The use of best management practices or BMPs to treat urban stormwater runoff has been pervasive for many years. Extensive research has been conducted to evaluate the performance of individual BMPs at specific locations; however, little research has been published that seeks to evaluate the impacts of small, distributed BMPs throughout a watershed at the regional level. To address this, a model is developed using EPA SWMM 5.0 for the Duck Pond watershed, which is located in Blacksburg, Virginia and encompasses much of the Virginia Polytechnic and State Institute's campus and much of the town of Blacksburg as well. A variety of BMPs are designed and placed within the model. Several variations of the model are created in order to test different aspects of BMP design and to test the BMP modeling abilities of EPA SWMM 5.0. Simulations are performed using one-hour design storms and yearlong hourly rainfall traces. From these simulations, small water quality benefits are observed at the system level. This is seen as encouraging, given that a relatively small amount of the total drainage area is controlled by BMPs and that the BMPs are not sited in optimal locations. As expected, increasing the number of BMPs in the watershed generally increases the level of treatment. The use of the half-inch rule in determining the required water quality volume is examined and found to provide reasonable results. The design storm approach to designing detention structures is also examined for a two-pond system located within the model. The pond performances are examined under continuous simulation and found to be generally adequate for the simulated rainfall conditions, although they do under-perform somewhat in comparison to the original design criteria. The usefulness of EPA SWMM 5.0 as a BMP modeling tool is called into question. Many useful features are identified, but so are many limitations. Key abilities such as infiltration from nodes or treatment in conduit flow are found to be lacking. Pollutant mass continuity issues are also encountered, making specific removal rates difficult to define. / Master of Science

Livestock best management practices (BMPs) such as streamside exclusion fencing are installed to reduce cattle impacts on stream water quality such as increases in bacteria through direct deposition and sediment through trampling. The main objective of this study is to assess the effects of different cattle management strategies on water quality. The project site was located near Keezletown, VA encompassing Cub Run and Mountain Valley Road Tributary streams. During two, one-week studies, eight automatic water samplers took two-hour composites for three periods: baseline, cattle access, and recovery. During the cattle access period, livestock were able to enter the riparian zone normally fenced off. Water samples were analyzed for E.coli, sediment, and nutrients to understand the short-term, high-density, or flash grazing, impact on water quality. Additional weekly grab and storm samples were collected. Results show that cattle do not have significant influence on pollutant concentrations except in stream locations where cattle gathered for an extensive period of time. Approximately three cattle in the stream created an increase in turbidity above baseline concentrations. E.coli and TSS concentrations of the impacted sites returned to baseline within approximately 6 to 20 hours of peak concentrations. Weekly samples show that flash grazing does not have a significant influence on pollutant concentrations over a two-year time frame. Sediment loads from storms and a flash grazing event showed similar patterns. Pollutant concentrations through the permanent exclusion fencing reach tended to decrease for weekly and flash grazing samples. / Master of Science

No / Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) play roles in embryonic development and postnatal remodelling of the skin. Many indications suggest that BMP signalling regulates keratinocyte proliferation and differentiation. Chronic wounds have been shown to exhibit high levels of BMP ligands; however, the effect of BMP pathway modulation on human skin healing remains undefined. A human ex vivo skin wound healing model was used to analyse the expression of BMP signalling pathway components during healing and to investigate the effects of BMPs and the BMP antagonist Noggin on skin repair. Additionally, the effects of BMP signalling on keratinocyte proliferation, apoptosis and migration were tested using in vitro flow cytometry and ‘scratch’ migration assays, respectively. BMP receptor-1B (BMPR-1B) and downstream signalling protein phosphorylated-Smad-1/5/8 were highly expressed in healing epidermis. Treatment of human skin with exogenous BMPs impaired wound closure by reducing keratinocyte proliferation and increasing apoptosis. The BMP antagonist Noggin negated the inhibitory effects of BMP ligands, and when used alone, Noggin reduced keratinocyte apoptosis in the wound bed. In vitro, BMP ligands suppressed keratinocyte proliferation whilst Noggin stimulated proliferation. Keratinocyte migration was slowed following BMP treatment; in contrast, migration was significantly accelerated due to inhibition of BMP activity by either Noggin or BMPR-1B silencing. BMP signalling is inherently involved in wound healing. BMPs slow skin repair by suppressing keratinocyte proliferation and migration. Thus, modulation of BMP signalling using BMP inhibitors such as Noggin may serve as a new approach to promote cutaneous wound repair. Level of evidence: Not ratable.

