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Die Knochenregeneration während der Frakturheilung beinhaltet das Zusammenspiel von Wachstumsfaktoren. In einigen Patienten kommt es zu einer verzögerten oder unvollständigen Heilung. Die Gründe hierfür sind bisher nicht komplett verstanden. Neutralisierende Autoantikörper (aAB) gegen Wachstumsfaktoren oder deren Rezeptoren könnten den Heilungsprozess verzögern und potentiell beeinträchtigen In dieser Arbeit wurden 265 Frakturpatienten analysiert. Autoantikörper gegen IGF1 Rezeptor, Insulin Rezeptor, BMP7, BMP2, IGF1 und (Pro)Insulin wurden in den Seren dieser Frakturpatienten gemessen. In Frakturpatienten wurden in 5% der Seren aAB gegen den IGF1R und in 6% gegen den IR gefunden. Das Auftreten von IGF1R- und IR-aAB wurde nicht induziert und war nicht mit dem Heilungsergebnis assoziiert. BMP7-aAB wurden in 1-2,5% gesunder Probanden und Frakturpatienten, die nicht mit rhBMP7 behandelt wurden, detektiert. Patienten, die mit rhBMP7 behandelt wurden, zeigten ein höheres Auftreten der BMP7-aAB Positivität mit 6% zum Zeitpunkt der Operation und 18% vier Wochen nach der Operation. BMP2-aAB wurden in 2% der gesunden Kontrollen und 6% der mit rhBMP7-behandelten Frakturpatienten entdeckt. Bei der Charakterisierung des biologischen Effekts der BMP7-aAB durch einen zell-basierten Reporter-Assay, zeigte sich ein neutralisierender Effekt in Proben mit hohem BMP7-aAB Titer. Als das wichtigste Kriterium für klinische Relevanz wurde die Konsolidierung untersucht. Das Vorhandensein von BMP-aAB wurde nicht signifikant mit der Konsolidierung in Zusammenhang gebracht. Zusammenfassend wurden neue diagnostische Assays zur Detektion von aAB gegen Wachstumsfaktoren und deren Rezeptoren generiert und angewandt um aAB in Seren von Frakturpatienten zu messen. Keiner der identifizierten aAB war negativ mit dem Heilungsprozess assoziiert. Bedenken bezüglich der Sicherheit von rhBMP7 Behandlungen sind berechtigt, da die Anwendung aAB gegen BMP7 induziert, die den BMP7-Signalweg blockieren. / Regeneration of bone during fracture healing includes concerted actions of growth factors. Some fractures show delayed healing or non-union due to as yet unknown reasons. Neutralizing autoantibodies (aAB) against growth factors or their receptors might influence and potentially impair the bone healing capacity. In this study, a cohort of 265 fracture patients with different treatment regimen and healing outcomes were analysed. Autoantibodies against IGF1 receptor, insulin receptor, BMP7, BMP2, IGF1 and (pro)insulin were measured in sera of these fracture patients. The prevalence of aAB against IGF1R and IR was 5% and 6% in fracture patients, respectively. The appearance of IGF1R- and IR-aAB was not induced by the surgical intervention and was unrelated to the healing outcome. BMP7-aAB were found in 1-2.5% of healthy subjects and in fracture patients that were not treated with rhBMP7. Patients that had received rhBMP7 treatment showed a higher incidence of BMP7-aAB positivity of 6% at surgery and 18% four weeks post surgery. BMP2-aAB were found in 2% of healthy controls and 6% of the fracture patients that were treated with rhBMP7. Characterizing the biological effect of BMP7-aAB in a cell-based reporter assay, a neutralizing effect was observed for samples with high titres. As the most relevant clinical outcome, the criterion consolidation was analysed defining whether the fracture gap was closed after six months or not. The presence of BMP-aAB was not significantly associated with the healing outcome. In summary, novel diagnostic assays for the detection and quantification of growth factor and receptor aAB were generated and used to determine aAB in sera from fracture patients. None of the identified aAB were negatively associated with the regeneration process or healing outcome. Ongoing concerns regarding the safety of rhBMP7 treatment are justified as the biological treatment induces aAB against BMP7 that block the BMP signal transduction.

