Orphan nuclear receptor subfamily NR4A their interplay with other nuclear receptors and functions in osteoblasts

Nurr1, NGFI-B and Nor1 (NR4A2, NR4A1 and NR4A3, respectively) belong to the NR4A subfamily of nuclear receptors. The NR4A receptors are orphan nuclear receptors which means that activating or repressing ligands for these receptors have not been found. NR4A expression is rapidly induced in response to various stimuli including growth factors and the parathyroid hormone (PTH). The studies concerning the NR4A receptors in the central nervous system have demonstrated that they have a major role in the development and function of the dopaminergic neurons of the midbrain and in regulating hypothalamus-pituitary-adrenal-axis. However, the peripheral functions of the NR4A family are largely unknown. Cultured mouse primary osteoblasts, a preosteoblastic cell line and several osteoblastic cell lines were used to investigate the role of NR4A receptors in osteoblasts. NR4A receptors were shown to directly bind to and activate the promoter of the osteopontin gene (OPN) in osteoblastic cells, thus regulating its expression. OPN is a major bone matrix protein expressed throughout the differentiation of preosteoblastic cells into osteoblasts. The activation of the OPN promoter was shown to be dependent on the activation function-1 located in the N-terminal part of Nurr1 and to occur in both monomeric and RXR heterodimeric forms of NR4A receptors. Furthermore, PTH was shown to upregulate OPN expression through the NR4A family. It was also demonstrated that the fibroblast growth factor-8b (FGF-8b) induces the expression of NR4A receptors in osteoblasts as immediate early genes. This induction involved phosphatidylinositol-3 kinase, protein kinase C, and mitogen activated protein kinase, which are all major pathways of FGF signalling. Nurr1 and NGFI-B were shown to induce the proliferation of preosteoblastic cells and to reduce their apoptosis. FGF-8b was shown to stimulate the proliferation of osteoblastic cells through the NR4A receptors. These results suggest that NR4A receptors have a role both in the differentiation of osteoblasts and in the proliferation and apoptosis of preosteoblast. The NR4A receptors were found to bind to the same response element on OPN as the members of the NR3B family of orphan receptors do. Mutual repression was observed between the NR4A receptors and the NR3B receptors. This repression was shown to be dependent on the DNA-binding domains of both receptor families, but to result neither from the competition of DNA binding nor from the competition for coactivators. As the repression was dependent on the relative expression levels of the NR4As and NR3Bs, it seems likely that the ratio of the receptors mediates their activity on their response elements. Rapid induction of the NR4As in response to various stimuli and differential expression of the NR3Bs can effectively control the gene activation by the NR4A receptors. NR4A receptors can bind DNA as monomers, and Nurr1 and NGFI-B can form permissive heterodimers with the retinoid X receptor (RXR). Permissive heterodimers can be activated with RXR agonists, unlike non-permissive heterodimers, which are formed by RXR and retinoic acid receptor or thyroid hormone receptor (RAR and TR, respectively). Non-permissive heterodimers can only be activated by the agonists of the heterodimerizing partner. The mechanisms behind differential response to RXR agonists have remained unresolved. As there are no activating or repressing ligands for the NR4A receptors, it would be important to find out, how they are regulated. Permissiviness of Nurr1/RXR heterodimers was linked to the N-terminal part of Nurr1 ligand-binding domain. This region has previously been shown to mediate the interaction between NRs and corepressors. Non-permissive RAR and TR, permissive Nurr1 and NGFI-B, and RXR were overexpressed with corepressors silencing mediator for retinoic acid and thyroid hormone receptors (SMRT), and with nuclear receptor corepressor in several cell lines. Nurr1 and NGFI-B were found to be repressed by SMRT. The interaction of RXR heterodimers with corepressors was weak in permissive heterodimers and much stronger in non-permissive heterodimers. Non-permissive heterodimers also released corepressors only in response to the agonist of the heterodimeric partner of RXR. In the permissive Nurr1/RXR heterodimer, however, SMRT was released following the treatment with RXR agonists. Corepressor release in response to ligands was found to differentiate permissive heterodimers from non-permissive ones. Corepressors were thus connected to the regulation of NR4A functions. In summary, the studies presented here linked the NR4A family of orphan nuclear receptors to the regulation of osteoblasts. Nurr1 and NGFI-B were found to control the proliferation and apoptosis of preosteoblasts. The studies also demonstrated that cross-talk with the NR3B receptors controls the activity of these orphan receptors. The results clarified the mechanism of permissiviness of RXR-heterodimers. New information was obtained on the regulation and functions of NR4A receptors, for which the ligands are unknown.