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

Objetivo: O propósito deste estudo é avaliar a regeneração óssea nos defeitos alveolares congênitos com proteína morfogenética óssea (rhBMP-2) em membrana de colágeno reabsorvível. Material e Métodos: Vinte e quatro pacientes com fissura labiopalatina completa unilateral, em momento ideal, foram distribuídos ao acaso entre os grupos controle (porção medular de ilíaco) e teste (rhBMP-2 em membrana de colágeno absorvível). Foram avaliados em 6 meses e 1 ano de pós-operatório por meio de radiografias periapicais e tomografias computadorizadas pela escala de Chelsea. Resultados: Encontraram-se índices de sucesso quanto à altura do septo ósseo semelhantes entre os grupos: em 6 meses, na análise radiográfica, 81,82% no controle e 91,67% no teste; em 1ano, 83,33% no controle e 100% no teste; na análise tomográfica, observaram-se 70% no controle e 91,67% no teste em 6 meses, 75% no controle e 100% no teste em 1 ano. Na análise volumétrica, os valores encontrados foram 45,34% de preenchimento ósseo da fissura no grupo controle aos 6 meses e 40,63% no teste, 53,33% no controle e 53,1% no teste em 1 ano. Não houve diferença estatisticamente significante. Conclusão: A rhBMP-2 em membrana reabsorvível de colágeno produziu resultados semelhantes ao enxerto autógeno de medula de crista ilíaca ao reparar defeitos alveolares de fissuras labiopalatinas completas. / Objective: The purpose of this study is to assess the bony regeneration of alveolar clefts using bone morphogenetic protein (rhBMP-2) in a collagen sponge carrier. Material and Methods: Twenty four patients with unilateral complete cleft lip and palate, in the very best moment, were randomly assigned to groups control (iliac crest cancellous bone) and test (rhBMP-2 in an absorbable collagen sponge). They were evaluated 6 months and 1 year postoperative with periapical radiographs and computed tomographs using Chelsea scale. Results: For control group, radiographic examination revealed success index for bone height 81,82% 6 months and 83,33% 1 year postoperative; for test group, findings were 91,67% first control and 100% 1 year. Computed tomograph revealed 70% 6 months and 75% 1 year for control group, 91,67% first control and 100% 1 year for bone height test group. Volume ratios were 45,34% bone filling 6 months and 53,33% 1 year for control group and 40,63% 6 months and 53,1% 1 year for test. There was no statistical significance. Conclusion: rhBMP-2 in a collagen sponge carrier induced bony regeneration close to that from iliac crest cancellous bone to repair complete alveolar clefts.

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.

