120 ratos (rattus norvegicus albinus, Wistar) machos, com aproximadamente 7 a 8 semanas. Os defeitos ósseos foram realizados na tíbia direita dos animais e preenchidos de acordo com a seguinte divisão: Grupo I- controle negativo, sem preenchimento; Grupo II- foi preenchido por Biogran®; Grupo III- preenchido por biovidro teste; Grupo IVcontrole positivo, com preenchimento com osso autógeno. Nos períodos de 7, 14, 21, 28, 49 e 70 dias pós-operatório, os animais foram eutanasiados e os processos de mineralização óssea e reparo foram analisados através de histomorfometria (% de osso neoformado no sítio do defeito). Níveis de BMP-2 foram mensurados através de ensaio de ELISA. Análise estatística foi realizada utilizando programa SPSS (versão 20.0, SPSS Inc., Chicago, USA). Os resultados da análise histológica demonstraram que, no controle negativo, houve neoformação óssea até os 14 dias (20,40%, p<0,001) e depois houve reabsorção em até 21 dias (6,60%, p<0,001). No grupo de Biogran®, houve uma neoformação óssea junto aos grãos em 7 dias (34,20%, p= 0,019) e que se manteve enquanto o material estava presente nos defeitos em 70 dias (15,67%, p= 0,048). O biovidro teste foi reabsorvido totalmente até 21 dias e os picos de osso neoformado foram observados em 7 (21,00%, p= 0,019) e 49 dias (15,60%, p= 0,036). Nesse grupo, células semelhantes a macrófagos, dispostas em lençol, foram visualizadas junto a tecido ósseo neoformado. Quanto ao controle positivo, o osso autógeno foi totalmente reabsorvido em até 14 dias e o pico de formação óssea se deu nesse 9 mesmo momento, em 14 dias, (40,80%, p<0,001), mostrando-se, nos tempos subsequentes, similar ao controle negativo. Células gigantes multinucleadas foram encontradas em áreas de remodelação óssea, junto ao Biogran® e ao biovidro teste. Os níveis de BMP-2 no grupo controle negativo se mostraram maiores nos tempos de 7 (418,80pg/mL, p= 0,871) e 28 dias (346,36 pg/mL, p= 0,035). No grupo Biogran® o pico de BMP-2 se deu em 7 dias (471,95 pg/mL, p= 0,871). O biovidro teste teve seu pico de liberação de BMP-2 em 7 dias também (471,39 pg/mL, p= 0,871). Já o controle positivo apresentou nível de BMP-2 em maior quantidade nos tempos de 7 (346,55 pg/mL, p= 0,871) e 21 dias (407,57 pg/mL, p= 0,300). O biovidro é um material degradável, e com características biológicas de osteoindução e osteocondução


The autogenous origin grafts are osteogenic and have the advantage of producing bone tissue originated from the body itself. Its limitation is the great surgical morbidity. The bioglass is a bioactive ceramic with unlimited availability that leads to a surgery with less morbidity. This study aimed to evaluate the bioglass test during bone repair and to compare it with other bone substitutes in experimental surgical defects in the tibia of rats. The bioglass test (CEELBIO, Belo Horizonte, Brazil), has been characterized and compared to the Biogran® (Biomet 3i Inovattions Inc., Palm Beach Gardens, USA) by spectroscopy in the infrared, scanning electron microscopy (SEM), EDS spectroscopy and Rx fluorescence. The study in vivo has been started after the structural analysis. 120 rats (Rattus norvegicus albinus, Wistar rats), with approximately 7 to 8 weeks, were used. The bone defects were performed in the right tibia of animals and filled according to the following breakdown: Group I- negative control without filling; Group II- filled by Biogran®; Group III- filled with bioglass test; IV- positive control group, with filling of autogenous bone. At 7, 14, 21, 28, 49 and 70 postoperative days, the animals were euthanized and bone mineralization processes and repair were analyzed by histomorphometry (% of newly formed bone in the defect site). BMP-2 levels were measured by ELISA assay. Statistical analysis was performed using SPSS (version 20.0, SPSS Inc., Chicago, USA). The results of the histological analysis showed that in the negative control there was no bone growth up to 14 days (20.40%, p <0.001) and after reabsorption, within 21 days (6.60%, p <0.001). In Biogran® group, there was new bone formation along the grain in 7 days (34.20%, p = 0.019) and that was maintained as the material was present in shortcomings in 70 days (15.67%, p = 0.048) . The test bioglass was completely reabsorbed within 21 days; new bone formation and peaks were observed in 7 (21.00%, p = 0.019) and 49 days (15.60%, p = 0.036). In this group, macrophage-like cells arranged in sheets, were viewed with the newly formed bone. On the positive control, autologous bone was completely reabsorbed within 14 days, and the peak bone formation occurred at the same moment, within 14 days (40.80%, p <0.001), showing, in subsequent occasions, similar to control negative. Multinucleated giant cells were found in areas of bone remodeling, with the Biogran® and test bioglass. BMP-2 levels in the negative control group were higher in 7 (418,80pg / ml, p = 11 0.871) and 28 days (346.36 pg / ml, p = 0.035). In Biogran® group peak BMP-2 occurred within 7 days (471.95 pg / ml, p = 0.871). The test bioglass had its peak BMP-2 release in 7 days as well (471.39 pg / ml, p = 0.871). The positive control showed BMP-2 level in greater quantities in 7 (346.55 pg / ml, p = 0.871) and 21 days (407.57 pg / ml, p = 0.300). The test bioglass is a biodegradable material, with biological characteristics of osteoinduction and osteoconduction


Subject(s)
Animals , Rats , Bone Regeneration , Ceramics/analysis , Glass/analysis , Osteogenesis/genetics , Fluorescence , Microscopy, Electrochemical, Scanning/statistics & numerical data , Spectrum Analysis/statistics & numerical data
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