Avaliação da diferenciação osteoblástica de células-tronco mesenquimais de ratos tratados cronicamente com bifosfonatos

Apesar dos bifosfonatos (BPs), fármacos antirreabsortivos, atuarem principalmente nos osteoclastos, a ação desses medicamentos em osteoblastos tem sido demonstrada em experimentos in vitro. Porém, na maioria desses experimentos, há exposição das culturas de osteoblastos aos BPs. Na presente investigação, foram avaliados osteoblastos diferenciados a partir de células-tronco mesenquimais (CTMs) de ratos tratados in vivo com alendronato de sódio (ALE: 1mg/ml/kg/semana), ácido zoledrônico (ZOL: 0,3mg/ml/kg/semana) ou solução salina (VEH: 0,009mg/ml/kg/semana) durante 13 semanas. As CTMs da medula óssea dos fêmures direitos dos animais foram cultivadas em meio osteogênico, na densidade de 5.000 células/200μl/poço. Após 21 dias de cultura, osteoblastos foram avaliados quanto à viabilidade celular e à formação de matriz mineralizada. Foram observadas viabilidades celulares semelhantes nos grupos BPs (ALE e ZOL) e superiores ao controle (VEH). Quanto à formação da matriz mineralizada, houve maior mineralização no grupo ZOL em relação ao grupo ALE, sendo ambas inferiores ao observado no grupo VEH. Os resultados obtidos sugerem que a exposição in vivo das CTMs ao ALE e ao ZOL influenciou a atividade dos osteoblastos in vitro. Ambos os medicamentos utilizados são BPs nitrogenados; contudo, na dose empregada, o ALE afetou mais significativamente a formação de matriz mineralizada

Jaw and Long Bone Marrows Have a Different Osteoclastogenic Potential

Osteoclasts, the multinucleated bone-resorbing cells, arise through fusion of precursors from the myeloid lineage. However, not all osteoclasts are alike; osteoclasts at different bone sites appear to differ in numerous respects. We investigated whether bone marrow cells obtained from jaw and long bone differed in their osteoclastogenic potential. Bone marrow cells from murine mandible and tibiae were isolated and cultured for 4 and 6 days on plastic or 6 and 10 days on dentin. Osteoclastogenesis was assessed by counting the number of TRAP+ multinucleated cells. Bone marrow cell composition was analyzed by FACS. The expression of osteoclast- and osteoclastogenesis-related genes was studied by qPCR. TRAP activity and resorptive activity of osteoclasts were measured by absorbance and morphometric analyses, respectively. At day 4 more osteoclasts were formed in long bone cultures than in jaw cultures. At day 6 the difference in number was no longer observed. The jaw cultures, however, contained more large osteoclasts on plastic and on dentin. Long bone marrow contained more osteoclast precursors, in particular the myeloid blasts, and qPCR revealed that the RANKL:OPG ratio was higher in long bone cultures. TRAP expression was higher for the long bone cultures on dentin. Although jaw osteoclasts were larger than long bone osteoclasts, no differences were found between their resorptive activities. In conclusion, bone marrow cells from different skeletal locations (jaw and long bone) have different dynamics of osteoclastogenesis. We propose that this is primarily due to differences in the cellular composition of the bone site-specific marrow

Osteoclast biology: in vivo study of the estrogen action on osteoclasts and in vitro study of the osteoclastogenesis from precursors of distinct bone sites

