Browsing by Subject "Osteoblasts"
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- PublicationOpen AccessActin cytoskeletal organization in human osteoblasts grown on different dental titanium implant surfaces(Murcia: F. Hernández, 2007) Salido, M.; Vilches, J.I.; Gutiérrez, J.L.; Vilches, J.The understanding of the cellular basis of osteoblastic cell-biomaterial interaction is crucial to the analysis of the mechanism of osseointegration. Cell adhesion is a complex process that is dependent on the cell types and on the surface microtopography and chemistry of the substrate. We have studied the role of microtopography in modulating cell adhesion, in vitro, using a human osteoblastic cell line for the assessment of actin cytoskeletal organization. Through application of CLSM combining reflection and fluorescence, 2D or 3D images of cytoskeleton were obtained. On smooth surfaces, Ti CP machined, predominantly planar bone cells with an axial ratio of 1.1 were randomly oriented, with stress fibers running in all directions, and thin filopodia. On TiCP Osseotite® surfaces the osteoblastic cells conformed to the irregular terrain of the sustrate with focal adhesion sites only established on the relative topographical peaks separated for a longer distance than in the machined surface, and defined wide lamellopodia and long filopodia, with enhanced expression of stress fibers, forming large clear focal contacts with the rough surface. The cytoskeletal organization of cells cultured on rough titanium supports an active role for the biomaterial surface in the events that govern osteoblastic cell adhesion. The results enforce the role of the rough sustrate surface in affecting osteoblastic cell adhesion and provide valuable information for the design of material surfaces that are required for the development of an appropriate osteogenic surface for osteoblastic anchorage, compared to machined surface, in dental implants.
- PublicationOpen AccessAn autoradiographic study of the uptake of tritiated proline by osteoblasts during hibernation(Murcia : F. Hernández, 1986) Steinberg, B.; Singh, I.J.; Mitchell, O.G.Twenty-four LSH and LVG strain golden hamsters, Mesocricetus auratus, were used. Experimental animals were maintained at 5 C and allowed to hibernate. Control animals were kept at 27 C. Six animals (3 experimental, 3 control) were injected subcutaneously with I pCi of 'H-proline/gm body wt. (Spec. act. 3 Ci/mM) after hibernation lasting 12 hours, I day, 3 days, or 7 days. Animals were killed I hour after injection and autoradiographs were prepared from 5 Frn thick ilecalcified sections of femurs. A greater number of endosteal cells were labeled than periosteal cells and also exhibited a greater magnitude of labeling throughout the study. Differences between endosteal and periosteal cells both in percentage of cells labeled and magnitude of labeling were maximum in control animals and progressively decreased with increasing periods of hibernation. A reduction in synthesis of matrix proteins during the early period of hibernation was seen and was attributed to a significant reduction both in average cell activity and in the number of active cells during hibernation. The latter phenomenon apparently made a large contribution to the reduced matrical synthesis. 'H-proline uptake by osteoblasts probably retlects the reduced requirements of matrical synthesis during hibernation.
- PublicationOpen AccessCellular and molecular basis of fibrous dysplasia(Murcia : F. Hernández, 2001) Marie, P.J.Recent advances have been made in the cellular and molecular mechanisms involved in monostotic and polyostotic fibrous dysplasia, a rare nonmalignant disease causing bone deformations and fractures. The molecular basis of fibrous dysplasia has been clarified when mutations affecting the stimulatory a subunit of G protein (Gs) have been found in dysplastic bone lesions. The histological analysis of dysplastic lesions revealed that the mutations in Gsa caused abnormalities in cells of the osteoblastic lineage and therefore in the bone matrix. Further in vitro analyses of bone cells from mutant dysplastic bone lesions revealed that the abnormal deposition of immature bone matrix in fibrous dysplasia results from decreased differentiation and increased proliferation of osteoblastic cells. Finally, the signaling pathway involved in these osteoblastic abnormalities has been identified. It is now apparent that the constitutive elevation in cAMP leve1 induced by the Gsa mutations leads to alterations in the expression of several target genes whose promoters contain cAMP-responsive elements, such as c-fos, c-jun, 11-6 and 11-11. This in turn affects the transcription and expression of downstream genes and results in the alterations of osteoblast recruitment and function in dysplastic bone lesions. These mechanisms provide a cellular and molecular basis for the alterations in bone cells and bone matrix in fibrous dysplasia.
