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Actual. osteol ; 18(1): 40-52, 2022. ilus, tab
Article in Spanish | LILACS, BINACIS, UNISALUD | ID: biblio-1396075


El "microbioma" no solo está constituido por los microbios, sino por todos los componen-tes que viven en el mismo hábitat conforman-do un nicho ecológico. Es decir, está conformado por los microorganismos (bacterias, hongos, protozoos, etc.), todo el espectro de moléculas producidas por ellos tales como sus componentes estructurales (ácidos nucleicos, proteínas, lípidos y glúcidos), meta-bolitos, toxinas, etc., y las moléculas producidas por el huésped. El microbioma intestinal (MI) ha emergido como un factor que tiene un gran efecto sobre la cantidad, calidad y fuerza del hueso. Las investigaciones revelan que la homeostasis ósea está ligada al micro-bioma saludable, mientras que la disbiosis (alteración en la biodiversidad microbiana) puede exacerbar la actividad osteoclástica y promover la osteoporosis. Los mecanismos potenciales involucrados en la interacción del microbioma intestinal y el hueso son la influencia del metabolismo del huésped, el mantenimiento de la integridad intestinal y regulación de la absorción de nutrientes, la regulación del eje intestino-sistema inmune y la modulación del sistema endocrino. Es decir que hay múltiples vías por las cuales el MI influye sobre el hueso, pero estos y otros mecanismos deben profundizarse más aún. También es necesario que se identifiquen y caractericen mejor los microorganismos que están asociados a las enfermedades óseas. El conocimiento de estos aspectos podría ser útil para el desarrollo de herramientas terapéuticas basadas en el MI que puedan mejorar la eficacia de los distintos tratamientos existentes. (AU)

The microbiome is not only constituted by microbes, but by all the components that live in the same habitat forming an ecological niche. It is conformed by the microorganisms ( bacteria, fungi, protozoa, etc), the entire spectrum of molecules produced by them (nucleic acids, proteins, lipid and carbohydrates, metabolites, toxins, etc) and the molecules produced by the host. The intestinal microbiome (IM) has emerged as a factor with great effects on the quantity, quality and strength of bone. The investigations reveal that bone homeostasis is linked to the healthy microbiome, while the dysbiosis (alteration in the microbial biodiversity) can exacerbate the osteoclastic activity and promote osteoporosis. The potential mechanisms involved in the interaction between IM and bone are the influence of the host metabolism, the maintenance of the intestinal integrity and regulation of the nutrient absorption, the regulation of the intestine/ immune system axis and the modulation of the endocrine system. That is, there are multiple ways through which IM influences on bone, but these and other mechanisms need to be further studied. It is also necessary to identify and characterize the microorganisms associated with the bone diseases. Knowledge of these aspects could be useful to develop therapeutical tools based on the IM that could improve the efficacy of the current treatments. (AU)

Humans , Osteoblasts/immunology , Osteoclasts/immunology , Bone and Bones/immunology , Dysbiosis/complications , Gastrointestinal Microbiome/immunology , Osteoblasts/metabolism , Osteoclasts/metabolism , Bone and Bones/metabolism , Intestines/immunology , Intestines/microbiology
Chinese Journal of Biotechnology ; (12): 1159-1172, 2022.
Article in Chinese | WPRIM | ID: wpr-927771


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.

Alkaline Phosphatase/metabolism , Animals , Electromagnetic Fields , Osteoblasts/metabolism , Osteogenesis/genetics , Rats , TRPP Cation Channels/physiology
Article in Chinese | WPRIM | ID: wpr-888119


The present study aimed to explore the effect of Erxian Decoction on proteomics of osteoblasts stimulated by hydrogen peroxide(H_2O_2) and its protective mechanism with the H_2O_2-induced cell model of oxidative stress. The primary osteoblasts were cultured from the skulls of newborn rats(within 24 hours) and divided into a control group, a model group, a Fosamax group, and an Erxian Decoction group. Blank serum was added in the control group and model group, and the drug-containing serum was added correspondingly to the remaining two groups. After 45 hours, H_2O_(2 )stimulation was conducted for three hours except for the control group, followed by protein extraction. Nano-LC-LTQ-Orbitrap system was used for protein detection, Protein Discovery for protein identification, and SIEVE for quantitative and qualitative analysis. Furthermore, following the blocking of PI3 K signaling pathway by LY294002(10 μmol·L~(-1)), a control group, a model group, an LY294002 group, an Erxian Decoction group, and an Erxian Decoction + LY294002 group were set up to observe the effect of Erxian Decoction on cell proliferation, alkaline phosphatase(ALP) activity, and the relative expression of BMP-2, OPG, p-Akt, p-FoxO1 of osteoblasts stimulated by H_2O_2 under LY294002 intervention. The results revealed that 78 differential proteins were discovered between the Erxian Decoction group and model group, which were involved in the regulation of PI3 K/Akt, glucagon, estrogen, insulin, and other signaling pathways. LY294002 blunted the promoting effect of Erxian Decoction on osteoblast proliferation and significantly down-regulated the expression of OPG and p-FoxO1, whereas its down-regulation on the expression of BMP-2 and p-Akt was not significant. Both LY294002 and Erxian Decoction increased the ALP activity of osteoblasts, which may be related to the cell state and the cell differentiation. The above results suggest that Erxian Decoction can protect osteoblasts stimulated by H_2O_2, with the PI3 K/Akt signaling pathway as one of the internal mechanisms.

