Altération de l’interaction entre la PTH et LRP6 dans les ostéoblastes humains arthrosiques via DKK2

L’ostéoarthrose (OA) se caractérise par une perte du cartilage articulaire, une sclérose osseuse, et une inflammation de la membrane synoviale. Des études in vivo et in vitro indiquent que les modifications du tissu osseux sont responsables de la perte du cartilage articulaire. Les ostéoblastes (Ob) OA présentent une réduction de la réponse à l’hormone parathyroïdienne (PTH), un signal anabolique important pour le tissu osseux, versus les Ob normaux. Le récepteur à la PTH (PTH-R) interagit avec le LRP6, le récepteur des ligands Wnts, et les antagonistes Dickkopf (DKK) bloquent cette interaction. Puisque le niveau de DKK2 est élevé en réponse au TGF-β1 dans les OA Ob, nous proposons que DKK2 altère l’interaction LRP6/PTH-R, le recyclage de PTH-R, et inhibe la réponse à la PTH. Nous avons utilisé des Ob OA et normaux humains en culture primaire. L’expression de PTH-R, LPR6 et DKK2 est mesurée par RT-PCR. Les niveaux protéiques de LRP6, PTH-R et DKK2 ont été déterminés par immunobuvardage. L’inhibition de DKK2 s’est effectuée par siRNA et l’AMP cyclique (AMPc) a été mesurée par ELISA. L’effet de TGF-β1 sur l’expression de DKK2 et PTH-R a été testé sur des cellules d’ostéosarcome SaOS-2. L’expression de PTH-R et LRP6 est similaire entre Ob normaux et OA, mais le niveau protéique de PTH-R est réduit dans les Ob OA. Par contre, l’expression et la production de DKK2 sont plus élevées dans les Ob OA. L’inhibition de DKK2 ne modifia pas l’expression de LRP6 et PTH-R dans les Ob OA mais a augmenté le niveau protéique de PTH-R détecté par immunobuvardage alors que celui de LRP-6 demeura inchangé. L’inhibition de DKK2 dans les Ob OA entraîna une augmentation de PTH-R dans la fraction membranaire et une diminution de la fraction intracellulaire. Les résultats de l'inhibition de la voie de clathrine par le triflupromazine ont montré une expression accrue du récepteur PTH dans la fraction membranaire qui peut être due à l'inhibition de sa dégradation par la voie de clathrine. L’induction de DKK2 dans les cellules SaOS-2 par TGF-β1 entraîna l’inhibition de PTH-R mais non celle de LRP6. L’inhibition de DKK2 dans les Ob OA a stimulé la production d’AMPc en réponse à la PTH par les Ob OA. En outre, le traitement de TGF-β1 dans les cellules SaOS-2 réduit la production d'AMPc en réponse à la PTH.Ces résultats démontrent que les niveaux élevés de DKK2, via l’inhibition de la signalisation Wnt/bcaténine, sont aussi responsables de l’altération de la réponse à la PTH observée dans les Ob OA. Le niveau élevé de DKK2 diminue spécifiquement l’affichage membranaire de PTH-R dans ces cellules et non son expression. Ces résultats suggèrent donc une altération du cross-talk entre LRP6 et PTH-R dans les Ob OA en lien avec leur niveau de DKK2.

Vers une meilleure compréhension de la pathogenèse de la maladie osseuse reliée à la fibrose kystique

