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 ReactionABSTRACT
SUMMARY: The purpose of this study was to investigate effects of diabetes mellitus (DM) on the alveolar bone with histopathological and immunohistochemical methods. Wistar rats were divided into two groups, control and diabetes group. Control group was fed standard rat chow and drinking water for 8 weeks. Single dose (Streptozotocin) STZ (55 mg/kg), was dissolved in sodium citrate buffer and introduced intraperitoneal injection. Diabetes group and control group were compared in terms of glucose values. The blood glucose concentration in diabetic rats was significantly high (p <0.05). In diabetes group; periodontal membrane and the dilation of blood vessels, hemorrhage has also been a significant increase in inflammatory cells. In the diabetes group, osteonectin showed positive expression in periodontal membrane and showed negative expression in osteocytes of alveolar bone. Osteopontin expression in fibroblast cells and periodontal membrane collagen fibrils was positive, alveolar cells, osteocytes and bone matrix bone was found positive. Diabetes results showed that there formed periodontitis; due to the increase in inflammation inhibiting bone formation delaying the development of early bone cells.
RESUMEN: El objetivo de este estudio fue investigar los efectos de la diabetes mellitus (DM) sobre el hueso alveolar con métodos histopatológicos e inmunohistoquímicos. Las ratas Wistar se dividieron en dos grupos, grupo control y grupo de diabetes. El grupo control fue alimentado con comida estándar y agua potable durante 8 semanas. La dosis única Streptozotocina (STZ) (55 mg/ kg), se disolvió en tampón de citrato de sodio y se introdujo mediante inyección intraperitoneal. El grupo diabetes y el grupo control se compararon en términos de valores de glucosa. La concentración de glucosa en sangre en ratas diabéticas fue significativamente alta (p <0,05). En el grupo diabetes hubo un aumento significativo de la membrana periodontal y dilatación de los vasos sanguíneos y hemorragia, con un aumento significativo de células inflamatorias. En el grupo diabetes, la osteonectina mostró una expresión positiva en la membrana periodontal además se observó expresión negativa en los osteocitos del hueso alveolar. La expresión de osteopontina en fibroblastos y fibrillas de colágeno en membrana periodontal fue positiva, las células alveolares, osteocitos y hueso de la matriz ósea dio positivo. Los resultados de la diabetes mostraron que existía periodontitis, debido al aumento de la inflamación que inhibió la formación ósea retardando el desarrollo de células óseas tempranas.
Subject(s)
Animals , Rats , Alveolar Process/metabolism , Alveolar Process/pathology , Diabetes Mellitus, Experimental/pathology , Blood Glucose , Blotting, Western , Diabetes Mellitus, Experimental/metabolism , Immunohistochemistry , Osteonectin/metabolism , Osteopontin/metabolism , Rats, WistarABSTRACT
Abstract This study aimed to investigate the influence of a three-dimensional cell culture model and bioactive glass (BG) particles on the expression of osteoblastic phenotypes in rat calvaria osteogenic cells culture. Cells were seeded on two-dimensional (2D) and three-dimensional (3D) collagen with BG particles for up to 14 days. Cell viability and alkaline phosphatase (ALP) activity was performed. Cell morphology and immunolabeling of noncollagenous bone matrix proteins were assessed by epifluorescence and confocal microscopy. The expressions of osteogenic markers were analyzed using RT-PCR. Mineralized bone-like nodule formation was visualized by microscopy and calcium content was assessed quantitatively by alizarin red assay. Experimental cultures produced a growing cell viability rate up to 14 days. Although ALP activity at 7 days was higher on BG cultures, cells on 3D and 3D+BG had an activity decrease of ALP at 14 days. Three-dimensional conditions favored the immunolabeling for OPN and BSP and the expression of ALP and COL I mRNAs. BG particles influenced positively the OC and OPN mRNAs expression and calcified nodule formation in vitro. The results indicated that the 3D cultures and BG particles contribute to the expression of osteoblastic phenotype and to differentiated and mineralized matrix formation.