Animals , Drugs, Chinese Herbal , Hydrogen Peroxide , Osteoblasts/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proteomics , Proto-Oncogene Proteins c-akt/metabolism , Rats , Signal Transduction
Braz. j. med. biol. res ; 51(9): e6948, 2018. tab, graf
Article in English | LILACS | ID: biblio-951763


It is increasingly evident that the microenvironment of bone can influence cancer phenotype in many ways that favor growth in bone. CD147, a transmembrane protein of the immunoglobulin (Ig) superfamily, was identified independently in different species and has many designations across different species. However, expression levels of CD147 mRNA in bone cancer have not been described. In this study, we have used real-time fluorescence quantification (RT-PCR) to demonstrate CD147 expression in malignant bone cancer and benign bone tumor tissues. The results suggested that the expression of CD147 gene was significantly up-regulated in malignant bone cancer. Moreover, we found that over-expressed RANKL progressively enhanced osteoclast formation up to 48 h, which suggested that RANKL could promote the formation of osteoclast, indicating that both CD147 and RANKL play important roles in the formation of osteoclasts. Furthermore, the expressions of four osteoclast specific expression genes, including TRACP, MMP-2, MMP-9 and c-Src, were analyzed using RT-PCR. The results indicated that four osteoclast-specific expression genes were detectable in all osteoclast with different treatments. However, the highest expression level of these four osteoclast-specific expression genes appears in the CD147+ RANKL group and the lowest expression level of these four osteoclast-specific expression genes appears with si-RANKL treatment. Characterization of the role of CD147 in the development of tumors should lead to a better understanding of the changes occurring at the molecular level during the development and progression of primary human bone cancer.

Humans , Male , Female , Middle Aged , Aged , Osteoclasts/metabolism , Bone Neoplasms/genetics , Up-Regulation , Basigin/genetics , RANK Ligand/metabolism , Osteoblasts/cytology , Osteoblasts/metabolism , Bone Neoplasms/secondary , Bone Neoplasms/therapy , Gene Expression Regulation, Neoplastic , Blotting, Western , Reverse Transcriptase Polymerase Chain Reaction
Actual. osteol ; 13(3): 207-213, Sept - DIc. 2017. ilus, graf
Article in English | LILACS | ID: biblio-1117111


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)

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
Actual. osteol ; 13(3): 225-232, Sept - DIc. 2017. ilus
Article in Spanish | LILACS | ID: biblio-1117386


El esqueleto es uno de los sistemas más grandes de un vertebrado y, como tal, es razonable especular que no puede funcionar aislado del resto del organismo. De hecho, sabemos que existen sistemas complejos de regulación cruzada entre el esqueleto y muchos otros órganos. Hoy poseemos herramientas que nos permiten realizar supresión genética en células o tejidos específicos. Esto nos ha permitido comprender cómo los órganos se comunican entre sí y ha revitalizado el concepto de fisiología del organismo como un todo. Efectivamente, los últimos años han sido testigos del descubrimiento de funciones inesperadas que ejerce el esqueleto y que afectan al organismo en su totalidad. Una de tales funciones reconocidas recientemente es el control del metabolismo energético, a través de la secreción de osteocalcina. La osteocalcina es una hormona producida por los osteoblastos que regula la secreción de insulina, la sensibilidad a esta hormona y el metabolismo energético. Los hallazgos iniciales suscitaron varias preguntas fundamentales sobre la naturaleza de la acción de la insulina sobre el hueso. Pero esto solo fue la punta del iceberg. Efectivamente, más adelante se descubrió, mediante el análisis de ratones que carecen del receptor de insulina (Ins R) solamente en osteoblastos, que la acción de la insulina sobre estas células favorecía la homeostasis de la glucosa en todo el cuerpo. Es importante destacar que esta función de la insulina en los osteoblastos opera mediante la regulación negativa de la carboxilación y la biodisponibilidad de la osteocalcina. Más aún, se observó que las vías de señalización de la insulina en los osteoblastos regulan positivamente no solo la formación sino también la resorción del hueso. Curiosamente, parece que las vías de señalización de la insulina en osteoblastos pueden inducir la activación de la osteocalcina mediante la estimulación de la actividad de los osteoclastos. De hecho, el bajo pH generado durante la resorción ósea es suficiente para desencadenar la descarboxilación (y subsiguiente activación) de la osteocalcina. En breve discutiremos dos nuevas proposiciones: 1) los osteoblastos son un blanco utilizado por la insulina para controlar la homeostasis de la glucosa en todo el organismo y 2) la resorción ósea desempeña un papel fundamental en la regulación de la activación de la osteocalcina. (AU)