- PublicationOpen AccessCytoprotection by pyruvate through an anti-oxidative mechanism in cultured rat calvarial osteoblasts(Murcia : F. Hernández, 2006) Moriguchi, N.; Hinoi, E.; Tsuchihashi, Y.; Fujimori, S.; Iemata, M.; Takarada, T.; Yoneda, Y.Although we have previously shown drastic cell death by pyruvate deficiency in osteoblasts at the proliferative stage, the exact mechanism remains unclear so far. Cell survivability was significantly decreased in rat calvarial osteoblasts cultured for 0 to 3 days in vitro (DIV) following replacement of the eutrophic a- modified minimum essential medium (a-MEM) with Dulbecco’s modified eagle medium (DMEM) for cultivation. The addition of pyruvate enriched in a- MEM, but not in MEM, entirely prevented cell death induced by the medium replacement throughout a culture period from 0 to 3 DIV. Both cysteine and reduced glutathione protected cell death in cells cultured for 3 DIV without significantly affecting that in cells cultured for 1 DIV, however, while none of lactate, acetate and insulin significantly prevented the cell death irrespective of the culture period up to 3 DIV. A marked increase was detected in intracellular reactive oxygen species (ROS) levels 4 h after the medium replacement. In osteoblasts cultured in a-MEM for 3 DIV, but not in those for 7 DIV, hydrogen peroxide (H2O2) markedly decreased cell survivability when exposed for 2 to 24 h. Furthermore, H2O2 was effective in significantly decreasing cell survivability in osteoblasts cultured in DMEM for 7 DIV. Pyruvate at 1 mM not only prevented cell death by H2O2, but also suppressed the generation of intracellular ROS in osteoblasts exposed to H2O2. These results suggest that pyruvate could be cytoprotective through a mechanism associated with the anti-oxidative property rather than an energy fuel in cultured rat calvarial osteoblasts.
- PublicationOpen AccessMitochondrial bioenergetics and distribution in living human osteoblasts grown on implant surfaces(Murcia : F. Hernández, 2009) Salido, Mercedes; Vilches Pérez J.Ignacio; González, Juan L.; Vilches, JoséOsseointegration of implants is crucial for the long-term success of oral implants. The periimplant bone formation by osteoblasts is strongly dependent on the local mechanical environment in the interface zone. Robust demands for energy are placed on osteoblasts during the adhesion process to solid surfaces, and mitochondria are capital organelles in the production of most of the ATP needed for the process. We have assessed the relationship between osteoblast differentiation and mitochondrial bioenergetics in living cells grown on two different titanium surfaces, in order to provide valuable information for the design of material surfaces required for the development of the most appropriate osteogenic surface for osteoblastic anchorage. Combined backscattered and fluorescence confocal microscopy showed that in flat cells grown on a machined surface, highly energized mitochondria were distributed along the cell body. In contrast, cells grown on the rough surface emitted long protrusions in search of surface roughness, with actin stress fibers clearly polarized and highly energized mitochondria clustered at focal adhesion sites. This report using normal human osteoblastic cells indicates that these cells are especially sensitive to surface cues through energy production that enhances the necessary adhesion required for a successful osseointegration.
- PublicationOpen AccessRegulation of human cranial osteoblast phenotype by FGF-2, FGFR-2 and BMP-2 signaling(Murcia : F. Hernández, 2002) Marie, P.J.; Debiais, F.; Haÿ, E.The formation of cranial bone requires the differentiation of osteoblasts from undifferentiated mesenchymal cells. The balance between osteoblast recruitment, proliferation, differentiation and apoptosis in sutures between cranial bones is essential for calvarial bone formation. The mechanisms that control human osteoblasts during normal calvarial bone formation and premature suture ossification (craniosynostosis) begin to be understood. Our studies of the human calvaria osteoblast phenotype and calvarial bone formation showed that premature fusion of the sutures in nonsyndromic and syndromic (Apert syndrome) craniosynostoses results from precocious osteoblast differentiation. We showed that Fibroblast Growth Factor-2 (FGF-2), FGF receptor-2 (FGFR-2) and Bone Morphogenetic Protein-2 (BMP-2), three essential factors involved in skeletal development, regulate the proliferation, differentiation and apoptosis in human calvaria osteoblasts. Mechanisms that induce the differentiated osteoblast phenotype have also been identified in human calvaria osteoblasts. We demonstrated the implication of molecules (N-cadherin, Il-1) and signaling pathways (src, PKC) by which these local factors modulate human calvaria osteoblast differentiation and apoptosis. The identification of these essential signaling molecules provides new insights into the pathways controlling the differentiated osteoblast phenotype, and leads to a more comprehensive view in the mechanisms that control normal and premature cranial ossification in humans.
