No description available.

Introduction: Mammalian cell recombinant bone morphogenetic protein (rBMP) synthesis is reported to be poor. The BMP pro-domain may be involved in folding, stability and secretion. Objectives: Investigate the effect of inhibition of pro-domain cleavage on rhBMP-2 production. Methods: CHO cells transfected with human BMP-2 (hBMP-2) were cultured in the presence of the proprotein convertase inhibitor 9DR in short (multi-well) and long-term (bioreactor) cultures. Mature and proBMP secretion was measured by ELISA and characterized by Western blot. BMP activity was determined by C2C12 bioassay. Results: 9DR significantly enhanced the yields of both pro- and mature hBMP-2 in short and long-term cultures, without any negative effects on cell growth or viability. The rhBMP-2 was biologically active. ProBMP-2 could be converted by exogenous furin treatment into mature BMP-2 as shown by Western blot. Conclusions: 9DR increases rhBMP-2 production and is a simple, but effective way to enhance the yield of active, mature BMP-2 in CHO cells.

Introduction: Mammalian cell recombinant bone morphogenetic protein (rBMP) synthesis is reported to be poor. The BMP pro-domain may be involved in folding, stability and secretion. Objectives: Investigate the effect of inhibition of pro-domain cleavage on rhBMP-2 production. Methods: CHO cells transfected with human BMP-2 (hBMP-2) were cultured in the presence of the proprotein convertase inhibitor 9DR in short (multi-well) and long-term (bioreactor) cultures. Mature and proBMP secretion was measured by ELISA and characterized by Western blot. BMP activity was determined by C2C12 bioassay. Results: 9DR significantly enhanced the yields of both pro- and mature hBMP-2 in short and long-term cultures, without any negative effects on cell growth or viability. The rhBMP-2 was biologically active. ProBMP-2 could be converted by exogenous furin treatment into mature BMP-2 as shown by Western blot. Conclusions: 9DR increases rhBMP-2 production and is a simple, but effective way to enhance the yield of active, mature BMP-2 in CHO cells.

Hepatic injuries due to viral infection and alcoholic abuse frequently lead to activation and proliferation of hepatic stellate cells (HSCs), concomitantly with tissue repairing and remodeling mechanism. Transforming growth factor-£]1 (TGF-£]1) is known to be one of the potent cytokines that directly upregulates synthesis of extracellular matrix, thereby aggravating liver fibrosis. Bone morphogenetic protein-2 (BMP-2), as a member of TGF-Hepatic injuries due to viral infection and alcoholic abuse frequently lead to activation and proliferation of hepatic stellate cells (HSCs), concomitantly with tissue repairing and remodeling mechanism. Transforming growth factor-£]1 (TGF-£]1) is known to be one of the potent cytokines that directly upregulates synthesis of extracellular matrix, thereby aggravating liver fibrosis. Bone morphogenetic protein-2 (BMP-2), as a member of TGF-£]1 superfamily, has been known to regulate cell proliferation, differentiation, and bone morphogenesis. Our previous study demonstrated that BMP-2 was downregulated in fibrotic liver of mice, supporting its antagonistic role in hepatic fibrogenesis. Downregulation of BMP-2 in fibrotic livers may lose its ability to antagonize the pro-fibrogenic action of TGF-£]1. The purpose of this study was to determine whether mutual regulatory mechanisms exist between BMP-2 and TGF-£]1. Treatment of recombinant protein, TGF-£]1, significantly suppressed BMP-2 expression in hepatocytes clone-9 and HSC-T6 cells. Moreover, treatment of BMP-2 in both cell also attenuated TGF-£]1protein levels in a dose-dependent manner. This finding supports that TGF-£]1 and BMP-2 mutually modulated their expression not only in vivo, but also in vitro. Western blotting analysis showed that TGF-£]1 and BMP-2 exerted different kinase phosphorylation profiles of signaling activities. However, the signal pathways and detail mechanisms of the interactions of BMP-2 and TGF-£]1 in the fibrogenic action will need to further evaluate.

