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1.
Braz. j. biol ; 84: e251970, 2024. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1345559

ABSTRACT

Abstract In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a "Michaelian" behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.


Resumo Para melhor compreender o processo de ossificação em anuros, nosso estudo foi conduzido em girinos e adultos de Lithobates catesbeianus. Nesse sentido, as propriedades cinéticas da fosfatase alcalina com p-nitrofenilfosfato (pNPP) e pirofosfato (PPi) foram caracterizadas, e as atividades enzimáticas das fosfatases ácida e ácida tartarato resistente foram avaliadas. Os extratos enzimáticos foram obtidos de fêmur de girinos e adultos, divididos em epífise e diáfise. Após a homogeneização as amostras foram submetidas à centrifugação diferencial para obter membrana celular e, em seguida, ao tratamento com fosfolipase C (PIPLC), para remover as proteínas de membrana ancoradas por fosfatidilinositol. A média da atividade específica da fosfatase alcalina, liberada pela PIPLC de Bacillus cereus, para a hidrólise de pNPP (pH 10,5) nas diferentes regiões do fêmur e idades dos animais foi de 1.142,57 U.mg-1, enquanto para a hidrólise do PPi (pH 8,0) foi de 1.433,82 U.mg-1. Entre os compostos testados para a atividade enzimática, o de maior influência foi o EDTA, inibindo aproximadamente 67% e 77% das atividades de pNPPase e PPase, respectivamente. Quanto aos parâmetros cinéticos, a enzima apresentou comportamento Michaeliano para a hidrólise dos dois substratos. O valor de Km foi de 0,6 mM para a atividade de pNPPase e variou de 0,01 a 0,11 para a atividade de PPase, indicando uma maior afinidade por esse substrato. O estudo da hidrólise de pNPP e PPi revelou que o pH ótimo aparente de atuação foi de 10,5 para o pNPP e 8,0 para o PPi, próximo ao fisiológico, sendo que esse é considerado o substrato natural da fosfatase alcalina. Os resultados demonstram que, apesar do tipo de ossificação que ocorre, a mesma enzima ou isoenzimas, atuam nos diferentes locais do osso e estágios de vida dos anuros. A similaridade dos estudos com os realizados com outros vertebrados apontam que os anuros podem ser considerados excelentes modelos animais para o estudo da calcificação biológica.


Subject(s)
Animals , Osteogenesis , Alkaline Phosphatase/metabolism , Rana catesbeiana , Bone and Bones/metabolism , Kinetics
2.
Braz. j. biol ; 83: e246592, 2023. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1339408

ABSTRACT

Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.


Resumo As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a análise. Não houve variações nas leituras entre as amostras estudadas, concluindo-se que a FT-Raman não atendeu às expectativas nas condições estudadas.


Subject(s)
Animals , Rats , Mesenchymal Stem Cells , Osteogenesis , Polyesters , Spectrum Analysis, Raman , Culture Media, Conditioned , Cell Proliferation , Tissue Scaffolds
3.
J. Health Biol. Sci. (Online) ; 10(1): 1-4, 01/jan./2022. ilus
Article in Portuguese | LILACS | ID: biblio-1368288

ABSTRACT

Introdução: Osteogênese Imperfeita (OI) é uma doença genética rara com fragilidade óssea. A classificação inclui muitos tipos. Além do risco de recorrência, o manejo pode variar com o tipo de OI. Relato do caso: Apresentamos um paciente do sexo masculino nascido com 39 semanas, de pais não consanguíneos e saudáveis. A hidrocefalia foi diagnosticada no pré-natal. Com 50 dias de vida, detectamos muitas fraturas e calos ósseos. O teste molecular identificou uma deleção em homozigose do éxon 4 do gene WNT1. Considerações finais: Concluímos que o caso apresentado tinha características clínicas de OI XV, e o teste molecular foi fundamental para o diagnóstico preciso e aconselhamento genético.