Les lamines A et C sont deux composants majeurs de la lamina nucléaire, réseau de filaments intermédiaires situé sous la membrane nucléaire interne. Les mutations du gène LMNA, codant les lamines A/C, ont été associées à de nombreuses pathologies humaines, appelées laminopathies, et affectant un ou plusieurs tissus dont le muscle. Les mécanismes physiopathologiques sous-jacents ne sont encore que partiellement élucidés. Les lamines A/C jouant un rôle crucial dans l’architecture nucléaire et l’organisation de la chromatine, l’hypothèse d’une altération de l’expression de facteurs de transcription ou de gènes tissus-spécifiques a été formulée. De plus, au niveau musculaire, il a été décrit que les lamines A/C jouent un rôle majeur dans la mise en place d’une différenciation musculaire efficace.Afin d’identifier des altérations potentielles au sein des voies de signalisation régulant la différenciation musculaire, nous avons utilisés un modèle de myoblastes murins conditionnellement immortalisés et comparés le profil d’expression entre les myoblastes sauvages et inactivés pour le gène Lmna (Lmna-/-). Nous avons donc identifiés deux altérations majeures de la voie BMP (Bone Morphogenetic Pathway) : la diminution de l’expression du ligand Bmp4 et l’augmentation de celle de Smad6, un inhibiteur intracellulaire de la voie. Cette surexpression de Smad6 est responsable d’une séquestration cytoplasmique des Smads 1, 5 et 8 phosphorylées et d’une diminution de l’expression des gènes cibles, Id1 et Id2. Les myoblastes Lmna-/- montrent une différenciation myogénique prématurée, phénotype réversible par des expériences d’ARN interférent ciblant Smad6. Enfin, nous avons montré que ces défauts sont retrouvés dans des myoblastes humains porteurs hétérozygotes de la mutation LMNA R310X.Ces résultats apportent un nouveau mécanisme physiopathologique des laminopathies musculaires et identifient une nouvelle cible thérapeutique potentielle / LMNA gene encodes lamins A and C, two major components of the nuclear lamina, a network of intermediate filaments underlying the inner nuclear membrane. LMNA mutations have been associated with a wide spectrum of human diseases collectively called “laminopathies” affecting one or several tissues, such as muscles. The physiopathological mechanisms underlying laminopathies remain unclear. Given the crucial role of lamins A/C in nuclear architecture and chromatin organization, the “gene regulation” hypothesis have been proposed. It suggests that LMNA mutations could alter in a tissue-specific manner transcription factors and/or genes expression. Moreover, lamins A/C have been described as important regulators in muscle differentiation regulation.To identify potential alterations in signaling pathways regulating muscle differentiation in LMNA-mutated myoblasts, we used a previously described model of conditionally immortalized murine myoblasts and compared gene expression profiles in wild-type and Lmna-/- H-2K myoblasts. We identified two major alterations of the Bone Morphogenetic Protein (BMP) pathway in Lmna-/- myoblasts: Bmp4 downregulation and Smad6 overexpression. We demonstrated that Smad6 overexpression lead to Smad1/5/8 sequestration in the cytoplasm and to the downregulation of their target genes, Id1 and Id2. As a consequence, Lmna-null myoblasts displayed a premature differentiation which could be rescued by downregulating Smad6 expression. Finally, we showed that these defects are relevant for human laminopathies as they are also present in myoblasts from a human patient carrying a LMNA+/Q310X mutation.Taken together, these results provide a potential mechanism for the muscle stem cell exhaustion and muscle atrophy observed in muscle laminopathies and identify a new therapeutical target likely to reverse pathological phenotypes