A variety of synthetic bone graft materials such as the polymer poly(lactic-co-glycolic acid) (PLGA) have been investigated as alternatives to current tissue based bone graft materials. In this study, efforts have been made to improve the tissue-PLGA interface by immobilizing bone morphogenetic protein-2 (BMP-2) in an oriented manner on scaffolds using covalently immobilized heparin. The results demonstrated a four-fold increase in covalently immobilized heparin compared to non-specific heparin attachment. Furthermore, the scaffolds with covalently attached heparin retained approximately three-fold more BMP-2 than did either scaffolds with no heparin attached or scaffolds with non-specific heparin attachment. The activity of scaffolds with BMP-2 immobilized in various manners was examined using an alkaline phosphatase assay on C3H10T1/2-seeded scaffolds. These results indicated approximately twice the amount of activity with scaffolds that had BMP-2 immobilized with covalently attached heparin than on scaffolds with adsorption of BMP-2 and a three-fold increase in activity when compared to scaffolds that had non-specific heparin attachment as the mechanism for BMP-2 immobilization. These results demonstrated that PLGA with covalently linked heparin has potential to immobilize BMP-2 in a specific orientation that is favorable for cell-receptor binding, leading to the more efficient use of the bone-growth factor.

No description available.

The local release of complexed siRNA from biomaterials enables targeted therapy of specific cells and tissues. This thesis focused on gelatin microparticles cross-linked (cGM) with an anhydride-containing oligomer (oPNMA) as a drug delivery system for siRNA. The siRNA-loaded cGM were aggregated with SaOS-2 cells or human mesenchymal stem cells (hMSC) to microtissues and stimulated with osteogenic supplements. Cell survival and tissue formation in microtissues could be improved by incorporating cGM in spheroid cultures. We observed hydroxyapatite deposition in the particles in dependence of medium and cell type. Osteogenic stimulation with BMP-2 and simultaneous silencing of BMP-2 antagonist chordin accelerated matrix mineralization of the microtissues. Higher cross-linking degree of cGM positively influenced chordin silencing and alkaline phosphatase (ALP) activity as a marker for osteogenic differentiation. These higher cross-linked cGM mineralized in an osteogenic medium within 8–9 days, in presence and absence of cells. The effects of pre-differentiated and chordin-silenced microtissues were investigated by simulation of in vivo conditions in an unstimulated co-culture system of hMSC and human peripheral blood mononuclear cells (hPBMC). Increased ALP activity and osteoprotegerin (OPG) secretion were observed after 14 days compared to co-cultures with siRNA-free controls. These results indicate that the pre-differentiated and silenced microtissues can induce osteogenic differentiation of surrounding unstimulated cells. Using the microtissue approach with siRNA complexed with tyrosine-modified low molecular weight polyethyleneimine (P10Y/P5Y) as transfection reagent was not successful. The results of this thesis indicate that the pre-differentiation of microtissues with BMP-2 in combination with chordin silencing stimulates and enhances osteogenic differentiation of other stem cells. As a combination of biomaterial, RNAi, and autologous cells, microtissues could be a promising approach to regenerating bone defects.:Chapter I: Controlled release of siRNA for bone tissue engineering Chapter II: Microtissues from mesenchymal stem cells and siRNA-loaded cross-linked gelatin microparticles for bone regeneration Chapter III: Mineralizing gelatin microparticles as cell carrier and drug delivery system for siRNA for bone tissue engineering Chapter IV: Tyrosine-modified polyethylenimines for siRNA transfection in microtissues Chapter IV: Final discussion

