Indiana University-Purdue University Indianapolis (IUPUI) / Fibroblast growth factor-23 (FGF23) is a hormone produced in bone that acts upon the kidney to control blood phosphate and 1,25-(OH)2 vitamin D concentrations. Chronic kidney disease-mineral bone disorder (CKD-MBD) is a major public health problem, affecting 1 in 8 individuals. These patients can have markedly elevated FGF23 at end stage disease which is associated with metabolic bone anomalies, left ventricular hypertrophy, as well as increased mortality (>6-fold). The FGF23 co-receptor αKlotho (αKL) is a membrane-bound protein (mKL) that forms heteromeric complexes with FGF receptors (FGFRs) to initiate intracellular signaling. It also circulates as a cleavage product of mKL (‘cleaved’, or cKL). Previously, a patient with increased plasma cKL from a balanced translocation between chromosomes 9 and 13 in the KLOTHO gene presented with metabolic bone disease and a complex endocrine profile, despite hypophosphatemia. The lack of a reliable cell model in which to study potential FGF23-cKL interactions is a major hurdle for the field of phosphate metabolism. The goal of the present studies was to test and characterize bone cell lines that may respond to FGF23 and/or cKL, permitting study of novel aspects of phosphate handling and control of FGF23 expression. It was confirmed that stable delivery of cKL via AAV2/8 to wild type (WT) and KL-KO mice resulted in highly elevated bone FGF23 mRNA. MC3T3 (mouse) and ROS (rat) osteoblastic cell lines were tested for p-ERK1/2 responses to control FGFs, as well as FGF23 and cKL, alone or in combination. Importantly, both cell lines demonstrated responsiveness to FGF23+cKL only, and not the individual factors. To test responsiveness at the cell level, EGR1 mRNA was tested as an index of FGFR activity and showed modest increases with the same treatments, supporting that other factors may be required for full transcriptional effects. The present studies show that MC3T3 have FGF-dependent signaling capabilities, and that the combination of FGF23+cKL is required for efficient MAPK signaling. These results demonstrated that cKL provision is permissive for efficient FGF23 signaling in bone, and revealed important implications for the regulation of FGF23 and cKL in Mendelian, and common, genetic disorders of phosphate handling and biomineralization.

Sabe-se que o flúor causa alterações químicas e celulares no osso, dependendo da dosagem e do tempo de exposição a esse elemento. Já foi demonstrado que alterações clínicas importantes, como a osteoporose, osteopetrose e esclerose óssea, podem ser geradas pela ingestão de flúor. Sabe-se que o flúor pode guiar os osteoblastos ao crescimento, sob doses e tempos de exposição específicos. A sensibilidade dos tecidos mineralizados ao fluoreto responde a mecanismos genéticos inerentes a cada população. Dentro de uma mesma espécie, diferentes linhagens apresentam um comportamento distinto frente a esse fármaco. Durante várias décadas, os estudos com fluoretos resumiram-se ao fluoreto de sódio, amplamente utilizado na prevenção das cáries dentárias. Recentemente, outras fontes de fluoretos como TiF4 e SnF2 têm sido buscadas, com intuito de potencializar as vantagens desse elemento. Porém não existem estudos ainda sobre o efeito do AlF3 sobre o tecido ósseo até a presente data. Desse modo, achamos oportuno investigar seu papel, quando administrado a células ósseas e para tal, investigamos a viabilidade e padrão de atividade das fosfatases desse sal (AlF3) quando administrado às células da linhagem pré-osteblástica MC3T3, em comparação ao NaF. Além disso, sabendo que diferentes células apresentam comportamento distinto ao tratamento com fluoretos, estudamos o papel do NaF quando administrado a duas linhagens celulares de camundongos com densidades ósseas distintas e com diferentes susceptibilidades ao fluoreto: C3H/HeJ e C57BL/6J (C3H e C57, alta e baixa densidades ósseas, respectivamente). As células também foram avaliadas quanto à viabilidade celular e ao perfil das fosfatases. Todos os resultados foram submetidos à Análise de Variância a três critérios e nos permitiu identificar que entre os sais de fluoreto, apenas o NaF promoveu alterações na viabilidade celular das células MC3T3. O AlF3 não exerceu influência na atividade da fosfatase alcalina quando consideramos as células pré-osteoblásticas, no entanto, o efeito desse fluoreto sobre a atividade das fosfatases ácidas totais e ácida resistente ao tartarato ocorre de modo dicotômico. Quando consideramos uma população celular heterogênea, coletada de calvária, como foi o caso das linhagens C3H e C57, verificamos que o NaF interferiu tanto sobre a proliferação, quanto a atividade das fosfatases de modo bastante distinto para ambas linhagens celulares. Assim concluimos que o NaF é capaz de exercer efeitos mais diversificados em diferentes tipos celulares, alterando viabilidade celular e atividade de enzimas; em contrapartida, o AlF3 produz efeitos mais especifícos e delimitados em pré-osteoblastos. / It is well known that fluoride causes chemical and cellular alteration on