Introduction: Osteogenesis Imperfecta (OI) is a rare genetic disease with bone fragility. The classification includes many types. In addition, the risk of a recurrence, the management can vary with the kind of OI. Case report: We report a male patient born at 39 weeks from non-consanguineous healthy parents. The patient was diagnosed with Hydrocephalus at prenatal. At 50 days of life, we detected many fractures and bone calluses. The molecular test identified a homozygous deletion of exon 4 of the WNT1 gene. Final considerations: We conclude this case had clinical features of OI XV, and the molecular test was fundamental for the precise diagnosis and the genetic counseling.


Subject(s)
Osteogenesis Imperfecta , Osteogenesis , Patients , Prenatal Care , Sex , Infant, Premature , Fractures, Bone , Genetic Counseling , Genetics , Genetic Diseases, Inborn , Hydrocephalus , Men
4.
Rev. ADM ; 79(3): 165-176, mayo-jun. 2022. ilus, tab
Article in Spanish | LILACS | ID: biblio-1378976

ABSTRACT

Introducción: El hueso, reservorio de minerales y moléculas orgánicas, es un tejido dinámico que detecta y se adapta a las cargas mecánicas de los órganos y tejidos del cuerpo, el cual mantiene la estructura ósea del esqueleto durante el crecimiento y a través de la vida del ser humano. Las células óseas son sensibles a las cargas mecánicas y microvibra- ciones que recibe el esqueleto. Objetivo: El propósito de este estudio fue realizar una revisión sistemática acerca de los efectos que ejerce la microvibración de alta frecuencia-baja intensidad, en osteocitos cultivados in vitro sobre la síntesis de factores solubles, con el propósito de entender si la microvibración tiene influencia en la aceleración del movimiento dentario. Material y métodos: Se realizó una búsqueda de artículos de revisión de osteocitos y otras células óseas in vitro, a través de la estrategia PICO (Paciente, Intervención, Comparación, Resultado [Outcome]), con el empleo de palabras clave como: «os- teocitos¼, «microvibración¼, «remodelación¼, «osteoclastogénesis¼, «citocinas¼ y «osteoblastos¼. Se estructuró por medio de PRISMA (informe de revisiones sistemáticas y meta-análisis). La captación de datos finales se hizo por medio del método de puntuación de calidad Jadad y Cochrane (modelo de correlación) como herramientas para evaluar el riesgo de sesgo de cada uno de los artículos. Se incluyeron 11 artículos con alta calidad metodológica. Resultados: La mayoría de los experimentos in vitro demostraron que la microvibración tuvo un aumento estadísticamente significativo en la proliferación y dife- renciación de las células madre mesenquimales (MSC), en osteoblastos (MC3T3-E1), en la expresión de proteínas para inducir osteogénesis y en los osteocitos (MLO-Y4). Asimismo, sobrerregularon la expresión de osteoprotegerina (OPG), prostaglandina (PGE2) y óxido nitroso (NO) al alterar y regular los factores solubles como las citocinas, factores de crecimiento y quimiocinas, de las demás células, además de mostrar una disminución en la actividad de los osteoclastos (RAW246.7) en la resorción ósea. Conclusión: La microvibración induce remodelación ósea. Los osteocitos son sensibles a los estímulos mecánicos y producen factores solubles para inducir la remodelación ósea, razón por la cual se emplea la microvibración como una terapia innovadora y prometedora, no invasiva y no farmacológica en la estimulación de la formación ósea de la superficie del hueso (AU)


Subject(s)
Humans , Osteogenesis , Vibration , Bone Remodeling , Osteocytes , Bone Resorption , Analysis of Variance , Cytokines , Culture Media , RANK Ligand
5.
Article in English | WPRIM | ID: wpr-929152

