Bone resorption involves osteoclast-mediated acidification via a vacuolar type H+-ATPase (V-ATPase) found in lysosomes and at the ruffled border membrane. V-ATPases are proton pumps that include the a3 subunit, one of four isoforms (a1-a4) in mammals. The a3 isoform is enriched in osteoclasts where it is essential for bone resorption. Over 50% of humans with osteopetrosis have mutations in the a3 subunit and a3 mutations in mouse also result in osteopetrosis. A mouse founder with an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This mouse bears a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit resulting in the replacement of a highly conserved amino acid, arginine 740, with serine (R740S). The heterozygous mice (+/R740S) exhibit high bone density but otherwise have a normal appearance, size and weight. Osteoblast parameters are unaffected whereas osteoclast number and marker expression are increased along with a decreased number of apoptotic osteoclasts. V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport along with wild type levels of ATP hydrolysis, indicating that the R740S mutation uncouples ATP hydrolysis from proton transport. The mutation however has no effect on ruffled border formation or polarization of +/R740S osteoclasts. Mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis than +/R740S mice and die by postnatal day 14. Similarly to the mouse models that lack the a3 subunit (oc/oc and Tcirg1-/-) R740S/R740S osteoclasts do not polarize and lack ruffled border membranes. However R740S/R740S osteoclasts exhibit unique phenotypic traits, including increased apoptosis and defective early stage autophagy. Intracellular and extracellular acidification is absent in R740S/R740S osteoclasts, providing evidence for a requirement for lysosomal acidification for cytoplasmic distribution of key osteoclast enzymes such as TRAP and other important osteoclast phenotypic traits. This work provides evidence that the a3 subunit of V-ATPases and the proton pumping function of a3-containing V-ATPases play a major role in osteoclast survival, maturation and function.

O processo de diferenciação e ativação de osteoclastos, essencial para a manutenção da homeostasia do tecido ósseo e também envolvido na patogênese de diversas patologias caracterizadas pela atividade osteolítica, depende de um sistema central de controle que envolve a ligação das moléculas RANK/RANKL. Além do sistema RANK/RANKL, moléculas co-estimulatórias de osteoclastos, tais como os complexos DAP-12, TREM-2 e SIRP1, e FcR, OSCAR e PIR-A, também apresentam um papel importante na geração e ativação de osteoclastos. Entretanto, a possível contribuição de tais moléculas para a progressão da doença periodontal (DP) permanece desconhecida, assim como o possível impacto de citocinas na modulação de sua expressão no microambiente periodontal. Nosso objetivo foi investigar, por RealTimePCR, o padrão de expressão de moléculas co-estimulatórias de osteoclastos (DAP-12, TREM-2 e SIRP1, e FcR, OSCAR e PIR-A) na periodontite crônica em humanos, além de avaliar a cinética de expressão destas moléculas e a sua modulação por citocinas (TNF-, IFN-, IL-17 e IL-10) ao longo do curso da DP em camundongos em camundongos C57Bl/6 wild-type (WT) e geneticamente modificados (TNFp55KO, IFNKO, IL17KO, IL10KO. Nossos resultados demonstram que nas lesões periodontais crônicas a expressão de todas as moléculas co-estimulatórias de osteoclastos apresentaram-se significativamente aumentadas quando comparadas às amostras controle. Com relação à periodontite experimental, verificamos que todas as moléculas co-estimulatórias alvo apresentavam aumento em sua expressão após a indução de doença quando comparado aos controles. Nos camundongos para TNFp55KO, IFNKO e IL17KO, observamos uma redução na severidade da DP (reabsorção óssea e quantidade de células inflamatórias) e na expressão de moléculas