bone tissue, depending on the dosage and time of exposure to this element. Several studies have been conducted to show that some bone diseases, such as osteoporosis, osteopetrosis and esclerosis, can occur as a consequence of the excessive fluoride uptake. Fluoride can lead osteoblast to proliferate, under certain conditions as time and dosage. The same way, excessive dosages of fluoride can alter protein activity in osteoblasts, by changing the expression of retated genes. Sensitivity of mineralized tissues to fluoride depends on a genetic background inherent to each population. Within the same specie, different lineages present distint behavior to the ingestion of this íon. Over several decades, many researches focused on the study of NaF as the only source of fluoride, once it is wide spread used to the prevention of dental caries. More recently, different sources of fluoride have been assessed, like TiF4 e SnF2</sub, with the main aim to enhance biochemical and physical benefits of this element. Despite of this, none of these formulations have been tested to evaluate their effect on the bone tissue. Thus, we believe it is advisable to investigate viability and phosphatases activities of pre-osteoblast MC3T3 cells treated with Aluminium fluoride, compared to NaF treatments. Besides this, facing the knowledge that different cell lines have distinct behavior when treated with fluoride, we used two inbred strains of mice with distint mineral bone densities, C3H/HeJ (C3H, high bone density) and C57BL/6J (C57, low bone density), to assess the viability and phosphatases activities after the treatment with NaF. Overall results were tested by 3 way- Anova. AlF3 did not exibit influence over on alkaline phosphatase when pre-osteoblast cells are considered, but the effect of this fluoride salt on the activity of total and tartarate resistant acid phosphatases ocurrs on a dicotomic way. When we consider a mixed cell population, collected from calvária as for the C3H and C57 assays, NaF did interfered as with the proliferation rate as phosphatase activities on a very distinct fashion between the two lineages. Thus, it is possible to conclude that NaF is able to exert variable effects in different cell types and enzyme activities; on the other hand, AlF3 effects are more specific and surrounded in pre-osteblastic cells.

Sabe-se que o flúor causa alterações químicas e celulares no osso, dependendo da dosagem e do tempo de exposição a esse elemento. Já foi demonstrado que alterações clínicas importantes, como a osteoporose, osteopetrose e esclerose óssea, podem ser geradas pela ingestão de flúor. Sabe-se que o flúor pode guiar os osteoblastos ao crescimento, sob doses e tempos de exposição específicos. A sensibilidade dos tecidos mineralizados ao fluoreto responde a mecanismos genéticos inerentes a cada população. Dentro de uma mesma espécie, diferentes linhagens apresentam um comportamento distinto frente a esse fármaco. Durante várias décadas, os estudos com fluoretos resumiram-se ao fluoreto de sódio, amplamente utilizado na prevenção das cáries dentárias. Recentemente, outras fontes de fluoretos como TiF4 e SnF2 têm sido buscadas, com intuito de potencializar as vantagens desse elemento. Porém não existem estudos ainda sobre o efeito do AlF3 sobre o tecido ósseo até a presente data. Desse modo, achamos oportuno investigar seu papel, quando administrado a células ósseas e para tal, investigamos a viabilidade e padrão de atividade das fosfatases desse sal (AlF3) quando administrado às células da linhagem pré-osteblástica MC3T3, em comparação ao NaF. Além disso, sabendo que diferentes células apresentam comportamento distinto ao tratamento com fluoretos, estudamos o papel do NaF quando administrado a duas linhagens celulares de camundongos com densidades ósseas distintas e com diferentes susceptibilidades ao fluoreto: C3H/HeJ e C57BL/6J (C3H e C57, alta e baixa densidades ósseas, respectivamente). As células também foram avaliadas quanto à viabilidade celular e ao perfil das fosfatases. Todos os resultados foram submetidos à Análise de Variância a três critérios e nos permitiu identificar que entre os sais de fluoreto, apenas o NaF promoveu alterações na viabilidade celular das células MC3T3. O AlF3 não exerceu influência na atividade da fosfatase alcalina quando consideramos as células pré-osteoblásticas, no entanto, o efeito desse fluoreto sobre a atividade das fosfatases ácidas totais e ácida resistente ao tartarato ocorre de modo dicotômico. Quando consideramos uma população celular heterogênea, coletada de calvária, como foi o caso das linhagens C3H e C57, verificamos que o NaF interferiu tanto sobre a proliferação, quanto a atividade das fosfatases de modo bastante distinto para ambas linhagens celulares. Assim concluimos que o NaF é capaz de exercer efeitos mais diversificados em diferentes tipos celulares, alterando viabilidade celular e atividade de enzimas; em contrapartida, o AlF3 produz efeitos mais especifícos e delimitados em pré-osteoblastos. / It is well known that fluoride causes chemical and cellular alteration on bone tissue, depending on