Resumo O objetivo deste estudo foi investigar a influência do modelo de cultura celular tridimensional e das partículas de vidro bioativo (BG) sobre a expressão fenotípica de culturas de células osteogênicas da calvária de ratos. As células foram mantidas em culturas sobre superfícies colágenas bi-dimensionais (2D) e em géis de colágeno tridimensional (3D) com e sem partículas de BG até 14 dias. Foram avaliadas: viabilidade celular, atividade de fosfatase alcalina (ALP), morfologia celular e imunomarcação de proteínas da matriz não-colágena do osso através de epifluorescência e microscopia confocal. As expressões de marcadores osteogênicos foram analisadas utilizando RT-PCR. A formação de nódulos mineralizados foi visualizada através de microscopia e o conteúdo de cálcio foi avaliado quantitativamente pelo Alizarina Red. As culturas experimentais produziram uma taxa crescente de viabilidade até 14 dias. Embora a atividade ALP em 7 dias tenha sido maior em culturas com BG, as células em 3D e 3D+BG apresentaram uma diminuição da atividade ALP aos 14 dias. As condições tridimensionais favoreceram a imunomarcação para OPN e BSP e a expressão de mRNAs para ALP e COL I. As partículas de BG influenciaram positivamente a expressão do mRNAs para OPN e OC e a formação de nódulos calcificados in vitro. Os resultados indicaram que as culturas em 3D e partículas BG contribuíram para a expressão do fenótipo osteoblástico e para a diferenciação e formação de matriz mineralizada.
Subject(s)
Animals , Biocompatible Materials , Glass , Osteoblasts/cytology , Osteogenesis , Skull/cytology , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Biomarkers/metabolism , Calcium/metabolism , Cell Culture Techniques , Cell Survival , Collagen Type I/genetics , Collagen Type I/metabolism , Fluorescent Antibody Technique, Indirect , Gene Expression Profiling , Integrin-Binding Sialoprotein/metabolism , Microscopy, Confocal , Microscopy, Fluorescence , Osteoblasts/enzymology , Osteoblasts/metabolism , Osteopontin/metabolism , Rats, Wistar , Real-Time Polymerase Chain Reaction , RNA, Messenger/genetics , Skull/enzymology , Skull/metabolism , Tissue ScaffoldsABSTRACT
The aim of this study was to evaluate the effects of melatonin healing in a tibial bone defect model in rats by means of histopathological and immunohistochemistry analysis. Twenty one male Wistar albino rats were used in this study. In each animal, bone defects (6 mm length ) were created in the tibias. The animals were divided into three groups. In group 1 control group (rats which tibial defects). Group 2 melatonin (10 mg/kg) + 14 days in the tibial defect group) was administered intraperitoneally to rats. Group 3 melatonin (10 mg/kg) + 28 days in the tibial defect group) was administered intraperitoneally to rats. Histopathological analysis of samples was performed to evaluate the process of osteoblastic activity, matrix formation, trabecular bone formation and myeloid tissue in bone defects. Immunohistochemical and immunoblot analysis demonstrated non-collagenous proteins (osteopontin and osteonectin) differences in tibial bone defects. The expression of osteopontin on tibia was increased by 14 days melatonin treatment. The expression of osteonectin on tibia was dramatically increased by 14 days melatonin treatment.
El objetivo fue evaluar por medio de análisis histopatológico e inmunohistoquímico los efectos cicatrizantes de la melatonina en un modelo de defecto óseo tibial en ratas. Se utilizaron 21 ratas albinas Wistar macho. En cada animal, se crearon defectos óseos en las tibias de 6 mm de longitud. Los animales se dividieron en tres grupos. El Grupo 1 correspondió al grupo control (defectos tibiales sin tratamiento). Al Grupo 2 se administró melatonina por vía intraperitoneal (10 mg/kg) 14 días posteriores al defecto tibial. Al Grupo 3 se administró melatonina por vía intraperitoneal (10 mg/kg) 28 días posteriores al defecto tibial. Se realizó un análisis histopatológico para evaluar los procesos de actividad osteoblástica, formación de matriz, formación de hueso trabecular y tejido mieloide en los defectos óseos. Los análisis inmunohistoquímicos y de inmunotransferencia mostraron diferencias de proteínas no colágenas (osteopontina y osteonectina). La expresión de osteopontina en defectos óseos tibiales se incrementó en el Grupo 2. La expresión de osteonectina en la tibia se incrementó fuertemente bajo el tratamiento con melatonina por 14 días.