co-estimulatórias, ao contrário do observado nos camundongos IL10KO. Entretanto, ao normalizarmos os níveis de expressão das moléculas co-estimulatórias de osteoclastos pelo número de células inflamatórias, verificamos que TNF- e IL-17 se mostram associados a uma maior expressão de moléculas co-estimulatórias, enquanto IFN- e IL-10 parecem regular negativamente a expressão de tais moléculas. Em termos gerais, demonstramos que a expressão de moléculas co-estimulatórias de osteoclastos se mostra aumentada na DP humana e experimental, e que citocinas parecem modular sua expressão por mecanismos diretos e indiretos, tais como a migração de células inflamatórias para os sítios de doença periodontal. / The osteoclast differentiation and activation are essential to bone tissue homeostasis and in the development of bone pathologies, which RANK/RANKL signaling molecules are the major osteoclastogenic factor. However, osteoclast co-stimulatory molecules, such as DAP-12, TREM-2, SIRP1, FcR, OSCAR and PIR-A, also present an important role in the osteoclastogenesis. However, the exact role and regulation of these molecules in human and mice periodontal diseases (PD) development have not completely known. Our aim was to investigate the pattern of osteoclast co-stimulatory expression (DAP-12, TREM-2, SIRP1, FcR, OSCAR and PIR-A) in human chronic periodontitis (CP), apart from analyze the kinetic of these molecules and their regulation by cytokines (TNF-, IFN-, IL-17 and IL-10) in the development of experimental periodontal disease in mice C57Bl/6 and knockout. Our results demonstrated that all osteoclast co-stimulatory molecules presented highly expressed in CP patients when compared with control. Similar results are presented about experimental PD, where all co-stimulatory molecules was presented highly expressed in infected mice when compared with control mice. We observed in TNFp55KO, IFNKO and IL17KO mice a decrease in PD scores and co-stimulatory molecules expression, the opposite of IL10KO mice. However, when we standardized the co-stimulatory molecules levels by the number of inflammatory cells, we found that TNF- and IL-17 are associated with increased expression of co-stimulatory molecules, while IFN- and IL-10 appear to negatively regulate the expression of such molecules. In conclusion, we demonstrated that osteoclast co-stimulatory molecules shown increased in human and experimental PD, and cytokines appear to modulate their expression by direct and indirect mechanisms, such as inflammatory cells migration to the PD infected tissue.

A movimentação dentária ortodôntica ocorre através de dois processos, nos quais o osso alveolar é reabsorvido nas áreas de pressão, enquanto que novo osso é formado na área de tração. O processo de reabsorção óssea ocorre pela ação de células multinucleadas, os osteoclastos. Os bisfosfonatos constituem um grupo de fármacos com propriedade de inibir a reabsorção óssea, foi utilizado no presente estudo com a finalidade de interferir na remodelação óssea induzida ortodonticamente. Para isso, força contínua de 15 cN foi aplicada aos primeiros molares superiores de ratos machos Wistar de 2 1/2 meses, utilizando uma biomecânica com fios superelásticos. Os animais foram divididos aleatoriamente em 4 grupos: 1) O grupo controle constituído por dezoito ratos, os quais foram injetados solução salina por 7 dias antes da instalação da biomecânica passiva, que permaneceu por 3, 10 e 18 dias; 2) Dezoito animais foram tratados com ALN (dose 2,5 mg/Kg) por 7 dias antes da instalação da biomecânica passiva que permaneceu por 3, 10 e 18 dias; 3) Dezoito animais foram tratados com alendronato com a mesma dose citada acima por 7 dias antes da instalação da biomecânica ativa que permaneceu por 3, 10 e 18 dias; 4) Dezoito animais foram injetados com solução salina 7 dias antes da instalação da biomecânica ativa que permaneceu por 3, 10 e 18 dias. As maxilas