Yung, Lai Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 93-115). / Abstract also in Chinese. / Declaration --- p.i / Acknowledgements --- p.ii / Abbreviations --- p.iii / Abstract in English --- p.v / Abstract in Chinese --- p.ix / Contents --- p.xi / Chapter Chapter I - --- Introduction / Chapter 1.1) --- Endothelial cells function --- p.1 / Chapter 1.2) --- Oxidative stress in the vascular wall --- p.2 / Chapter 1.2.1) --- Sources of ROS --- p.3 / Chapter 1.2.2) --- Actions of ROS --- p.3 / Chapter 1.2.2.1) --- Impaired endothelium-dependent vasodilatation --- p.3 / Chapter 1.2.2.2) --- VSMC migration --- p.4 / Chapter 1.2.2.3) --- Programmed cell death (cell apoptosis) --- p.4 / Chapter 1.3) --- Endothelial cell apoptosis --- p.7 / Chapter 1.3.1) --- Apoptosis and cardiovascular diseases --- p.7 / Chapter 1.3.2) --- Mechanisms of endothelial cells apoptosis --- p.7 / Chapter 1.3.2.1) --- What are caspases? --- p.8 / Chapter 1.3.2.2) --- Death receptor-mediated apoptosis --- p.9 / Chapter 1.3.2.3) --- Mitochondria-dependent pathway --- p.9 / Chapter 1.3.3) --- Regulations of endothelial cells apoptosis --- p.10 / Chapter 1.3.3.1) --- Oxidative stress --- p.10 / Chapter 1.3.3.2) --- Shear Stress --- p.11 / Chapter 1.3.3.3) --- Growth factors --- p.12 / Chapter 1.3.3.4) --- NO --- p.12 / Chapter 1.3.3.5) --- Inflammatory mediators --- p.13 / Chapter 1.4) --- Mitogen activated kinases signaling in apoptosis --- p.15 / Chapter 1.5) --- Bone morphogenic proteins (BMPs) --- p.17 / Chapter 1.5.1) --- BMPs functions and cardiovascular system --- p.17 / Chapter 1.5.2) --- BMPs signaling pathways --- p.18 / Chapter 1.5.2.1) --- Smad-dependent pathway --- p.18 / Chapter 1.5.2.2) --- MAPKs and SAPKs pathways --- p.19 / Chapter 1.5.2.3) --- Antagonists of BMPs signaling --- p.20 / Chapter 1.5.3) --- BMP4 and cardiovascular diseases --- p.20 / Chapter 1.6) --- "Justification, long-term significance and objectives of the present project" --- p.23 / Chapter Chapter II - --- Methods and Materials / Chapter 2.1) --- Animal handling --- p.24 / Chapter 2.2) --- Endothelial cell isolation and culture --- p.24 / Chapter 2.2.1) --- Primary culture of rat endothelial cells --- p.24 / Chapter 2.2.2) --- Culture of human umbilical cord vein endothelial cells… --- p.25 / Chapter 2.3) --- Apoptosis assessment --- p.25 / Chapter 2.3.1) --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay --- p.25 / Chapter 2.3.2) --- Cell death detection ELISA kit --- p.26 / Chapter 2.3.3) --- Flow cytometry --- p.27 / Chapter 2.4) --- Western blot analysis --- p.28 / Chapter 2.4.1) --- Sample preparation --- p.28 / Chapter 2.4.2) --- SDS-PAGE and transfer --- p.28 / Chapter 2.5) --- DHE fluorescence --- p.29 / Chapter 2.6) --- "Drugs, chemicals and other reagents" --- p.30 / Chapter 2.6.1) --- Drugs and chemicals used in the present experiments --- p.30 / Chapter 2.6.2) --- Reagents for Western blot analysis --- p.30 / Chapter 2.6.3) --- Primary antibodies --- p.33 / Chapter 2.7) --- Small interfering RNA experiment --- p.34 / Chapter 2.8) --- Statistical analysis --- p.34 / Chapter Chapter III - --- BMP4 induces endothelial