Mesenchymale Stammzellen können sich durch den Einfluss verschiedener Transkriptionsfaktor zu Osteoblasten, Adipozyten, Chondrozyten oder Myoblasten differenzieren. Während sie sich unter Runx2-Einfluss entlang der osteoblastären Linie differenzieren, entwickeln sie sich bei vorliegendem PPARγ entlang des adipogenen Differenzierungswegs. Das Gleichgewicht zwischen beiden Faktoren und ihr Zusammenspiel stellen einen wichtigen Bereich in der Osteoporoseforschung dar. In dieser Dissertation wurde durch Runx2-Suppression bzw. Runx2-Überexpression die Rolle dieses Faktors in pHOB und SCP1-Zellen erfasst und die Interaktion zwischen Runx2 und PPARγ untersucht. Der Runx2-Knockdown’ erfolgte mittels RNA-Interferenz, die Runx2-Überexpression durch ein Runx2 exprimierendes Plasmid. In RT-PCRs wurden mRNA-Messungen durchgeführt. Die Proteinbestimmung erfolgte im ‚Westernblot’. Der funktionelle Einfluss der Runx2-Überexpression auf die PPARγ-Transkription wurde durch Kotransfektion des an Luziferase gekoppelten PPARg-Promotorgens erfasst. Die funktionelle Aktivität des PPARg-Proteins wurde durch die Transfektion des an Luziferase gekoppelten PPRE-Gens gemessen. Promotoraktivität und Funktionalität der Proteine wurden in Luziferase-Reportergenassays erfasst. Unter basalen Kulturbedingungen differenzierten sich pHOB osteogen. Durch zweimalige siRunx2-Transfektion gelang auf mRNA-Ebene eine suffiziente Runx2-Suppression über 29 Tage auf durchschnittlich 10,1%. Neben einer Steigerung der PPARγ-mRNA nach sieben Tagen konnte darunter auch eine Suppression der osteogenen Differenzierungsmarker OC und AP beobachtet werden. Ein ‚Rescue’ der supprimierten Runx2-Genexpression konnte durch osteogene Stimulation nicht erreicht werden. In den Runx2-/PPARγ-Interaktionsversuchen wurden SCP1-Zellen adipogen stimuliert, um die PPARγ2-mRNA und PPARγ-Promotoraktivität zu erhöhen. Darunter konnte ebenfalls eine gesteigerte Funktionalität des PPARγ-Proteins beobachtet werden. Durch Runx2-Überexpression wurde in SCP1-Zellen die PPARγ-Promotoraktivität und somit der Beginn der mRNA-Synthese gehemmt. Die PPARγ2-mRNA hingegen blieb unbeeinflusst. Die zentrale Rolle des Runx2 in der osteogenen Differenzierung scheint durch den Einfluss auf die osteogenen Marker OC und AP in pHOB bestätigt zu werden. Auch der Einfluss auf die adipogene Differenzierung erfolgt über Runx2. Im Rahmen dieser Dissertation konnte erstmalig die Hemmung des PPARγ-Promotors durch Runx2 beschrieben werden. Hierdurch werden die PPARγ-Transkription und somit voraussichtlich die Interaktion zwischen Adipogenese und Osteogenese beeinflusst.