foram fixadas com 4% de formaldeído + 0,1% de glutaraldeído, descalcificadas em EDTA a 4,13% e incluídas em parafina ou resina Spurr. Os cortes foram corados com HE para análise morfológica. Alguns cortes foram submetidos à imuno-histoquímica para detecção de RANKL e OPG. Foi utilizado o método TRAP, marcador de osteoclastos e microscopia eletrônica de transmissão para análise ultraestrutural. Alguns espécimes tiveram a cortical óssea vestibular do primeiro molar superior congelada em nitrogênio líquido para análise da expressão de RANKL por Western Blotting. O ALN inibiu a reabsorção óssea e radicular de todos os grupos tratados. As células clásticas apresentaram-se em estado latente. No grupo da movimentação ortodôntica o osso alveolar foi remodelado e com 18 dias a superfície radicular apresentou-se reabsorvida e o TRAP revelou clastos ativos, achados confirmados pela microscopia eletrônica de transmissão. A expressão de RANKL, molécula ativadora de células clásticas, nao foi inibida pela droga. A expressão de OPG foi aumentada nos animais tratados. Os resultados demonstram que o uso de alendronato sódico na movimentação ortodôntica não interfere no recrutamento dos osteoclasto, ele aparentemente inibe sua ativação, o que pode interferir no processo de remodelação óssea e talvez diminua a quantidade de movimentação dentária. / Orthodontic tooth movement occurs through two processes in which the alveolar bone is resorbed in the pressure areas, whereas new bone is formed in the tension area. The bone resorption occurs by multinucleated cell, the osteoclasts. The bisphosphonates are drugs with capability to inhibit clastic activity were used in the present study in order to interfere with the bone remodeling induced orthodontic. For this continuous force of 15 cN was applied to the first molars of Wistar male rats of 2 1/2 months, using a biomechanical with superelastic wire. The animals were randomly divided into 4 groups: 1) The control group consisted of eighteen mice, which received sterile saline solution saline for 7 days prior to installation of passive biomechanics, which remained for 3, 10 and 18 days; 2) Eighteen animals were treated with ALN (dose 2.5 mg / kg) for 7 days prior to installation of the passive biomechanical to remain for 3, 10 and 18 days; 3) Eighteen animals were treated with alendronate with the same dose quoted above for 7 days prior to the biomechanical installation that remains active for 3, 10 and 18 days; 4) Eighteen animals were injected with sterile saline solution 7 days prior to the biomechanical installation that remains active for 3, 10 and 18 days. The maxillae were fixed with 4% formaldehyde + 0.1% glutaraldehyde, decalcified in EDTA 4.13% and embedded in paraffin or Spurr resin. The specimens were morphologically analyzed in HE stained sections. Some stained sections were used for immunolabeling for RANKL and OPG. The osteoclasts were marked by tartrate-resistant acid phosphatase (TRAP) histochemistry. The ultrathin sections were examined in a trasnmission electron micrsocpe. Some specimens were frozen in liquid nitrogen for protein extraction and Western Blotting protein expression analyzes. The ALN inhibited bone resorption and root of all the treated groups. The clastic cells present in a latent state. In the orthodontic movement group alveolar bone was remodeled with 18 days to root surface presented itself reabsorbed and the TRAP revealed clasts assets, findings confirmed by transmission electron microscopy. Expression of RANKL activating molecule clastic cells was not inhibited by the drug. The OPG expression was increased in treated animals. The results demonstrate that the use of alendronate in the orthodontic movement does not interfere with osteoclast recruitment, it apparently inhibits their activation, which can interfere in the bone remodeling process and may reduce the amount of tooth movement.