ABSTRACT

Aging of craniofacial skeleton significantly impairs the repair and regeneration of trauma-induced bony defects, and complicates dental treatment outcomes. Age-related alveolar bone loss could be attributed to decreased progenitor pool through senescence, imbalance in bone metabolism and bone-fat ratio. Mesenchymal stem cells isolated from oral bones (OMSCs) have distinct lineage propensities and characteristics compared to MSCs from long bones, and are more suited for craniofacial regeneration. However, the effect of epigenetic modifications regulating OMSC differentiation and senescence in aging has not yet been investigated. In this study, we found that the histone demethylase KDM4B plays an essential role in regulating the osteogenesis of OMSCs and oral bone aging. Loss of KDM4B in OMSCs leads to inhibition of osteogenesis. Moreover, KDM4B loss promoted adipogenesis and OMSC senescence which further impairs bone-fat balance in the mandible. Together, our data suggest that KDM4B may underpin the molecular mechanisms of OMSC fate determination and alveolar bone homeostasis in skeletal aging, and present as a promising therapeutic target for addressing craniofacial skeletal defects associated with age-related deteriorations.


Subject(s)
Aging , Cell Differentiation , Facial Bones/physiology , Humans , Jumonji Domain-Containing Histone Demethylases/genetics , Mesenchymal Stem Cells/cytology , Osteogenesis , Osteoporosis
6.
Article in English | WPRIM | ID: wpr-929149

ABSTRACT

Sphingosine-1-phosphate (S1P) is an important lipid mediator that regulates a diverse range of intracellular cell signaling pathways that are relevant to tissue engineering and regenerative medicine. However, the precise function of S1P in dental pulp stem cells (DPSCs) and its osteogenic differentiation remains unclear. We here investigated the function of S1P/S1P receptor (S1PR)-mediated cellular signaling in the osteogenic differentiation of DPSCs and clarified the fundamental signaling pathway. Our results showed that S1P-treated DPSCs exhibited a low rate of differentiation toward the osteogenic phenotype in association with a marked reduction in osteogenesis-related gene expression and AKT activation. Of note, both S1PR1/S1PR3 and S1PR2 agonists significantly downregulated the expression of osteogenic genes and suppressed AKT activation, resulting in an attenuated osteogenic capacity of DPSCs. Most importantly, an AKT activator completely abrogated the S1P-mediated downregulation of osteoblastic markers and partially prevented S1P-mediated attenuation effects during osteogenesis. Intriguingly, the pro-inflammatory TNF-α cytokine promoted the infiltration of macrophages toward DPSCs and induced S1P production in both DPSCs and macrophages. Our findings indicate that the elevation of S1P under inflammatory conditions suppresses the osteogenic capacity of the DPSCs responsible for regenerative endodontics.


Subject(s)
Cell Differentiation , Cell Proliferation , Cells, Cultured , Dental Pulp/metabolism , Lysophospholipids , Osteogenesis , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Sphingosine/analogs & derivatives , Stem Cells
7.
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 , DNA Methylation , Dental Pulp , Genomic Imprinting , Humans , Osteogenesis/genetics , Stem Cells
8.
Article in English | WPRIM | ID: wpr-929137

ABSTRACT

Ginsenoside Rb1, the effective constituent of ginseng, has been demonstrated to play favorable roles in improving the immunity system. However, there is little study on the osteogenesis and angiogenesis effect of Ginsenoside Rb1. Moreover, how to establish a delivery system of Ginsenoside Rb1 and its repairment ability in bone defect remains elusive. In this study, the role of Ginsenoside Rb1 in cell viability, proliferation, apoptosis, osteogenic genes expression, ALP activity of rat BMSCs were evaluated firstly. Then, micro-nano HAp granules combined with silk were prepared to establish a delivery system of Ginsenoside Rb1, and the osteogenic and angiogenic effect of Ginsenoside Rb1 loaded on micro-nano HAp/silk in rat calvarial defect models were assessed by sequential fluorescence labeling, and histology analysis, respectively. It revealed that Ginsenoside Rb1 could maintain cell viability, significantly increased ALP activity, osteogenic and angiogenic genes expression. Meanwhile, micro-nano HAp granules combined with silk were fabricated smoothly and were a delivery carrier for Ginsenoside Rb1. Significantly, Ginsenoside Rb1 loaded on micro-nano HAp/silk could facilitate osteogenesis and angiogenesis. All the outcomes hint that Ginsenoside Rb1 could reinforce the osteogenesis differentiation and angiogenesis factor's expression of BMSCs. Moreover, micro-nano HAp combined with silk could act as a carrier for Ginsenoside Rb1 to repair bone defect.