Mesenchymale Stammzellen bieten eine interessante Option in der regenerativen Medizin, da sie praktisch unlimitiert verfügbar sind. Um das Verhalten von humanen MSC zu studieren, werden Untersuchungen momentan an immundefizienten Mäusen durchgeführt, deren Verwendung kostenintensiv und aufwendig ist. Fra-gestellung war, ob durch Immunisolation (Alginat, Dialyseschlauch, Diffusionskammer) die Knorpel erhaltenden -, bzw. bildenden Eigenschaften von MSC-Konstrukten ebenso gut in immunkompetenten Mäusen untersucht werden können. Gleichzeitig sollte geprüft werden, ob die mit einer Immunabschirmung einhergehende Reduktion der Zellversorgung und damit die Annäherung an die Gelenksituation ihre Mineralisierung vermindern kann und ob Mauszellen für eine Veränderung der vordifferenzierten Knorpelpellets verantwortlich sind. Hierzu wurden hBMSC chondrogen differenziert. Die Zellpellets wurden mit Alginat, dem Dialyseschlauch oder der Diffusionskammer verkapselt und parallel zu unver-kapselten Kontrollpellets subkutan in immundefiziente SCID-Mäuse sowie in immunkompetente BDF1-Mäuse implantiert. Die Explantate wurden mit Alzianblau-, Alizarinrot-, Kollagen Typ II-Färbungen, sowie einer ALU in-situ Hybridisierung mar-kiert und mittels Histologiescore doppelt blind bewertet (MannWhitneyU). Überra-schenderweise zeigten die unverkapselten Kontrollen in den BDF1-Mäusen weder Zeichen von Inflammation noch von Destruktion und 4/5 der Pellets waren auf Kol-lagen Typ-II und Alzianblau positiv. Gleichzeitig war der Grad der Mineralisierung in den BDF1-Mäusen gegenüber SCID-Mäusen reduziert (p = 0,03). Durch Alginat wurde die Mineralisierung in den BDF1 Mäusen (0/8) völlig verhindert, während in den SCID-Mäusen noch 7/8 der Pellets Kalzifizierung zeigten (p = 0,001). Die Verkapselung mit Alginat verglichen mit der Kontrolle führte in beiden Mausstämmen zu höheren Scores für Kollagen Typ II (SCID: p = 0,013, BDF1: p = 0,042) und zeigte gleichzeitig eine Reduktion der Mineralisierung (SCID: p = 0,018, BDF1: p = 0,031). In SCID-Mäusen war außerdem der Alzianblau-Wert gegenüber den Kontrollen erhöht (p = 0,003). Die Diffusionskammer erwies sich als ungeeignet, da die Pellets ihre knorpeligen Eigenschaften verloren. Durch die Verwendung des Dialyseschlauches konnte lediglich in der SCID-Maus eine Erhöhung der Kollagen Typ II (p = 0,03) und eine Reduktion der Kalzifizierung (p = 0,004) erreicht werden. Sowohl im Alginatbead in der BDF1-Maus (1/3 Spendern), als auch im Dialyseschlauch mit Kollagenmembran (2/3 Spendern) konnte eine erfolgreiche in vivo Chondrogenese durchgeführt werden. Zur Untersuchung der in vivo Stabilität knorpeliger MSC-basierter Konstrukte stellt die BDF1-Maus eine attraktive, kostengünstige Alternative mit einer gegenüber der SCID-Maus verringerten Mineralisierungsrate dar. Die in vitro gebildete knorpelige Extrazellulärmatrix erzeugt dabei bereits eine Immunisolation, welche die Transplantatdestruktion verhindert. Ob ein intaktes lymphozytäres System die Knorpelstabilität gegenüber defizienten Immunsystemen begünstigt, muss durch die Untersuchung weiterer Ansätze belegt werden. Im Gegensatz zur Diffusionskammer bietet Alginat das richtige Maß an Versorgungsreduktion, um die Stabilisierung des Knorpelphänotyps der Konstrukte zu ermöglichen.