Les patients atteints de la fibrose kystique (FK) ont désormais un âge médian de survie dépassant la cinquantaine. Par contre, avec ce vieillissement surviennent de nouvelles complications dont l'une des plus prévalente est la maladie osseuse associée à la FK. Les souris dont le Cftr est invalidé génétiquement présentent une densité osseuse amoindrie qui découle d’un débalancement du remodelage osseux caractérisé par une diminution de la formation et une augmentation de la résorption osseuse. L'observation que plusieurs modèles murins FK ont un phénotype ostéopénique et ce, même en absence de certains facteurs étiologiques (inflammation chronique, prise de glucocorticoïdes, insuffisance pancréatique etc.) laisse croire que le Cftr, le gène muté dans la FK, joue un rôle non-négligeable dans le métabolisme osseux. Le présent projet étudiera l’impact de l’absence du CFTR, sur les ostéoblastes (Ob) et ostéoclastes (Oc) dans un modèle murin de FK, soit les souris Cftr-/- de souche BALB/c. De plus, les Ob, sont reconnus comme ayant un effet modulateur sur le microenvironnement leucocytaire de la moelle osseuse (MO). Ce projet visera également à investiguer l’impact de l’absence du CFTR sur la niche leucocytaire de la MO. Nos résultats de densitométrie osseuse et de microtomographie à rayons X ont confirmé que les souris Cftr -/- ont une densité osseuse et un contenu minéral osseux abaissé, une diminution du volume osseux trabéculaire, un nombre amoindri de travées osseuses et une plus grande séparation entre les travées comparé aux souris Cftr+/+. Afin de mieux comprendre ce phénotype osseux, nous avons vérifié et confirmé que l’expression génique et protéique du CFTR est présente chez des Ob dérivés de la MO, mais est absent au niveau des Oc dérivés de la MO. Ces observations corroborent nos résultats portant sur la différenciation des cellules osseuses où nous avons démontré que seule la différenciation et fonction ostéoblastique sont affectées par l'absence du CFTR. Ce défaut ostéoblastique semble influencer négativement la leucopoïèse puisque nous observons une quantité moindre de cellules T, de macrophages et de cellules dendritiques chez les souris Cftr -/- vs. Cftr +/+. À la lumière de ces résultats, l'absence du CFTR semble avoir un impact important sur les ostéoblastes et la moelle osseuse.

Apatite-Polymer Composite Particles for Controlled Delivery of BMP-2: In Vitro Release and Cellular Response

Bone morphogenetic protein-2 (BMP-2) has the ability to induce osteoblast differentiation of undifferentiated cells, resulting in the healing of skeletal defects when delivered with a suitable carrier. We have applied a versatile delivery platform comprising a novel composite of two biomaterials with proven track records – apatite and poly(lactic-co-glycolic acid) (PLGA) – to the delivery of BMP-2. Sustained release of this growth factor was tuned with variables that affect polymer degradation and/or apatite dissolution, such as polymer molecular weight, polymer composition, apatite loading, and apatite particle size. The effect of released BMP-2 on C3H10T1/2 murine pluripotent mesenchymal cells was assessed by tracking the expression of osteoblastic makers, alkaline phosphatase (ALP) and osteocalcin. Release media collected over 100 days induced elevated ALP activity in C3H10T1/2 cells. The expression of osteocalcin was also upregulated significantly. These results demonstrated the potential of apatite-PLGA composite particles for releasing protein in bioactive form over extended periods of time.

Natural products as alternative treatments for metabolic bone disorders and for maintenance of bone health

Bone metabolism involves a complex balance between the deposition of matrix and mineralization and resorption. There is now good evidence that dietary components and herbal products can influence these processes, particularly by inhibiting bone resorption, thus having beneficial effects on the skeleton. For example, it has been reported that a number of common vegetables, including onion, garlic and parsley, can inhibit bone resorption in ovariectomized rats. Essential oils derived from sage, rosemary, thyme and other herbs inhibit osteoclast activity in vitro and in vitro and leading to an increase in bone mineral density. Soya, a rich source of isoflavones, has shown promising results and epidemiological evidence to support a use in maintaining bone health, and various traditional herbal formulae in Chinese and Ayurvedic medicine also have demonstrable effects in pharmacological models of osteoporosis. Recently, cannabinoids have been described as having positive effects on osteoblast differentiation, and the presence of cannabinoid receptors in bone tissue indicates a more complex role in bone metabolism than previously thought. The first part of this review briefly discusses normal bone metabolism and disorders caused by its disruption, with particular reference to osteoporosis and current pharmacological treatments. The effects of natural products on bone and connective tissue are then discussed, to include items of diet, herbal extracts and food supplements, with evidence for their efficacy outlined. Copyright (c) 2006 John Wiley & Sons, Ltd.

Effects of the Association between a Calcium Hydroxide Paste and 0.4% Chlorhexidine on the Development of the Osteogenic Phenotype In Vitro

The present study aimed to evaluate whether the association between a calcium hydroxide paste (Calen paste) and 0.4% chlorhexidine (CHX) affects the development of the osteogenic phenotype in vitro. With rat calvarial osteogenic cell cultures, the following parameters were assayed: cell morphology and viability, alkaline phosphatase activity, total protein content, bone sialoprotein immunolocalization, and mineralized nodule formation. Comparisons were carried out by using the nonparametric Kruskal-Wallis test (level of significance, 5%). The results showed that the association between Calen paste and 0.4% CHX did not affect the development of the osteogenic phenotype. No significant changes were observed in terms of cell shape, cell viability, alkaline phosphatase activity, and the total amount of bone-like nodule formation among control, Calen, or Calen + CHX groups. The strategy to combine Ca(OH)(2) and CHX to promote a desirable synergistic antibacterial effect during endodontic treatment in vivo might not significantly affect osteoblastic cell biology. (J Endod 2008;34:1485-1489)