The skeleton is one of the biggest systems in a vertebrate animal and, as such, it is reasonable to speculate that it cannot function isolated from the rest of the organism. In fact, we know that complex systems exist for the cross-regulation between the skeleton and several other organs. Today, we have the tools that allow us to perform genetic suppression in specific cells or tissues. This has allow us understand the mechanisms by which the organs communicate with each other and has revitalized the concept of organismal physiology as a whole. Studies conducted in recent years have uncovered unexpected functions performed by the skeleton. One of these is the control of global energy metabolism, through the secretion of osteocalcin, a protein produced by osteoblasts that acts as a hormone regulating insulin secretion, insulin sensitivity and energy expenditure. The evidence comes from the analysis of mice lacking insulin receptor (InsR) exclusively in osteoblasts. These mice have a global metabolic phenotype demonstrating that the action of insulin in osteoblasts promotes the homeostasis of glucose throughout the body. This action of insulin in osteoblasts is mediated by the negative regulation of the carboxylation (and bioavailability) of osteocalcin. The decarboxylation (and activation) of osteocalcin, in turn, occurs in the osteoclastic resorption pit. Briefly: the osteoblast is a target used by insulin to control the homeostasis of glucose throughout the body and bone resorption is the mechanism that regulates the activation of osteocalcin. (AU)

Humans , Animals , Mice , Osteocalcin/biosynthesis , Energy Metabolism , Insulin/biosynthesis , Osteoblasts/metabolism , Osteogenesis , Skeleton/physiology , Skeleton/metabolism , Bone Resorption/metabolism , Receptor, Insulin/metabolism , Signal Transduction , Osteocalcin/metabolism , Decarboxylation , Insulin Secretion , Glucose/biosynthesis , Glucose/metabolism , Insulin/metabolism
Braz. dent. j ; 28(3): 307-316, May-June 2017. tab, graf
Article in English | LILACS | ID: biblio-888646


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.

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 Scaffolds
Actual. osteol ; 13(1): 58-66, Ene - Abr. 2017. ilus
Article in English | LILACS | ID: biblio-1118913


Connexins (Cxs) are a family of transmembrane proteins that form gap junctions and hemi-channels, which mediate cell-cell communication between neighboring cells and the respective extracellular milieu in different tissues. Most tissues and cell types throughout the body express one or more Cx proteins, highlighting its importance in regulating cell growth, differentiation, adhesion, migration, cell death and others. Moreover, Cx can propagate intracellular signals through its C-terminus domain, and thus function beyond a mere channel. Cx43 is the most highly expressed and most well studied Cx in bone and musculoskeletal tissues, although Cx40, Cx45, Cx46 and more recently, the Cx37 have been described in bone tissue, along with Cx26, Cx32 and Cx39 in other musculoskeletal tissues. Here, we discuss the basic structure of gap junctions and the role of the Cxs in musculoskeletal tissue, with special focus on Cx37. (AU)

Las conexinas (Cxs) son una familia de proteínas transmembrana que forman uniones en hendidura y hemicanales encargados de mediar la comunicación entre células vecinas y el respectivo medio extracelular en diferentes tejidos. La mayoría de los tejidos y células expresan una o más proteínas conexina, jugando un papel importante en la regulación de la proliferación celular, diferenciación, adhesión, migración y muerte celular, entre otras funciones. Además de actuar como un canal, las conexinas pueden propagar señales intracelulares a través del dominio C-terminal. La Cx43 es la conexina mas expresada y mejor estudiada en el tejido óseo y el músculo, aunque las Cx40, Cx45, Cx46, y mas recientemente Cx37, son también detectadas en el hueso. A su vez la expresión de la Cx26, Cx32 y Cx39 ha sido observada en otros tejidos músculoesqueléticos. En este manuscrito describimos la estructura básica de las uniones tipo gap y el papel que las Cxs, y en especial la Cx37, tienen en tejidos músculo-esqueléticos. (AU)

Humans , Bone and Bones/metabolism , Bone Resorption/prevention & control , Connexins/physiology , Osteoblasts/metabolism , Osteocytes/metabolism , Tendons/metabolism , Signal Transduction/physiology , Cartilage/metabolism , Cell Communication/physiology , Cell Physiological Phenomena , Gap Junctions/drug effects , Gap Junctions/physiology , Connexin 43/physiology , Muscle, Skeletal/metabolism , Bone Density Conservation Agents/therapeutic use , Ligaments/metabolism , Anti-Arrhythmia Agents/adverse effects
Clinics ; 70(11): 738-742, Nov. 2015. tab
Article in English | LILACS | ID: lil-766147