Os bisfosfonatos são drogas capazes de inibir a reabsorção óssea por meio de seu efeito direto sobre as células ósseas, interferindo na dinâmica dos tecidos mineralizados. O alendronato (ALN), um tipo de bisfosfonato nitrogenado, foi utilizado com o objetivo de investigar os seus efeitos sobre os tecidos dentários e periodontais após luxação lateral de molares com as raízes em desenvolvimento. Ratos Wistar com 21 dias de idade tiveram os segundos molares superiores luxados lateralmente. Doses diárias de 2,5 mg / kg de ALN começaram no dia seguinte à luxação; os controles receberam solução salina estéril. As maxilas foram fixadas, descalcificadas e incluídas em parafina ou em resina Spurr 7, 14 e 21 dias pós-luxação. Os cortes foram corados com H & E, incubados por histoquímica TRAP e imuno marcados para osteopontina (OPN), bem como para análise ultraestrutural. Após 21 dias, o ápice dos molares luxados sem ALN estava aberto e desorganizado, coberto por uma camada irregular de cemento celular. Os molares luxados dos animais tratados com ALN apresentaram alguns locais de anquilose, bem como lacunas de reabsorção na superfície do cemento. Os osteoclastos TRAP positivos foram mais numerosos no grupo ALN, apesar de sua aparência latente e sua localização, afastados das trabéculas ósseas, em relação aos controles, achado que foi confirmado com a análise ultraestrutural. A imunomarcação de OPN revelou uma linha grossa imunopositiva na dentina, que deve ter surgido a partir do momento da luxação, enquanto que as amostras tratadas com ALN não apresentaram alterações na dentina. Os resultados indicam que o alendronato inibe algumas alterações na dentina e na formação do cemento, induzidas pelo trauma dental de luxação. / Bisphosphonates are drugs that inhibit bone resorption through its direct effect on bone cells, interfering with the dynamics of mineralized tissues. Alendronate (ALN), a nitrogenated bisphosphonate, was used in order to investigate their effects on dental and periodontal tissues after lateral dislocation of molars with developing roots. Twenty one days old Wistar rats had their second molars laterally l. Daily doses of 2.5 mg / kg ALN started the day following the dislocation, while controls received saline solution. The maxillae were fixed, decalcified and embedded in paraffin or in Spurr resin after 7, 14 and 21 days post-dislocation. The sections were stained with H & E, incubated for TRAP, immunolabeled for osteopontin (OPN), and ultrastructurally analyzed by transmission electron microscopy. After 21 days, the apex of the luxated molar without ALN was open and disorganized, covered by an irregular layer of cellular cementum. The luxated molar from ALN-treated animals showed some areas of ankylosis and resorption lacunae on the cementum surface. TRAP-positive osteoclasts were more numerous in the ALN group, despite their latent appearance compared to controls, a finding that was ultrastructurally confirmed. OPN immunostaining revealed a thick immunopositive line in dentin, which must be resultant from the moment of dislocation, while the samples treated with ALN showed no changes in dentin. The results indicate that alendronate inhibits some changes in dentin and cementum formation induced by dental trauma of lateral luxation.

L'autotaxine (ATX) est une protéine sécrétée par différents tissus y compris le foie, le tissu adipeux et l'os. L'ATX possède une activité lysophopholipase D responsable du clivage de la lysophosphatidyl-choline (LPC) en acide lysophosphatidique (LPA), facteur de croissance affectant la prolifération cellulaire, la différenciation et la migration. Il a été démontré que les effets biologiques du LPA pourraient être la conséquence de la production locale d'ATX dans un tissu ou une cellule donnée. Nous avons récemment démontré que le LPA contrôle l'ostéoclastogenèse et l'activité de résorption ostéoclastique. De plus, nous avons démontré que l'expression de Enpp2 (gène de l'ATX) augmente au cours de l'ostéoclastogenèse. Le but de notre étude était d'évaluer l'implication de l'ATX produite par les ostéoclastes au cours de l'ostéoclastogenèse et dans le contrôle de la masse osseuse, en particulier en conditions inflammatoires.La production et le rôle de l'ATX au cours de l'ostéoclastogenèse ont été analysés sur les