Activateurs de plasminogène et résorption osseuse

Le rôle des activateurs de plasminogène dans les processus de résorption osseuse est jusqu’à ce jour sujet à spéculations. Il est clairement établi que ces processus complexes sont dépendants d’une part du recrutement, de la migration et de l’activation de précurseurs de cellules ostéoclastiques à partir des vaisseaux sanguins jusqu’aux surfaces osseuses minéralisées et que le système activateur de plasminogène / plasmide est impliquée dans les processus de migration de nombreux types cellulaires physiologiques ou pathologiques. D’autre part, ces processus sont aussi dépendants de l’action d’une enzyme spécifique, la collagénase, sécrétée sous une forme latente activable par la plasmine. au vu de ces données, nous avons tout d’abord analysé la production d’activateur de plasminogène au sein d’un tissu osseux intact dans lequel la résorption est induite par le PTH ou inhibée par la calcitonine. Les résultats obtenus lors de culture de calvariae de souris embryonnaires de 19 jours ont validé, au niveau du tissu osseux, les observations de stimulation du t-PA par la PTH réalisées dans des systèmes de culture de cellules ostéoblastiques isolées. Ils ont montré de plus que cette activité d’activateur de plasminogène de type tissulaire, t-PA, stimulée par le PTH est à 98% associée au tissu même, qu’elle suit une courbe de stimulation similaire à celle d’autres paramètres de la résorption mais que contrairement à celle-ci elle n’est pas inhibée par la calcitonine et qu’enfin, elle s’accompagne de la libération dans les milieux conditionnés d’une faible activité de type urokinase. Considérant les hypothèses émises quant aux rôles éventuels de ces activateurs lors des processus de résorption, c’est-à-dire, soit un rôle dans les phases préalables à la résorption lors des étapes de recrutement et de migration des précurseurs d’ostéoclastes soit un rôle dans l’activation de la procollagénase , nous avons tenté d’établir la signification physiologique des premiers résultats et en particulier, nous avons recherché si l’activation du plasminogène en plasmine est nécessaire soit aux phénomènes préparatoires à la résorption (recrutement et migration des précurseurs d’ostéoclastes) soit à al résorption ostéoclastique proprement dite. En utilisant un modèle de culture de métatarses de souris fœtales au 17ème jour de leur développement, décrit par Burger (1982), nous disposions d’un système de culture dans lequel la phase de résorption ostéoclatique proprement dite est totalement dépendante d’une phase préparatoire. Après avoir adapté ce modèle à nos conditions de culture en milieu liquide plutôt qu’en milieu semi-solide, nous l’avons caractérisé par l’usage d’agent s hormonaux ou pharmacologiques connus pour être des inducteurs (PTH, vit. D) ou des inhibiteurs (calcitonine, NaF, biphosphonate) de résorption osseuse. Nous avons ensuite analysé les activateurs de plasminogène et observé que l’u-PA est l’activateur majeur produit par les métatarses entiers de même que par les diaphyses considérées isolément. Finalement, nous avons entrepris d’évaluer le rôle du système activateur de plasminogène / plasmine dans la résorption osseuse par le biais d’expériences visant soit à inhiber l’activité de la plasmine par addition d’inhibiteurs physiologiques ou pharmacologiques soit à dépléter le système de culture en plasminogène. Les résultats obtenus semblent bien exclure la nécessité de l’activation du plasminogène en plasmine pour le bon déroulement des diverses étapes qui, faisant suite à l’extravasation des précurseurs d’ostéoclastes au niveau du périoste, aboutissent à la résorption de l’os par des ostéoclastes matures. Il reste cependant à établir la signification physiologique de la forte stimulation de la production de t-PA obtenue dans le tissu osseux (calvariae) sous l’effet des agents inducteurs de résorption, - une réponse due sans doute essentiellement aux ostéoblastes. Cette signification pourrait bien se trouver dans des effets qui se manifestent indépendamment de la présence de plasminogène et de l’activité catalytique de la plasmineStudies on culture of isolated osteoblasts have established that various bone-resorbing hormones increase the expression of tissue plasminogen activator (t-PA) by these cells. The bone resorption processes are known to be dependent, on the one hand, on the recruitment and the migration of osteoclast precursor cells from the blood vessels to mineralized bone surfaces and it is known also that the plasminogen activator / plasmin cascade may be involved in some physiological or