Role of Eda and Troy pathways in ectodermal organ development

Several organs of the embryo develop as appendages of the ectoderm, the outermost layer of the embryo. These organs include hair follicles, teeth and mammary glands, which all develop as a result of reciprocal tissue interactions between the surface epithelium and the underlying mesenchyme. Several signalling molecules regulate ectodermal organogenesis the most important ones being Wnts, fi broblast growth factors (Fgfs), transforming growth factor -βs (Tgf-βs) including bone morphogenetic proteins (Bmps), hedgehogs (Hhs), and tumour necrosis factors (Tnfs). This study focuses on ectodysplasin (EDA), a signalling molecule of the TNF superfamily. The effects of EDA are mediated by its receptor EDAR, an intracellular adapter protein EDARADD, and downstream activation of the transcription factor nuclear factor kappa-B (NF-кB). Mice deficient in Eda (Tabby mice), its receptor Edar (downless mice) or Edaradd (crinkled mice) show identical phenotypes characterised by defective ectodermal organ development. These mouse mutants serve as models for the human syndrome named hypohidrotic ectodermal dysplasia (HED) that is caused by mutations either in Eda, Edar or Edaradd. The purpose of this study was to characterize the ectodermal organ phenotype of transgenic mice overexpressing of Eda (K14-Eda mice), to study the role of Eda in ectodermal organogenesis using both in vivo and in vitro approaches, and to analyze the potential redundancy between the Eda pathway and other Tnf pathways. The results suggest that Eda plays a role during several stages of ectodermal organ development from initiation to differentiation. Eda signalling was shown to regulate the initiation of skin appendage development by promoting appendageal cell fate at the expense of epidermal cell fate. These effects of Eda were shown to be mediated, at least in part, through the transcriptional regulation of genes that antagonized Bmp signalling and stimulated Shh signalling. It was also shown that Eda/Edar signalling functions redundantly with Troy, which encodes a related TNF receptor, during hair development. This work has revealed several novel aspects of the function of the Eda pathway in hair and tooth development, and also suggests a previously unrecognized role for Eda in mammary gland development.

Molecular basis for the development of diagnostic methods and specific treatment modalities for idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease with unknown aetiology and poor prognosis. IPF is characterized by alveolar epithelial damage that leads tissue remodelling and ultimately to the loss of normal lung architecture and function. Treatment has been focused on anti-inflammatory therapies, but due to their poor efficacy new therapeutic modalities are being sought. There is a need for early diagnosis and also for differential diagnostic markers for IPF and other interstitial lung diseases. The study utilized patient material obtained from bronchoalveolar lavage (BAL), diagnostic biopsies or lung transplantation. Human pulmonary fibroblast cell cultures were propagated and asbestos-induced pulmonary fibrosis in mice was used as an experimental animal model of IPF. The possible markers for IPF were scanned by immunohistochemistry, RT-PCR, ELISA and western blot. Matrix metalloproteinases (MMPs) are proteolytic enzymes that participate in tissue remodelling. Microarray studies have introduced potential markers that could serve as additional tools for the assessment of IPF and one of the most promising was MMP 7. MMP-7 protein levels were measured in the BAL fluid of patients with idiopathic interstitial lung diseases or idiopathic cough. MMP-7 was however similarly elevated in the BAL fluid of all these disorders and thus cannot be used as a differential diagnostic marker for IPF. Activation of transforming growth factor (TGF)-ß is considered to be a key element in the progression of IPF. Bone morphogenetic proteins (BMP) are negative regulators of intracellular TGF-ß signalling and BMP-4 signalling is in turn negatively regulated by gremlin. Gremlin was found to be highly upregulated in the IPF lungs and IPF fibroblasts. Gremlin was detected in the thickened IPF parenchyma and endothelium of small capillaries, whereas in non-specific interstitial pneumonia it localized predominantly in the alveolar epithelium. Parenchymal gremlin immunoreactivity might indicate IPF-type interstitial pneumonia. Gremlin mRNA levels were higher in patients with end-stage fibrosis suggesting that gremlin might be a marker for more advanced disease. Characterization of the fibroblastic foci in the IPF lungs showed that immunoreactivity to platelet-derived growth factor (PDGF) receptor-α and PDGF receptor-β was elevated in IPF parenchyma, but the fibroblastic foci showed only minor immunoreactivity to the PDGF receptors or the antioxidant peroxiredoxin II. Ki67 positive cells were also observed predominantly outside the fibroblastic foci, suggesting that the fibroblastic foci may not be composed of actively proliferating cells. When inhibition of profibrotic PDGF-signalling by imatinib mesylate was assessed, imatinib mesylate reduced asbestos-induced pulmonary fibrosis in mice as well as human pulmonary fibroblast migration in vitro but it had no effect on the lung inflammation.