ostéoclastes murins et humains. De plus, l'ATX a été spécifiquement ciblée dans les ostéoclastes avec l'utilisation des souris Enpp2fl/fl Ctsk-Cre et par le traitement pharmacologique des souris par un anti-ATX. La perte osseuse en conditions inflammatoires a été évaluée dans le modèle d'inflammation induite par le lipopolysaccharide (LPS) et dans les modèles de souris arthritiques par transfert de sérum K/BxN et avec la lignée transgénique surexprimant le TNF humain hTNF. La production de l'ATX par les ostéoclastes est cruciale pour l'activité de résorption osseuse ostéoclastique in vitro. Les souris Enpp2fl/fl Ctsk-Cre ne présentent pas de phénotype osseux en conditions physiologiques mais sont protégées de la perte osseuse systémique et des érosions osseuses observées en conditions arthritiques. De plus, l'ihibition pharmacologique de l'ATX protège les souris de la perte osseuse systémique et des érosions focales observées dans le modèle hTNF+/- .L'inhibition pharmacologique et génétique de l'ATX améliore la perte osseuse locale et systémique inflammatoire en inhibant la résorption osseuse. L'ATX semble être une cible thérapeutique prometteuse dans la prévention de la perte osseuse associée à l'inflammation / Autotaxin (ATX) is a secreted protein produced by various tissues including the liver, adipose tissue and bone. ATX exhibits a lysophospholipase D activity responsible for cleavage of lysophosphatidyl-choline (LPC) in lysophosphatidic acid (LPA) that in turn acts as a growth factor, affecting cell proliferation, differentiation, and migration. It has been shown that biological effects of LPA could be the direct consequence of local production of ATX in a given tissue or cell type. We showed that LPA controls osteoclastogenesis and osteoclast bone resorption activity. In addition, we observed that Enpp2 (ATX gene) was upregulated during osteoclastogenesis. The aim of the study was to evaluate if ATX produced by osteoclasts could play a role in osteoclastogenesis and bone mass control, especially in inflammatory conditions.The role of ATX on osteoclastogenesis and osteoclast activity were analysed by murine and human assays. ATX was targeted by conditional inactivation of Ennp2 (ATX gene) in osteoclasts (Enpp2fl/fl Ctsk-Cre) and by treatment with anti-ATX. Arthritic and erosive diseases were studied in arthritis models using human tumor necrosis factor transgenic (hTNF+/-) mice and K/BxN serum-treated mice. Systemic bone loss was analysed in the Lipopolysaccharide (LPS)-induced inflammation model. Joint inflammation and osteoclasts were assessed by histology and bone mass and bone erosion by micro-CT.ATX production by osteoclasts was observed and was revealed as a crucial factor controlling osteoclast activity. Inhibition of ATX led to reduced bone resorption in vitro. Then, we generated Enpp2fl/fl Ctsk-Cre+/- mice allowing ATX depletion specifically in mature osteoclasts. These mice showed no specific bone phenotype under physiological conditions but displayed significant protection against systemic bone loss and almost an absence of bone erosion formation after challenge with LPS and K/BxN serum transfer. Pharmacological inhibition of ATX significantly mitigated focal and systemic TNF-induced bone loss. Pharmacological and genetic inactivation of ATX ameliorates systemic and focal inflammatory bone loss by inhibiting bone resorption. ATX emerges as a new promising therapeutic target for the prevention of inflammatory associated bone loss

The use of nanoparticles for disease treatment is an increasingly popular area of research. The potential for multi-functionality allows nanoparticles to be used as transport and delivery vehicles for drugs and as diagnostic aides, among other applications, to address the unmet needs of many disease treatments. One such class of disease is osteoporosis including severe disorders, like Paget’s disease, Osteogenesis Imperfecta and Legg Calve Perthes disease. In this dissertation, we discuss a nanoparticle system consisting of gold nanoparticles surface functionalized with primary amine bisphosphonates, which is a classification of pharmaceuticals that is common in the treatment of osteoporosis. Functionalized nanoparticles allow for greater intracellular concentrations of pharmaceutical, while the properties of the