pathological cell migrations. On the other hand, bone resorption requires the action of collagenase, released as an inactive precursor, procollagonase, and this zymogen is known to be activatable by plasmin. On this basis, it may thus be speculated that Pas might participate in the bone resorption processes. In the present work, we first investigated the production of Pas that may occur in organized bone tissue which resorption was induced by PTH or inhibited by calcitonin. The results obtained from culture of 19 day-old fetal mouse calvariae have validated, at the level of explanted bone tissue, the observations on the PTH induction of t-PA done previously in culture systems of isolated osteoblast-like cells. In our studies, tissue (t-PA, 70 kD) and urokinase (u-PA, 48 kD) type PA were detected essentially in the tissue extracts of the explanted bones, with only 1-2% of the total activity released in the surrounding culture media. PTH induced bone resorption and stimulated in parallel the accumulation of PA in the tissue; other bone-reosrbing agents, 1.25-dihydroxyvitamin D3 and prostaglandin E2, had similar effects. Densitometric scanning if the zymograms of the bone extracts indicated that PTH stimulated only the production of t-PA and had no effect on that of u-PA. However, PTH also enhanced the release of the u-PA (both the 48 kD and the 29 kD forms) from the bones into the media. Although inhibiting bone resorption, calcitonin had no effect on the PTH-induced accumulation of PA in bone or on the release of t-PA, but it prevented the PTH-induced accumulation of 29 kD u-PA in the culture fluids. Thus these studies support the view that t-PA and possibly also u-PA may have a role in the physiology of bone. In a second series of experiments, we tried to establish the physiological significance of these findings and particularly to evaluate whether the activation of plasminogen into plasmin is necessary for either the preliminary phases to bone resorption (recruitment and migration of osteoclast precursor cells) or the actual bone resorption processes. At the time of explantation, the calvaria used in our first series of experiments contain already osteoclasts in contact with mineralized bone surfaces and are thus not suitable t study possible interferences with the preparatory phases of resorption. Therefore a model was used that involved 17 day-old fetal mouse metatarsals that were explanted and cultured under conditions in which they pursue in vitro for a few days their normal bone remodeling. As described by Burger (1982), the osteoclastic resorption phase is, in this model, totally dependent of a preparatory phase of osteoclasts in their mineralized zones but contain the osteoclast mononuclear precursors in their periosteum. These precursors then migrate from the periosteum through the bone collar and into the calcified cartilage, where they further differentiate into active multinuclear osteoclasts and resorb the mineralized matrix. Of the mineral has been prelabeled in the culture medium. This model was adapted first to our culture conditions, particularly to the use of a liquid medium. It was then characterized with the use of various agents known to be either inducers (PTH, vit. D) or inhibitors (calcitonin, NaF, bisphosphonates) of bone resorption. We further analyzed the PAs present in the explants and observed that u-PA is the main PA present in the whole metatarsals or in their diaphysis. Zymograms indicated however that t-PA contrary to u-PA, is significantly stimulated by PTH, as had been observed already in the calvaria model. We investigated the possible involvement of the PA / plasmin cascade in the bone resorption processes by culturing the metatarsals either under conditions in which plasmin activity was inhibited by physiological (α2-antiplasmin, α1-antiplasmin) or pharmacological (aprotinin) inhibitors or under conditions in which plasminogen was depleted from the system. Our results led us to conclude that plasminogen activation and plasmin activity are not necessary to allow the normal evolution of the different stages of the resorptive processes which follow the extravasation of osteoclast precursor cells in the periosteum and end up with the osteoclastic resorption o the mineralized matrix. The physiological significance of the PTH-induced t-PA accumulation observed in bone tissue ion our cultures remains thus to be established. Its role may possibly be related to PA effects independent of either plasminogen presence or plasmin activityThèse de doctorat en sciences biomédicales (biochimie - biologie cellulaire) -- UCL, 199