BACKGROUND Adult bone marrow derived MSCs had been explored to treat myocardial infarction (MI) and heart failure, for which various beneficial paracrine effects had been suggested. Since MSCs in vitro express anti-inflammatory cytokines, we tested the hypothesis that changes in the pro-/anti-inflammatory cytokine ratio in the infarct microenvironment may provide such a paracrine mechanism to improve early cardiac function following acute coronary occlusion. / Methods Rats (n=88) underwent acute left coronary artery ligations and were randomized into groups M and C and then injected with culture media or MSCs, respectively. These rats underwent blinded echocardiography to evaluate left ventricular ejection fractions (LVEF). Real Time PCR was used to compare cytokine gene expression for IL-1beta, IL-6, IL-8 (pro-inflammatory) and IL-10 (anti-inflammatory) at various times. Extra-cellular matrix (ECM) deposition and inflammatory cell infiltration were also analyzed. / Results As early as 12 hours, the ratio of pro-/anti-inflammatory cytokine gene expression in group C was significantly lower than group M. Similar results were found at 24 hours, 1 and 2 weeks, respectively. LVEF improved significantly in group C (M=62% vs C=68% at 12 hours* , M=66% vs C=75% at 24 hours*, M=57% vs C=75% at 1 week *, and M=52% vs C=70% at 2 weeks*, *p<0.01). The ratio of MMP-2/TIMP1 levels was lower in the Group C at all time frames, reaching significance at 12 and 24 hours and 2 weeks. In group C, histopathological analysis revealed significantly less ECM deposition (M=1.95% vs C=0.75% at 24 hours*, M=19.30% vs C=9.36% at 1 week*, M=24.46% vs C=7.57% at 2 weeks*, *p<0.01). This was associated with significantly decreased inflammatory cell infiltration after 24 hours. / Conclusions The current data suggests that MSCs therapy decreases the pro-/anti-inflammatory cytokine ratio in the infarct microenvironment. This is associated with improved cardiac function, reduced ECM deposition, and decreased inflammatory cell infiltration. This paracrine mechanism of MSCs therapy may explain the early functional improvement after MI before cell transdifferentiation or other mechanisms takes place.

<p>Activation of Wnt signaling pathways is critical to a variety of developmental events across all animal taxa. These highly evolutionarily conserved pathways are also important in the adult organism for maintaining homeostasis of self-renewing tissues. Because of its role in such important physiological processes, deregulation of Wnt signaling can have severe consequences; indeed, inappropriate activation of this pathway has been implicated in multiple human diseases, including cancer.</p><p>Upon binding their cellular receptors, canonical Wnt ligands, like Wnt 3A, stimulate the stabilization, accumulation, and nuclear translocation of a multifunctional cellular protein &#946;catenin, the consequence of which is induction of &#946;catenin-dependent transcription. This work describes the identification and characterization of two Wnt3A-stimulated intracellular signaling pathways activated in parallel to &#946;catenin stabilization: the RhoA pathway and the ERK pathway. These two auxiliary pathways do not affect &#946;catenin stability, accumulation, or subcellular localization; rather, they modulate &#946;catenin -dependent transcriptional activity through other mechanisms. As a result of their influence on &#946;catenin-dependent transcription, these pathways instruct cell fate decisions in C3H10T1/2 mesenchymal stem cells, in particular inhibition of adipogenesis and promotion of osteoblastogenesis.