gold nanoparticles provide the ability to track the pharmaceutical and enhance imaging. We have synthesized and characterized bisphosphonate functionalized gold nanoparticles of controlled size of approximately 15 nm, which are suitable for cellular uptake, and functionalized the surface using self-assembly with pamidronate and alendronate. In one major finding of this study, inductively coupled plasma mass spectrometry was used to estimate approximate surface density of the bisphosphonates on the gold nanoparticles. This resulted in concentrations of approximately 0.65 molecules per nm2 (approximately 154 Å2/molecule) for pamidronate functionalized on gold, and approximately 2.6 molecules per nm2 (approximately 39 Å2/molecule) for alendronate functionalized on gold. This allows for more accurate estimates of pharmaceutical concentrations, during in vitro and in vivo studies. Additionally, we investigated the effects of bisphosphonate functionalized gold nanoparticles on the viability and morphology of osteoclast and osteoblast cells in vitro. We found that attaching the bisphosphonates to the surface of the nanoparticles leads to increased apoptotic effects of the bisphosphonates on the osteoclast cells compared to free bisphosphonates. Further, we showed bisphosphonate functionalized gold nanoparticles may have an effect on nuclei morphology that may provide an additional means of modulating bone resorption rather than just through influencing viability. Further we showed that it may be possible to target concentrations that are safe for osteoblasts, which is critical in determining potential treatment concentrations. These viability results bring to light a number of potential considerations into the optimization of potential treatments, such as dosing concentrations. Finally, detailed results are given on effects of bisphosphonate functionalized gold nanoparticles on important behavior and activity of osteoclast and osteoblast cells in vitro. We showed that while using concentrations below the toxicity threshold, some of the normal activity of the cells could be maintained. RANKL and ALP expression in osteoblasts were maintained when removing viability as a variable. Additionally, bone nodule formation was also maintained for osteoblasts and co-cultured in vitro systems. Finally, we showed that the introduction of bone in the in vitro studies adds a new degree of consideration as to the interaction of the bisphosphonates with the hydroxyapatite surface. This strong interaction with bone is an important consideration in further developing potential treatments for osteoporotic disease. This dissertation provides insights into the use of bisphosphonate functionalized gold nanoparticles as a potential treatment and means of study for bone remodeling disorders.

Skeletal metastasis is a lethal component of many advanced cancers including prostate, the second most common cancer among men. Patients whose prostate cancer is localized and detected early benefit from multiple treatment options ranging from active surveillance to radiation and surgery, resulting in a 5-year survival rate of nearly 100%. Unfortunately, the prognosis and survival for patients with advanced metastatic disease is much worse due to the highly aggressive nature of the disease and a paucity of treatment options. Understanding the mechanisms and interactions that occur between metastatic cancer cells and the bone will enable the future treatment landscape for bone metastatic prostate cancer to expand, thereby improving patient outcomes. Our current knowledge of how metastatic prostate cancer cells interact with the bone is summarized in a model known as the “vicious cycle.” Numerous fundamental vicious cycle factors have been identified, including parathyroid hormone-related protein (PTHrP), while additional elements, such as matrix metalloproteinases (MMPs), are progressively being discovered and added to the model. PTHrP is a critical regulator of bone resorption and augments osteolysis in skeletal malignancies. In Chapter 2, we report that the mature PTHrP1-36 hormone is processed by MMPs to yield a stable product, PTHrP1-17. PTHrP1-17 retains the ability to signal through PTH1R to induce calcium flux and ERK phosphorylation but not cyclic AMP production or CREB phosphorylation. Notably, PTHrP1-17 