Techniques d'évaluation du potentiel anticarie des matériaux libérateurs de fluorures.

Clinical evaluation of anticariogenic potential of fluoride releasing materials seems to be the best way to evaluate products effectiveness. This is an expensive, lengthy and difficult process to realise. Yet there is a need to determine the relative effectiveness of different materials so that clinicians can make rational decision on what materials to use. Fluoride release measurement and fluoride uptake in dental tissues, antibacterial effect evaluation and caries-like lesion inhibition constitute a part of different model investigations use to study fluoride releasing materials. Even if the results of these different in vitro model systems may not be directly transposed to clinical reality, they give some useful information to determine anticariogenic potential of some dental materials. Three materials are analyzed: glass ionomer cements (conventional and resin-modified), polyacid modified resin composites and fluorated resin composites. Results obtained are different for the three kind of materials. Glass ionomer cements show much more anticariogenic properties than polyacid modified resin composites and fluorated resin composites. These two last products are more difficult to distinguish. It is probably due to their similar chemical properties. Even if these products don't have a large anticariogenic effect, it could be enough to prevent secondary caries. The anticariogenic action is principally due to the fluoride release of the materials. That's why it is necessary to define the acceptable level of fluoride release, which is required to have anticariogenic action

Les acquis d’apprentissage (« learning outcomes ») : approches contextuelles et travaux en cours dans le secteur des sciences de la santé

A l’aube de la réforme annoncée des études médicales – qui diminuera la durée de la formation initiale du médecin de sept à six ans – sommes-nous au clair sur le résultat attendu au terme de ce nouveau cursus de base ? Si nous reconnaissons implicitement qu’après six ou sept ans nous voulons former de « bons médecins de base », comment le définir explicitement ? Quels sont les critères qui caractérisent le « bon médecin » ? Un professionnel capable de « bien soigner » ses patients ou un praticien réflexif, autonome, capable de développer de nouvelles connaissances tout au long de sa carrière, … ? Avant de revoir l’architecture et le contenu d’un programme de formation ramené à six ans, il est important de définir le « profil de sortie » de ce nouveau « médecin de base ». En d’autres termes, quels sont les savoirs, aptitudes et compétences que nous souhaitons voir maîtriser par tout étudiant au terme du master en médecine et ce, préalablement à toute formation spécialisée ? Avant toute définition de contenus ou d’activités de formation, l’explicitation des « acquis attendus de l’apprentissage » (« learning outcomes ») est une étape incontournable de la planification curriculaire dans une approche-programme centré sur l’apprentissage de l’étudiant

Radiopacity of resin-based materials measured in film radiographs and storage phosphor plate (Digora)

This study compared the radiopacity of 41 resin-based materials using conventional dental x-ray film (Ultraspeed-D) and a digital system (Digora) based on storage phosphor plate technology. For the film-based technique, optical density measurements were carried out using an X-Rite densitometer. Al equivalents (mm) were calculated as described in the literature using a calibration curve of Optical Density versus the thickness of aluminum. Regarding the digital system after exposures of 0.16 and 0.32 seconds, the images were exported to an image processing software (NIH Image Engineering). An approach similar to that used for optical density was used to generate a calibration curve for gray pixel values. Linear correlations were found between the percentage of fillers by weight and x-ray film radiopacity and the Digora system, and the same coefficient of estimation was recorded (r=0.60; p! 0.05). A linear correlation was also observed between the conventional x-ray film technique and the Digora system (r=0.93; pless than or equal to0.05). Using two different exposure times did not affect the radiopacity. Considerable differences were found among materials of the same category. Flowable resin composites were more radiopaque than dentin, while microfine composites were "radiolucent." Most of the available resin-based materials were more radiopaque than enamel. The radiopacity of resin composites depended on their fillers (percentage and type). Using elements with low atomic numbers (Si) resulted in radiolucent materials, while adding elements with high molecular numbers (Ba, Y, Yb), resulted in radiopaque resin composites. Despite the numerous benefits offered by the digital imaging system (low irradiation dose, instant image, image manipulation), the conventional x-ray film technique seems to be more accurate for radiopacity measurements

Dynamic and static moduli of elasticity of resin-based materials.

OBJECTIVES: The purpose of this study was to assess and compare the elastic moduli of 34 resin-based materials using a dynamic and a static method. The effect of water storage was also studied up to 6 months. METHODS: Five samples of each material were prepared according to ISO-4049. The dynamic moduli were first determined non-destructively from the fundamental period of the vibrating specimen, then the static moduli were determined by a three-point bending test. The percentages of fillers by weight were determined by ashing in air at 900 degrees C. RESULTS: Low values were obtained with flowable composites as well as with two packable resin composites. Correlations were found between the static and the dynamic modulus of elasticity (r = 0.94; p = 0.0001) as well as between the weight percentage of fillers and the moduli of elasticity (r = 0.82; p < 0.05 for static modulus and r = 0.90; p < 0.05 for the dynamic modulus) both at 24h. Water storage significantly affected both static and dynamic moduli of elasticity (ANOVA two factors; p < 0.05). SIGNIFICANCE: The low moduli of the flowable composites do not allow their use in posterior cavities under high stress. However, this does not exclude their use for minimally-invasive Class I cavities when the opposing tooth is stabilized to a large amount on the natural enamel.The Grindosonic method is very useful and simple for determining the dynamic moduli although it gives higher values than the static one. The elastic modulus evolution of resin-based materials after water storage is unpredictable since different patterns were observed as a function of time