Transforming Growth Factor-beta superfamily:evaluation as breast cancer biomarkers and preventive agents

The Transforming Growth Factor-beta (TGFbeta) superfamily of cytokines is comprised of a number of structurally-related, secreted polypeptides that regulate a multitude of cellular processes including proliferation, differentiation and neoplastic transformation. These growth regulatory molecules induce ligand-mediated hetero-oligomerization of distinct type II and type I serine/threonine kinase receptors that transmit signals predominantly through receptor-activated Smad proteins but also induce Smad-independent pathways. Ligands, receptors and intracellular mediators of signaling initiated by members of the TGFbeta family are expressed in the mammary gland and disruption of these pathways may contribute to the development and progression of human breast cancer. Since many facets of TGFbeta and breast cancer have been recently reviewed in several articles, except for discussion of recent developments on some aspects of TGFbeta, the major focus of this review will be on the role of activins, inhibins, BMPs, nodal and MIS-signaling in breast cancer with emphasis on their utility as potential diagnostic, prognostic and therapeutic targets.

Rôle des récepteurs Toll-like et de la protéine adaptatrice MyD88 dans la régulation de l’hepcidine et le développement des hyposidérémies associées à l’inflammation

Le fer est un oligo-élément nécessaire pour le fonctionnement normal de toutes les cellules de l'organisme et joue un rôle essentiel dans de nombreuses fonctions biologiques. Cependant, le niveau de fer dans le corps doit être bien réglé, sinon la carence en fer entraine des divers états pathologiques tels que l'anémie et la diminution de l’immunité. D'autre part, une surcharge en fer potentialise la multiplication des germes, aggrave l’infection et la formation de radicaux libres ayant des effets toxiques sur les cellules et leurs composants, ce qui favorise les maladies cardio-vasculaires, l'inflammation et le cancer. L'hepcidine (HAMP), un régulateur négatif de l'absorption du fer, induit la dégradation de la ferroportine (FPN), le seul exportateur connu de fer ce qui réduit sa libération par les macrophages et inhibe son absorption gastro-intestinale. HAMP est synthétisé principalement par les hépatocytes, mais aussi par les macrophages. Cependant, il y a très peu de données sur la façon dont HAMP est régulé au niveau des macrophages. Plus récemment, nous avons constaté que l’induction de l’hepcidin dans le foie par le polysaccharide (LPS) est dépendante de la voie de signalisation médiée par « Toll-like receptor 4 » (TLR4). Grâce au TLR4, le LPS induit l'activation des macrophages qui sécrètent de nombreuses différentes cytokines inflammatoires, y compris Interleukine 6 (IL-6), responsable de l'expression de HAMP hépatique. Dans le premier chapitre de la présente étude, nous avons étudié la régulation de HAMP dans la lignée cellulaire macrophagique RAW264.7 et dans les macrophages péritonéaux murins stimulés par différents ligands des TLRs. Nous avons constaté que TLR2 et TLR4 par l'intermédiaire de la protéine adaptatrice « myeloid differentiation primary response gene 88 » (MyD88) activent l'expression de HAMP dans les cellules RAW264.7 et les macrophages péritonéaux sauvages murins, tandis que cette expression a été supprimée dans les macrophages isolés des souris TLR2-/-, TLR4-déficiente ou MyD88-/-. En outre, nous avons constaté que la production d'IL-6 par les cellules RAW264.7 stimulées avec du LPS a été renforcée par l’ajout des quantités élevées de fer dans le milieu de culture. Au cours de l’inflammation, le niveau de HAMP est fortement augmenté. Ainsi, lorsque l'inflammation persiste, l’expression de