Subject(s)
Alginates/pharmacology , Animals , Bone Regeneration , Cell Differentiation , Durapatite/pharmacology , Ginsenosides , Osteogenesis , Rats , Silk/pharmacology , Tissue Scaffolds
9.
Article in English | WPRIM | ID: wpr-929133

ABSTRACT

Neural crest-derived mesenchymal stem cells (MSCs) are known to play an essential function during tooth and skeletal development. PRX1+ cells constitute an important MSC subtype that is implicated in osteogenesis. However, their potential function in tooth development and regeneration remains elusive. In the present study, we first assessed the cell fate of PRX1+ cells during molar development and periodontal ligament (PDL) formation in mice. Furthermore, single-cell RNA sequencing analysis was performed to study the distribution of PRX1+ cells in PDL cells. The behavior of PRX1+ cells during PDL reconstruction was investigated using an allogeneic transplanted tooth model. Although PRX1+ cells are spatial specific and can differentiate into almost all types of mesenchymal cells in first molars, their distribution in third molars is highly limited. The PDL formation is associated with a high number of PRX1+ cells; during transplanted teeth PDL reconstruction, PRX1+ cells from the recipient alveolar bone participate in angiogenesis as pericytes. Overall, PRX1+ cells are a key subtype of dental MSCs involved in the formation of mouse molar and PDL and participate in angiogenesis as pericytes during PDL reconstruction after tooth transplantation.


Subject(s)
Animals , Cell Differentiation , Mesenchymal Stem Cells , Mice , Molar , Osteogenesis/physiology , Periodontal Ligament
10.
Article in English | WPRIM | ID: wpr-929132

ABSTRACT

Distraction osteogenesis (DO) is widely used for bone tissue engineering technology. Immune regulations play important roles in the process of DO like other bone regeneration mechanisms. Compared with others, the immune regulation processes of DO have their distinct features. In this review, we summarized the immune-related events including changes in and effects of immune cells, immune-related cytokines, and signaling pathways at different periods in the process of DO. We aim to elucidated our understanding and unknowns about the immunomodulatory role of DO. The goal of this is to use the known knowledge to further modify existing methods of DO, and to develop novel DO strategies in our unknown areas through more detailed studies of the work we have done.


Subject(s)
Bone Regeneration/physiology , Bone and Bones , Osteogenesis/physiology , Osteogenesis, Distraction/methods , Tissue Engineering
11.
Article in English | WPRIM | ID: wpr-928654

ABSTRACT

Magnetic nanoparticles (MNP) have been widely used as biomaterials due to their unique magnetic responsiveness and biocompatibility, which also can promote osteogenic differentiation through their inherent micro-magnetic field. The MNP composite scaffold retains its superparamagnetism, which has good physical, mechanical and biological properties with significant osteogenic effects and . Magnetic field has been proved to promote bone tissue repair by affecting cell metabolic behavior. MNP composite scaffolds under magnetic field can synergically promote bone tissue repair and regeneration, which has great application potential in the field of bone tissue engineering. This article summarizes the performance of magnetic composite scaffold, the research progress on the effect of MNP composite scaffold with magnetic fields on osteogenesis, to provide reference for further research and clinical application.