</p><p>Expression microarray analysis and biochemical and pharmacological techniques were used to further characterize the two Wnt3A-stimulated auxiliary pathways in C3H10T1/2 cells. Remarkably, each pathway influences &#946;catenin function via a novel mechanism. In the Wnt3A/RhoA pathway, Wnt3A-stimulated trimeric G proteins activate a RhoA-ROCK-SRF cascade. Activated SRF can cooperate with &#946;catenin to enhance the induction of Wnt3A target genes, like Ctgf, that also contain SRF binding sites within regulatory elements. In the Wnt3A/ERK pathway, Wnt3A transactivates the EGFR in a concentration-dependent manner, leading ultimately to ERK activation, which interacts with and promotes &#946;catenin/Tcf4 interaction and enhances induction of &#946;catenin/Tcf4 target genes. </p><p>These data emphasize the complexity of Wnt signaling and have intriguing implications regarding cross-regulation of the pathway, especially in stem cells. Also, since not all cells are capable of responding to Wnt3A by activation of these auxiliary pathways, this work identifies novel mechanisms that could underlie cell type-specific responses to Wnts and provides mechanistic insight into cellular responses to Wnt concentration gradients. Moreover, this work identifies novel transcriptional mechanisms important for promoting osteogenic cell fate specification, which could ultimately provide new therapeutic targets in disease states with bone loss or ineffective bone formation.</p> / Dissertation

The Snail1 transcriptional repressor is a key factor responsible in triggering epithelial to mesenchymal transition. Although Snail1 is widely expressed in early development, it is limited in adult animals to a subset of mesenchymal cells where it has a largely unknown function. In this project we have demonstrated that Snail1 is required to maintain mesenchymal stem cells (MSCs). This effect is associated to the responsiveness to TGF-[beta]1 which showed a strong Snail1 dependence. Snail1-depletion in conditional knock-out adult animals caused a significant decrease in the number of bone marrow-derived MSCs. In culture, Snail1-deficient MSCs prematurely differentiated to osteoblasts or adipocytes and, in contrast to controls, were resistant to the TGF-[beta]1-induced differentiation block. TGF-[beta]1 was unable to up-regulate most of its targets in Snail1 KO MSCs, an effect that was related, but not limited, to defective PTEN repression and Akt activation. Correspondingly, an analysis of human sarcomas also showed enhanced expression of Snail1 in undifferentiated tumors, which was strongly associated with high expression of TGF-[beta] and poor outcome. These results not only demonstrate a new role for Snail1 in TGF-[beta] response and MSC maintenance but also suggest the involvement of MSCs in sarcoma generation. / El repressor transcripcional Snail1 ha estat descrit principalment com el responsable de la inducció de la transició epiteli mesènquima. Encara que Snail1 s’expressa durant les etapes més primerenques del desenvolupament embrionari, la seva expressió en adults es veu limitada en un conjunt de cèl•lules mesenquimals sense saber-se la seva funció. En aquest projecte hem demostrat que Snail1 es requereix per mantenir el fenotip més indiferenciat de les cèl•lules mare del mesènquima. Aquesta funció la fa en part, per la capacitat de resposta de la citoquina TGF-[beta] la qual mostra una força dependència amb Snail1. Quan s’elimina Snail1 en ratolins adults provoca una clara disminució en el nombre de cèl•lules mare de la medul•la òssia. Aquestes cèl•lules en cultiu presenten una clara diferenciació prematura a osteoblasts i adipòcits. Pel contrari, tractaments amb TGF-[beta]1 aturen la diferenciació. El TGF-[beta]1 es incapaç de incrementar moltes dianes en cèl•lules mare del mesènquima aïllades del ratolí deficient per snail1, aquest efecte en part es degut a la repressió de PTEN i l’activació de AKT. L’anàlisi de sarcomes humans ens ha mostrat una alta expressió de Snail1, el qual també es troba associada amb una alta expressió de TGF-[beta] i baixa supervivència. Aquests resultats no només demostren una nova funció per Snail1 en resposta a TGF-[beta] i el manteniment de les MSC, sinó que també suggereix que Snail1 podria participar en la generació del sarcoma.