the dosage and time of exposure to this element. Several studies have been conducted to show that some bone diseases, such as osteoporosis, osteopetrosis and esclerosis, can occur as a consequence of the excessive fluoride uptake. Fluoride can lead osteoblast to proliferate, under certain conditions as time and dosage. The same way, excessive dosages of fluoride can alter protein activity in osteoblasts, by changing the expression of retated genes. Sensitivity of mineralized tissues to fluoride depends on a genetic background inherent to each population. Within the same specie, different lineages present distint behavior to the ingestion of this íon. Over several decades, many researches focused on the study of NaF as the only source of fluoride, once it is wide spread used to the prevention of dental caries. More recently, different sources of fluoride have been assessed, like TiF4 e SnF2</sub, with the main aim to enhance biochemical and physical benefits of this element. Despite of this, none of these formulations have been tested to evaluate their effect on the bone tissue. Thus, we believe it is advisable to investigate viability and phosphatases activities of pre-osteoblast MC3T3 cells treated with Aluminium fluoride, compared to NaF treatments. Besides this, facing the knowledge that different cell lines have distinct behavior when treated with fluoride, we used two inbred strains of mice with distint mineral bone densities, C3H/HeJ (C3H, high bone density) and C57BL/6J (C57, low bone density), to assess the viability and phosphatases activities after the treatment with NaF. Overall results were tested by 3 way- Anova. AlF3 did not exibit influence over on alkaline phosphatase when pre-osteoblast cells are considered, but the effect of this fluoride salt on the activity of total and tartarate resistant acid phosphatases ocurrs on a dicotomic way. When we consider a mixed cell population, collected from calvária as for the C3H and C57 assays, NaF did interfered as with the proliferation rate as phosphatase activities on a very distinct fashion between the two lineages. Thus, it is possible to conclude that NaF is able to exert variable effects in different cell types and enzyme activities; on the other hand, AlF3 effects are more specific and surrounded in pre-osteblastic cells.

This dissertation presents material design strategies to investigate cell-biomaterial interactions on specific biocompatible polymers and polymer blends by using mouse pre-osteoblastic MC3T3 cells aiming for potential applications in bone tissue engineering. Chapter 1 reviews some related background knowledge including polymeric biomaterials for tissue engineering, cell-biomaterial interaction, synthetic photo-crosslinkable and degradable polymers, and the effect of surface features on osteoblast cell responses. Chapter 2 presents photo-crosslinkable composites of poly(propylene fumarate) (PPF), an injectable and biodegradable polyester, and methacryl-polyhedral oligomeric silsesquioxane (mPOSS), which has eight methacryl groups tethered with a cage-like hybrid inorganic-organic nanostructure, for bone tissue engineering applications. Blending mPOSS with PPF was found to decrease the viscosity of PPF, expedite photo-crosslinking process, increase tensile modulus and accelerate hydrolytic degradation of crosslinked PPF/mPOSS while it did not significantly alter surface wettability, protein adsorption, and cell response. Chapter 3 demonstrates a polymer blend composed of amorphous PPF and semicrystalline poly(ε-caprolactone) (PCL), a widely used biocompatible and biodegradable polymer, in both uncrosslinked and photo-crosslinked forms. Thermal, rheological, mechanical properties as well as surface hydrophilicity and morphology can be well controlled by crosslinking density and crystallinity. Distinct cell attachment, spreading, and proliferation have been found to PPF/PCL blends in the presence or absence of cross-links. Chapter 4 and 5 describe the crystallization induced banded spherulitic morphologies in PPF/PCL blends and PCL homo-blends and their preliminary biological evaluation. Thermal properties, crystallization kinetics, and surface morphology of these blends can be regulated by isothermal crystallization temperature and composition. Surface roughness has been found to play an important role in influencing protein adsorption and cell response. Chapter 6 introduces a newly synthesized biodegradable elastomer, poly(ε-caprolactone) triacrylate (PCLTA), with two different molecular weights resulting in distinct mechanical properties at physiological temperature. Using replica molding from silicon wafers, photo-crosslinked PCLTA substrates with concentric micro-grooves have been successfully fabricated. MC3T3 cell attachment, proliferation, and differentiation could be better supported by stiffer substrates while not significantly influenced by micro-groove dimensions. Cell orientation, nuclei shape and localization, mineralization, and gene expression level of osteocalcin have been found to be more significant on narrower micro-grooves when groove depth was 10 μm.