cell apoptosis in ROS related p38 MAPK and JNK mediated caspase-3 dependent pathway / Chapter 3.1) --- Introduction --- p.35 / Chapter 3.2) --- Methods and materials --- p.39 / Chapter 3.2.1) --- Isolation and culture of endothelial cells --- p.39 / Chapter 3.2.2) --- Drugs treatment --- p.39 / Chapter 3.2.3) --- Assay for cell apoptosis --- p.40 / Chapter 3.2.3.1) --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay --- p.40 / Chapter 3.2.3.2) --- Cell death detection ELISA kit --- p.41 / Chapter 3.2.3.3) --- Flow cytometric analysis --- p.41 / Chapter 3.2.4) --- Western blot analysis --- p.41 / Chapter 3.2.5) --- Dihydroethidium (DHE) staining --- p.42 / Chapter 3.2.6) --- Statistical analysis --- p.42 / Chapter 3.3) --- Results --- p.43 / Chapter 3.3.1) --- Dose- and time-dependent effect of BMP4 --- p.43 / Chapter 3.3.2) --- Role of caspases in apoptosis of RAECs and HUVECs --- p.43 / Chapter 3.3.3) --- Roles of BMP4 and ROS in endothelial cell apoptosis --- p.44 / Chapter 3.3.3.1) --- Noggin antagonism of BMP4-induced effect --- p.44 / Chapter 3.3.3.2) --- NAD(P)H oxidase-mediated ROS production --- p.44 / Chapter 3.3.3.3) --- Inhibition of endothelial cell apoptosis by ROS scavengers --- p.45 / Chapter 3.3.4) --- Roles of MAPKs/SAPKs in BMP4-induced endothelial cell apoptosis --- p.45 / Chapter 3.3.5) --- Relationship between ROS and MAPKs/SAPKs --- p.46 / Chapter 3.3.6) --- Relationship between p38 MAPK and JNK --- p.46 / Chapter 3.4) --- Discussion --- p.82 / Chapter 3.4.1) --- Caspase-dependent pathways --- p.82 / Chapter 3.4.2) --- Oxidative stress --- p.85 / Chapter 3.4.3) --- Role of MAPKs activation in BMP4-induced endothelial cell apoptosis --- p.87 / Chapter 3.4.4) --- ROS mediates BMP4-induced activation of MAPKs --- p.88 / Chapter 3.4.5) --- Role of p38 MAPK in the activation of JNK 1 --- p.89 / Chapter 3.5) --- Concluding remarks --- p.91 / References --- p.93 / Publications and Awards --- p.116

Une des causes d’échec les plus importantes dans la prise en charge des cancers est la rechute, reflet de la persistance de cellules souches cancéreuses. Les leucémies aiguës myéloïdes représentent la forme principale de leucémie aiguë chez l’adulte et sont caractérisées par une prolifération excessive de cellules immatures et un défaut d’apoptose. En haut de la hiérarchie clonale, les cellules souches leucémiques (CSL) au travers de leurs capacités fonctionnelles participent à l’initiation et la maintenance de la maladie. Ces cellules sont régulées à la fois de façon extrinsèque au travers du microenvironnement et de façon intrinsèque notamment les facteurs de transcription.Notre équipe travaille sur le récepteur à la vitamine D (VDR) et son ligand la vitamine D (VD) et a mis en évidence une synergie d’action entre chélation martiale et VD afin de lever le blocage de différenciation des LAM avec une toxicité réduite (Callens et al, JEM 2010). Une étude clinique rétrospective a été conduite mettant en évidence qu’un taux de vitamine D élevé chez les patients atteints de LAM avant tout traitement leur confère un meilleur pronostic (Paubelle, Zylbersztejn et al, Plos One 