Periodontal disease is initiated by bacterial plaque that induces a chronic inflammatory condition with subsequent leukocyte infiltration, osteoclast activation and alveolar bone resorption. The ideal treatment for periodontal disease is the complete regeneration of the lost periodontium. Resolvin E1 (RvE1) is an endogenous anti-inflammatory lipid mediator derived from omega-3 fatty acids. Animal studies showed that topical treatment of periodontitis with RvE1 significantly decreased osteoclast counts, prevented alveolar bone loss, and restored lost periodontal tissues including bone. It is not known if RvE1 directly impacts osteoblast functions and bone formation. The objective of this study was to determine RvE1 mechanism of action on osteoblasts. Results showed that topical RvE1 treatment prevented the progression of ligature-induced periodontitis in mice compared to the vehicle. RvE1 receptor, chemR23, was expressed in murine calvaria osteoblasts and the expression was not changed in the inflammatory environment with or without RvE1 treatment. RvE1 treatment resulted in a significant increase in the ALP activity after 2 and 7 days of treatment compared to the control as well as in the inflammatory milieu. Similarly, RvE1 treatment enhanced murine calvaria osteoblasts mineralization in vitro compared to the inflammatory environment. The proliferation of mouse calvaria osteoblasts was significantly increased with RvE1 treatment after 2 and 10 days of treatment compared to the control. Results for evaluating the impact of RvE1 on OPG/RANKL axis showed that RvE1 treatment markedly elevated OPG production compared to the IL-6/IL-6R treatment and decreased RANKL. Overall, RvE1 increased the OPG/RANKL ratio to favor bone formation. RvE1 treatment resulted in a significant increase in the phosphorylation of AKT, ERK1/2 and ribosomal S6 (rS6) kinase. Those pathways were further confirmed via using specific pharmacological inhibitors which showed a significant reduction in OPG production and in the osteoblast proliferation as well. In conclusion, RvE1 has a positive anabolic impact in ligature-induced periodontitis model via direct actions on osteoblasts. RvE1 increases the OPG/RANKL production ratio favoring the bone formation through a pathway that includes phosphorylation of AKT, ERK1/2 and rS6 kinase. The data suggest that RvE1 stimulates bone formation in inflammatory conditions by directly modulating both the osteoclast and osteoblast functions.

Since bone tissues grow in intimate contact with implant surfaces in vivo, there is a need to investigate how bone cells respond to mechanical loads adjacent to implants under well characterized loading conditions that stimulate the bone-implant surface. Thus, the objective of this study was to demonstrate an effective means for applying known, uniform, cyclic strain to cells growing on implant materials in vitro. A cell culture strain plate device was developed based on the application of the four-point bending principle. The device uses a small electric motor to drive belts attached to shafts which turn a set of cams. The cams are attached to pins which connect to a titanium plate which rests over arched supports. When deflected and depending on which set of cams are used, strains generated range from around 200 to 1000 ìstrain. UMR-106 osteoblast-like cells were cultured on the titanium plate, and the plate was deflected at three strain magnitudes at 1.5 Hz for durations of 4 and 24 hours. Strain gages recorded average maximum strain levels of 182 ± 3, 366 ± 9, and 984 ± 7µstrain. The strain device, with attached cells, was tested in an amiable bioenvironment. Results from strain gages indicated a uniform strain field existed within the center region of the plate and culture area. Cells in the test plates stained viable, exhibited similar morphology to controls, and were assayed for alkaline phosphatase (ALP) activity, total protein production, and calcium deposition. Results also indicated that stretched cells exhibited increases in proliferation, as well as changes in ALP activity vs. unstrained controls. Thus, the device was successful in distinguishing differences in cell response to mechanical perturbations and may be used to investigate how cells respond to strains at implant-bone interfaces.

Bone and bone cells have been shown to respond to mechanical forces placed upon them. Particularly, strain plays an important role in osteogenic differentiation of marrow cells around artificial implants in bone. These strains, depending on their magnitude, duration, and repetition, can alter the proliferation and matrix synthesis of osteoblasts. To test how strain parameters influence osteoblast behavior, a four-point bending apparatus was used to impose cyclic strain on osteogenic stem cells isolated from rats and seeded on titanium plates. Cells were stimulated at 1 Hz for 15 minutes daily and compared to an unstrained control. Stimulation occurred at two magnitudes: 400 and 1000 micro-strain, and three levels of repetition: one, three, and five consecutive days. DNA, protein, alkaline phosphatase, and calcium levels were measured to determine the proliferation and matrix synthesis activity of the cells. No statistically significant effect was found for the tested parameters under these conditions.