Les lésions de cartilage ne cicatrisent pas spontanément et la réparation de ce tissu est un challenge. Les techniques chirurgicales restant insatisfaisantes, la thérapie cellulaire et l'ingénierie tissulaire sont maintenant envisagées. La transplantation de chondrocytes autologues (TCA) existe déjà mais cette procédure nécessite l'amplification des chondrocytes qui s'accompagne d'une perte du phénotype différencié (dont l'indicateur est le collagène de type II), au profit d'un phénotype fibroblastique (dont l'indicateur est le collagène de type I, retrouvé dans les tissus fibreux). La TCA conduit donc à une greffe de chondrocytes dédifférenciés produisant un fibrocartilage, dont les propriétés mécaniques sont différentes du cartilage hyalin natif. L'objectif de mes travaux était de développer un nouveau kit d'ingénierie tissulaire du cartilage par association de chondrocytes humains, de biomatériaux et d'une sélection de facteurs solubles. Nous avons utilisé le cocktail FGF-2/insuline (FI) pour l'amplification cellulaire et le cocktail BMP-2/insuline/T3 (BIT) pour redifférencier les chondrocytes dans des éponges de collagène. Nos résultats ont montré que cette combinaison permet la synthèse d'une matrice cartilagineuse dans les supports collagène. Cependant, cette synthèse s'est trouvée favorisée en périphérie des éponges cultivées en conditions statiques. Nous avons ensuite utilisé un bioréacteur pour perfuser les éponges et nos résultats ont révélé alors un dépôt plus homogène de cartilage dans ces supports. De manière très intéressante, nous avons aussi observé l'arrêt de l'expression du collagène de type I. Ainsi, notre approche multifactorielle combinant des chondrocytes humains, des biomatériaux collagène, une combinaison FI-BIT et une culture en perfusion permet la reconstruction d'un cartilage non fibrotique / Cartilage lesions are irreversible and cartilage repair is challenging. Actual surgical techniques remain unsatisfactory and therefore, cell therapy and tissue engineering approaches are now considered. The Autologous Chondrocytes Transplantation (ACT) already exists but this procedure requires chondrocytes amplification. During this amplification, a dedifferentiation process occurs: chondrocytes lose their differentiated phenotype (characterized by type II collagen) towards a fibroblastic phenotype (characterized by type I collagen, a component of fibrous tissues). ACT leads to the graft of dedifferentiated chondrocytes, hence provoking the production of a fibrocartilage that presents different mechanical properties than native hyaline cartilage. The aim of my work was to develop a new kit of tissue engineering for cartilage repair using human chondrocytes, biomaterials and a selection of soluble factors. We used a cocktail of FGF-2 and insulin (FI) for cell amplification and a cocktail of BMP-2, insulin and T3 (BIT) for chondrocyte redifferentiation in collagen sponges. Our results showed that the combination allows the synthesis of a cartilaginous matrix in collagen scaffolds. However, matrix production is favored in periphery of the sponges cultivated in static conditions. A perfusion bioreactor was then used to perfuse the sponges and our results revealed a more homogeneous deposition of cartilage in the scaffolds. Very interestingly, we also noticed a stop of type I collagen expression. Thus, our multifactorial approach combining human chondrocytes, collagen scaffold, the combination FI-BIT and culture under perfusion allows the reconstruction of a non-fibrotic cartilage

O desenvolvimento ontogenético em peixes é considerado como uma das etapas mais importantes no fornecimento de informações para biologia do desenvolvimento, aquicultura e estudos taxonômicos. A Proteína Óssea Morfogenética 2 (BMP-2) é considerada uma molécula essencial como regulador no desenvolvimento embrionário e na formação óssea, sendo ainda pouco estudada em peixes. Neste trabalho, foram comparadas todas as fases do desenvolvimento de três espécies de peixes importantes da bacia do rio Pardo, curimbatá (Prochilodus hartii), piabanha (Brycon sp) e piau (Leporinus steindachneri). A superfície ovocitária, estádios e duração do desenvolvimento embrionário/larval foram classificados com a utilização de microscópio estereoscópio, havendo variações entre as espécies no desenvolvimento. A transformação dos alevinos também apresentaram variações, com 16 dias pós-eclosão em P. hartii, 5 dias em Brycon sp e 14 dias em L. steindachneri. Na imunohistoquímica, BMP-2 foi identificada na blastocele nas fases de blástula e gástrula; na formação da vesícula óptica, notocorda e somitos, na fase embrionária das espécies estudadas. Na fase larval e de juvenil, BMP-2 foi identificada na formação de brânquias, olhos, coração, estômago, intestino, fígado, nadadeiras, músculos e na ossificação em todas as espécies estudadas. Indivíduos adultos foram sacrificados, e depois de realizada a biometria, dissecados para mensuração dos órgãos internos e documentação fotográfica. Observou-se que o estômago de P. hartii tem forma de \"U\" com presença de uma estrutura semelhante a uma moela, enquanto em Brycon sp tem forma de \"J\" com presença de cecos pilóricos aderidos no piloro e em L. steindachneri, o mesmo possui forma de \"Y\" sem presença de cecos pilóricos. Esqueletos foram preparados através da técnica de maceração com insetos dermestídeos, sendo observadas diferenças, sendo interessante descrever que em P. hartii foram encontrados 4 ossos infraorbitais, 6 em Brycon sp e 4 em L. steindachneri. Conclui-se que os exemplares de P. hartii possuem hábito alimentar iliófago, enquanto Brycon sp e L. steindachneri são considerdados onívoros, baseado nos achados anatômicos e a expressão de BMP-2 está ligada com a morfogênese e organogênese na embriologia das espécies estudadas. / The ontogenetic development in fish is considered as one of the most important steps in providing information to developmental biology, aquaculture and taxonomic studies. The Bone Morphogenetic Protein 2 (BMP-2) is considered as a molecule essential for regulaton of embryonic development and bone formation, and has been poorly studied in fish. In this work, we have compared all the development phases of three important species of fish from the Pardo River basin, Curimbata (Prochilodus hartii) Piabanha (Brycon sp) and Piau (Leporinus steindachneri). The surface oocyte, stage and duration of embryonic/larval development were classified by using a stereoscopic microscope, and showed variations between the studied species. The transformation of the juveniles also showed variations, with 16 days post-hatching in P. hartii, 5 days in Brycon sp and 14 days in L. steindachneri. In immunohistochemistry, BMP-2 was identified in the blastocoel of the blastocyst and gastrula stages; forming the optic vesicle, notochord and somites on embryo of the ivestigated species. In larval and juvenil stages, BMP-2 has been identified in the formation of gills, eyes, heart, stomach, intestine, liver, fins, muscle and ossification in the species studies. Adults were sacrificed, dissected for biometrics measurements of the internal organs and photo documentation. The stomach of P. hartii showed a \"U\" shape with the presence of a structure similar to a gizzard, whereas in Brycon sp it was shaped like a \"J\" with the presence of pyloric caeca and in L. steindachneri it presented a \"Y\" form without the presence of pyloric caeca. Skeletons were prepared by retting technique with Dermestides beetles, interesting differences were observed between numbers of bones, where 4 infraorbital bones were found in P. hartii, 6 in Brycon sp and 4 in L. steindachneri. It is concluded that the specimens of P. hartii show ilyophagous eating habits while Brycon sp and L. steindachneri were considered omnivorous based on anatomical findings and that expression of BMP-2 is linked with morphogenesis and organogenesis of the studied species.