Introdução: O osso é um tecido mineralizado que está sob a influência de diversos fatores sistêmicos, locais e ambientais. Entre os fatores sistêmicos, o estrógeno é um hormônio bem conhecido por exercer uma função inibitória sobre a reabsorção óssea. Devido ao fato de o osso alveolar de ratas jovens sofrer contínua e intensa remodelação para acomodar os dentes em formação e erupção, ele constitui um adequado modelo in vivo para estudar a possível ação do estrógeno sobre os osteoclastos. Objetivo: Na tentativa de investigar a possibilidade do estrógeno induzir a morte de osteoclastos, foi examinado o osso alveolar de ratas jovens tratadas com estrógeno. Métodos: Quinze ratas de 22 dias foram divididas em grupos: Estrógeno (GE), Sham (GS) e Controle (GC). Os animais do GE receberam, durante 7 dias, injeção intramuscular diária de 0,125mg/100g de estrógeno (Benzoginoestril-ap ® ) diluído em óleo de milho. Os animais do GS receberam o óleo utilizado como veículo de diluição. Após 8 dias, fragmentos contendo osso alveolar foram removidos e processados para microscopia de luz e microscopia eletrônica de transmissão. Os cortes foram corados em hematoxilina/eosina (HE), e o método do TRAP (fosfatase ácida resistente ao tartarato) foi utilizado como marcador de osteoclastos. Foi realizada a análise quantitativa do número de osteoclastos TRAP-positivos/mm de superfície óssea. Para detecção de apoptose, cortes foram submetidos ao método do TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling). Os métodos TUNEL/TRAP combinados foram também utilizados. Resultados: O número de osteoclastos TRAP-positivos/mm de superfície óssea foi significantemente reduzido no GE comparado ao GC e ao GS. No GE, foram observados osteoclastos TRAP-positivos exibindo núcleos TUNEL-positivos. Além disso, imagens ultraestruturais revelaram osteoclastos encolhidos exibindo núcleos com massas conspícuas e tortuosas de cromatina condensada, típicos de apoptose. Conclusões: Os resultados reforçam a idéia de que o estrógeno inibe a reabsorção óssea promovendo a redução do número de osteoclastos, indicando, portanto, que essa redução pode ser, pelo menos em parte, uma consequência da apoptose de osteoclastos.Introduction: Bone is a mineralized tissue that is under the influence of several systemic, local and environmental factors. Among systemic factors, estrogen is a hormone well known for its inhibitory function on bone resorption. As alveolar bone of young rats undergoes continuous and intense remodeling to accommodate the growing and erupting tooth, it is a suitable in vivo model for using to study the possible action of estrogen on bone. Purpose: In an attempt to investigate the possibility that estrogen may induce the death of osteoclasts, we examined the alveolar bone of estrogen-treated rats. Methods: Fifteen, 22-d-old female rats were divided into estrogen, sham and control groups. The estrogen group received estrogen and the sham group received corn oil used as the dilution vehicle. After 8 d, fragments containing alveolar bone were removed and processed for light microscopy and transmission electron microscopy. Sections were stained with hematoxylin and eosin and tartrate-resistant acid phosphatase (TRAP)–an osteoclast marker. Quantitative analysis of the number of TRAP-positive osteoclasts per mm of bone surface was carried out. For detecting apoptosis, sections were analyzed by the Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL) method; TUNEL/TRAP combined methods were also used. Results: The number of TRAP-positive osteoclasts per mm of bone surface was significantly reduced in the estrogen group compared with the sham and control groups. TRAP-positive osteoclasts exhibiting TUNEL-positive nuclei were observed only in the estrogen group. In addition, in the estrogen group the ultrastructural images revealed shrunken osteoclasts exhibiting nuclei with conspicuous and tortuous masses of condensed chromatin, typical of apoptosis. Conclusions: Our results reinforce the idea that estrogen inhibits bone resorption by promoting a reduction in the number of osteoclasts, thus indicating that this reduction may be, at least in part, a consequence of osteoclast apoptosisCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP: 04/09898-0Capes: BEX:1174/08-8TEDEBV UNIFESP: Teses e dissertaçõe

Jaw and long bone marrow derived osteoclasts differ in shape and their response to bone and dentin

Increasing evidence suggests the existence of osteoclast diversity. Here we investigated whether precursors obtained from marrow of the mandibula or long bone could give rise to phenotypically different osteoclasts. Formation of multinucleated cells was assessed after culturing mouse marrow cells of the two bone types with macrophage colony stimulating factor (M-CSF) and receptor activator of NF kappa B ligand (RANKL) for up to 10 days on plastic, bone or dentin. Two times more osteoclasts formed from long bone marrow cells on bone compared to dentin, whereas higher numbers of jaw osteoclasts formed on dentin. Resorption of dentin or bone was similar for osteaclasts formed from both types of precursors. in contrast to jaw marrow derived osteoclasts, long bone osteoclasts predominantly had a multi-compartmented shape, with at least two nuclei containing compartments per cell. Osteoclasts on bone contained two times more actin rings than osteoclasts on dentin, regardless of their precursor origin. However, the area per osteoclast covered by actin rings was similar (20%) for both substrates. This study suggests that marrow cells obtained from different bones give rise to different osteoclasts. the substrate on which the osteoclasts are generated plays a role in steering their formation rather than their resorption. (C) 2011 Elsevier Inc. All rights reserved.Univ Amsterdam, Acad Ctr Dent Amsterdam ACTA, VU Univ Amsterdam, Dept Periodontol,Res Inst MOVE, NL-1081 LA Amsterdam, NetherlandsUniv Amsterdam, Acad Ctr Dent Amsterdam ACTA, VU Univ Amsterdam, Dept Oral Cell Biol,Res Inst MOVE, NL-1081 LA Amsterdam, NetherlandsUniversidade Federal de São Paulo, Dept Morphol & Genet, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Morphol & Genet, São Paulo, BrazilWeb of Scienc

Increased apoptosis in osteoclasts and decreased RANKL immunoexpression in periodontium of cimetidine-treated rats