2013) . Nous poursuivons donc ce projet sur l’étude la voie VD/VDR dans la maintenance des cellules souches hématopoïétiques et sa dérégulation dans la LAM. Mon projet doctoral a pour objectif de déterminer l’implication du microenvironnement tumoral (voie des BMP et du VDR) dans le maintien des cellules souches leucémiques de LAM. Notre hypothèse de travail est que le récepteur à la vitamine D en plus de son rôle différenciant connu, aurait un impact sur le maintien des cellules souches hématopoïétiques normales et de LAM et que son mécanisme d’action passerait par la voie des Bone Morphogenetic Protein. Nous avons dans un premier temps démontré l’importance du VDR dans la régulation des CSH et pu tester l’intérêt de l’emploi de son ligand afin de cibler spécifiquement les cellules souches leucémiques dans des modèles pré-cliniques. Enfin nous avons pu confirmer la dérégulation de cette voie dans des cellules primaires de LAM et la régulation de ce récepteur par la voie des Bone Morphogenetic Protein. Ces travaux ouvrent de nouvelles perspectives dans la compréhension dans la biologie des CSH et de leur dérégulation dans la LAM / One of the most important causes of failure in the management of cancer is relapse, due to cancer stem cells persistence. Acute Myeloid Leukemias (AML) are the major form of acute leukemia in adults and are characterized by excessive proliferation of immature cells and apoptosis defect. At the top of the clonal hierarchy, leukemic stem cells (LSC) through their functional abilities participate in the initiation and maintenance of the disease. These cells are regulated both extrinsically mechanisms through the microenvironment and intrinsically by transcription factors.Our team is working on the Vitamin D Receptor (VDR) and its ligand Vitamin D (VD) and has demonstrated a synergistic action between iron chelation and VD in order to lift the differentiation blocking of AML with reduced toxicity (Callens et al, JEM 2010). A retrospective clinical study was conducted showing that a high vitamin D level in patients with AML before any treatment gives them a better prognosis (Paubelle, Zylbersztejn et al, Plos One 2013). We are continuing this project on the study of the VD/VDR pathway in the maintenance of hematopoietic stem cells (HSC) and its deregulation in AML. My project aims to determine the involvement of the tumor microenvironment (BMP and VDR pathway) in the maintenance of AML-LSC. Our working hypothesis is that the vitamin D receptor, in addition to its known differentiating role, would have an impact on the maintenance of normal HSC and AML and that its mechanism of action would be through the Bone Morphogenetic Protein pathway. We first demonstrated the importance of VDR in the regulation of HSCs and tested the interest of the use of its ligand to specifically target LSC in pre-clinical models. Finally we were able to confirm the deregulation of this pathway in primary AML cells and the regulation of this receptor by the Bone Morphogenetic Protein pathway. These works open up new perspectives in the understanding in CSH biology and their deregulation in AML.