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Mesenchymal stem cells are the stem cells that replace the bone, fat and cartilage tissues of the human body. In addition, these cells can form muscles, ligaments and neurons. This wide multipotency has made mesenchymal stem cells of particular interest in the fields of tissue engineering and regenerative medicine. Furthermore, mesenchymal stem cells can modulate the immune system by reducing factors that increase inflammation and immune recognition. This immune recognition suppression has resulted in their application as part of bone marrow transplantation in the prevention of 'graft versus host‘ disease. There are hundreds of on-going clinical trials using these cells for the treatment of autoimmune diseases such as type I diabetes, arthritis and multiple sclerosis. The increasing importance of these cells has brought in to focus the culture methods used to for their expansion and manipulation. Currently, animal derived components are used as surfaces for their growth and as components in the culture media. This exposes these cells to animal pathogens and antigens that can be passed to the recipients of these cells. In the first part of this thesis, polymer microarrays were employed to identify alternatives to the biological surfaces currently used for mesenchymal stem cell culture. This platform allowed hundreds of polyacrylates/acrylamides and polyurethanes to be simultaneously scrutinised to identify surfaces that could support their growth and maintain their stem cell characteristics. Identified polymer surfaces were monitored in long-term culture (10 passages) and were shown to retain the cell phenotype and capacity to differentiate, thus providing chemically defined substrates for long-term mesenchymal stem cell culture. In the second part of this thesis, a 'smart‘ polymer microarray of hydrophilic cross-linked polymers (hydrogels) were used to remove another key biological component of culture, trypsin. These 'smart‘ hydrogels modulated their properties depending on the temperature. Hydrogels that could trigger mesenchymal stem cell release after a reduction in temperature were identified. A unique passaging system using a modest temperature reduction for 1h was developed as a passaging method. Cells were maintained and monitored for 10 passages using this novel enzyme free passaging method. Analysis of the mesenchymal stem cell phenotype and differentiation capacity revealed this method superior than conventional culturing methods. In the final part of this thesis, a 'knowledge-based‘ small molecule library was designed, which could potentially yield small molecules to manipulate/enhance the mesenchymal stem cell state without the use of biological components. The key protein pathways that control the stem cell state were examine with the bioinformatics tool GeneGo was used to identify compounds that affected these pathways, resulting in selection of 200 small molecules. The effect of the small molecules on the mesenchymal phenotype was examined and 5 small molecules were identified that enhanced the phenotype of these cells. The anti-inflammatory properties associated with the hit compounds led to the investigation of their effects on key surface proteins associated with the immune-modulatory state of the cells. In this preliminary study, two of the small molecules, estriol and spermine, increased the expression of a key mesenchymal stem cell marker STRO-1 and down regulated ICAM-1, a critical component of the immune modulation capacity of this cell type.