Subject(s)
Cell Differentiation , Magnetite Nanoparticles , Osteogenesis , Tissue Engineering , Tissue Scaffolds
12.
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 , Calcium Carbonate/metabolism , Core Binding Factor Alpha 1 Subunit/metabolism , Exosomes/metabolism , Humans , Male , Osteogenesis/genetics , RNA, Messenger/metabolism , Rabbits
13.
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)
Alkaline Phosphatase/metabolism , Animals , Electromagnetic Fields , Osteoblasts/metabolism , Osteogenesis/genetics , Rats , TRPP Cation Channels/physiology
14.
Article in English | WPRIM | ID: wpr-922535

ABSTRACT

OBJECTIVE@#In traditional Chinese medicine, the herbal pair, Radix Achyranthis Bidentatae (RAB) and Eucommiae Cortex (EC), is widely used to treat osteoporosis. Herein, we determined whether this herbal pair can be used to ameliorate glucocorticoid (GC)-induced osteoporosis (GIOP) and find its optimal dosage in zebrafish.@*METHODS@#The characteristics of the aqueous extract of RAB and EC were separately characterized using high-performance liquid chromatography. Osteoporosis was induced in 5-day post-fertilization zebrafish larvae by exposing them to 10 μmol/L dexamethasone (Dex) for 96 h. Seven combinations of different ratios of RAB and EC were co-administered. Treatment efficacy was determined by calculating zebrafish vertebral area and sum brightness, via alizarin red staining, and by detecting alkaline phosphatase (ALP) activity. Multiple regression analysis was conducted to test the optimal dosage ratio.@*RESULTS@#According to the Chinese Pharmacopoeia (2015), β-ecdysone (β-Ecd) is a major bioactive marker in RAB extract, while pinoresinol diglucoside (PDG) is the major marker in EC extract. Both of β-Ecd and PDG content values aligned with the Chinese Pharmacopoeia standards. Treatment with 10 μmol/L Dex reduced zebrafish vertebral area, sum brightness, and ALP activity, but RAB and EC attenuated these effects. Combining 50 µg/mL RAB and 50 µg/mL EC was optimal for preventing GIOP in zebrafish. Reverse transcription-quantitative polymerase chain reaction was used to evaluate the mRNA expression of osteogenesis-related genes. A treatment of 10 μmol/L Dex decreased runt-related transcription factor 2 (Runx2), osteogenic protein-1 (OP-1), bone γ-carboxyglutamic acid-containing protein (BGLAP), and β-catenin levels. This effect was counteracted by RAB and EC co-treatment (P < 0.05). Additionally, the effect of using the two herbal extracts together was better than single-herb treatments separately. These results demonstrated that RAB and EC preserve osteoblast function in the presence of GC. The best mass ratio was 1:1.@*CONCLUSION@#RAB and EC herbal pair could ameliorate GC-induced effects in zebrafish, with 1:1 as the optimal dosage ratio.


Subject(s)
Animals , Glucocorticoids , Medicine, Chinese Traditional , Osteogenesis , Osteoporosis/prevention & control , Zebrafish
15.
Braz. j. oral sci ; 20: e211512, jan.-dez. 2021. ilus
Article in English | LILACS, BBO | ID: biblio-1254424

ABSTRACT

Aim: Several systemic diseases, such as periodontitis and apical periodontitis, can cause extensive bone resorption. Host defense peptides may have the potential for the development of novel therapies for the bone resorption process. This study evaluated the potential of host defense peptides clavanins A, MO, and LL-37 in in vitro osteoclastogenesis. Methods: RAW 264.7 cultures were stimulated with recombinant of receptor activator of nuclear factor kappa B ligand in the presence of different tested concentrations of host defense peptides, besides calcium hydroxide and doxycycline. Cellular viability, nitric oxide production, and a number of differentiated osteoclast-like cells were also evaluated. Results: Results showed that none of the substances were cytotoxic, except for 128 µg.mL-1 of doxycycline after 3 days. Host defense peptides, calcium hydroxide, and doxycycline did not interfere in nitric oxide production or downregulated it. An exception was observed in the presence of 2 µg.mL-1 of doxycycline, in which nitric oxide production was up-regulated. All host defense peptides were capable of reducing osteoclast-like cell differentiation. Conclusion: Host defense peptides clavanins A and MO demonstrated to be potential suppressors of osteoclastogenesis in vitro without interfering in cellular viability and nitric oxide production. These promising results need to be further analyzed in in vivo models of bone resorption