- PublicationOpen AccessTreatment with tacrolimus enhances alveolar bone formation and decreases osteoclast number in the maxillae: A histomorphometric and ultrastructural study in rats(Murcia : F. Hernández, 2008) Andia, Denise Carleto; Nassar, Carlos Augusto; Oehlmeyer Nassar, Patricia; Rodrigues Guimarães, Morgana; Cerri, Paulo Sérgio; Spolidorio, Luis CarlosRecent studies have suggested that tacrolimus monotherapy is a beneficial therapeutic alternative for the normalization of cyclosporin-induced bone loss in animal models and humans. The mechanism accounting for this action is unclear at present. In the present study, we attempted to determine the effect of tacrolimus monotherapy on alveolar bone using histological, histomorphometrical and transmission electron microscopy (TEM). Groups of rats (n=10 each) were treated with either tacrolimus (1mg/kg/day, s.c.) or drug vehicle for 60 days. Fragments containing maxillary molars were processed for light microscopy to investigate the alveolar bone volume, trabecular separation, number of osteoclasts and osteoblasts, and transmission electron microscopy to investigate their ultrastructural basic phenotype. Treatment with tacrolimus monotherapy during 60 days may induce increases in alveolar bone volume (BV/TV,%; P<0.05) and a non-significant decrease in trabecular separation (Tb.Sp,mm; P>0.05), represented by a decrease in osteoclast number (N.Oc/BS; P<0.05) and maintenance of osteoblast number (N.Ob/BS; P>0.05). Osteoblasts were often observed as a continuous layer of active cells on the bone surface. Osteoclasts appeared to be detached from the resorbed bone surface, which was often filled by active osteoblasts and collagen-rich matrix. Moreover, osteoclasts in the treated group were frequently observed as inactive cells (without ruffled border, clear zone and detached from the bone surface). Within the limits of the present study, we conclude that tacrolimus leads to an increase in alveolar bone formation, which probably exerts action on osteoclasts. Tacrolimus could, therefore, play a crucial role in the control of both early osteoclast differentiations from precursors, as well as in functional activation.
- PublicationOpen AccessZoledronate promotes bone formation by blocking osteocyte-osteoblast communication during bone defect healing(Universidad de Murcia. Departamento de Biología Celular e Histología, 2018) Cui, Pingping; Liu, Hongrui; Sun, Jing; Amizuka, Norio; Sun, Qinfeng; Li, MinqiNitrogen-containing bisphosphonates (NBPs) are potent antiresorptive drugs and their actions on osteoclasts have been studied extensively. Recent studies have suggested that N-BPs also target bone-forming cells. However, the precise mechanism of N-BPs in osteoblasts is paradoxical, and the specific role of osteocytes is worthy of in-depth study. Here, we investigated the cellular mechanisms of N-BPs regulating bone defect healing by zoledronate (ZA). Bone histomorphometry confirmed an increase in new bone formation by systemic ZA administration. ZA induced more alkaline phosphatase-positive osteoblasts and tartrate-resistant acid phosphatase-positive osteoclasts residing on the bone surface. Inexplicably, ZA increased SOST expression in osteocytes embedded in the bone matrix, which was not compatible with the intense osteoblast activity on the bone surface. ZA induced heterogeneous osteocytes and disturbed the distribution of the osteocytic-canalicular system (OLCS). Furthermore, according to the degree of OLCS regularity, dentin matrix protein 1 reactivity had accumulated around osteocytes in the ZA group, but it was distributed evenly in the OLCS of the control group. The control group showed a dense array of the gap junction protein connexin 43. However, connexin 43 was extremely sparse after ZA administration. In summary, ZA treatment reduces gap junction connections and blocks cellular communication between osteocytes and osteoblasts. Retaining SOST expression in osteocytes leads to activation of the Wnt signaling pathway and subsequent bone formation.