OBJECTIVES: To explore the effects of serum from patients with ankylosing spondylitis on the canonical Wnt/β-catenin pathway and to assess whether the serum has an osteogenic effect in MG63 cells. METHODS: MG63 cells were cultured with serum from 45 ankylosing spondylitis patients, 30 healthy controls, or 45 rheumatoid arthritis patients. The relative PPARD, fra-1, MMP7, OPG and RANKL mRNA levels were measured using quantitative real-time polymerase chain reaction. Associations between gene expression and patient demographics and clinical assessments were then analyzed. RESULTS: MG63 cells treated with serum from ankylosing spondylitis patients had higher PPARD, fra-1, MMP7 and OPG gene expression than did cells treated with serum from controls or rheumatoid arthritis patients (all p<0.05). RANKL expression was higher in MG63 cells treated with serum from patients with ankylosing spondylitis or rheumatoid arthritis than in those treated with serum from controls (both p<0.05). The OPG/RANKL ratio was also higher in MG63 cells treated with serum from ankylosing spondylitis patients than in those treated with serum from controls (p<0.05). No associations were found between the expression of the five genes and the patient demographics and clinical assessments (all p>0.05). CONCLUSIONS : Serum from ankylosing spondylitis patients increases PPARD, fra-1, MMP7, OPG and RANKL expression and the OPG/RANKL ratio in MG63 cells; these effects may be due to the stimulatory effect of the serum on the Wnt pathway.

Adult , Female , Humans , Male , Middle Aged , Young Adult , Arthritis, Rheumatoid/blood , Osteoblasts/metabolism , Serum , Spondylitis, Ankylosing/blood , Cells, Cultured , Culture Media , Cytokines/metabolism , Gene Expression , /metabolism , PPAR delta/metabolism , Proto-Oncogene Proteins c-fos/metabolism , RANK Ligand/metabolism , Real-Time Polymerase Chain Reaction , Wnt Signaling Pathway/genetics , beta Catenin/genetics
Biol. Res ; 48: 1-8, 2015. graf, tab
Article in English | LILACS | ID: biblio-950829


BACKGROUND: Tridaxprocumbens flavonoids (TPFs) are well known for their medicinal properties among local natives. Besides traditionally used for dropsy, anemia, arthritis, gout, asthma, ulcer, piles, and urinary problems, it is also used in treating gastric problems, body pain, and rheumatic pains of joints. TPFs have been reported to increase osteogenic functioning in mesenchymal stem cells. Our previous study showed that TPFs were significantly suppressed the RANKL-induced differentiation of osteoclasts and bone resorption. However, the effects of TPFs to promote osteoblasts differentiation and bone formation remain unclear. TPFs were isolated from Tridax procumbens and investigated for their effects on osteoblasts differentiation and bone formation by using primary mouse calvarial osteoblasts. RESULTS: TPFs promoted osteoblast differentiation in a dose-dependent manner demonstrated by up-regulation of alkaline phosphatase and osteocalcin. TPFs also upregulated osteoblast differentiation related genes, including osteocalcin, osterix, and Runx2 in primary osteoblasts. TPFs treated primary osteoblast cells showed significant upregulation of bone morphogenetic proteins (BMPs) including Bmp-2, Bmp-4, and Bmp-7. Addition of noggin, a BMP specific-antagonist, inhibited TPFs induced upregulation of the osteocalcin, osterix, and Runx2. CONCLUSION: Our findings point towards the induction of osteoblast differentiation by TPFs and suggested that TPFs could be a potential anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis.

Animals , Mice , Osteoblasts/drug effects , Osteogenesis/drug effects , Flavonoids/pharmacology , Cell Differentiation/drug effects , Asteraceae/chemistry , Osteoblasts/cytology , Osteoblasts/metabolism , Skull/cytology , Skull/drug effects , Transcription Factors/genetics , Flavonoids/analysis , Calcification, Physiologic/drug effects , Osteocalcin/drug effects , Osteocalcin/genetics , Up-Regulation/genetics , Bone Morphogenetic Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Alkaline Phosphatase/drug effects , Alkaline Phosphatase/metabolism , Core Binding Factor Alpha 1 Subunit/genetics , Core Binding Factor Alpha 1 Subunit/metabolism , Primary Cell Culture , Sp7 Transcription Factor , Medicine, Traditional , Mice, Inbred C57BL
Braz. j. med. biol. res ; 47(9): 759-765, 09/2014. tab, graf
Article in English | LILACS | ID: lil-719322


The monoamine serotonin (5-hydroxytryptamine, 5-HT), a well-known neurotransmitter, also has important functions outside the central nervous system. The objective of this study was to investigate the role of 5-HT in the proliferation, differentiation, and function of osteoblasts in vitro. We treated rat primary calvarial osteoblasts with various concentrations of 5-HT (1 nM to 10 µM) and assessed the rate of osteoblast proliferation, expression levels of osteoblast-specific proteins and genes, and the ability to form mineralized nodules. Next, we detected which 5-HT receptor subtypes were expressed in rat osteoblasts at different stages of osteoblast differentiation. We found that 5-HT could inhibit osteoblast proliferation, differentiation, and mineralization at low concentrations, but this inhibitory effect was mitigated at relatively high concentrations. Six of the 5-HT receptor subtypes (5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, and 5-HT2C) were found to exist in rat osteoblasts. Of these, 5-HT2A and 5-HT1B receptors had the highest expression levels, at both early and late stages of differentiation. Our results indicated that 5-HT can regulate osteoblast proliferation and function in vitro.