No description available.

This study aimed at guiding osteoblast cells on biodegradable polymer carriers with well-defined surface microtopography and chemistry, and investigating the effect of cell alignment on osteoblast phenotype expression. A blend of two different polyesters, one being natural in origin (PHBV) and the other synthetic (P(L/DL)LA), was used to form a film with parallel macro- (250 &micro / m wide) or microgrooves (27 &micro / m wide) on its surface, by solvent casting on patterned templates. The micropatterned Si template was produced by photolithography, while the Teflo macropatterned template was lathe cut. Fibrinogen (Fb) was adsorbed or immobilized via epichlorohydrin spacer/crosslinker on the film surfaces to enhance cell attachment by increasing the surface hydrophilicity and by providing RGD amino acid sequence for integrin binding. Surface hydrophilicity was assessed by water contact angle measurements. Adsorption of Fb caused an increase in hydrophilicity, while the opposite was achieved with its covalent immobilization. Fb was homogeneously immobilized throughout the whole micropatterned film surface with amount of 153.1 &plusmn / 42.4 g Fb/cm2, determined with the Bradford assay, while it was adsorbed within the grooves of the micropattern. Surface characteristics of the films were studied with Scanning Electron (SEM) and Light microscopy. Osteoblast cells derived from rat bone marrow were seeded on the polymeric films with different surface topography and chemistry and were grown for one and three weeks. Osteoblast proliferation on the films was determined with Cell Titer 96 TM Non-Radioactive Cell Proliferation (MTS) test. Alkaline Phosphatase (ALP) assay and tetracycline labelling of mineralized matrix were carried out to determine osteoblast phenotype expression on different surfaces. SEM and fluorescence microscopy were used to evaluate the cell alignment. Osteoblasts on the micropatterned films with adsorbed Fb aligned along the groove axis with a mean deviation angle of 13.1o, while on the unpatterned films deviation from horizontal axis was 63.2o and cells were randomly distributed. Cell alignment did not affect cell proliferation. However, the highest ALP specific activity and the most homogeneous mineral distribution were obtained on the Fb adsorbed micropatterned films.

Periodontitis is a chronic inflammatory disease initiated by gram negative anaerobic bacteria. These bacteria possess pathogen-associated molecular patterns (PAMPs) that interact with various receptors including Toll like receptors (TLRs). Bacterial DNA (bDNA) is one of the PAMPs mainly recognized by TLR9. Interaction of bDNA and its receptors leads to activation of inflammatory signaling pathways potentially resulting in periodontal bone destruction. The aim of this study was to determine the production of IL- 6 and IL-8 in response to periodontal bDNA from human osteoblastic cells (MG-63). MG- 63 cells were stimulated in duplicate for 20 hours with 100ng/μl of bDNA from various pathogens including Porhyromonas gingivalis, Esherichia coli, Streptococcus sanguinis, Aggregatibacter actinomycetemcomitans as well as heat killed whole bacteria (1:100). E.coli LPS (10ng/μl) was used as a positive control in each experiment. To block TLR9 signaling, further experiments were carried out by treating MG-63 cells with chloroquine (10ng/μl) for 2 hours at 37ºC prior to stimulations. Cytokine levels were determined using enzyme linked-immunosorbent assay. Although IL-6 and IL-8 production was increased in response to periodontal bDNA in MG-63 cells, the results were not significant compared to unstimulated controls. As expected, E.coli DNA, E.coli LPS and heat killed whole bacteria stimulated significantly increased cytokine production (p<0.05). Blocking TLR9 with chloroquine did not affect the amount of cytokine production in bDNA stimulated cells suggesting that TLR9 may not be operant in triggering IL-6 and IL-8 production from MG- 63 cells. In conlusion, periodontal bDNA did not trigger significantly increased IL-6 and IL-8 production from MG-63 cells. Considering the involvement of several inflammatory mediators in periodontal bone destruction, further studies are warranted to assess the production of other cytokines in response to periodontal bDNA in human osteoblastic cells.