Hypercholesterolemia is a risk factor for osteoporosis but the underlying mechanism is unknown. Previous evidence suggests that osteoporosis results from an impaired regulation of osteoblasts by fluid pressure fluctuations in the bone matrix. Recently, our laboratory showed that enhanced cholesterol in the cell membrane, due to hypercholesterolemia, alters leukocyte mechanosensitivity. We predict a similar link between osteoblasts and hypercholesterolemia leading to osteoporosis. Specifically, we hypothesize that extracellular cholesterol modifies the osteoblast sensitivity to pressure. MC3T3-E1 cells were exposed to hydrodynamic pressures regimes (mean=40mmHg, amplitude=0-20mmHg, frequency=1Hz) for 1-12 hours. To assess the impact of membrane cholesterol enrichment, cells were pre-treated with 0-50 µg/mL cyclodextran:cholesterol conjugates. We assessed the pressure effects on mitosis and F-actin stress fiber formation (SFF) of cells. Exposure of cells to 50/30 mmHg pressure transiently increased the number of cells in the S- and G2M-phases of mitosis after 6 and 12 hours, respectively. Relative to controls, osteoblast-like cells exposed to all pressures exhibited significantly (p

L'efficacité des matériaux utilisés pour favoriser la régénération osseuse dépend entre autres de leur capacité d'interaction avec le tissu environnant. Ainsi, des peptides d'adhésion contenant la séquence Arginine-Glycine-Acide aspartique (RGD) sont parmi les plus utilisés pour favoriser 1'attachement des cellules osseuses au matériau. Les facteurs de croissance, dont les protéines morphogénétiques osseuses (BMPs), jouent également un rôle clé dans le processus de différenciation et de fonctionnement des cellules osseuses. La BMP-2 approuvée par la Food and Drug Administration aux États-Unis dans le cadre d'un système de libération est déjà actuellement utilisée cliniquement pour accroître la régénération osseuse. De plus, la BMP-9 a récemment suscité un grand intérêt en raison de son potentiel ostéogénique supérieur à celui de la BMP-2. Néanmoins, peu d'études se sont intéressées à l'influence des peptides d'adhésion sur la réponse cellulaire aux BMPs. Le principal objectif de ce travail de doctorat a donc été de fonctionnaliser un polymère par des peptides d'adhésion et de déterminer son impact sur la capacité de préostéoblastes de souris MC3T3-E1 à répondre à la BMP-2 et à la BMP-9. Cette étude s'est tout d'abord intéressée à la synthèse et à la caractérisàtion d'un film de polycaprolactone (PCL) fonctionnalisé par un peptide dérivé de la sialoprotéine osseuse qui contient 15 acides aminés dont la séquence RGD (pRGD). La fonctionnalisation a consisté en une hydrolyse alcaline du film PCL suivie d'un greffage covalent du pRGD. Les films PCL hydrolysés ont adsorbé des protéines sériques adhésives fibronectine et vitronectine mais sans favoriser l'étalement des préostéoblastes MC3T3-E1. En absence de sérum, les films PCL-pRGD ont permis un étalement plus important des préostéoblastes MC3T3-E1 par rapport au PCL fonctionnalisé par le pRGE (peptide négatif) ou au PCL hydrolysé. Le PCL-pRGD a augmenté le niveau de phosphorylation de la FAK (Tyr 397 ), évènement nécessaire chez les préostéoblastes murins pour permettre leur différenciation en ostéoblastes matures. Ainsi, seules les cellules adhérant au PCL-pRGD ont répondu à la BMP-2 via une activation de la voie canonique des Smads. Dans un deuxième temps, la stimulation des préostéoblastes par la BMP-2 et/ou BMP-9 a été déterminée sur PCL-pRGD. L'EC50 pour chacune des BMPs a tout d'abord été évaluée afin de choisir la concentration optimale à utiliser lors de la combinaison des BMP-2/BMP-9. Tandis que la BMP-2 a induit une phosphorylation des Smad1/5/8 dès 30 min, la BMP-9 a engendré un retard d'activation à 4 h. Cette observation était concomitante avec une diminution de la ?