Animals , Calcification, Physiologic/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Osteoblasts/drug effects , Serotonin/pharmacology , DNA Primers , Gene Expression , Osteoblasts/cytology , Osteoblasts/metabolism , Primary Cell Culture , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Receptors, Serotonin/metabolism , Serotonin/metabolism
Braz. j. med. biol. res ; 46(10): 831-838, 24/set. 2013. tab, graf
Article in English | LILACS | ID: lil-688557


Wear particles are phagocytosed by macrophages and other inflammatory cells, resulting in cellular activation and release of proinflammatory factors, which cause periprosthetic osteolysis and subsequent aseptic loosening, the most common causes of total joint arthroplasty failure. During this pathological process, tumor necrosis factor-alpha (TNF-α) plays an important role in wear-particle-induced osteolysis. In this study, recombination adenovirus (Ad) vectors carrying both target genes [TNF-α small interfering RNA (TNF-α-siRNA) and bone morphogenetic protein 2 (BMP-2)] were synthesized and transfected into RAW264.7 macrophages and pro-osteoblastic MC3T3-E1 cells, respectively. The target gene BMP-2, expressed on pro-osteoblastic MC3T3-E1 cells and silenced by the TNF-α gene on cells, was treated with titanium (Ti) particles that were assessed by real-time PCR and Western blot. We showed that recombinant adenovirus (Ad-siTNFα-BMP-2) can induce osteoblast differentiation when treated with conditioned medium (CM) containing RAW264.7 macrophages challenged with a combination of Ti particles and Ad-siTNFα-BMP-2 (Ti-ad CM) assessed by alkaline phosphatase activity. The receptor activator of nuclear factor-κB ligand was downregulated in pro-osteoblastic MC3T3-E1 cells treated with Ti-ad CM in comparison with conditioned medium of RAW264.7 macrophages challenged with Ti particles (Ti CM). We suggest that Ad-siTNFα-BMP-2 induced osteoblast differentiation and inhibited osteoclastogenesis on a cell model of a Ti particle-induced inflammatory response, which may provide a novel approach for the treatment of periprosthetic osteolysis.

Animals , /metabolism , Osteoblasts/metabolism , RNA, Small Interfering/metabolism , Titanium/adverse effects , Tumor Necrosis Factor-alpha/metabolism , Adenoviridae/genetics , /genetics , Bone Resorption/genetics , Cell Differentiation , Cell Line , Gene Expression , Genetic Vectors/genetics , Osteoblasts/cytology , Osteoblasts/drug effects , RNA, Small Interfering/genetics , Tumor Necrosis Factor-alpha/genetics
Braz. j. med. biol. res ; 46(8): 676-680, ago. 2013. graf
Article in English | LILACS | ID: lil-684529


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.

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 , Transfection
Indian J Biochem Biophys ; 2013 Feb; 50(1): 19-25
Article in English | IMSEAR | ID: sea-147282


R-spondin (Rspo)s proteins are a new group of Wnt/beta-catenin signaling agonists. These signaling molecules are known to be involved in the developmental stages of skeletal system. Recent studies in various murine osteoblast models have proposed that Rspo1 may interact with Wnt signaling pathway to induce differentiation in osteoblasts. Though findings in murine osteoblasts implicate a synergestic role of Rspo1 with Wnt signaling, still no study has addressed the similar role in more clinically applicable osteoblast models i.e., human cell lines or primary cells. Therefore, in the present study, we investigated the possible role of Rspo1 during differentiation process of human in vitro osteoblast cell models like primary osteoblasts or human osteoprogenitor cell line hFOB1.19 along with murine preosteoblast cell line MC3T3 E-1. Our results showed increase in Rspo1 at transcript level during differentiating phase of human primary osteoblasts and human FOB1.19 cells. We also found that Rspo1 (100 ng/mL) acts additively with Wnt3a to activate Wnt signaling, as confirmed by luciferase activity after transfection of TOPFLASH construct to hFOB1.19 cells. Similar additive role of Rspo1 and Wnt3a was apparent in alkaline phosphatase (ALP) activity analysis of human primary cells. Moreover, a reduction in ALP activity was observed with knock-down of Rspo1 by transfected shRNA in hFOB1.19 cells. These results suggested the possibility of autocrine regulation by Rspo1 on the osteogenic activities in human in vitro osteoblast models. Furthermore, these results were corroborated in MC3T3-E1, murine osteoblast cell model. Osteoblastic differentiation was induced by transfection of Rspo1 which was confirmed by increased ALP staining and qRT-PCR analysis of osteogenic markers, such as Runx2 and osteocalcin. In conclusion, present study highlights the role of Rspo1 in bone remodeling where it activates Wnt signaling to induce differentiation, as shown in human as well murine in vitro osteoblast cell models.