published_or_final_version / Anatomy / Master / Master of Philosophy

This thesis presents deeper insights into bone applicable biomaterials’ design. Poor affinity of BMP-2 towards scaffolds required supra-physiological dose administration. Though molecules containing sulfate could sustain BMP-2 release, side effects occurred due to BMP-2 supra-dose, or these sulfate-containing biomolecules. Improved affinity between BMP-2 and scaffolds was first witnessed by using an acidic carrier (paper I). Hyaluronic acid (HA) hydrazone derived hydrogels having a pH of 4.5-loaded BMP-2 showed sustained release of bioactive BMP-2 in vitro and enhanced bone formation in vivo, while pH 7 HA hydrogels showed Fickian behavior and less bone formation in vivo. Computational evaluation revealed stronger electrostatic interactions between BMP-2, and HA were predominant at pH 4.5, whereas, weaker Van der Waals interactions played a key role at pH 7. During the pre-bone formation phase, endogenous cell responses to pH 4.5 and 7 with or without BMP-2 were investigated. HA hydrogels exhibited extraordinary biocompatibility and recruitment of neutrophils, monocytes, macrophages and stromal cells regardless of hydrogels’ pH and BMP-2 presence.  The different inflammatory responses to HA hydrogels were observed (Appendix). Thiol derivatives can cleave the disulfide bond of BMP-2 to generate inactive monomeric BMP-2. In paper II, thiol-acrylate chemistry-based HA hydrogels (HA-SH) were compared to hydrazone-based HA hydrogels as BMP-2 carriers. Thiol modified HA disrupted BMP-2 integrity and bioactivity. HA-SH hydrogels with BMP-2 exhibited less bioactive BMP-2 release in vitro and induced less bone formation in vivo. Accumulated evidence has shown great osteogenic potential of lithium ions (Li). In paper III, we coordinated Li onto HA-PVA hydrazone hydrogels (Li-gel); Li-gel enhanced 3D cultured hMSCs osteogenic differentiation and induced higher bone formation in CAM defect model. Instead of BMP-2 protein, delivery of BMP-2-coding-plasmid can produce BMP-2 over a long term at a closer physiological level. Yet, efficient gene delivery reagents are needed. In paper IV, two novel gene delivery nanoplexes were developed by post coating DNA-nanoplexes with chondroitin sulfate (CS). To ensure the stability, aldehyde-modified CS (CS-CHO) reacted with free amines of pDNA/PEI complexes. We provided first evidence that CS-CHO coated nanoplexes controlled the release from endosomes, which is essential for higher transfection efficiency.

Secreted Phosphoprotein-24 (Spp24) was initially isolated and characterized as a component of bovine cortical bone matrix. Subsequent characterization has shown it is multiply phosphorylated and homologous to cystatin and TGF-β receptor type II. Spp24 is a minor component of the serum fetuin mineral complex that binds calcium-phosphate minerals and prevents their deposition. The TGF-β receptor homology domain binds BMP-2 weakly in vitro and enhances BMP-2’s osteogenic effects in vivo. The ability of Spp24 to affect BMP activity suggests an important role for Spp24 as a native, bioactive componentof bone that regulates bone development. Spp24 was highly up-regulated in rat cortical kidneys following a low calcium diet regime. Tissue distribution of both Spp24 protein and RNA showed that while Spp24 accumulates in bone, a majority is produced at distant sites, namely the liver and kidney. Additionally, Spp24 was present in more tissues than previously believed. Spp24 migrates to a number of different molecular weights, suggesting multiple, alternative posttranslational modifications may generate subtly different forms of the protein. Theexpression of Spp24 in the kidney may be regulated to counteract changes in serum mineral levels. Additionally, homology in the Spp24 sequence suggests that it, like other bone and dentine matrix proteins, may interact with mineral as an important influencer of mineral calcification. Utilizing microarray analysis of primary bone marrow-derived mesenchymal stem cells transduced with Spp24 and control viruses we examined changes elicited by the overexpression of Spp24. A change in overall morphology was observed for cellstransduced with the Spp24 similar to changes described in cells undergoing osteoblasticdifferentiation. Nodule formation was also seen in the Spp24 transduced cells. Microarray results showed key markers of osteoblast differentiation, CBFA1/RUNX2 and osterix(OSX), were not up-regulated although there were distinguishable changes in the gene expression profile of mesenchymal stem cells. The cells appeared to be blocked from differentiation into a number of mesenchymal lineages: adipocytes, myocytes andchondrocytes. The changes appeared to prime cells for signals that activate osteoblastdifferentiation by blocking other pathways and altering internal signaling response pathways to those signals. This document was created in Microsoft Word 2003.