It has been demonstrated that histamine interferes with the recruitment, formation and activity of osteoclasts via H1- and H2-receptors. Cimetidine is a H2-receptor antagonist used for treatment of gastric ulcers that seems to prevent bone resorption. in this study, a possible cimetidine interference was investigated in the number of alveolar bone osteoclasts. the incidence of osteoclast apoptosis and immunoexpression of RANKL (receptor activator of nuclear factor ?B ligand) was also evaluated. Adult male rats were treated with 100 mg kg-1 of cimetidine for 50 days (CimG); the sham group (SG) received saline. Maxillary fragments containing the first molars and alveolar bone were fixed, decalcified and embedded in paraffin. the sections were stained by H&E or submitted to tartrate-resistant acid phosphatase (TRAP) method. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) method and immunohistochemical reactions for detecting caspase-3 and RANKL were performed. the number of TRAP-positive osteoclasts, the frequency of apoptotic osteoclasts and the numerical density of RANKL-positive cells were obtained. Osteoclast death by apoptosis was confirmed by transmission electron microscopy (TEM). in CimG, TRAP-positive osteoclasts with TUNEL-positive nuclei and caspase-3-immunolabeled osteoclasts were found. A significant reduction in the number of TRAP-positive osteoclasts and a high frequency of apoptotic osteoclasts were observed in CimG. Under TEM, detached osteoclasts from the bone surface showed typical features of apoptosis. Moreover, a significant reduction in the numerical density of RANKL-positive cells was observed in CimG. the significant reduction in the number of osteoclasts may be due to cimetidine-induced osteoclast apoptosis. However, RANKL immunoexpression reduction also suggests a possible interference of cimetidine treatment in the osteoclastogenesis.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fed Univ São Paulo UNIFESP, Dept Morphol & Genet, São Paulo, BrazilUniv Estadual Paulista, UNESP, Sch Dent, Dept Morphol,Lab Histol & Embryol, BR-14801903 Araraquara, SP, BrazilFed Univ São Paulo UNIFESP, Dept Morphol & Genet, São Paulo, BrazilFAPESP: FAPESP - 2007/59374-6FAPESP: 2010/03571-0FAPESP: 2010/10391-9Web of Scienc

Structural and functional changes in the alveolar bone osteoclasts of estrogen-treated rats

This study investigated structural and functional features of apoptotic alveolar bone osteoclasts in estrogen-treated rats. For this purpose, 15 female rats 22 days old were divided into three groups: Estrogen (EG), Sham (SG) and Control (CG). The rats of EG received daily intramuscular injection of estrogen for 7 days. The SG received only the oil vehicle. Maxillary fragments containing alveolar bone were removed and processed for light and transmission electron microscopy. Area (OcA) and number of nuclei (OcN) and bone resorption surface per TRAP-positive osteoclasts (BS/OC) were obtained. Vimentin, caspase-3 and MMP-9 immunoreactions, TUNEL/TRAP and MMP-9/TUNEL combined reactions were performed. In EG, the OcA, OcN and BS/Oc were reduced. Moreover, osteoclasts showed cytoplasm immunolabelled by caspase-3 and a different pattern of vimentin expression in comparison with CG and SG. MMP-9 expression was not affected by estrogen and the TUNEL-positive osteoclasts were MMP-9-immunolabelled. In EG, ultrastructural images showed that apoptotic osteoclasts did not exhibit ruffled borders or clear zones and were shedding mononucleated portions. TRAP-positive structures containing irregular and dense chromatin were partially surrounded by fibroblast-like cells. In conclusion, the reduction in the BS/Oc may be due to reduction in OcA and OcN; these effects seem to be related to vimentin disarrangement rather than to an interference of estrogen with osteoclast MMP-9 expression. Osteoclast apoptosis involves caspase-3 activity and vimentin degradation; these cells release portions containing one apoptotic nucleus and, subsequently, undergo fragmentation, giving rise to apoptotic bodies.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