L'athérosclérose est une complication cardiovasculaire majeure des maladies liées à une augmentation du stress oxydatif, comme le diabète de type 2 et le syndrome métabolique. Dans ces situations, les lipoprotéines de faible densité (LDL) subissent une oxydation et leur forte absorption induit une accumulation de cholestérol dans les macrophages sous-endothéliaux. D'autre part, les LDL oxydées sont enrichies en produits d'oxydation du cholestérol appelés oxystérols, dont certains sont impliqués dans la capacité des LDL oxydées à induire un stress oxydant cellulaire et une cytotoxicité, principalement par apoptose. Le Bis(Monoacylglycéro)Phosphate (BMP) est un phospholipide unique, localisé préférentiellement dans les endosomes tardifs, compartiment cellulaire clef dans le métabolisme du cholestérol dérivé des LDL. Lors de travaux antérieurs, l'équipe a démontré le rôle prépondérant du BMP dans la régulation de l'homéostasie du cholestérol dans les macrophages. L'objectif de ce travail a été de décrypter les mécanismes moléculaires intervenant dans le trafic intracellulaire du cholestérol. Ainsi, le BMP régule l'efflux de cholestérol par les HDL (high density lipoproteins) grâce à des mécanismes impliquant les LXRs (liver X receptors) et les transporteurs ABCA1/ABCG1 (ATP binding cassette-type A1/G1). De plus, notre étude indique que le BMP exerce également un rôle protecteur contre l'effet pro-apoptotique des LDL oxydées via la réduction de la production intracellulaire d'oxystérols. Comme une partie du trafic intracellulaire des stérols au sein des macrophages est régulé par OSBP (oxysterol binding protein) et ses protéines dérivées, les ORPs (OSBP-related proteins), nous montrons dans ce rapport que l'action de protection du BMP contre les effets cytotoxiques des oxystérols est fortement diminuée dans des cellules où la protéine ORP11 est supprimée, suggérant que le BMP exerce son rôle protecteur via un mécanisme utilisant la fonction d'ORP11 dans le transport intracellulaire de stérols / Atherosclerosis is a major cardiovascular complication in increased oxidative stress-related diseases such as type 2 diabetes and metabolic syndrome. In these situations, the low density lipoproteins (LDL) undergo oxidation and their high uptake induces cholesterol accumulation in subendothelial macrophages. On the other hand, oxidized LDL are enriched in cholesterol oxidation products called oxysterols, some of them are involved in the ability of oxidized LDL to induce cellular oxidative stress and cytotoxicity, mainly by apoptosis. Bis(Monoacylglycero)Phosphate (BMP) is a unique phospholipid localized preferentially in late endosomes, a central cellular compartment of LDL-cholesterol metabolism. In previous work, the team demonstrated the leading role of BMP in regulation of cholesterol homeostasis in macrophages. The aim of this work was to characterize the molecular mechanisms involved in the intracellular trafficking of cholesterol. Thus, BMP regulates cholesterol efflux to HDL (high density lipoproteins) by mechanisms involving liver X receptors (LXRs) and ABCA1/ABCG1 (ATP binding cassette-type A1/G1) transporters. Moreover, we report BMP also exerts a protective role against the pro-apoptotic effect of oxidized LDL via a reduced production of intracellular pro-apoptotic oxysterols.As part of macrophage intracellular sterol traffic is regulated by oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs), we show that protective action of BMP against cytotoxic oxysterol effects in ORP11-silenced cells, was markedly abrogated, suggesting BMP exerts its protective role via a mechanism involving the function of ORP11 in intracellular sterol transport

La compréhension de la régulation de l’AMH et de son récepteur spécifique, l’AMHR2, par les BMPs a amené un éclairage nouveau sur leur rôle dans le développement folliculaire et la régulation du taux d’ovulation. Nos résultats se basent sur l’étude de 4 modèles ovins porteurs de mutations Fec, mutations qui affectent des membres de la famille des BMPs, à savoir leur ligand BMP15 ou leur récepteur. Ces mutations se traduisent phénotypiquement par une baisse de l’expression de l’AMH ou de son récepteur dans les cellules folliculaires, et des ovulations multiples chez les brebis porteuses. D’un point de vue mécanistique, nos résultats ont mis en évidence une régulation de l’AMH et de l’AMHR2 par les BMPs in vivo et in vitro, passant par le récepteur BMPR1B, et s’exerçant sur l’activité transcriptionnelle du promoteur de l’AMH via SMAD1 et SF1. Cette régulation a également été en partie mise en évidence dans l’espèce porcine avec une observation supplémentaire dans ce modèle : un taux d’ovulation naturellement très élevé est associé à une faible production ovarienne d’AMH. Ces observations mettent en exergue le rôle possible de l’AMH dans la régulation du taux d’ovulation. / The understanding of the regulation of AMH and its specific receptor, AMHR2, by the BMPs, brought a new highlight on their role in the regulation of follicular development and the control of ovulation rate. Our results are based on the study of 4 sheep models carrying Fec mutations which affect different members of the BMPs family, namely their ligand BMP15 or their receptor, Mutations result phenotypically in a low expression of AMH or AMHR2 in the granulosa cells of ovarian follicles, and multiple ovulations in carrier ewes.. From a mechanistic point of view, the results demonstrated the in vivo and in vitro regulation of AMH and AMHR2 by BMPs, acting through the BMPR1B receptor and enhancing the transcriptional activity of the AMH promoter via SMAD1 and SF1. This regulation has also been partially demonstrated in swine with an additional observation in this model: a naturally high ovulation rate is associated with a low ovarian production of AMH. In conclusion, these observations show a possible role of the AMH in the regulation of ovulation rate.

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.