Subject(s)
Osteogenesis , Bone Resorption , Antimicrobial Cationic Peptides , Nitric Oxide
16.
Braz. j. biol ; 81(1): 53-61, Feb. 2021. tab, graf
Article in English | LILACS | ID: biblio-1153329

ABSTRACT

Abstract The aim of this study was to analyze the biological behavior and osteogenic potential of magnesium (Mg) substituted hydroxyapatite (HA) microspheres, implanted in a critical bone defect, considering that this ion is of great clinical interest, since it is closely associated with homeostasis and bone mineralization. For the purpose of this study, 30 rats were used to compose three experimental groups: GI - bone defect filled with HA microspheres; GII - bone defect filled with HA microspheres replaced with Mg; GIII - empty bone defect; evaluated at biological points of 15 and 45 days. The histological results, at 15 days, showed, in all the groups, a discrete chronic inflammatory infiltrate; biomaterials intact and surrounded by connective tissue; and bone neoformation restricted to the borders. At 45 days, in the GI and GII groups, an inflammatory response of discrete granulomatous chronic type was observed, and in the GIII there was a scarce presence of mononuclear inflammatory cells; in GI and GII, the microspheres were seen to be either intact or fragmented, surrounded by fibrous connective tissue rich in blood vessels; and discrete bone neoformation near the edges and surrounding some microspheres. In GIII, the mineralization was limited to the borders and the remaining area was filled by fibrous connective tissue. It was concluded that the biomaterials were biocompatible and osteoconductive, and the percentage of Mg used as replacement ion in the HA did not favor a greater bone neoformation in relation to the HA without the metal.


Resumo O objetivo deste estudo foi analisar o comportamento biológico de microesferas de hidroxiapatita (HA) substituída com magnésio (Mg) durante o reparo de defeito ósseo crítico, tendo em vista que este íon é de grande interesse clínico, pois está intimamente associado à homeostasia e à mineralização óssea. Para tanto, utilizou-se 30 ratos para compor três grupos experimentais: GI - defeito ósseo preenchido com microesferas de HA; GII - defeito ósseo preenchido com microesferas de HA substituída com Mg; GIII (controle) - defeito ósseo vazio; avaliados nos pontos biológicos de 15 e 45 dias. Os resultados histológicos evidenciaram, aos 15 dias, discreto infiltrado inflamatório crônico e neoformação óssea restrita às bordas, em todos os grupos. Nos grupos GI e GII, os biomateriais mantiveram-se íntegros e circundados por tecido conjuntivo frouxo. Aos 45 dias, notou-se resposta inflamatória do tipo crônica granulomatosa discreta nos grupos GI e GII, e no GIII presença escassa de células inflamatórias mononucleares. As microesferas implantadas no GI e GII mantiveram-se, em sua maioria, íntegras e envolvidas por tecido conjuntivo fibroso. Notou-se discreta neoformação óssea próxima às bordas e circunjacente a algumas microesferas. No GIII, a mineralização limitou-se às bordas e a área remanescente foi preenchida por tecido conjuntivo fibroso. Conclui-se que os biomateriais foram biocompatíveis, bioativos, osteocondutores e apresentaram biodegradação lenta, indicando seu grande potencial para em aplicações clínica como biomaterial de preenchimento.