SUBSTITUTOS ÓSSEOS ALÓGENOS E XENÓGENOS COMPARADOS AO ENXERTO AUTÓGENO: REAÇÕES BIOLÓGICAS

A instalação do implante para reabilitação de pacientes desdentados é dificultada pela insuficiência de volume ósseo, sendo necessária uma reconstrução óssea prévia. O osso autógeno continua sendo o biomaterial “padrão ouro”, pois apresenta-se eficaz no processo de regeneração óssea, contendo células viáveis, não transmitindo doenças infeciosas ou desencadeando reações imunológicas. Além disso, apresenta rápida incorporação e consolidação. Por outro lado, esse tipo de enxerto apresenta desvantagens como maior morbidade e disponibilidade limitada. Diante da constante busca por substitutos ósseos que possam apresentar propriedades semelhantes às do osso autógeno, mas que não necessitem de um segundo sítio cirúrgico, tem aumentado bastante o uso de biomateriais alógenos (provenientes de indivíduos da mesma espécie, porém, geneticamente diferentes) e xenógenos (proveniente de espécie diferente) em reabilitações implantossuportadas. Porém, por serem provindos de outro indivíduo ou de outra espécie, a possibilidade de induzirem uma reação imunológica pode ser questionada. Deste modo, esta revisão de literatura teve como propósito comparar os implantes alógeno e xenógeno ao enxerto autógeno, quanto às suas características biológicas. Foi também avaliado o risco dos substitutos alógeno e xenógeno desencadearem reação imunológica. Os dados encontrados na literatura confirmam que enxerto autógeno apresenta as propriedades biológicas mais favoráveis. Porém, quando bem indicados, os implantes alógeno e xenógeno podem evitar a morbidade de um segundo sítio cirúrgico doador de enxerto autógeno. Em relação às possíveis reações imunológicas, parece haver um protocolo bastante rígido de tratamento e preparo dos implantes alógenos e xenógenos. Por outro lado, embora a utilização dos mesmos tenha mostrado resultados clínicos satisfatórios, faltam informações sobre a composição final e a estrutura microscópica desses biomateriais

Rehabilitation of the Maxillary Arch After Bone Graft Using Immediate Loading With Implant-Supported Fixed Restoration

Moderate and controlled loading environments support or enhance osteogenesis, and, consequently, a high degree of bone-to-implant contact can be acquired. This is because when osteoprogenitor cells are exposed to limited physical deformation, their differentiation into osteoblasts is enhanced. Then, some range of microstrain is considered advantageous for bone ingrowth and osseointegration. The primary stability has been considered one of the main clinical means of controlling micromotion between the implant and the forming interfacial tissue, which helps to establish the proper mechanical environment for osteogenesis. Based on the biological aspects of immediate loading (IL), the objective of this study is to present a clinical case of maxillary arch rehabilitation using immediate loading with implant-supported fixed restoration after bone graft. Ten dental implants were placed in the maxilla 6 months after the autogenous bone graft, removed from the mandible (bilateral oblique line and chin), followed by the installation of an immediate-load fixed cross-arch implant-supported restoration because primary stability was reached for 8 implants. In addition, instructions about masticatory function and how it is related to interfacial micromotion were addressed and emphasized to the patient. The reasons for the IL were further avoidance of an interim healing phase, a potential reduction in the number of clinical interventions for the patient, and aesthetic reasons. After monitoring the rehabilitation for 8 years, the authors can conclude that maxillary IL can be performed followed by a well-established treatment planning based on computed tomography, providing immediate esthetics and function to the patient even when autogenous bone graft was previously performed in the maxilla

Aspectos biológico-celulares da osseointegração baseados nas superfícies de implantes

Osseointegration involves a cascade of biological events, which can be accelerated by modifying the micro and/or nanometric topography of dental implant surfaces. Considering that different treatment types modify the titanium surface giving it a more pronounced rough topography, and physicochemical changes that appear to positively influence the osseointegration process, a literature review was made on the main types of surface treatments and their influence on the biological and cellular aspects of osseointegration, with publications dating from 1969 until the present moment. Although the precise role of the implant surface on the osseointegration of dental implants is not completely clear, the specific effects of implant surface on bone regeneration, initial kinetics, and evolution of mechanical properties have shown to be quite promising. Thus, based on dental implant surface modifications, osseointegration can be defined as a process by which rigid asymptomatic fixation of an alloplastic material can be achieved and kept in close contact with bone tissue, being resistant to early and late functional loads. This process can be modulated by an appropriate treatment of the alloplastic material surface.A Osseointegração envolve uma cascata de eventos biológico-celulares, que podem ser acelerados por meio da modificação micro e/ou nanométrica da topografia da superfície dos implantes dentais. Considerando-se que diferentes tipos de tratamentos modificam a superfície do titânio, conferindo-lhe uma topografia mais rugosa, além de alterações físico-químicas que parecem influenciar positivamente a osseointegração, foi realizada uma revisão da literatura sobre os principais tipos de tratamentos de superfícies de implantes de titânio e sua influência na Osseointegração do ponto de vista biológico-celular. Para a realização deste estudo foi feito um levantamento bibliográfico de publicações que datam de 1969 até o presente. Apesar do papel preciso da topografia e da química de superfície sobre a osseointegração de implantes dentários não estar completamente elucidado, os efeitos específicos da superfície de implantes sobre regeneração óssea inicial, cinética e evolução das propriedades mecânicas têm se mostrado bastante promissores. Assim, baseado nas modificações das superfícies de implantes dentais, a Osseointegração pode ser definida como um processo pelo qual a fixação rígida e assintomática de um material aloplástico pode ser alcançada e mantida em íntimo contato com o tecido ósseo, apresentando resistência às cargas funcionais precoces e tardias, podendo este processo ser modulado pelo adequado tratamento da superfície do material aloplástico