Animals , Cell Differentiation , Cell Line , Humans , Mice , Osteoblasts/cytology , Osteoblasts/metabolism , Osteogenesis/physiology , Thrombospondins/metabolism , Wnt Proteins/metabolism , Wnt Signaling Pathway/physiology
J. appl. oral sci ; 20(6): 628-635, Nov.-Dec. 2012. ilus
Article in English | LILACS | ID: lil-660633


Bone morphogenetic protein type 2 (BMP-2) is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. OBJECTIVES: This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and β-glycerophosphate. MATERIAL AND METHODS: Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2) or absence (ASCs+OM) of BMP-2. The alkaline phosphatase (ALP) activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II), osteonectin, and osteocalcin were evaluated by qPCR. Results: ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. CONCLUSIONS: We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity), intermediate (osteonectin and osteocalcin), or final (calcium deposition) phases, suggesting that the exogenous addition of BMP-2 did not improve the in vitro osteogenesis process of human ASCs.

Humans , Adipose Tissue/cytology , /pharmacology , Cell Differentiation/drug effects , Glycerophosphates/pharmacology , Osteogenesis , Stem Cells/drug effects , Analysis of Variance , Alkaline Phosphatase/physiology , Ascorbic Acid/metabolism , Ascorbic Acid/pharmacology , Blotting, Western , /metabolism , /metabolism , /metabolism , Cells, Cultured , Glycerophosphates/metabolism , Osteoblasts/metabolism , Polymerase Chain Reaction , RNA, Messenger/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Time Factors
Braz. j. med. biol. res ; 45(8): 693-700, Aug. 2012. ilus, tab
Article in English | LILACS | ID: lil-643657


Tissue transglutaminase (type II, TG2) has long been postulated to directly promote skeletal matrix calcification and play an important role in ossification. However, limited information is available on the expression, function and modulating mechanism of TG2 during osteoblast differentiation and mineralization. To address these issues, we cultured the well-established human osteosarcoma cell line SAOS-2 with osteo-inductive conditioned medium and set up three time points (culture days 4, 7, and 14) to represent different stages of SAOS-2 differentiation. Osteoblast markers, mineralization, as well as TG2 expression and activity, were then assayed in each stage. Furthermore, we inhibited TG activity with cystamine and then checked SAOS-2 differentiation and mineralization in each stage. The results showed that during the progression of osteoblast differentiation SAOS-2 cells presented significantly high levels of osteocalcin (OC) mRNA, bone morphogenetic protein-2 (BMP-2) and collagen I, significantly high alkaline phosphatase (ALP) activity, and the increased formation of calcified matrix. With the same tendency, TG2 expression and activity were up-regulated. Furthermore, inhibition of TG activity resulted in a significant decrease of OC, collagen I, and BMP-2 mRNA and of ALP activity and mineralization. This study demonstrated that TG2 is involved in osteoblast differentiation and may play a role in the initiation and regulation of the mineralization processes. Moreover, the modulating effects of TG2 on osteoblasts may be related to BMP-2.

Humans , Calcification, Physiologic/physiology , Cell Differentiation/physiology , Osteoblasts/cytology , Transglutaminases/physiology , /metabolism , Cell Line, Tumor , Collagen/metabolism , Osteoblasts/metabolism , Osteocalcin/metabolism , RNA, Messenger/metabolism , Time Factors
Braz. j. med. biol. res ; 44(7): 618-623, July 2011. ilus
Article in English | LILACS | ID: lil-595709


Taurine has positive effects on bone metabolism. However, the effects of taurine on osteoblast apoptosis in vitro have not been reported. The aim of this study was to investigate the activity of taurine on apoptosis of mouse osteoblastic MC3T3-E1 cells. The data showed that 1, 5, 10, or 20 mM taurine resulted in 16.7, 34.2, 66.9, or 63.75 percent reduction of MC3T3-E1 cell apoptosis induced by the serum deprivation (serum-free α-MEM), respectively. Taurine (1, 5, or 10 mM) also reduced cytochrome c release and inhibited activation of caspase-3 and -9, which were measured using fluorogenic substrates for caspase-3/caspase-9, in serum-deprived MC3T3-E1 cells. Furthermore, taurine (10 mM) induced extracellular signal-regulated kinase (ERK) phosphorylation in MC3T3-E1 cells. Knockdown of the taurine transporter (TAUT) or treatment with the ERK-specific inhibitor PD98059 (10 μM) blocked the activation of ERK induced by taurine (10 mM) and abolished the anti-apoptotic effect of taurine (10 mM) in MC3T3-E1 cells. The present results demonstrate for the first time that taurine inhibits serum deprivation-induced osteoblast apoptosis via the TAUT/ERK signaling pathway.