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) coordinate tissue development and postnatal remodelling by regulating proliferation, differentiation and apoptosis. However, their role in wound healing remains unclear. To study this, transgenic mice overexpressing Smad1 (K14-caSmad1) or the BMP antagonist Noggin (K14-Noggin) were utilised, together with human and mouse ex vivo wound healing models and in vitro keratinocyte culture. In wild-type mice, transcripts for Bmpr-1A, Bmpr-II, Bmp ligands and Smad proteins were decreased following tissue injury, whilst Bmpr-1B expression was up-regulated. Furthermore, immunohistochemistry revealed a down-regulation of BMPR-1A in hair follicles adjacent to the wound in murine skin, whilst in murine and human wounds, BMPR-1B and phospho-Smad-1/5/8 expression was pronounced in the wound epithelial tongue. K14-caSmad1 mice displayed retarded wound healing, associated with reduced keratinocyte proliferation and increased apoptosis, whilst K14-Noggin mice exhibited accelerated wound healing. Furthermore, microarray analysis of K14-caSmad1 epidermis revealed decreased expression of distinct cytoskeletal and cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myo5a versus controls. Human and mouse keratinocyte proliferation and migration were suppressed by BMP-4/7 both in vitro and ex vivo, whilst they were stimulated by Noggin. Additionally, K14-caSmad1 keratinocytes showed retarded migration compared to controls when studied in vitro. Furthermore, Bmpr-1B silencing accelerated migration and was associated with increased expression of Krt16, Krt17 and Myo5a versus controls. Thus, this study demonstrates that BMPs inhibit proliferation, migration and cytoskeletal re-organization in epidermal keratinocytes during wound healing, and raises a possibility that BMP antagonists may be used for the future management of chronic wounds.

A desmineralização óssea superficial tem se demonstrado favorável à consolidação de enxertos e ao comportamento celular, entretanto os mecanismos envolvidos ainda não estão esclarecidos. Os subsídios para o embasamento biológico da desmineralização, proporcionado por publicações anteriores, sugeriram que modificações na superfície óssea teriam influenciado o comportamento de pré-osteoblastos em cultura. Assim, este estudo objetivou comparar o efeito de duas concentrações de ácido cítrico na desmineralização de superfícies ósseas onde foram cultivadas células pré-osteoblásticas (MC3T3-E1), e analisar parâmetros de superfície comparando superfícies desmineralizadas a não desmineralizadas. Setenta amostras ósseas bicorticais foram removidas das calvárias de 35 ratos e divididas em grupos para as análises: 1) Microscopia Eletrônica de Varredura (MEV) para avaliação da área de recobrimento e espessura da camada de células sobre as amostras (n = 15) durante 24, 48 e 72 horas: Grupo AC.10 amostras desmineralizadas por 30 segundos com ácido cítrico 10 %; Grupo AC.50 amostras desmineralizadas por 30 segundos com ácido cítrico 50 %; e Grupo C (controle) amostras não desmineralizadas; 2) Microscopia Confocal para análise da área de expressão e intensidade de fluorescência das BMP-2, -4 e -7: AC.10 seis amostras desmineralizadas conforme item 1); AC.50 seis amostras desmineralizadas conforme item 1); C três amostras não desmineralizadas; 3) Microscopia Confocal para análise da rugosidade superficial média (Ra e Sa): Grupos AC.10 e AC.50 com cinco amostras cada, desmineralizadas conforme o item 1), sendo cada amostra seu próprio controle (análises antes e depois da desmineralização). Também foram avaliadas as distâncias entre picos (P-P) e entre picos e vales (P-V) antes e depois da desmineralização; 4) Microscopia Eletrônica de Varredura / Espectroscopia de Energia Dispersiva (MEV / EDS) para análise da composição superficial: mesmas amostras do item 3) foram avaliadas antes e depois da desmineralização quanto à porcentagem atômica (%A) de carbono, oxigênio, magnésio, fósforo, enxofre e cálcio. Análises estatísticas foram feitas adotando nível de significância de 95 %. Amostras desmineralizadas apresentaram células morfologicamente em estágios mais avançados de diferenciação do que as não desmineralizadas. A área de recobrimento superficial foi