Subject(s)
Animals , Rats , Melatonin/pharmacology , Tibial Fractures/drug therapy , Tibia/drug effects , Disease Models, Animal , Melatonin/administration & dosage , Osteonectin/drug effects , Osteonectin/metabolism , Osteopontin/drug effects , Osteopontin/metabolism , Rats, Sprague-Dawley , Tibial Fractures/pathology , Tibia/pathology , Wound Healing/drug effectsABSTRACT
Reactive proliferations of the gingiva comprise lesions such as pyogenic granuloma (PG), inflammatory fibroepithelial hyperplasia (IFH), peripheral ossifying fibroma (POF), and peripheral giant cell lesion. Osteopontin (OPN) has a dual role, it promotes mineralization when it is bound to solid substrate, and on the other hand, it inhibits mineralization when it is seen in association with solution. Objectives The study aimed to evaluate the expression of osteopontin in normal gingival tissue and different types of focal reactive proliferations of gingival tissue, and its role in the development of calcification within it. Material and Methods The presence and distribution of osteopontin was assessed using immunohistochemistry in five cases of normal gingival tissue and 30 cases of focal reactive proliferations of gingiva. Results There was no expression of osteopontin in normal subjects. Few cases of pyogenic granuloma, inflammatory fibroepithelial hyperplasia, and all the cases of peripheral ossifying fibroma showed positivity for osteopontin in the inflammatory cells, stromal cells, extracellular matrix, and in the calcifications. Conclusion The expression of osteopontin in all the cases of peripheral ossifying fibroma speculates that the majority of the cases of peripheral ossifying fibroma originate from the periodontal ligament cells. The treatment modalities for peripheral ossifying fibroma should differ from other focal reactive proliferations of gingiva. .
Subject(s)
Humans , Gingiva/metabolism , Gingival Diseases/metabolism , Osteopontin/metabolism , Bone Neoplasms/metabolism , Case-Control Studies , Fibroma, Ossifying/metabolism , Giant Cell Tumors/metabolism , Granuloma, Pyogenic/metabolism , Hyperplasia/metabolism , Immunohistochemistry , Reference ValuesABSTRACT
Multipotent mesenchymal stromal cells (MSCs) were first isolated from bone marrow and then from various adult tissues including placenta, cord blood, deciduous teeth, and amniotic fluid. MSCs are defined or characterized by their ability to adhere to plastic, to express specific surface antigens, and to differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Although the molecular mechanisms that control MSC proliferation and differentiation are not well understood, the involvement of microRNAs has been reported. In the present study, we investigated the role of miR-125b during osteoblastic differentiation in humans. We found that miR-125b increased during osteoblastic differentiation, as well as Runx2 and ALPL genes. To study whether the gain or loss of miR-125b function influenced osteoblastic differentiation, we transfected MSCs with pre-miR-125b or anti-miR-125b and cultured the transfected cells in an osteoblastic differentiation medium. After transfection, no change was observed in osteoblastic differentiation, and Runx2, OPN, and ALPL gene expression were not changed. These results suggest that the gain or loss of miR-125b function does not influence levels of Runx2, OPN, and ALPL during osteoblastic differentiation.
Subject(s)
Female , Humans , Male , Alkaline Phosphatase/metabolism , Cell Differentiation/physiology , Core Binding Factor Alpha 1 Subunit/metabolism , MicroRNAs/metabolism , Osteoblasts/cytology , Osteopontin/metabolism , Alkaline Phosphatase/genetics , Antigens, Differentiation/isolation & purification , Bone Marrow Cells/cytology , Core Binding Factor Alpha 1 Subunit/genetics , Gene Expression/physiology , Leukocytes, Mononuclear/cytology , Mesenchymal Stem Cells/cytology , MicroRNAs/genetics , Osteoblasts/metabolism , Osteogenesis/physiology , Osteopontin/genetics , Primary Cell Culture , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
Osteopontin (OPN) is phosphorylated sialic acid –rich non-collagenous bone matrix protein. OPN is found in several biological fluids including human plasma, serum, breast milk and urine.OPN was named for its function as a bridge between cells and minerals. OPN has been implicated as an important factor in bone remodeling. Osteopontin is expressed in immune cells, including macrophages, neutrophils, dendritic cells with varying kinetics. OPN influences cell mediated immunity and has Th 1 cytokine functions .OPN is overexpressed in cancers of lung, breast, colorectal, stomach and ovary.OPN is found in atheromatous plaques within arteries. OPN plays an important role during both acute and chronic inflammation. OPN is upregulated in tissues during several pathological process including atherosclerosis, valve stenosis, myocardial infarction and rheumatic arthritis. OPN is a key cytokine regulating tissue repair. OPN’s plasma levels are elevated in overweight and obesity.