-caténine. Par contre, aucune différence n'a été observée avec la BMP-2 et la BMP-9 en termes d'activation des MAPKinases. Néanmoins, tant la BMP-2 que la BMP-9 ont été capables d'induire la différenciation des préostéoblastes MC3T3-E1 sur PCL-pRGD telle que mis en évidence par la synthèse d' ARNm codant pour Dlx5, Ostérix ou l'ostéocalcine et l'augmentation de l'activité de l'alcaline phosphatase à 72h. Mais l'utilisation d'une combinaison de la BMP-2 avec la BMP-9 stabilisant la ?-caténine n'a pas permis d'obtenir un effet additif sur la différenciation, des activations semblables à celles de la BMP-2 utilisée seule ayant été alors observées. En résumé, l'utilisation de BMP-2 ou de BMP-9 en combinaison avec une surface fonctionnalisée par des peptides d'adhésion semble être une voie prometteuse pour favoriser la différenciation cellulaire. Cependant, cette étude montre également que la BMP-2 et la BMP-9 induisent des voies d'activation antagonistes au contact du PCL-pRGD. Elle met donc l'emphase sur la nécessité de mieux comprendre l'influence de la fonctionnalisation de matériaux par des peptides d'adhésion sur la réponse aux facteurs de croissance.

Transforming growth factor-β1 (TGF-β1), an abundant growth factor in bone matrix, has been shown to be involved in bone formation and fracture healing. The mechanism of action of the osteogenic effect of TGF-β1 is not clearly understood. In this study, we found that the addition of TGF-β1 to murine osteoblastic MC3T3-E1 cells induced vascular endothelial growth factor (VEGF) mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of TGF-β1. The induction was superinduced by cycloheximide and blocked by actinomycin D. Ro 31-8220, a protein kinase C inhibitor, abrogated the induction. In addition, curcumin, an inhibitor for transcription factor AP-1, also blocked the induction. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-κB. Transient transfection experiment showed that VEGF promoter activity increased 3.6-fold upon TGF-β1 stimulation. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 4 h after TGF-β1 stimulation. Our results therefore suggest that at least part of the osteogenic activity of TGF-β1 may be attributed to the production of VEGF.

Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine that induces apoptosis in a number of cell systems, including osteoblasts. Transforming growth factor β1 (TGF-β1) is an abundant growth factor that is known to stimulate bone formation. This study was designed to examine the role of TGF-β1 on TNF-α-induced apoptosis in murine osteoblastic MC3T3-E1 cells. Total RNA was extracted from MC3T3-E1 cells treated with 20 ng/ml of TNF-α, 10 ng/ml of TGF-β1, or combination, for 6 h. TNF-α exerted a variety of effects on the apoptotic gene expression in osteoblasts. Ribonuclease protection assays (RPA) revealed that TNF-α upregulated the mRNA levels of caspase-1, -7, -11, -12, and FAS. Western blot analysis showed enhanced processing of caspase-1, -7, -11, and -12, with the appearance of their activated enzymes 24 h after TNF-α treatment. In addition, caspase-3-like activity was significantly activated following TNF-α treatment. Levels of cleaved poly(ADP-ribose) polymerase and FAS protein were also elevated by TNF-α. Finally, Hoechst staining, terminal deoxynucleotidyl-transferase nick-end labeling (TUNEL) assay, and oligonucleosome ELISA all indicated that TNF-α induced apoptosis. In contrast, the addition of TGF-β1 attenuated all of the aforementioned effects of TNF-α. Our results demonstrate that TGF-β1 can decrease TNF-α-induced apoptosis in murine osteoblasts at least in part by attenuating TNF-α-induced caspase gene expression.