significantemente maior após 24 horas de cultura nos grupos teste do que no controle e a espessura da camada de células também foi maior nos grupos teste às 48 e 72 horas. Houve significantemente maior expressão de BMP-2 e -7 nos grupos teste do que no controle e, apenas AC.10 demonstrou maior expressão de BMP-4 do que os demais grupos, sem significância em relação a AC.50. Os parâmetros de superfície Ra e Sa foram inconclusivos, mas P-P e P-V diminuíram consideravelmente após a desmineralização para distâncias compatíveis com superfícies favoráveis à adesão e diferenciação celular. A análise da composição química superficial revelou diminuição da %A de enxofre e magnésio nos grupos teste. A concentração do ácido, embora não tenha apresentado diferença significante para a maioria das análises, pareceu ter influência positiva nos resultados para o ácido cítrico 10 %. Concluiu-se que a desmineralização superficial parece promover a proliferação e diferenciação celular, proporcionando superfícies com características de composição e topografia que favorecem o comportamento celular verificado. / The superficial bone demineralization has proved to be a favorable procedure for bone grafts consolidation and cell behavior, however the underlying mechanisms have not been clarified yet. Therefore, this study aimed to compare the effect of two concentrations of citric acid on demineralization of bone surfaces where pre-osteoblastic cells (MC3T3-E1) were cultivated, and analyze surface parameters comparing demineralized bone surfaces with non-demineralized surfaces. Seventy bicortical bone samples were harvested from the calvaria of 35 rats and divided into groups as follows: 1) Scanning Electron Microscopy (SEM) to evaluate the coating area and thickness of cells layers cultured on the samples (n = 15) for 24, 48, and 72 hours: Group CA.10 samples demineralized for 30 seconds with 10 % citric acid; Group CA.50 samples demineralized for 30 seconds with 50 % citric acid, and Group C (control) non-demineralized samples; 2) Confocal Microscopy for analysis of expression area and intensity of fluorescence of BMP-2, -4, and -7: CA.10 six samples demineralized as item 1); CA.50 six samples demineralized as item 1); Group C three non-demineralized samples; 3) Confocal Microscopy for surface mean roughness analysis (Ra and Sa): Groups CA.10 and CA.50 made up of five samples each and demineralized according to item 1), each sample was its own control (analysis before and after demineralization). The distances between peaks (P-P) and between peaks and valleys (P-V) were also evaluated before and after demineralization; 4) Scanning Electron Microscopy / Energy dispersive Spectroscopy (SEM / EDS) to analyze the surface composition: the same samples of item 3) were evaluated before and after demineralization for atomic percentage (%A) of carbon, oxygen, magnesium, phosphorus, sulfur and calcium. Statistical test was made by adopting the 95 % significance level. Demineralized samples showed cells with morphology in the later stages of differentiation than non-demineralized ones. The coating surface area by cells was significantly higher after 24 hours of culture in the test groups than in the control and the thickness of the layers were also greater in the test groups at 48 and 72 hours of evaluation. There was significantly higher expression of BMP-2 and -7 in test groups than in the control group, and only the CA.10 group showed higher BMP-4 expression than the other groups, but the difference was not statistically significant compared to the CA.50 group. Ra and Sa surface parameters were inconclusive, however P-P and P-V decreased considerably after demineralization to distances compatible with surfaces favorable to cell adhesion and cell differentiation. The chemical composition analysis of the surfaces revealed a decrease in the %A for sulfur and magnesium in test groups. Although the acid concentration did not shown significant difference for most analysis, it seemed to have a positive influence for the results with citric acid 10 %. It was concluded that the surface demineralization seems to promote cell proliferation and differentiation, providing surfaces with composition and topography that can favor observed cell behavior.