HAMP continue à être activée par des cytokines pro-inflammatoires conduisant à une hyposidérémie. Malgré que cette dernière soit considérée comme une défense de l'hôte pour priver les micro-organismes de fer, celle ci cause un développement d'anémies nommées anémies des maladies chroniques. Ainsi, dans le deuxième chapitre de la présente étude, nous avons étudié l'implication des TLRs et leurs protéines adaptatrices MyD88 et TIR-domain-containing adapter-inducing interferon-β (TRIF) dans le développement des hyposidérémies. En utilisant des souris déficientes en MyD88 et TRIF, nous avons montré que les voies de signalisations MyD88 et TRIF sont essentielles pour l’induction de HAMP par le LPS. Malgré l'absence de HAMP, les souris déficientes ont été capables de développer une hyposidérémie, mais la réponse des souris déficientes en MyD88 a été très légère, ce qui indique l'exigence de cette protéine pour assurer une réponse maximale au LPS. En outre, nous avons constaté que la signalisation MyD88 est nécessaire pour le stockage du fer au niveau de la rate, ainsi que l'induction de lipocaline 2 (LCN2), qui est une protéine impliquée dans la fixation du fer pour limiter la croissance bactérienne. Indépendamment de MyD88 ou TRIF, l'activation de TLR4 et TLR3 a conduit, au niveau de la rate, à une diminution rapide de l’expression de FPN et du « Human hemochromatosis protein » (HFE) qui est une protéine qui limite la séquestration du fer cellulaire à partir de la circulation. Cependant, malgré cette baisse d’expression, le manque de la signalisation MyD88 a altéré de manière significative la réponse hyposidérémique. En établissant le rôle des TLRs et de la protéine adaptatrice MyD88 dans la diminution du taux du fer sérique au cours de la réponse inflammatoire, nous avons remarqué qu’en réponse au surcharge en fer les souris déficientes en MyD88 accumulent de manière significative plus de fer hépatique par rapport aux souris sauvages, et cela indépendamment des TLRs. Ainsi, dans le troisième chapitre de la présente étude, nous avons étudié le phénotype observé chez les souris déficientes en MyD88. Nous avons trouvé que l'expression de HAMP chez ces souris a été plus faible que celle des souris de type sauvage. Pour cela, nous avons exploré la signalisation à travers la voie du « Bone Morphogenetic Proteins 6 » (BMP6) qui est considérée comme étant la voie fondamentale de la régulation de HAMP en réponse aux concentrations du fer intracellulaires et extracellulaires et nous avons trouvé que l'expression protéique de Smad4, un régulateur positif de l'expression de HAMP, est significativement plus faible chez les souris MyD88-/- par rapport aux souris sauvages. En outre, on a montré que MyD88 interagit avec « mothers against decapentaplegic, Drosophila, homolog 4 » (Smad4) et que cette interaction est essentielle pour l’induction de HAMP à travers la voie BMP6. En conclusion, notre étude montre que l'expression de HAMP dans les macrophages est régulée principalement par TLR2 et TLR4 à travers la voie MyD88 et que l'accumulation du fer dans les macrophages peut affecter les niveaux des cytokines pro-inflammatoires. En outre, nos analyses démontrent que le développement d’hyposidérémie en réponse au LPS se produit par l'intermédiaire d’un mécanisme dépendant de MyD88 qui est dissociée de la production de cytokines et de HAMP. En plus, nos recherches montrent que MyD88 est nécessaire pour l'expression de Smad4 et cela pour garantir une réponse optimale à travers la signalisation BMP6, conduisant ainsi à une expression adéquate de HAMP. Enfin, la protéine MyD88 joue un rôle crucial dans, la régulation de HAMP au niveau des macrophages, la diminution du taux du fer sérique en réponse au LPS et le maintien de l'homéostasie du fer.