Animals , Cattle , Mice , Apoptosis/drug effects , Apoptosis/physiology , Extracellular Signal-Regulated MAP Kinases/metabolism , Membrane Glycoproteins/metabolism , Membrane Transport Proteins/metabolism , Osteoblasts/drug effects , Taurine/pharmacology , Analysis of Variance , Caspase 9/metabolism , /metabolism , Osteoblasts/metabolism , RNA, Messenger/metabolism
Braz. dent. j ; 22(3): 179-184, 2011. ilus
Article in English | LILACS | ID: lil-595640


The bone-biomaterial interface has been characterized by layers of afibrillar extracellular matrix (ECM) enriched in non collagenous proteins, including osteopontin (OPN), a multifunctional protein that in bone controls cell adhesion and ECM mineralization. Physical and chemical aspects of biomaterial surfaces have been demonstrated to affect cell-ECM-substrate interactions. The present paper described the ability of oxidative nanopatterning of titanium (Ti) surfaces to control extracellular OPN deposition in vitro. Ti discs were chemically treated by a mixture of H2SO4/H2O2 for either 30 min [Nano(30') Ti] or 4 h [Nano(4h) Ti]. Non-etched Ti discs were used as control. Primary osteogenic cells derived from newborn rat calvarial bone were plated on control and etched Ti and grown under osteogenic conditions up to 7 days. High resolution scanning electron microscopy revealed that treated Ti discs exhibited a nanoporous surface and that areas of larger nanopits were noticed only for Nano(4h) Ti. Large extracellular OPN accumulation were detectable only for Nano(4h) Ti, which was associated with OPN-positive cells with typical aspects of migrating cells. At day 3, quantitative results in terms of areas of OPN labeling were as follows: Nano(4h) Ti > Nano(30') Ti > Control Ti. In conclusion, chemically nanostructured Ti surfaces may support the enhancement of endogenous extracellular OPN deposition by osteogenic cells in vitro depending on the etching time, a finding that should be taken into consideration in strategies to biofunctionalize implant surfaces with molecules with cell adhesion capacity.

A interface osso-implante é caracterizada pela presença de uma camada de matriz extracellular (MEC) afibrilar rica em proteínas não-colágenas, incluindo osteopontina (OPN), cujas funções no tecido ósseo estão relacionadas à adesão celular e ao controle do processo de mineralização da MEC (crescimento de cristais). Aspectos físicos e químicos das superfícies de biomateriais podem afetar as interações célula-MEC-substrato. O objetivo do presente estudo foi demonstrar a capacidade de aspectos nanotopográficos de superfície de titânio (Ti) de controlar a deposição extracelular de OPN in vitro. Discos de Ti foram tratados quimicamente por solução de H2SO4/H2O2 durante 30 min [Nano(30') Ti] ou 4 h [Nano(4h) Ti]. Superfícies de Ti não tratadas foram usadas como controle. Células osteogênicas primárias derivadas de calvárias de ratos recém-nascidos foram plaqueadas sobre os discos de Ti e cultivadas em condições osteogênicas por até 7 dias. Microscopia eletrônica de varredura de alta resolução revelou que os discos de Ti tratados quimicamente exibiam superfície nanoporosa, com áreas de nanoporos maiores para Nano(4h) Ti. Apenas para esse grupo detectavam-se acúmulos extensos de OPN extracelular, os quais se distribuíam em áreas adjacentes a células OPN-positivas, com aspectos morfológicos típicos de células em migração. Em conclusão, a nanoestruturação química de superfície de Ti pode favorecer o aumento da deposição extracelular de OPN endógena por células osteogênicas in vitro, dependendo do tempo de condicionamento utilizado, o que deve ser considerado no desenvolvimento de estratégias para funcionalizar superfícies de implantes com moléculas com reconhecido efeito no processo de adesão celular.

Animals , Rats , Biocompatible Materials/chemistry , Dental Materials/chemistry , Extracellular Matrix Proteins/pharmacokinetics , Nanoparticles/chemistry , Osteopontin/pharmacokinetics , Titanium/chemistry , Adsorption , Animals, Newborn , Acid Etching, Dental/methods , Cells, Cultured , Cell Adhesion/physiology , Cell Movement/physiology , Hydrogen Peroxide/chemistry , Materials Testing , Microscopy, Electron, Scanning , Nanotechnology , Oxidation-Reduction , Osteoblasts/metabolism , Osteoblasts/physiology , Osteogenesis/physiology , Rats, Wistar , Surface Properties , Sulfuric Acids/chemistry , Time Factors
Braz. dent. j ; 21(6): 491-498, 2010. ilus, tab
Article in English | LILACS | ID: lil-572294


The aim of this study was to investigate the effects of low-level laser therapy (LLLT) by using gallium aluminum arsenide (GaAlAs) diode laser on human osteoblastic cells grown on titanium (Ti). Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured on Ti discs for up to 17 days. Cells were exposed to LLLT at 3 J/cm2 (wavelength of 780 nm) at days 3 and 7 and non-irradiated cultures were used as control. LLLT treatment did not influence culture growth, ALP activity, and mineralized matrix formation. Analysis of cultures by epifluorescence microscopy revealed an area without cells in LLLT treated cultures, which was repopulated latter with proliferative and less differentiated cells. Gene expression of ALP, OC, BSP, and BMP-7 was higher in LLLT treated cultures, while Runx2, OPN, and OPG were lower. These results indicate that LLLT modulates cell responses in a complex way stimulating osteoblastic differentiation, which suggests possible benefits on implant osseointegration despite a transient deleterious effect immediately after laser irradiation.