Family with sequence similarity 20 (FAM20) consists of three members: FAM20A, FAM20B and FAM20C. Mutations in FAM20 family have been linked to developmental disorders involving bones, cartilage and teeth. FAM20A mutations in humans are associated with amelogenesis imperfecta with gingival fibromatosis and enamel renal syndrome. Fam20a knockout (KO) mouse showed growth retardation. The aim of this study was to characterize the skeletal and dental phenotypes using Fam20a KO mouse. Our results showed that body size and bone length of KO mice were smaller than those of WT. The microcomputed tomography (μCT) analyses of trabecular and cortical bones in KO displayed lower bone volume, thinner trabeculae and thinner bone cortex as compared to WT. Histological examination of KO growth plate demonstrated disorganized chondrocyte zones and extended hypertrophic zone. qRT-PCR results showed downregulation of several osteoblast differentiation markers in KO long bone. Immunohistochemical examination demonstrated reduced chondrocyte proliferation, apoptosis and increased collagen X expression in KO growth plate. Our data showed a lower number of osteoblasts and osteoclasts in KO as compared to WT. In vitro study, Fam20a KO showed a lower number of bone marrow stromal cells and osteoprogenitors. In vitro mineralization was impaired in KO osteoblasts. Fam20a KO had hypoplastic enamel, delayed tooth eruption and gingival overgrowth. The µCT results demonstrated that enamel in Fam20a KO was not fully mineralized and enamel matrix was detached from dentin. Scanning electron microscopy displayed absence of decussation patterns in Fam20a KO enamel. Histological examination of maxillary first molar at differentiation stage showed no difference between WT and KO. At the secretory stage, Fam20a KO ameloblasts were short and non-polarized as compared to WT. Immunohistochemical analysis showed diffuse staining pattern of amelogenin in Fam20a KO first molar compared to WT. Western blot analysis demonstrated that amelogenin proteolytic process was impaired in KO and showed slower migration pattern of MMP20. In conclusion, endochondral ossification defects and reduced number of osteoblasts and their precursors led to the bone phenotype in Fam20a KO. Amelogenin processing defects caused amelogenesis imperfecta phenotype in KO. Our study indicated that Fam20a plays a role in skeletal development and amelogenesis.

No abstract available

Breast cancer most commonly metastasizes to the bone, where it perpetuates the vicious cycle leading to osteolytic lesions. This occurs when secreted factors from breast cancer cells disrupt bone homeostasis by deregulation of osteoblast bone formation, and enhance osteoclast bone degradation thereby releasing bone matrix bound growth factors leading to further tumor growth. Although the use of osteoclast targeting agents, such as bisphosphonates and RANK-L inhibitors, are common practice for the treatment of bone metastasis, they have not been shown to increase patient survival. We therefore sought to investigate the role of focal adhesion kinase (FAK), a potential therapeutic target, in the treatment of breast cancer mediated osteolysis. FAK is a non-receptor tyrosine kinase known to directly regulate tumor progression and metastasis; it is also expressed in all of the cell types involved in breast cancer mediated osteolysis. Thus, we hypothesized that the inhibition of FAK would restore normal bone homeostasis, as well as mediate direct anti-tumor activity. FAK depletion resulted in the decrease of expression of several osteolytic factors secreted by breast cancer cells. However, the use of FAK depleted breast cancer conditioned media did not prevent breast cancer mediated osteoclastogenesis in an osteoblast/osteoclast coculture. In monoculture however, using the FAK inhibitor PF-271, we have shown that FAK inhibition leads to increased apoptosis of mature osteoclasts, and their decreased ability to degrade mineralized bone matrix, perhaps in part due to reduced expression of lytic factors such as tartrate resistant acid phosphatase and cathepsin K. Further, FAK inhibition in osteoblast monoculture led to a decrease in their ability to express the maturation factor alkaline phosphatase, and also inhibited their ability to induce mineralization. This inhibition may be due in part to the specific effects of FAK inhibition using PF-271, which may result in decreased levels of p53 in treated osteoblasts. These results suggests that the pharmacological inhibition of FAK can effect all three cell types involved in the vicious cycle of bone metastasis, and as such could be a beneficial therapeutic for patients with bone metastasis resulting in prevention of bone degradation along with direct inhibition of tumor growth. However, it may require further evaluation in animal models to determine if observed effects on osteoblast activity in vitro also occurs in vivo with possible detrimental effects on restoration of damaged bone.