Subject(s)
Atherosclerosis , Humans , Inflammation , Obesity , Osteopontin/blood , Osteopontin/chemistry , Osteopontin/immunology , Osteopontin/metabolism , Osteopontin/physiology , Osteopontin/urine , Vascular CalcificationABSTRACT
OBJECTIVE@#To investigate the changes of collagen fibers and the expression of osteopontin in the left ventricle in cases of hypertrophic cardiomyopathy (HCM), along with the significance of their potential forensic application.@*METHODS@#Fifteen cases of HCM, 15 cases of coronary heart disease with cardiac hypertrophy and 20 cases of traffic accidents were selected as HCM group, coronary heart disease group and control group, respectively. Collagen volume fraction and osteopontin expression were observed and compared by HE staining, Masson trichrome staining and immunohistochemistry methods. Imaging and statistical methods were used for quantitative analysis.@*RESULTS@#Collagen volume fraction in left ventricle of HCM and coronary heart disease were significantly higher than that in the control group (P < 0.05), which was not significantly different between the HCM group and the coronary heart disease group. The integral light density value of osteopontin in left ventricular cardiomyocytes of the HCM group and the coronary heart disease group were significantly higher than that of the control group (P< 0.05), and the value of the HCM group was also significantly higher than that of coronary heart disease group (P < 0.05).@*CONCLUSION@#The increased contents of collagen fibers and the overexpression of osteopontin may play an important role in myocardial fibrosis, and they can be used as markers in aid of diagnosing sudden death due to HCM.
Subject(s)
Female , Humans , Male , Cardiomyopathy, Hypertrophic/physiopathology , Case-Control Studies , Collagen/metabolism , Coronary Disease/physiopathology , Death, Sudden, Cardiac/etiology , Fibrosis , Forensic Pathology , Heart Ventricles/pathology , Immunohistochemistry , Myocardium/pathology , Osteopontin/metabolism , Staining and LabelingABSTRACT
The molecules that constitute the extracellular matrix are important in several functions related to tissue support and cell-cell, cell-extracellular matrix interaction. Among the macromolecules that constitute the mentioned matrix we find osteopontin, fibrinogen and collagen. The present study was undertaken to analyze the rol of osteopontin, fibrinogen and collagen in uterine-placental interface during normal porcine gestation. Uterine and placental tissues from crossbred gilts of 30 (n=5), 60 (n=5), 70 (n=5) and 114 (at term, n=5) days of gestation were used. Macroscopic analysis of the embryos/fetuses allowed us to determine their gestational age by means of the crown-rump lenght. Haematoxylin-Eosin and Masson's Trichrome dyes along with light microscopy were used to structure analysis of every selected period of gestation. A spacial and temporal study of osteopontin and fibrinogen was performed through immunohistochemical technique. Determination of collagen fibers was carried out through Picrosirius red technique and polarizing microscopy. Results were expressed as semi-quantitative. Higher expression of osteopontin was observed at early periods of gestation, mainly in uterine and placental villi, endometrial gland epithelium and histotroph. Fibrinogen expressed abundantly in fetal mesenchyme in every period analyzed and in fetal and maternal vessels at Day 70. Negative expression of collagen fibers was observed in villi, however increasing expression of thick fibers throughout pregnancy was detected in uterine stroma and myometrium. These results confirm the importance of osteopontin, fibrinogen and collagen in the support of uterine and placental structures and in the suitable maintenance of pregnancy.