Este estudo analisou a influência de diferentes superfícies de titânio na adesão, proliferação e diferenciação de células semelhantes a osteoblastos de rato (osteo-1) em culturas, na presença ou não da proteína morfogenética óssea recombinante-2 (rhBMP-2). As células osteo-1 foram cultivadas sobre as seguintes superfícies de titânio: 1. superfície lisa, 2. superfície desgastada com partículas de areia e condicionamento ácido (SLA) e 3. superfície desgastada com partículas de areia e condicionamento ácido sob proteção de nitrogênio e armazenadas em solução isotônica de cloreto de sódio (SLActive), na presença ou não de 20 ng/ml de rhBMP-2. Foram analisadas a adesão celular em 24 horas, o conteúdo total de proteínas, o conteúdo de colágeno e a atividade de fosfatase alcalina em 7, 14 e 21 dias e a formação de nódulos calcificados em 21 dias. Os resultados mostraram que a adesão não foi influenciada nem pelo tipo de superfície nem pelo tratamento com rhBMP-2 (p=0,0936). Quando relacionamos o conteúdo total de proteínas ao número total de células, percebemos que a proliferação não foi influenciada pelo tipo de superfície de titânio, porém a adição de rhBMP-2 levou a uma redução estatisticamente significante na superfície SLA aos 21 dias (p=0,0000). Em relação à diferenciação, pudemos observar que o tipo de superfície não influenciou o conteúdo total de proteínas, o conteúdo de colágeno e a formação de nódulos calcificados em quaisquer dos períodos analisados. A atividade de fosfatase alcalina somente foi influenciada pelo tipo de superfície aos 14 dias, onde o grupo C/SLAactive apresentou valores inferiores ao grupo C/Liso (p=0,0000). A adição de rhBMP-2 promoveu uma maior influência sobre o processo de diferenciação, levando a uma redução estatisticamente significante no conteúdo total de proteínas na superfície SLA aos 21 dias (p=0,0000), a um aumento estatisticamente significante no conteúdo de colágeno na superfície SLActive no período de 7 dias (p=0,0005) e a uma diminuição estatisticamente significante na atividade de fosfatase alcalina na superfície lisa nos períodos de 14 e 21 dias, na superfície SLA aos 14 dias e na superfície SLActive aos 21 dias (p=0,0000). Somente a formação de nódulos calcificados não sofreu influência da adição de rhBMP-2. / This study has analyzed the influence of different titanium surfaces in the adhesion, proliferation and differentiation of rat osteoblast-like cells (osteo-1 culture), in the presence or not, of the recombinant bone morphogenetic protein-2 (rhBMP-2). The osteo-1 cells were grown on the following titanium surfaces: 1. smooth surface; 2. coarse grit-blasted and acid-etched surface (SLA); and 3. coarse grit-blasted and acid-etched surface under nitrogen protection, and stored in sodium chloride isotonic solution (SLActive), in the presence or not, of 20 ng/ml of rhBMP-2. It was analyzed the cell adhesion in 24 hours, the total protein content, the collagen content, and the alkaline phosphatase in 7, 14 and 21-day periods, and also the formation of calcified nodules in 21 days. The results showed that the adhesion was neither influenced by the surface type, nor by the treatment with rhBMP-2 (p=0.0936). When we related the total protein content to the total number of cells, we noticed that the proliferation was not influenced by the titanium surface type; however, the addition of rhBMP-2 led to a statistically significant reduction on the SLA surface at 21 days (p=0.0000). Concerning the differentiation, we could observe that the surface type did not influence the total content of proteins, the collagen content and the formation of calcified nodules in any of the analyzed periods. The alkaline phosphatase activity was only influenced by the surface type at 14 days, where the group C/SLActive presented lower values than the group C/Smooth (p=0.0000). The addition of rhBMP- 2 promoted a bigger influence over the differentiation process, thus leading to a statistically significant reduction in the total protein content on the SLA surface at 21 days (p=0.0000), a statistically significant increase in the collagen content on the surface SLActive in the 7-day period (p=0.0005), a statistically significant reduction in the alkaline phosphatase activity on the smooth surface in the 14 and 21-day periods, on the SLA surface at 14 days, and on the SLActive surface at 21 days (p=0.0000). Only the formation of calcified nodules did not undergo influence of the rhBMP-2 addition.