Immunohistochemical, tomographic and histological study on onlay iliac grafts remodeling

The information concerning the molecular events taking place in onlay bone grafts are still incipient. The objective of the present study is to correlate the effects of perforation of resident bone bed on (1) the timing of onlay autogenous graft revascularization; (2) the maintenance of volume/density of the graft (assessed through tomography); and (3) the occurrence of bone remodeling proteins (using immunohistochemistry technique) delivered in the graft. Thirty-six New Zealand White rabbits were subjected to iliac crest onlay bone grafting on both sides of the mandible. The bone bed was drill-perforated on one side aiming at accelerating revascularization, whereas on the other side it was kept intact. After grafts fixation and flaps suture all animals were submitted to tomography on both mandible sites. Six animals were sacrificed, respectively, at 3, 5, 7, 10, 20 and 60 days after surgery. A second tomography was taken just before sacrifice. Histological slides were prepared from each grafted site for both immunohistochemistry analysis [osteopontin, osteocalcin, type I collagen and vascular endothelial growth factor (VEGF) anti-bodies] and histometric analysis. The values on bone volume measured on tomography showed no statistic significance (P >= 0.05) between perforated and intact sites. Grafts placed on perforated beds showed higher bone density values compared with non-perforated ones at 3 days (P <= 0.05). This correlation was inverted at 60 days postoperatively. The findings from VEGF labeling revealed a tendency for earlier revascularization in the perforated group. The early revascularization of bone grafts accelerated the bone remodeling process (osteocalcin, type I collagen and osteopontin) that led to an increased bone deposition at 10 days. The extended osteoblast differentiation process at intermediate stages in the perforated group cooperated for a denser bone at 60 days.

Deiodinase-mediated thyroid hormone inactivation minimizes thyroid hormone signaling in the early development of fetal skeleton

Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To Study this, pregnant once were made hypothyroid and fetuses harvested at embryonic days (E) 12.5, 14.5, 16.5 and 18.5. Despite a marked reduction in fetal tissue concentration of both T4 and T3, bone development, as assessed at the distal epiphyseal growth plate of the femur and vertebra, was largely preserved Lip to E16.5. Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrophic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocyres, in addition to a slight delay in endochondral and intramembranous ossification. This Suggests that LIP to E 16.5, thyroid hormone signaling in bone is kept to a minimum. In fact, measuring the expression level of the activating and inactivating iodothyronine deiodinases (D2 and D3) helped understand how this is achieved. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly (similar to 10-fold) at E18.5, and even more at 14 days after birth (P14). In contrast. D2 mRNA expression increased significantly by E18.5 and markedly (similar to 2.5-fold) by P14. The reciprocal expression levels of D2 and D3 genes during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time Suggest that coordinated reciprocal deiodinase expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development. (c) 2008 Elsevier Inc. All rights reserved.

Potential antitumoral properties of a new copper complex with santonic acid

A new copper(II) complex of santonic acid [Cu(2)(sant)(4)(H(2)O)(2)]center dot 21/2H(2)O has been prepared and characterized by electronic, vibrational, EPR spectral studies, and stability determinations in solution. The presence of two antiferrromagnetically coupled copper centers in the solid state was detected by EPR. The dinuclear Cu(II) complex crystallizes in the tetragonal P4(3)2(1)2 space group, with a = b = 14.498(3), c = 64.07(1) angstrom. Biological studies indicate that the complex displays interesting potential antitumoral actions. (C) 2008 Elsevier Ltd. All rights reserved.

Estudo das características físico-químicas e biológicas pela adesão de osteoblastos em superfícies de titânio modificadas pela nitretação em plasma

SILVA, J. S. P. Estudo das características físico-químicas e biológicas pela adesão de osteoblastos em superfícies de titânio modificadas pela nitretação em plasma. 2008. 119 f. Tese (Doutorado) - Faculdade de Medicina, Universidade de São Paulo. São Paulo, 2008.