promotes osteoblast migration and mineralization in vitro, and systemic administration of PTHrP1-17 augments ectopic bone formation in vivo. Further, in contrast to PTHrP1-36, PTHrP1-17 does not affect osteoclast formation/function in vitro or in vivo. Finally, immunoprecipitation-mass spectrometry analyses using PTHrP1-17-specific antibodies establish that PTHrP1-17 is indeed generated by cancer cells. Thus, MMP-directed processing of PTHrP disables the osteolytic functions of the mature hormone to promote osteogenesis, indicating important roles for this mechanism in bone remodeling in normal and disease contexts. MMPs have traditionally been associated with cancer progression based on their extracellular matrix degrading activities. However, it has become evident that their regulation of non-extracellular matrix substrates can exert both contributive and protective effects during tumorigenesis. Previous studies of matrix metalloproteinase-3 (MMP-3) have demonstrated tissue dependent pro- and anti-tumorigenic effects, but despite elevated expression, its roles have not been explored in bone metastatic prostate cancer. In Chapter 3, we show that tumor-derived MMP-3 contributes to prostate tumor growth in bone. In vitro, we observe that silencing MMP-3 reduces prostate cancer cell proliferation. Further, we found increased levels of IGFBP3, a known MMP-3 substrate, and decreased IGF-1R, ERK, and AKT phosphorylation in the MMP-3 silenced cells. Notably, we also observe reduced tumor growth and proliferation in in vivo intratibial models when tumor-derived MMP-3 expression is silenced. These data suggest that increased MMP-3 expression by prostate cancer cells contributes to their proliferation in bone by regulating the activity of the IGF/IGF-1R signaling axis. Taken together, our studies indicate that MMPs possess important functional roles in bone metastatic prostate cancer. We believe that elucidation of these mechanisms and their contributions to the vicious cycle of bone metastasis will offer novel opportunities to design effective therapeutic treatment options.

Na ossificação endocondral, osteoclastos (Oc) reabsorvem os remanescentes de cartilagem, e osteoblastos (Ob) depositam matriz óssea. Bisfosfonatos (Bps) inibem a ação dos Oc. Foi avaliado o efeito dos Bps alendronato (Aln) e etidronato (Etn) em joelhos de ratos jovens (in vivo) e na cultura primária de Oc (in vitro). O material in vivo foi analisado por MEV, MET e ML (morfologia e histoquímica para TRAP). RNA foi extraído para análise por RT-PRC e proteínas para análise por WB, que também foram extraídos após o tratamento da cultura com Bps. O tratamento com Etn revelou lâmina epifiseal desorganizada com extensa área de cartilagem; a MEV mostrou pouco osso trabecular com lacunas de reabsorção, que não foram observadas com Aln. O Aln revelou numerosos Oc TRAP-positivos latentes, confirmados por MET. In vivo os Bps diminuem a expressão dos genes analisados; In vitro o Aln diminui somente a expressão de Runx2, menos expresso com Etn, assim como Spp1. A expressão proteica variou entre os grupos. Aln é o mais potente em inibir os Oc enquanto o Etn atua sobre os Ob. / In endochondral ossification, clastic cells (Oc) resorb the calcified cartilage, while osteoblasts (Ob) form new bone. Bisphosphonates (Bps) inhibit the action of Oc. The effect of the Bps alendronate (Aln) and etidronate (Etn) on the knees of young rats (in vivo) and in primary cultures of Oc (in vitro) was evaluated. The specimens were analyzed by SEM, TEM, and LM or TRAP histochemistry. RNA was extracted to analysis by RT-PRC and protein to analysis by WB. RNA and protein were also extracted after the treatment of cultures with Bps. Rats treated with Etn exhibited a disorganized epiphyseal plate containing large area of cartilage; SEM showed few bone trabeculae with resorption lacunae, which were not observed in Aln specimens. Aln showed numerous latent Oc by TRAP histochemistry and TEM. In vivo, the Bps decreased the expression of all analyzed genes; in vitro, Aln decreased only the expression of Runx2 as well as SPP1, which expression was less with Etn. Protein expression varied among the groups. Aln is more potent for inhibiting the Oc, while Etn acts on Ob.

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