Des résultats d’expériences cliniques de reconstruction de l’os maxillaire faites à partir de la greffe d’une "pâte osseuse" gélifiée par l’ajout de facteur tissulaire ont été le primum movens de ce travail. Cette "pâte osseuse", faite d’os en poudre et de plasma enrichi en plaquette (PRP) à laquelle on ajoute du facteur tissulaire, est un modèle à la fois de la coagulation et de la régénération osseuse. Pour analyser des effets de la coagulation, nous avons utilisé un modèle connu : la culture primaire de cellules endothéliales (HUVEC). Les effets in vitro des facteurs de coagulation, dénommés protéases de la coagulation, pris séparément, ont été bien étudiés dans ces cellules, néanmoins aucune information sur l’effet combiné de ces protéases ou du plasma en coagulation n’était connue. Nous avons mesuré la "signalisation calcium" comme réponse cellulaire aux différents agents et ces mesures de la signalisation calcium ont été complétées par la mesure d’une autre réponse biologique, à savoir la sécrétion de cytokines pro-inflammatoires (IL-6 et IL-8). Pour l’étude de la régénération osseuse, la signalisation calcium a été mesurée sur une lignée d’ostéosarcomes humains (SaOS-2), stimulée par des protéases de la voie extrinsèque de la coagulation (facteur VIIa, facteur Xa et thrombine). Comme réponse biologique complémentaire, nous avons évalué l’effet des protéases d’intérêt sur l’apoptose induite par l’absence de sérum dans le milieu de culture. Les premiers travaux, réalisés sur les HUVEC, nous ont permis de montrer que le facteur Xa et la thrombine induisaient des signaux calcium différents sur ces cellules en mono couches, alors que le complexe facteur tissulaire – facteur VIIa ne provoquait aucune signalisation calcium. Nous avons également pu montrer une addition des signaux calcium induits par le facteur Xa et la thrombine. L’activation in situ du facteur Xa et de la thrombine à partir de leur zymogène a permis à la fois de confirmer les résultats précédents et de se rapprocher de l’in vivo. Finalement, au plus proche de l’in vivo, les expériences faites avec du plasma en coagulation ont également permis de détecter un signal calcium. La réponse biologique (sécrétion d’IL-6 et d’IL-8) en aval du signal calcium a confirmé les résultats calcium. En ce qui concerne la régénération osseuse étudiée à partir de SaOS-2, nous avons démontré l’expression du facteur tissulaire sur la lignée SaOS-2 et nous avons montré que le facteur VIIa, le facteur Xa et la thrombine induisaient tous des signaux calcium. Ces signaux présentaient des caractéristiques propres suivant la ou les protéase(s) utilisée(s) pour la stimulation. Les mesures ont également permis de caractériser, sur ces cellules, les récepteurs activés par les protéases d’intérêt, à savoir les "protease-activated receptors" 1 et 2 (PAR-1 et PAR-2). Comme réponse biologique, nous avons mesuré la diminution de l’apoptose induite par les protéases en absence de sérum dans le milieu de culture. Il a ainsi été montré que seule l’activation du récepteur PAR-1 permettait de diminuer l’apoptose. Finalement, nous avons caractérisé la voie suivie, qui passait par la phosphoinositide 3-kinase et la voie des MAPK Raf/MEK/ERK 1/2. En conclusion, cette thèse a permis de montrer, d’une part, que le facteur Xa et la thrombine provoquent des réponses calciques et proinflammatoires additifs dans les cellules endothéliales et, d’autre part, que le complexe facteur tissulaire – facteur VIIa, le facteur Xa et la thrombine induisent des signaux calcium caractéristiques dans les ostéosarcomes par l’activation des récepteurs PAR, l’activation de PAR-1 diminuant l’apoptose induite par l’absence de sérum dans le milieu de culture.