Las moléculas que constituyen la matriz extracelular son importantes en varias funciones relacionadas con el soporte del tejido y la interacción célula-célula, célula-matriz extracelular. Entre las macromoléculas que constituyen la matriz mencionada se encuentra la osteopontina, el fibrinógeno y el colágeno. Este estudio se realizó para analizar el rol de la osteopontina, el fibrinógeno y el colágeno en la interface útero-placentaria durante la gestación porcina normal. Tejidos uterinos y la placentarios de hembras porcinas cruzadas de 30 (n=5), 60 (n=5), 70 (n=5) y 114 (a término, n=5) días de gestación fueron utilizados. El análisis macroscópico de los embriones/fetos nos permitió determinar la edad gestacional por medio de la longitud cráneo-rabadilla. Tinciones de Hematoxilina-Eosina y Tricrómico de Masson con microscopía de luz se utilizó para estructurar el análisis de cada periodo de tiempo seleccionado de la gestación. Un estudio espacial y temporal de la osteopontina y el fibrinógeno se realizó mediante técnicas de inmunohistoquímica. La determinación de fibras colágenas se llevó a cabo a través de la técnica Picrosirius rojo por microscopia de polarización. Los resultados se expresaron como semi-cuantitativos. La expresión de osteopontina se observó en los primeros períodos de gestación, principalmente en las vellosidades del útero y la placenta, epitelio de las glándulas endometriales e histotrofos. El fibrinógeno se expresa abundantemente en mesénquima fetal en todos los períodos analizados y en los vasos fetales y maternos el día 70. Una expresión negativa de fibras colágenas se observó en las vellosidades, sin embargo, un aumento de expresión de las fibras gruesas durante la gestación se detectó en el estroma uterino y el miometrio. Estos resultados confirman la importancia de la osteopontina, fibrinógeno y colágeno en el soporte de las estructuras del útero y placenta, así como el mantenimiento adecuado durante la gestación.
Subject(s)
Animals , Female , Pregnancy , Collagen/metabolism , Fibrinogen/metabolism , Osteopontin/metabolism , Placenta/metabolism , Swine , Extracellular Matrix , Immunohistochemistry , Pregnancy, Animal/metabolism , Uterus/metabolismABSTRACT
The maxilla and masseter muscles are components of the stomatognathic system involved in chewing, which is frequently affected by physical forces such as gravity, and by dental, orthodontic and orthopedic procedures. Thyroid hormones (TH) are known to regulate the expression of genes that control bone mass and the oxidative properties of muscles; however, little is known about the effects of TH on the stomatognathic system. This study investigated this issue by evaluating: i) osteoprotegerin (OPG) and osteopontine (OPN) mRNA expression in the maxilla and ii) myoglobin (Mb) mRNA and protein expression, as well as fiber composition of the masseter. Male Wistar rats (~250 g) were divided into thyroidectomized (Tx) and sham-operated (SO) groups (N = 24/group) treated with T3 or saline (0.9 percent) for 15 days. Thyroidectomy increased OPG (~40 percent) and OPN (~75 percent) mRNA expression, while T3 treatment reduced OPG (~40 percent) and OPN (~75 percent) in Tx, and both (~50 percent) in SO rats. Masseter Mb mRNA expression and fiber type composition remained unchanged, despite the induction of hypo- and hyperthyroidism. However, Mb content was decreased in Tx rats even after T3 treatment. Since OPG and OPN are key proteins involved in the osteoclastogenesis inhibition and bone mineralization, respectively, and that Mb functions as a muscle store of O2 allowing muscles to be more resistant to fatigue, the present data indicate that TH also interfere with maxilla remodeling and the oxidative properties of the masseter, influencing the function of the stomatognathic system, which may require attention during dental, orthodontic and orthopedic procedures in patients with thyroid diseases.