Regenerative medicine and cell therapies hold great potential to treat a variety of medical conditions. Product characterisation of these therapies is particularly difficult as they pose regulatory challenges due to donor heterogeneity and the lack of standardised lot release tests that can reliably predict in vivo function. Human mesenchymal stem cells (hMSCs), also called multipotent stem cells or mesenchymal stromal cells, are a viable option in cell therapies due to their immunosuppressive and pro-angiogenic functions. Currently there are no standardised methods or potency assays to quantify these properties. To address this, five individual hMSCs lines from different donors were created and characterised based upon growth rate, differentiation capability and extracellular surface protein expression. A novel multiparameter flow cytometry method to characterise the cells based upon extracellular surface markers was developed that supports high-throughput and high-content analyses. Three candidate lines were taken forward and assessed in multiple in vitro bioassays that examined the hMSC immunosuppressive response to a defined inflammatory environment, effect on T-cell proliferation, and effect on a mixed lymphocyte population. Next, the angiogenic properties were assessed using human umbilical vein endothelial cells (HUVECs) tube formation as a model for cardiac regeneration. This involved utilising automated time lapse microscopy techniques coupled with image analysis software to quantify endothelial to tube formation. Further analysis of the hMSC secretome revealed differences in the levels of pro-angiogenic cytokines such as vascular endothelial growth factor, hepatocyte growth factor and IL-8. Significant differences in angiogenic potency were found between the hMSC lines. This thesis highlights the need to develop specific assays that reflect the intended clinical action. Taken together, these quantitative approaches provide valuable tools to measure hMSC quality and potency, and supports continued efforts to improve characterisation strategies for cellular therapies.

Submitted by MARINA LANDIM E ALVARENGA null (marina@fmvz.unesp.br) on 2016-06-10T20:23:13Z No. of bitstreams: 1 dissertacao final.pdf: 2069659 bytes, checksum: f8308625d294fd21d8e9fadd7f344c09 (MD5) / Rejected by Ana Paula Grisoto (grisotoana@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: O arquivo submetido está sem a ficha catalográfica. A versão submetida por você é considerada a versão final da dissertação/tese, portanto não poderá ocorrer qualquer alteração em seu conteúdo após a aprovação. Corrija esta informação e realize uma nova submissão contendo o arquivo correto. Agradecemos a compreensão. on 2016-06-15T18:05:22Z (GMT) / Submitted by MARINA LANDIM E ALVARENGA null (marina@fmvz.unesp.br) on 2016-06-26T01:29:11Z No. of bitstreams: 1 dissertacao final ficha.pdf: 2083568 bytes, checksum: 389a90011381796dbfd42b7a5be2f131 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-06-27T14:25:14Z (GMT) No. of bitstreams: 1 alvarenga_ml_me_bot.pdf: 2083568 bytes, checksum: 389a90011381796dbfd42b7a5be2f131 (MD5) / Made available in DSpace on 2016-06-27T14:25:14Z (GMT). No. of bitstreams: 1 alvarenga_ml_me_bot.pdf: 2083568 bytes, checksum: 389a90011381796dbfd42b7a5be2f131 (MD5) Previous issue date: 2016-04-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A utilização de terapias celulares no tratamento de lesões musculoesqueléticas cresce mundialmente em larga escala e é cada vez mais aceita. As células tronco mesenquimais (CTMs) são facilmente isoladas e expandidas in vitro, sendo tipicamente obtidas do próprio paciente (autóloga). Contudo, o uso de células provenientes de um animal doador (alogênica) e submetidas ao cultivo e a criopreservação, cria a oportunidade de iniciar o tratamento imediatamente após o diagnóstico. Há evidencias de que as CTMs possuem capacidade de imunomodulação, interagindo com os linfócitos T de diversas maneiras, além de não estimularem a indução de uma resposta imune complexa por não expressarem MHC II. Contudo, ainda há falta de consenso entre os estudos quanto a imunogenicidade das CTMs. Desta forma, o presente estudo objetivou avaliar a segurança da administração das células alogênicas em equinos através da análise de uma possível inflamação decorrente do implante alogênico de CTMs em músculo hígido de equinos, por meio de exame físico, ultrassonográfico, termográfico, histopatológico e pela expressão gênica de IL-1 beta e TNF- alfa. Os resultados demostraram que aplicação de CTM alogênicas é um processo seguro uma vez que não houveram alterações clínicas significativas, contudo uma segunda aplicação de CTM alogênicas de um mesmo doador levou ao aumento significativo da expressão gênica de TNF- alfa, podendo caracterizar uma resposta imune celular mais acentuada. Nossos resultados contribuíram para pesquisas futuras sobre tratamento de lesões musculoesqueléticas em equinos com CTMs, através da criação de bancos de células oriundas de doadores saudáveis. / The use of cell therapy for treatment of musculoskeletal injuries in horses is increasing in large scale worldwide. The mesenchymal stem cells (MSCs) are easily isolated and expanded in vitro, making them promising for use in clinical trials. Typically, these cells are obtained from the own patient (autologous), however, cell from a donor horse (allogeneic) expanded in vitro and cryopreserved create the opportunity of immediate treatment after diagnosis. There is evidence that MSCs have immunomodulatory capacity by interacting with T lymphocytes in several ways, and do not stimulate a complex immune response for not expressing MHC II. However, there is still a lack of consensus among the studies on the immunogenicity of MSCs. Thus, this study aimed to evaluate the safety of administration of allogeneic mesenchymal stem cells in horses. For such a possible inflammation reaction to the implantation was analysed through physical examination, ultrasound, thermography, histopathology and gene expression of IL 1beta and TNF-alpha. The results showed that the implantation of allogeneic MSCs is a safe process since there were no significant clinical changes in the animals, but a second application of allogeneic MSCs from the same donor led to significant increase in gene expression of TNF-alpha, which can characterize a cellular immune response. Our results contribute to future research on treatment of musculoskeletal injuries in horses using MSCs, by creating banks of cells derived from healthy donors.