Fluoride release from glass-ionomer cements, compomers and resin composites.

The short and long-term fluoride release of 16 products (seven conventional glass-ionomers, five light-activated glass-ionomers, two polyacid-modified resin composites and two resin composites) commercialized as fluoride-releasing materials were measured. A potential link between the material type and its level of fluoride release was researched. The fluoride release was evaluated after different time intervals. Initial fluoride release from all materials was highest during the first 24 h and decreased sharply over the first week. Some groups of materials appeared to be significantly different after, respectively, 7 and 91 days. However, it was impossible to correlate the fluoride release of the materials by their type (conventional or resin-modified glass-ionomers, polyacid-modified resin composite and resin composite) except if we compared the products from the same manufacturer. The link between fluoride release and an acid-base reaction seems to be confirmed. The glass-ionomer composition (glass particles and polyacid's type, powder/liquid ratio) should have more influence on fluoride release than material type

Considerations for the Restoration of Endodontically Treated Molars

The restoration of endodontically treated teeth must be considered as integral part of the endodontic treatment, since it plays a major role in the long-term success of the procedure, as well as in tooth longevity. In order to maximize the chances of success, the specifi cities of endodontically treated molars need to be carefully considered, as well as the recent advances in adhesion, digital technologies, and biomaterials. This chapter will critically discuss important aspects such as reinforcement, restoration retention, the need for a ferrule and/or for posts, and the importance of cuspal coverage. Best-practice recommendations will be made, with particular focus on tissue preservation

Bone resorption and response to calcium-regulating hormones in the absence of tissue or urokinase plasminogen activator or of their type 1 inhibitor

Plasminogen activators (PA) are implicated in cell migration and tissue remodeling, two components of the bone resorption processes. Using mice with inactivated tissue PA (tPA), urokinase PA (uPA), or type 1 PA inhibitor (PAI-1) genes, we evaluated,whether these processes, or their stimulation by parathyroid hormone (PTH) or 1,25-dihydroxyvitamin (1,25[OH]D-2(3)) are dependent on these genes. Two culture models were used, one involving 19-day fetal calvariae, to evaluate the direct resorptive activity of osteoclasts, and the other involving Ca-45-labeled 17-day fetal metatarsals, in which this activity depends on preliminary (pre)osteoclast migration. PTH similarly increased (about 10-fold) PA activity in calvariae from mild-type tPA(+/+) and uPA(+/+) or deficient uPA(-/-) and PAI(-/-) mice; it affected only tPA, not uPA. In tPA(-/-) bones, the low PA levels, due to uPA, were not influenced by PTH. Calcitonin did not affect PA responses to PTH. No differences were observed between tPA(+/+), tPA(-/-), uPA(+/+), and uPA(-/-) calvariae for any parameter related to hone resorption (development of lacunae, release of calcium and lysosomal enzymes, accumulation of collagenase, loss of hydroxyproline), indicating similar responses to PTH or calcitonin. The progressive Ca-45 release was largely similar in cultures of tPA(+/+), tPA(-/-), uPA(+/+) uPA(-/-), PAI(+/+), or PAI(-/-) metatarsals and it was similarly enhanced by PTH or 1,25(OH)(2)D-3. However, uPA(-/-) metatarsals released Ca-45 at a slower rate at the beginning of the cultures, suggesting an impaired recruitment of the (pre)osteoclasts, which migrate at that time from the periosteum into the calcified cartilage. Thus, it appears that the direct resorptive activity of the osteoclasts does not necessitate the presence of either tPA or uPA, but uPA is likely to facilitate the migration of the (pre)osteoclasts toward the mineralized surfaces. Although considerably enhanced by PTH, tPA does not mediate the actions of PTH (nor of 1,25[OH]D-2(3)) evaluated in these models