Subject(s)
Animals , Male , Rats , Masseter Muscle/drug effects , Maxilla/drug effects , Myoglobin/metabolism , Osteopontin/metabolism , Osteoprotegerin/metabolism , Thyroid Hormones/physiology , Triiodothyronine/pharmacology , Blotting, Northern , Hyperthyroidism/physiopathology , Masseter Muscle/anatomy & histology , Masseter Muscle/metabolism , Maxilla/metabolism , Myoglobin/genetics , Osteopontin/genetics , Osteoprotegerin/genetics , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , RNA , RNA, Messenger/metabolism , Thyroidectomy , Thyroid Hormones/metabolismABSTRACT
OBJETIVO: O objetivo deste estudo foi avaliar o efeito da T3 na expressão da osteocalcina, osteopontina e colágeno I durante a diferenciação osteogênica das células-tronco mesenquimais (CTM). MATERIAIS E MÉTODOS: As células da medula óssea de ratas Wistar jovens foram extraídas, cultivadas e separadas em cinco grupos: controle (indiferenciado), diferenciado (estímulo osteogênico) e diferenciado com T3 (10-3 nM, 10-2 nM e 100 nM). Para cada grupo, foram cultivadas quatro amostras que foram analisadas por RT-PCR tempo real aos 7, 14 e 21 dias, para quantificação dos transcritos gênicos para osteocalcina, osteopontina e colágeno I. RESULTADOS: Todos os grupos diferenciados sem T3 ou com T3 independentemente da concentração apresentaram expressão de colágeno I significativamente menor e expressão de osteocalcina e osteopontina significativamente maior em comparação a das CTM indiferenciadas. Mas o grupo T3 100 nM apresentou concentração de osteocalcina mais elevada e semelhante à da cultura de osteoblastos. CONCLUSÃO: Conclui-se que a triiodotironina não altera a expressão de osteopontina e de colágeno pelas CTM, mas aumenta a expressão da osteocalcina durante a diferenciação osteogênica in vitro, sendo esse efeito dose-dependente.
OBJECTIVE: The aim of this study was to evaluate the effect of T3 on the expression of osteocalcin, osteopontin and collagen I during osteogenic differentiation of mesenchymal stem cells (MSC). MATERIALS AND METHODS: The bone marrow cells of Wistar rats with 30 days of age were extracted, cultured and separated into five groups: control (undifferentiated), differentiated (osteogenic stimulus) and differentiated with T3 (10-3 nM, 10-2 nM and 100 nM). For each group, four samples were cultured and were analyzed by real time RT-PCR at 7, 14 and 21 days for quantification of gene transcripts for osteocalcin, osteopontin and collagen I. RESULTS: All the different groups without T3 or with T3 regardless of the concentration, showed the collagen I expression significantly lower expression, and osteocalcin and osteopontin expression significantly greater than that of undifferentiated MSC. Nevertheless, the group T3 100 nM showed higher expression of osteocalcin and a similar expression of the osteoblast culture. CONCLUSION: In conclusion, the triiodothyronine does not affect the expression of osteopontin and collagen I, but increases ostecalcin expression during osteogenic differentiation in vitro of the MSC, and this effect is dose-dependent.
Subject(s)
Animals , Female , Rats , Bone Marrow Cells/drug effects , Bone and Bones/drug effects , Cell Differentiation/drug effects , Mesenchymal Stem Cells , Osteogenesis/drug effects , Triiodothyronine/pharmacology , Bone Marrow Cells/metabolism , Bone Matrix/metabolism , Bone and Bones/metabolism , Collagen/metabolism , Disease Models, Animal , Mesenchymal Stem Cells , Osteocalcin/metabolism , Osteopontin/metabolism , Proteins/metabolismABSTRACT
Purpose: To evaluate the ability of macroporous tricalcium phosphate cement (CPC) scaffolds to enable the adhesion, proliferation, and differentiation of mesenchymal stem cells derived from human bone marrow. Methods: Cells from the iliac crest of an adult human donor were processed and cultured on macroporous CPC discs. Paraffin spheres sized between 100 and 250µm were used as porogens. Cells were cultured for 5, 10, and 15 days. Next, we assessed cells' behavior and morphology on the biomaterial by scanning electron microscopy. The expression levels of the BGLA and SSP1 genes and the alkaline phosphatase (ALP) activity were quantified by the quantitative real-time polymerase chain reaction technique (QT-PCR) using the fluorophore SYBR GREEN®. Results: QT-PCR detected the expression of the BGLA and SSP1 genes and the ALP activity in the periods of 10 and 15 days of culture. Thus, we found out that there was cell proliferation and differentiation in osteogenic cells. Conclusion: Macroporous CPC, with pore sized between 100 and 250µm and developed using paraffin spheres, enables adhesion, proliferation, and differentiation of mesenchymal stem cells in osteogenic cells and can be used as a scaffold for bone tissue engineering.