Influence of rhBMP-2 on bone formation and osseointegration in different implant systems after sinus-floor elevation. An in vivo study on sheep

Background Several studies have reported certain bone morphogenic proteins (BMPs) to have positive effects on bone generation Although some investigators have studied the effects of human recombinant BMP (rhBMP-2) in sinus augmentation in sheep, none of these studies looked at the placement of implants at the time of sinus augmentation Furthermore, no literature could be found to report on the impact that different implant systems, as well as the positioning of the implants had on bone formation if rhBMP-2 was utilized in sinus-lift procedures Purpose The aim of this study was to compare sinus augmentation with rhBMP-2 on a poly-D, L-lactic-co-glycolic acid gelatine (PLPG) sponge with sinus augmentation with autologous pelvic cancellous bone in the maxillary sinus during the placement of different dental Implants Materials and methods Nine adult female sheep were submitted to bilateral sinus-floor elevation In one side (test group) the sinus lift was performed with rhBMP-2 on a PLPG-sponge, while the contralateral side served as the control by using cancellous bone from the iliac crest Three different implants (Branemark (R), 31 (R) and Straumann (R)) were inserted either simultaneously with the sinus augmentation or as a two staged procedure 6 weeks later The animals were sacrificed at 6 and 12 weeks for histological and histomorphometrical evaluations during which bone-to-implant contact (BIC) and bone density (BD) were evaluated Results BD and BIC were significantly higher at 12 weeks in the test group if the Implants were placed at the time of the sinus lift (p < 0 05) No difference was observed between the different implant systems or positions Conclusions The use of rhBMP-2 with PLPG-sponge increased BIC as well as BD in the augmented sinuses if compared to autologous bone Different implant systems and positions of the implants had no effect on BIC or BD (C) 2010 European Association for Cranio-Maxillo-Facial Surgery

Effect of meloxicam and diclofenac sodium on peri-implant bone healing in rats

Background: This study evaluated the effects of diclofenac sodium and meloxicam on peri-implant bone healing. Methods: Thirty male rats were divided into three groups: the control group (CG) received no drug; the diclofenac sodium group (DSG) received 1.07 mg/kg twice a day for 5 days; and the meloxicam group (MG) received 0.2 mg/kg daily for 5 days. A screw-shaped titanium implant was placed in the tibia. Fluorochromes, oxytetracycline (OxT), calcein (CA), and alizarin (AL), were injected at 7, 14, and 21 days, respectively, after implantation, and the animals were sacrificed 28 days after implant placement. The percentages of OxT-, CA-, and AL-labeled bone as well as the percentages of bone-to-implant contact (BIC), cortical bone area (CBA), and trabecular bone area (TBA) within the implant threads were evaluated. Results: Bone healing was delayed in the DSG during the first 14 days after implant placement (OxT-labeled bone: DSG: 5.3% +/- 7.3% versus CG: 13.2% +/- 9.8%, P= 0.002, and versus MG: 14.4% +/- 13.1%, P = 0.05). The percentages of BIC (DSG: 49.6% +/- 21.9%; MG: 67.1% +/- 22.8%; and CG: 68.1% +/- 22.8%) and CBA (DSG: 63.7% +/- 21.2%; MG: 82.7% +/- 12.4%; CG: 84.9% +/- 10.6%) were lower in the DSG compared to the MG and CG (P<0.001). The percentage of TBA was significantly greater in the DSG compared to the MG and CG (DSG: 36.3% +/- 21.2% versus MG: 17.3% +/- 12.7% and versus CG: 15.1% +/- 10.6%; P<0.001). Conclusion: Diclofenac sodium seemed to delay peri-implant bone healing and to decrease BIC, whereas meloxicam had no negative effect on peri-implant bone healing.

Functional Analysis of saxophone, the Drosophila Gene Encoding the BMP Type I Receptor Ortholog of Human ALK1/ACVRL1 and ACVR1/ALK2

In metazoans, bone morphogenetic proteins (BMPS) direct a myriad of developmental and adult homeostatic evens through their heterotetrameric type I and type II receptor complexes. We examined 3 existing and 12 newly generated mutations in the Drosophila type I receptor gene, saxophone (sax), the ortholog of the human Activin Receptor-Like. Kinasel and -2 (ALK1/ACVR1 and ALK2/ACVR1) genes. Our genetic analyses identified two distinct classes of sax alleles. The first class consists of homozygous viable gain-of-function (GOF) alleles that exhibit (1) synthetic lethality in combination with mutations in BMP pathway components, and (2) significant maternal effect lethality that can be rescued by an increased dosage of the BMP encoding gene, dpp(+). In contrast, the second class consists of alleles that are recessive lethal and do not exhibit lethality in combination with mutations in other BMP pathway components. The alleles in this second class are clearly loss-of-function (LOF) with both complete and partial loss-of-function mutations represented. We find that one allele in the second class of recessive lethals exhibits dominant-negative behavior, albeit distinct from the GOF activity of the first class of viable alleles. On the basis of the fact that the first class of viable alleles can be reverted to lethality and on our ability to independently generate recessive lethal sat mutations, our analysis demonstrates that sax is an essential gene. Consistent with this conclusion, we find that a normal sax transcript is produced by sax(P), a viable allele previously reported to be mill, and that this allele can be reverted to lethality. Interestingly, we determine that two mutations in the first: class of sax alleles show the same amino acid substitutions as mutations in the human receptors ALK1/ACVR1-1 and ACVR1/ALK2, responsible for cases of hereditary hemorrhagic telangiectasia type 2 (HHT2) and fibrodysplasia ossificans progressiva (FOP), respectively. Finally, the data presented here identify different functional requirements for the Sax receptor, support the proposal that Sax participates in a heteromeric receptor complex, and provide a mechanistic framework for future investigations into disease states that arise from defects in BMP/TGF-beta signaling.