Este estudo teve como objetivo investigar o efeito do laser diodo de gálio-alumínio-arsênio (GaAlAs) em células osteoblásticas humanas cultivadas sobre discos de Ti. Para tanto, células osteoblásticas foram obtidas por digestão enzimática de osso alveolar humano e cultivadas sobre discos de Ti por 17 dias. As células foram submetidas à irradiação no 3º e 7º dias na dose de 3 J/cm2 e comprimento de onda de 780 nm e células não irradiadas foram usadas como controle. A irradiação não alterou a proliferação celular, atividade de ALP e formação de matriz mineralizada. Microscopia por epifluorescência indicou que após 24 h da aplicação do laser, as culturas irradiadas apresentaram áreas sem células, que mais tarde foram repovoadas por células em fase de proliferação e menos diferenciadas. O laser aumentou a expressão gênica relativa da ALP, OC, BSP e BMP-7 e reduziu a de RUNX2, OPN e OPG. Os resultados indicam que a terapia com laser modula de forma complexa as respostas celulares, estimulando a diferenciação osteoblástica. Assim, é possível sugerir possíveis benefícios do laser na osseointegração de implantes de Ti apesar do efeito deletério às células imediatamente após a irradiação.

Humans , Bone Matrix/growth & development , Gene Expression/radiation effects , Low-Level Light Therapy , Osseointegration/radiation effects , Osteoblasts/radiation effects , Analysis of Variance , Alkaline Phosphatase/biosynthesis , Alkaline Phosphatase/genetics , /biosynthesis , /genetics , Cells, Cultured/radiation effects , Collagen Type I/biosynthesis , Collagen Type I/genetics , Core Binding Factor Alpha 1 Subunit/biosynthesis , Core Binding Factor Alpha 1 Subunit/genetics , Integrin-Binding Sialoprotein/biosynthesis , Integrin-Binding Sialoprotein/genetics , Intercellular Adhesion Molecule-1/biosynthesis , Intercellular Adhesion Molecule-1/genetics , Lasers, Semiconductor/therapeutic use , Osteoblasts/metabolism , Osteocalcin/biosynthesis , Osteocalcin/genetics , Osteopontin/biosynthesis , Osteopontin/genetics , Osteoprotegerin/biosynthesis , Osteoprotegerin/genetics , RANK Ligand/biosynthesis , RANK Ligand/genetics , Statistics, Nonparametric , Titanium
Article in English | IMSEAR | ID: sea-51558


BACKGROUND AND OBJECTIVE: The enamel matrix derivative (EMD) has been used in combination with barrier membranes to optimize regeneration in vertical osseous defects. However, the osteoblast response when exposed to the EMD/barrier membrane combination has not yet been evaluated. The osteoblast behavior when exposed to a combination of regenerative materials must be evaluated to fully understand their effect on bone regeneration. Therefore, the present study was undertaken to estimate the initial adhesion and alkaline phosphatase (ALP) activity of an osteoblast cell line (SaOS-2) when exposed to four commercially available resorbable membranes and determine if the addition of EMD had any modulatory effect on osteoblast behavior. MATERIALS AND METHODS: 5 x 104 SaOS-2 cells between passages 7-10 were cultured in two 24-well culture plates. Plate A was used for the adhesion assay and Plate B was used for the ALP assay. A MTT (3-[4, 5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide) assay was done after 24 hours to determine the adhesion of the osteoblastic cells to four barrier membranes: 1) a non cross-linked porcine Type I and III collagen membrane (BG), 2) a weakly cross-linked Type I collagen membrane (HG), 3) a glutaraldehyde cross-linked bovine Type I collagen (BM), and 4) a resorbable polymer membrane (CP). Osteoblast differentiation was studied using an ALP assay with p-nitro phenyl phosphate as the substrate at 24 hours, 72 hours, and 1 week. A total of 50 microg/ml of EMD dissolved in 10 mM acetic acid was added into each well and the entire experimental protocol outlined above was repeated. RESULTS: The osteoblast adhesion to collagen barriers showed a statistically insignificant reduction following the addition of EMD. Adhesion to the polymer barrier, although significantly lower when compared with collagen barriers, was unaffected by the addition of EMD. ALP activity after 1 week among the various groups was as follows: EMD alone (75.59+/-2.5)>EMD/BG(64.78+/-3.04)>EMD/HG(55.40+/-3.89) approximately EMD/BM(54.75+/-4.17)>BG (51.32+/-2.76)>HG(49.92+/-2.4)>BM(48.14+/-1.4)>Control(46.29+/-1.39)>EMD/CP (37.46+/-3.54)>CP(32.12+/-1.49) CONCLUSION: There was no additive effect on osteoblast adhesion/ALP activity following exposure to an EMD/polymer combination. EMD/collagen positively influences osteoblast differentiation in a time dependent manner.

Absorbable Implants , Alkaline Phosphatase/metabolism , Animals , Bone Regeneration/physiology , Cattle , Cell Adhesion/drug effects , Cell Differentiation/drug effects , Cell Line, Tumor , Dental Enamel Proteins/pharmacology , Fibrillar Collagens , Humans , Membranes, Artificial , Osteoblasts/drug effects , Osteoblasts/metabolism , Polymers , Swine