Subject(s)
Animals , Rats , Durapatite , Magnesium , Osteogenesis , Biocompatible Materials , Bone Regeneration
17.
Braz. dent. j ; 32(1): 26-33, Jan.-Feb. 2021. graf
Article in English | LILACS, BBO | ID: biblio-1180720

ABSTRACT

Abstract The limited options for bone repair have led to an extensive research of the field and the development of alloplastic and xenogeneic grafts. The purpose of this study was to evaluate bone repair with two bone substitutes: deproteinized bovine bone (DBB) and biphasic calcium phosphate ceramic (BCP) in critical-size defect. A total of 8-mm defects were made in the parietal bones of rabbits (n=12). The animals were divided into three experimental groups: sham (defect filled with a blood clot), DBB (defect filled with DBB), and BCP (defect filled with BCP). After the experimental periods of 15 and 45 days, the animals were euthanized and submitted to histomorphometric analysis. The total defect area, mineralized tissue area, biomaterial area, and soft tissue area were evaluated. A greater amount of immature bone tissue and biomaterial particles were observed in the BCP group compared to DBB and sham at 45 days (p<0.05). There was no difference in the qualitative pattern of bone deposition between DBB and BCP. However, the sham group did not show osteoid islands along with the defect, presenting a greater amount of collagen fibers as well in relation to the DBB and BCP groups. There was a greater number of inflammatory cells in the DBB at 45 days compared to BCP and sham groups. In conclusion, BCP and DBB are options for optimizing the use of bone grafts for maxillofacial rehabilitation. Bone defects treated with BCP showed greater deposition of bone tissue at 45 days.


Resumo As opções limitadas para reparo ósseo levaram ao desenvolvimento de abrangente pesquisa na área de enxertos aloplásticos e xenogênicos. O objetivo deste estudo foi avaliar o reparo ósseo com dois substitutos ósseos: osso bovino desproteinizado (DBB) e cerâmica fosfática de cálcio bifásica (BCP) em defeito de tamanho crítico. Material e métodos: defeitos críticos de 8 mm foram feitos nos ossos parietais de coelhos (n=12). Os animais foram divididos em três grupos experimentais: sham (defeito preenchido com coágulo sanguíneo), DBB (defeito preenchido com DBB) e BCP (defeito preenchido com BCP). Após os períodos experimentais de 15 e 45 dias, os animais foram sacrificados e submetidos à análise histomorfométrica. Foram avaliadas a área total de defeitos, área de tecidos mineralizados, área de biomateriais e área de tecidos moles. Resultados: maior quantidade de tecido ósseo imaturo e de partículas de biomaterial foram observados no grupo BCP em comparação aos grupos DBB e sham aos 45 dias (p<0,05). Não houve diferença no padrão qualitativo de deposição óssea entre DBB e BCP. Ainda, o grupo sham não apresentou ilhas osteóides ao longo do defeito, apresentando maior quantidade de fibras colágenas em relação aos grupos DBB e BCP. Houve maior quantidade de células inflamatórias no DBB aos 45 dias em comparação aos grupos BCP e sham. Conclusões: BCP e DBB são opções para otimizar o uso de enxertos ósseos na reabilitação de pacientes. Defeitos ósseos tratados com BCP mostraram maior deposição de tecido ósseo aos 45 dias.


Subject(s)
Animals , Rabbits , Bone Substitutes , Hydroxyapatites , Osteogenesis , Bone Matrix , Bone Regeneration , Calcium Phosphates , Cattle , Ceramics
18.
Article in English | WPRIM | ID: wpr-922463

ABSTRACT

Guided bone regeneration (GBR) uses resorbable and non-resorbable membranes as biological barriers. This study compared the differences in hard tissue stability between GBR using evidence-based digital titanium mesh and resorbable collagen membranes during implant placement. A total of 40 patients (65 implant sites) were enrolled and divided into two groups: resorbable membrane and digital titanium mesh groups. The alveolar bone was analyzed at two- and three-dimensional levels using cone-beam computed tomography and by reconstructing and superimposing the hard tissues at four time points: preoperatively, postoperatively, before second-stage surgery, and 1 year after loading. The use of digital titanium mesh showed less alveolar bone resorption in vertical and horizontal directions two-dimensionally before the second-stage surgery and 1 year after loading. Regarding volumetric stability, the percentage of resorption after 6 months of healing with resorbable membrane coverage reached 37.5%. However, it was only 23.4% with titanium mesh. Although postoperative bone volume was greater at all labial sites with resorbable membrane than with digital titanium mesh, after substantial bone resorption within 1 year of loading, the labial bone thickness at the upper part of implants was thinner with resorbable membrane than with digital titanium mesh. Furthermore, digital titanium meshes made according to ideal bone arch contour reduced soft tissue irritation, and the exposure rate was only 10%. Therefore, although both resorbable membrane and digital titanium mesh in GBR were able to successfully reconstruct the bone defect, digital titanium meshes were better at maintaining the hard tissue volume in the osteogenic space.