Objetivo: Avaliar a capacidade de suportes tridimensionais macroporosos de cimento de fosfato de cálcio (CFC), de permitir a adesão, proliferação e diferenciação de células-tronco mesenquimais derivadas da medula óssea humana. Métodos: células obtidas da crista ilíaca de um doador humano adulto foram processadas e cultivadas sobre suportes de CFC, macroporosos, que tiveram como corpo gerador de poros, microesferas de parafina, com tamanho entre 100 e 250µm. Os períodos de cultura estabelecidos foram de cinco, 10 e 15 dias. Após estes períodos, o comportamento e a morfologia das células junto ao biomaterial foram avaliados por meio de Microscopia Eletrônica de Varredura. Os níveis de expressão dos genes BGLA e SSP1 bem como a atividade da Fosfatase Alcalina (ALP) foram quantificados pela técnica de PCR em Tempo Real (QT-PCR) utilizando o fluoróforo SYBR Green®. Resultados: O QT-PCR detectou a expressão dos genes BGLA e SSP1 e a atividade da fosfatase alcalina nos períodos de 10 e 15 dias de cultura. No período de cinco dias, não foi observada a expressão de nenhum dos genes investigados. Conclusão: O CFC, macroporoso, com tamanho de poros entre 100 e 250µm, criados por meio da utilização de microesferas de parafina, permite a adesão, proliferação e diferenciação de células-tronco mesenquimais em células osteogênicas, podendo ser utilizado como arcabouço para engenharia de tecido ósseo.
Subject(s)
Adult , Humans , Biocompatible Materials , Bone and Bones , Bone Cements , Calcium Phosphates , Mesenchymal Stem Cells , Tissue Scaffolds , Tissue Engineering/methods , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Biocompatible Materials/chemistry , Bone Cements/chemistry , Cell Differentiation , Cell Proliferation , Calcium Phosphates/chemistry , Extracellular Matrix/metabolism , Gene Expression , Mesenchymal Stem Cells , Microscopy, Electron, Scanning , Osteogenesis , Osteocalcin/genetics , Osteocalcin/metabolism , Osteocytes/cytology , Osteopontin/genetics , Osteopontin/metabolism , Polymerase Chain Reaction/methods , Time Factors , Tissue Scaffolds/chemistryABSTRACT
Pulmonary adenocarcinoma is a common malignancy that often involves calcification; however, bone formation in primary lung adenocarcinoma is extremely rare. In ten cases of primary pulmonary adenocarcinoma with heterotopic ossification, we detected immunoreactivity against TGF-beta1, osteopontin, osteocalcin and Runx2 in the fibroblastic stroma and tumor cells within the area of ossification. Our results suggest that in primary pulmonary adenocarcinoma, heterotopic ossification occurs via intramembranous bone formation. To our knowledge, only 11 other cases of pulmonary adenocarcinoma with heterotopic ossification have been reported. Here, we present ten cases of pulmonary adenocarcinoma showing heterotopic ossification with a description of previously published results and the histogenesis of heterotopic bone formation.
Subject(s)
Aged , Female , Humans , Male , Middle Aged , Adenocarcinoma/diagnosis , Core Binding Factor Alpha 1 Subunit/metabolism , Lung Neoplasms/diagnosis , Ossification, Heterotopic/diagnosis , Osteocalcin/metabolism , Osteopontin/metabolism , Tomography, X-Ray Computed , Transforming Growth Factor beta1/metabolismABSTRACT
Pulmonary adenocarcinoma is a common malignancy that often involves calcification; however, bone formation in primary lung adenocarcinoma is extremely rare. In ten cases of primary pulmonary adenocarcinoma with heterotopic ossification, we detected immunoreactivity against TGF-beta1, osteopontin, osteocalcin and Runx2 in the fibroblastic stroma and tumor cells within the area of ossification. Our results suggest that in primary pulmonary adenocarcinoma, heterotopic ossification occurs via intramembranous bone formation. To our knowledge, only 11 other cases of pulmonary adenocarcinoma with heterotopic ossification have been reported. Here, we present ten cases of pulmonary adenocarcinoma showing heterotopic ossification with a description of previously published results and the histogenesis of heterotopic bone formation.