Proteoliposomes Harboring Alkaline Phosphatase and Nucleotide Pyrophosphatase as Matrix Vesicle Biomimetics

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Kinetic Analysis of Substrate Utilization by Native and TNAP-, NPP1-, or PHOSPHO1-Deficient Matrix Vesicles

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of low-level laser therapy (685 nm) at different doses in osteogenic cell cultures

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

In vitro biocompatibility of a novel membrane of the composite poly(vinylidene-trifluoroethylene)/barium titanate

This study was aimed at investigating the in vitro biocompatibility of a novel membrane of the composite poly(vinylidene-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT). Osteoblastic cells were obtained from human alveolar bone fragments and cultured under standard osteogenic condition until subconfluence. First passaged cells were cultured on P(VDF-TrFE)/BT and expanded polytetrafluoroethylene (e-PTFE - control) membranes in 24-well plates. Cell adhesion and spreading were evaluated at 30 min, and 4 and 24 h. For proliferation assay, cells were cultured for 1, 7, and 10 days. Cell viability was detected by trypan blue at 7 and 10 days. Total protein content and alkaline phosphatase (ALP) activity were measured at 7, 14, and 21 days. Cultures were stained with Alizarin red at 21 days, for detection of mineralized matrix. Data were compared by ANOVA and Student t test. Cell attachment (p = 0.001), cell number (p = 0.001), and ALP activity (p = 0.0001) were greater on P(VDF-TrFE)/BT. Additionally, doubling time was greater on P(VDF-TrFE)/BT (p = 0.03), indicating a decreased proliferation rate. Bone-like nodule formation took place only on P(VDF-TrFE)/BT. The present results showed that both membranes are biocompatible. However, P(VDF-TrFE)/BT presented a better in vitro biocompatibility and allowed bone-like nodule formation. Therefore, P(VDF-TrFE)/BT could be an alternative membrane to be used in guided tissue regeneration. (c) 2006 Wiley Periodicals, Inc.

Response of rat bone marrow cells to commercially pure titanium submitted to different surface treatments

Objectives. Alterations in the commercially pure titanium (cpTi) surface may be undertaken to improve its biological properties. The aim of this study is to investigate the biocompatibility of cpTi submitted to different surface treatments.Methods. The cpTi surfaces were prepared so that machined and blasted surfaces, either acid etched or not, were compared using rat bone marrow cells cultured to differentiated into osteoblast. For attachment evaluation, cells were cultured for 4 and 24 h. Cell morphology was evaluated after 3 days. After 7, 14, and 21 days cell proliferation was evaluated. Total protein content and alkaline phosphatase (ALP) activity were evaluated after 14 and 21 days. For bone-like nodule formation, cells were cultured for 21 days. Data were compared by analysis of variance.Results. Cell attachment, cell morphology, cell proliferation, and ALP activity were not affected by surface treatments. Total. protein content was reduced by blasted and acid etched surface. Bone-Like nodule formation was significantly reduced by blasted, acid etched, and a combination of both blasted and acid etched surfaces.Conclusions. Based on these results, it can be suggested that cpTi surfaces that were submitted only to machining treatment favor the final event of osteoblastic differentiation of the rat bone marrow cells, evidenced by increased bone-Like nodule formation. (C) 2003 Elsevier B.V. Ltd. All rights reserved.

Osteoblasts engulf apoptotic bodies during alveolar bone formation in the rat maxilla

During bone formation, as in other tissues and organs, intense cellular proliferation and differentiation are usually observed. It has been described that programmed cell death, i.e., apoptosis, takes place in the control of the cellular population by removing of the excessive and damaged cells. Although it is generally accepted that apoptotic bodies are engulfed by professional phagocytes, the neighboring cells can also take part in the removal of apoptotic bodies. In the present study, regions of initial alveolar bone formation of rat molars were examined with the aim to verify whether osteoblasts are capable of engulfing apoptotic bodies, such as professional phagocytes. Rats aged 11-19 days were sacrificed and the maxillary fragments containing the first molar were removed and immersed in the fixative solution. The specimens fixed in glutaraldehyde-formaldehyde were processed for light microscopy and transmission electron microscopy. For the detection of apoptosis, the specimens were fixed in formaldehyde, embedded in paraffin, and submitted to the TUNEL method. The results revealed round/ovoid structures containing dense bodies on the bone surface in close contact to osteoblasts and in conspicuous osteoblast vacuoles. These round/ovoid structures showed also positivity to the TUNEL method, indicating that bone cells on the bone surface are undergoing apoptosis. Ultrathin sections showed images of apoptotic bodies being engulfed by osteoblasts. Occasionally, the osteoblasts exhibited large vacuoles containing blocks of condensed chromatin and remnants of organelles. Thus, these images suggest that osteoblasts are able to engulf and degrade apoptotic bodies. (c) 2005 Wiley-Liss, Inc.