Immunohistochemical detection of estrogen receptor beta in alveolar bone cells of estradiol-treated female rats: possible direct action of estrogen on osteoclast life span

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influência da técnica cirúrgica guiada para implantes sobre o aquecimento ósseo, a viabilidade celular imediata e a deformação de fresas após osteotomia para implantes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biological evaluation of the bone healing process after application of two potentially osteogenic proteins: an animal experimental model

doi: 10.1111/j.1741-2358.2011.00526.x Biological evaluation of the bone healing process after application of two potentially osteogenic proteins: an animal experimental model Objective: The aim of this work was to analyse qualitatively and quantitatively the newly formed bone after insertion of rhBMP-2 and protein extracted from Hevea brasiliensis (P-1), associated or not with a carrier in critical bone defects created in Wistar rat calvarial bone, using histological and histomorphometrical analyses. Materials and methods: Eighty-four male Wistar rats were used, divided into two groups, according to the period of time until the sacrifice (2 and 6 weeks). Each one of these groups was subdivided into six groups with seven animals each, according to the treatments: (1) 5 mu g of pure rhBMP-2, (2) 5 mu g of rhBMP-2/monoolein gel, (3) pure monoolein gel, (4) 5 mu g of pure P-1, (5) 5 mu g of P-1/monoolein gel and (6) critical bone defect controls. The animals were euthanised and the calvarial bone tissue removed for histological and histomorphometrical analyses. Result and conclusion: The results showed an improvement in the bone healing process using the rhBMP-2 protein, associated or not with a material carrier in relation to the other groups, and this process demonstrated to be time dependent.

DEVELOPMENT OF AN EXPERIMENTAL MODEL OF INFECTED BONE VOID IN THE ULNA OF RABBITS

Objective: Develop a model that allowed the study of bone regeneration in infection conditions. Method: A 15 mm defect was surgically created in the rabbit ulna and inoculated with 5x10(8) colony-forming units (CFU) of S. aureus. Surgical debridement was performed two weeks after and systemic gentamicin was administered for four weeks. Animals were followed up to 12 weeks to evaluate infection control and bone regeneration. Result: Bone regeneration was inferior to 25% of the defect in radiological and histological analysis. Conclusion: Infected bone defect of 15 mm in the rabbit ulna was unable to achieve full regeneration without further treatment. Level of Evidence V, Experimental Study.

Influence of low-level laser associated with osteogenic proteins recombinant human BMP-2 and Hevea brasiliensis on bone repair in Wistar rats

This study analyzed the newly formed bone tissue after application of recombinant human BMP-2 (rhBMP-2) and P-1 (extracted from Hevea brasiliensis) proteins, 2 weeks after the creation of a critical bone defect in male Wistar rats treated or not with a low-intensity laser (GaAlAs 780 nm, 60 mW of power, and energy density dose of 30 J/cm2). The animals were divided into two major groups: (1) bone defect plus low-intensity laser treatment and (2) bone defect without laser irradiation. The following subgroups were also analyzed: (a) 5 mu g of pure rhBMP-2; (b) 5 mu g of pure P-1 fraction; (c) 5 mu g of rhBMP-2/monoolein gel; (d) 5 mu g of P-1 fraction/monoolein gel; (e) pure monoolein gel. Comparisons of the groups receiving laser treatment with those that did not receive laser irradiation show differences in the areas of new bone tissue. The group treated with 5 mu g of rhBMP-2 and laser irradiation was not significantly different (P >0.05) than the nonirradiated group that received the same treatment. The irradiated, rhBMP-2/monoolein gel treatment group showed a lower area of bone formation than the nonirradiated, rhBMP-2/gel monoolein treatment group (P < 0.001). The area of new bone tissue in the other nonirradiated and irradiated groups was not significantly different (P > 0.05). Furthermore, the group that received the 5 mu g of rhBMP-2 application showed the greatest bone formation. We conclude that the laser treatment did not interfere with the area of new bone tissue growth and that the greatest stimulus for bone formation involved application of the rhBMP-2 protein. Microsc. Res. Tech. 2011. (c) 2011 Wiley Periodicals, Inc.