La surface naturelle des prothèses, tels que des implants métalliques, n'est pas idéale pour l'obtention d'une bonne ostéo-intégration. Par conséquent, l'amélioration des propriétés de surface pour les rendre ostéo-condutrices ou ostéo-inductrices est souhaitable. La délivrance contrôlée de protéines ostéo-inductrices de la famille des bone morphogenetic proteins (BMPs) par la surface des matériaux implantables permettrait une formation osseuse optimisée et plus rapide autour de l'implant. En particulier, la BMP-2 est importante dans la phase initiale de la différenciation vers l'os. En raison de leur similarité avec les tissus naturels, l'utilisation des revêtements de biopolymères qui ont une bonne affinité avec les molécules bioactives, semble prometteuse pour le chargement et la délivrance de BMP-2. L'équipe a déjà mis au point un film à base des biopolymères hyaluronane et de poly(L-lysine), en utilisant la technique d'assemblage couche par couche. Ce film constitue un réservoir qui permet de présenter la BMP-2 "liée à la matrice". La bioactivité in vitro et les propriétés ostéo-inductrices in vivo de ces films ont déjà prouvées. Dans ce travail, nous avons cherché à mieux comprendre l'interaction de la BMP-2 avec le film et l'interaction des cellules avec la BMP-2. Tout d'abord, nous avons étudié la structure de la BMP-2 piégée dans les films et l'avons comparé à celle en solution; puis nous avons évalué l'impact du séchage, du stockage à long terme et de la stérilisation sur la structure du film et sa bioactivité. Enfin, nous avons étudié l'internalisation de la BMP-2 par les cellules en fonction de la réticulation du film et avons étudié la relation entre internalisation et voies de signalisation. / The natural surface of bulk prostheses materials, such as metallic implants, is not suitable for successful osteointegration of implants. Therefore, improving the surface to render it osteoconductive and osteoinductive is needed. The controlled delivery of osteoinductive bone morphogenetic proteins (BMPs) from the surface of implantable materials would enable faster and better bone formation around the implant. In particular, BMP-2 plays an important role in the early phase of differentiation of stems cells in bone cells. The coating of natural polymers that have a high affinity for BMP-2 would enable BMP retention and localized delivery at the implant surface. Using the layer-by-layer technique, we have developed a coating made of the biopolymers hyaluronan and poly(L-Lysine), which acts as a reservoir to trap BMP-2 and to present them to cells in a "matrix-bound" manner. The in vitro bioactivity and in vivo osteoinductive properties of BMP-2-loaded films have previously been proved. In this work, the aim is to further understand the interaction of the BMP-2 with the film and the uptake of BMP-2 by the cells. First, the secondary structure of matrix-bound BMP-2 was studied and compared to its structure in solution. Second, the impact of drying, long term storage and sterilization on film structure and bioactivity were assessed. Finally, we investigated if and how matrix-bound BMP-2 is internalized by the cells from the different cross-linked films, the internalization route and its relation to BMP-2 signaling.