Este estudo in vitro teve como objetivo avaliar os efeitos moleculares do TGF-&beta;1, com diferentes períodos de suplementação, sobre a formação do fenótipo osteogênico das células MC3T3-E1, comparando-os com células tratadas com AA+&beta;-GP suplementados com Dex e/ou TGF-&beta;1, sem e com a neutralização dos receptores de TGF-&beta;1. A expressão gênica do próprio TGF-&beta;1 e Smad3 foram analisadas, bem como, a diferenciação das células osteogênicas e a biomineralização. As células tratadas com TGF-&beta;1 sem neutralização de receptores apresentam efeito inibitório nos estágios mais avançados da diferenciação dos osteoblastos e da biomineralização in vitro, mas expressarem alguns marcadores importantes envolvidos na mineralização. Observaram-se nódulos de mineral em todos os tratamentos das células que tiveram os receptores de TGF-&beta;1 neutralizados, mas houve uma diminuição na expressão de alguns genes. Os resultados confirmam a complexidade da via de sinalização do TGF-&beta;1, mostrando que existem lacunas para que seja entendido o mecanismo dessa molécula na biologia osteoblástica. / This in vitro study aimed to evaluate the molecular effects of TGF-&beta;1, with different supplementation time periods on the establishment of MC3T3-E1 cells, comparing with cells treated with AA+&beta;-GP supplemented with Dex and/or TGF-&beta;1, without or with neutralization of TGF-&beta;1 receptors. The gene expression of the TGF-&beta;1 and Smad3 were analyzed, as well as the osteoblast differentiation and biomineralization. The cells treated with TGF-&beta;1 without neutralization of receptors have had inhibitory effect on some important stages of osteoblast differentiation and biomineralization in vitro, but expressed some important mineralization markers. Mineral nodules were observed in all treatments of cells with their TGF-&beta;1 receptors neutralized, but there was a decrease in the expression of some important genes. The results confirm the complexity of the pathway signaling of TGF-&beta;1, showing that there are gaps for understand the mechanisms of this molecule in the biology of osteoblasts.

Este estudo in vitro teve como objetivo avaliar os efeitos moleculares do TGF-&beta;1, com diferentes períodos de suplementação, sobre a formação do fenótipo osteogênico das células MC3T3-E1, comparando-os com células tratadas com AA+&beta;-GP suplementados com Dex e/ou TGF-&beta;1, sem e com a neutralização dos receptores de TGF-&beta;1. A expressão gênica do próprio TGF-&beta;1 e Smad3 foram analisadas, bem como, a diferenciação das células osteogênicas e a biomineralização. As células tratadas com TGF-&beta;1 sem neutralização de receptores apresentam efeito inibitório nos estágios mais avançados da diferenciação dos osteoblastos e da biomineralização in vitro, mas expressarem alguns marcadores importantes envolvidos na mineralização. Observaram-se nódulos de mineral em todos os tratamentos das células que tiveram os receptores de TGF-&beta;1 neutralizados, mas houve uma diminuição na expressão de alguns genes. Os resultados confirmam a complexidade da via de sinalização do TGF-&beta;1, mostrando que existem lacunas para que seja entendido o mecanismo dessa molécula na biologia osteoblástica. / This in vitro study aimed to evaluate the molecular effects of TGF-&beta;1, with different supplementation time periods on the establishment of MC3T3-E1 cells, comparing with cells treated with AA+&beta;-GP supplemented with Dex and/or TGF-&beta;1, without or with neutralization of TGF-&beta;1 receptors. The gene expression of the TGF-&beta;1 and Smad3 were analyzed, as well as the osteoblast differentiation and biomineralization. The cells treated with TGF-&beta;1 without neutralization of receptors have had inhibitory effect on some important stages of osteoblast differentiation and biomineralization in vitro, but expressed some important mineralization markers. Mineral nodules were observed in all treatments of cells with their TGF-&beta;1 receptors neutralized, but there was a decrease in the expression of some important genes. The results confirm the complexity of the pathway signaling of TGF-&beta;1, showing that there are gaps for understand the mechanisms of this molecule in the biology of osteoblasts.