Subject(s)
Bone Regeneration , Bone Resorption , Humans , Osteogenesis , Surgical Mesh , Titanium
19.
Article in English | WPRIM | ID: wpr-888704

ABSTRACT

Nanomaterial-based drug sustainable release systems have been tentatively applied to bone regeneration. They, however, still face disadvantages of high toxicity, low biocompatibility, and low drug-load capacity. In view of the low toxicity and high biocompatibility of polymer nanomaterials and the excellent load capacity of hollow nanomaterials with high specific surface area, we evaluated the hollow polydopamine nanoparticles (HPDA NPs), in order to find an optimal system to effectively deliver the osteogenic drugs to improve treatment of bone defect. Data demonstrated that the HPDA NPs synthesized herein could efficiently load four types of osteogenic drugs and the drugs can effectively release from the HPDA NPs for a relatively longer time in vitro and in vivo with low toxicity and high biocompatibility. Results of qRT-PCR, ALP, and alizarin red S staining showed that drugs released from the HPDA NPs could promote osteogenic differentiation and proliferation of rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. Image data from micro-CT and H&E staining showed that all four osteogenic drugs released from the HPDA NPs effectively promoted bone regeneration in the defect of tooth extraction fossa in vivo, especially tacrolimus. These results suggest that the HPDA NPs, the biodegradable hollow polymer nanoparticles with high drug load rate and sustainable release ability, have good prospect to treat the bone defect in future clinical practice.


Subject(s)
Animals , Bone Regeneration , Indoles , Nanoparticles , Osteogenesis , Pharmaceutical Preparations , Polymers , Rats
20.
Acta cir. bras ; 36(6): e360605, 2021. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1284912

ABSTRACT

ABSTRACT Purpose To evaluate and compare two types of different scaffolds in critical bone defects in rats. Methods Seventy male Wistar rats (280 ± 20 grams) divided into three groups: control group (CG), untreated animals; biomaterial group 1 (BG1), animals that received the scaffold implanted hydroxyapatite (HA)/poly(lactic-co-glycolic) acid (PLGA); and biomaterial group 2 (BG2), animals that received the scaffolds HA/PLGA/Bleed. The critical bone defect was induced in the medial region of the skull calotte with the aid of an 8-mm-diameter trephine drill. The biomaterial was implanted in the form of 1.5 mm thick scaffolds, and samples were collected after 15, 30 and 60 days. Non-parametric Mann-Whitney test was used, with the significance level of 5% (p ≤ 0.05). Results Histology revealed morphological and structural differences of the neoformed tissue between the experimental groups. Collagen-1 (Col-1) findings are consistent with the histological ones, in which BG2 presented the highest amount of fibers in its tissue matrix in all evaluated periods. In contrast, the results of receptor activator of nuclear factor kappa-Β ligand (Rank-L) immunoexpression were higher in BG2 in the periods of 30 and 60 days, indicating an increase of the degradation of the biomaterial and the remodeling activity of the bone. Conclusions The properties of the HA/PLGA/Bleed scaffold were superior when compared to the scaffold composed only by HA/PLGA.


Subject(s)
Animals , Male , Rats , Biocompatible Materials , Tissue Scaffolds , Osteogenesis , Bone Regeneration , Rats, Wistar , Polylactic Acid-Polyglycolic Acid Copolymer
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