O objetivo desse estudo foi investigar a participação de integrina &alpha;1&beta;1 e microRNAs (miRs) no potencial osteogênico de superfícies de titânio (Ti) com nanotopografia. Discos de Ti previamente polidos foram tratados quimicamente com H2SO4/H2O2 para obtenção de nanotopografia, que foi observada por microscopia eletrônica de varredura. Para o estudo da participação da integrina &alpha;1&beta;1, células-tronco mesenquimais (CTMs) de ratos foram cultivadas em condições osteogênicas e não osteogênicas sobre superfícies de Ti com nanotopografia e sem tratamento químico (controle). O resultados mostraram que a nanotopografia de Ti aumentou a proliferação celular, a atividade de fosfatase alcalina (Alp) e regulou positivamente a expressão gênica de marcadores da diferenciação osteoblástica em CTMs cultivadas tanto em condições osteogênicas quanto em condições não osteogênicas. Além disso, uma maior expressão gênica para as integrinas &alpha;1 e &beta;1 foi observada em culturas crescidas sobre nanotopografia em condições não osteogênicas em relação ao Ti controle. O uso de obtustatina, um inibidor de integrina &alpha;1&beta;1, reduziu os efeitos da nanotopografia sobre os marcadores osteoblásticos, indicando a participação da via de sinalização dessa integrina nos efeitos da nanotopografia sobre CTMs. Para investigar a participação de miRs no efeito osseoindutor da nanotopografia de Ti, foram utilizadas CTMs humanas e células préosteoblásticas de camundongos da linhagem MC3T3-E1. A análise em larga escala da expressão de miRs revelou que 60 miRs foram regulados positivamente (no mínimo, 2x maior), enquanto 58 miRs foram regulados negativamente (no mínimo, 2x menor) em CTMs crescidas sobre a nanotopografia. Três desses miRs, miR-4448, -4708 e -4773, cuja expressão foi significativamente reduzida pela nanotopografia de Ti (no mínimo, 5x menor), afetaram a diferenciação osteoblástica de CTMs. Esses miRs atuam diretamente sobre SMAD1 e SMAD4, proteínas transdutoras da sinalização da proteína óssea morfogenética 2 (Bmp-2), conhecida por sua capacidade osseoindutora. Além disso, verificou-se que a sobreexpressão de miR-4448, -4708 e -4773 em células pré-osteoblásticas MC3T3-E1 inibiu a expressão gênica e proteica de SMAD1 e SMAD4 e, consequentemente, a expressão gênica de marcadores ósseos. Esses dados sugerem a influência do circuito miR-SMAD-Bmp-2 sobre o efeito osseoindutor da nanotopografia. Conjuntamente, os achados do presente estudo mostraram que o efeito da nanotopografia de Ti sobre a diferenciação osteoblástica resulta de um mecanismo regulatório complexo, do qual fazem parte as vias de sinalização da integrina &alpha;1&beta;1 e da Bmp-2, com a participação de miRs. Esses resultados podem representar um avanço para o desenvolvimento de novas modificações de superfície, com o objetivo de acelerar e/ou melhorar o processo de osseointegração. / The aim of this study was to investigate the role of the &alpha;1&beta;1 integrin and microRNAs (miRs) on the osteogenic potential of titanium (Ti) with nanotopography. Polished Ti discs were chemically treated with H2SO4/H2O2 to generate nanotopography, which was observed under scanning electron microscopy. For the study related to the &alpha;1&beta;1 integrin, rat mesenchymal stem cells (MSCs) were cultured under osteogenic and non-osteogenic conditions on Ti with nanotopography and non-treated Ti discs (control). Nanotopography increased cell proliferation and alkaline phosphatase (Alp) activity and upregulated the gene expression of bone markers in cells cultured under osteogenic and non-osteogenic conditions. Furthermore, the gene expression of &alpha;1 and &beta;1 integrins was higher in cells cultured on nanotopography under non-osteogenic conditions compared with control. Obtustatin, an inhibitor of &alpha;1&beta;1 integrin, reduced the higher gene expression of the bone markers induced by nanotopography. These results indicate that &alpha;1&beta;1 integrin signaling pathway determines the osteoinductive effect of nanotopography on MSCs. The role of miRs in the osteogenic potential of Ti with nanotopography was evaluated using human MSCs and MC3T3-E1 mouse pre-osteoblastic cells. The miR sequencing analysis revealed that 60 miRs were upregulated (> 2 fold), while 58 miRs were downregulated (< 2 fold) in MSCs grown on nanotopography. Three miRs, miR-4448, -4708 and -4773, which were significantly downregulated (< 5 fold) by nanotopography, affected the osteoblast differentiation of MSCs. These miRs directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (Bmp-2) osteogenic signal, which were upregulated by nanotopography. Overexpression of miR-4448 - 4708 and 4773 in MC3T3-E1 cells noticeably inhibited gene and protein expression of SMAD1 and SMAD4 and by targeting them, these miRs repressed gene expression of key bone markers. These results suggest that a miR-SMAD-Bmp-2 circuit acts in the Ti nanotopography-mediated osteoblast differentiation. Taken together, our data showed that the osteoblast differentiation induced by Ti with nanotopography is governed by a complex regulatory network involving a crosstalk between &alpha;1&beta;1 integrin and Bmp-2 signaling pathways with participation of miRs.