L51P - A BMP2 variant with osteoinductive activity via inhibition of Noggin

Bone morphogenetic proteins (BMP) have to be applied at high concentrations to stimulate bone healing. The limited therapeutic efficacy may be due to the local presence of BMP antagonists such as Noggin. Thus, inhibiting BMP antagonists is an attractive therapeutic option. We hypothesized that the engineered BMP2 variant L51P stimulates osteoinduction by antagonizing Noggin-mediated inhibition of BMP2. Primary murine osteoblasts (OB) were treated with L51P, BMP2, and Noggin. OB proliferation and differentiation were quantified with XTT and alkaline phosphatase (ALP) assays. BMP receptor dependent intracellular signaling in OB was evaluated with Smad and p38 MAPK phosphorylation assays. BMP2, Noggin, BMP receptor Ia/Ib/II, osteocalcin, and ALP mRNA expressions were analyzed with real-time PCR. L51P stimulated OB differentiation by blocking Noggin mediated inhibition of BMP2. L51P did not induce OB differentiation directly and did not activate BMP receptor dependent intracellular signaling via the Smad pathway. Treatment of OB cultures with BMP2 but not with L51P resulted in an increased expression of ALP, BMP2, and Noggin mRNA. By inhibiting the BMP antagonist Noggin, L51P enhances BMP2 activity and stimulates osteoinduction without exhibiting direct osteoinductive function. Indirect osteoinduction with L51P seems to be advantageous to osteoinduction with BMP2 as BMP2 stimulates the expression of Noggin thereby self-limiting its own osteoinductive activity. Treatment with L51P is the first protein-based approach available to augment BMP2 induced bone regeneration through inhibition of BMP antagonists. The described strategy may help to decrease the amounts of exogenous BMPs currently required to stimulate bone healing.

Gene array of primary human osteoblasts exposed to enamel matrix derivative in combination with a natural bone mineral

OBJECTIVES The application of an enamel matrix derivative (EMD) for regenerative periodontal surgery has been shown to promote formation of new cementum, periodontal ligament, and alveolar bone. In intrabony defects with a complicated anatomy, the combination of EMD with various bone grafting materials has resulted in additional clinical improvements, but the initial cellular response of osteoblasts coming in contact with these particles have not yet been fully elucidated. The objective of the present study was to evaluate the in vitro effects of EMD combined with a natural bone mineral (NBM) on a wide variety of genes, cytokines, and transcription factors and extracellular matrix proteins on primary human osteoblasts. MATERIAL AND METHODS Primary human osteoblasts were seeded on NBM particles pre-coated with versus without EMD and analyzed for gene differences using a human osteogenesis gene super-array (Applied Biosystems). Osteoblast-related genes include those transcribed during bone mineralization, ossification, bone metabolism, cell growth and differentiation, as well as gene products representing extracellular matrix molecules, transcription factors, and cell adhesion molecules. RESULTS EMD promoted gene expression of various osteoblast differentiation markers including a number of collagen types and isoforms, SMAD intracellular proteins, osteopontin, cadherin, alkaline phosphatase, and bone sialoprotein. EMD also upregulated a variety of growth factors including bone morphogenetic proteins, vascular endothelial growth factors, insulin-like growth factor, transforming growth factor, and their associated receptor proteins. CONCLUSION The results from the present study demonstrate that EMD is capable of activating a wide variety of genes, growth factors, and cytokines when pre-coated onto NBM particles. CLINICAL RELEVANCE The described in vitro effects of EMD on human primary osteoblasts provide further biologic support for the clinical application of a combination of EMD with NBM particles in periodontal and oral regenerative surgery.