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PURPOSE This study evaluated the bone-forming potential of the demineralized human dentin matrix by performing histologic and morphometric analyses. The immunolabeling of osteopontin, a determinant protein for bone repair, was also evaluated. MATERIALS AND METHODS Wistar rats were selected and submitted to the extraction of the right and left second molars. Tooth sockets were separated into two groups: the control group (right), which was filled with the blood clot, and the experimental group (left), which was filled with demineralized human dentin matrix. Animals were sacrificed at 5, 10, and 21 days. Histologic and histoquantitative analyses (analyses of variance [ANOVA] and Tukey's test) were performed, as well as immunostaining for osteopontin as an osteogenesis indicator. RESULTS After 5 days, demineralized human dentin matrix was incorporated by new trabeculae. After 10 days, connective tissue organization and new trabeculae were observed in the experimental group, and intense staining for osteopontin close to demineralized human dentin matrix was observed in the experimental group. After 21 days, the experimental group was showing mature trabeculae. A statistical difference was observed (P < .05). There was a higher number of trabeculae in the experimental groups in all periods of analysis. The presence of osteopontin was observed more intensely at 10 days close to demineralized human dentin matrix. CONCLUSION This study indicates that demineralized human dentin matrix implanted in tooth sockets induces the acceleration of osteogenesis.

Thrombocytopenia-absent radius (TAR) syndrome is a congenital malformation in which affected individuals present reductions in the number of platelets, hypoplasia, or absence of radial bone unilaterally or bilaterally. Hematologic, skeletal, cardiac (particularly tetralogy of Fallot and septal-atrial defects), and gastrointestinal anomalies are most commonly associated with TAR syndrome. Skeletal changes result in a higher risk of dental and craniofacial trauma in patients with the syndrome. Thus, it is important for the dentist to be aware of the characteristics of TAR syndrome and its clinical management for better care of these patients. The objective of this study is to describe a case report of a 26-year-old patient with TAR syndrome with a history of trauma and root fracture of tooth 11 and alveolar bone ridge. During anamnesis, root fractures requiring the extraction of the 11 tooth, alveolar bone ridge fracture in the adjacent region, and dental trauma were observed. A hematological evaluation and blood and radiological examinations were performed. Osseointegrated implant was performed using the guided surgery and flapless technique, as well as prosthetic rehabilitation in the affected region. This report discusses the importance of careful planning, such as the use of incisions and conservative surgery, techniques for alveolar ridge preservation, gingival manipulation, and prosthesis confection. The patient was attended by a hematologist throughout the treatment. Key words:TAR syndrome, absent radii and thrombocytopenia, dental implants, oral surgery.

This experimental study aimed to evaluate the effects of a three-dimensional matrix of chitosan-gelatin (CG) associated with 1% hyaluronic acid (HA) on gingival healing and repairing of intrabuccal bone defects in rats.Standardized bone defects were created in the region of the upper 1st molars of rats. Study groups were created according to bone defects (n=6/group) treatment: Control group (CO); blood clot; HA group; CG group, and HA+CG group. After 7 and 21 days, the animals were sacrificed for histological and histomorphometric analysis. Bone formation was quantified as the percentage of newly synthesized collagen, visualized by Gomori's trichromic. Clinical/macroscopic evaluation was based on predetermined scores of gingival healing.Treatment with HA improved gingival healing at day 7, but no statistical differences were found among groups at day 21. The morphometric analysis demonstrated better results after the treatment of bone defects with both HA and CG on day 21. The three-dimensional structure of CG prevented the invasion of epithelial tissue into the defect, preserving its original volume.Isolated use of a chitosan-gelatin osteoconductive matrix promoted greater bone deposition and preserved the volume of the surgical site, irrespective of the presence of hyaluronic acid.

The composition and morphology of the internal and external surfaces of membranes are important for preventing migration of epithelial and connective cells, and allow the installation of osteogenic cells for bone growth. The objective of this study was to analyze the morphology and composition of three types of commercially available resorbable collagen membranes.Three types of collagen membranes, with different compositions and coming from different animals, were used: 1) Dental Surgidry F (bovine collagen type I); 2) Bio-Gide® (porcine collagen type I and III); and 3) OsseoGuard™ (bovine collagen type I). These membranes were analyzed using scanning electron microscopy and energy dispersive spectrometry.The membranes showed distinct superficial architectures, porosities and chemical compositions. The membranes exhibited different surfaces and thicknesses, ranging from 0.32 mm to 0.75 mm. The chemical compositions exhibited a high percentage of niobium (Nb) in the Surgidry and OsseoGuard™ membranes; the Bio-Gide® membrane showed a greater proportion of calcium and aluminum relative to other elements.Different types of resorbable collagen membranes exhibit different morphologies and chemical composition, which could lead to differences in the mode and time of resorption of the membranes used for guided tissue regeneration procedures.

Objective In this work we evaluated the bone-forming potential of BMP4, TGFβ1 and BMP4/TGFβ1 mixed by performing histological and morphometric analysis. We also evaluated the immunolabelling of fibronectin (FN) and collagen type III (Col III), two determinant proteins for the early phase of bone repair. Design Histological, histomorphometric and immunohistochemistry analysis were used to evaluate new bone and blood vessels formation as well as fibronectin and collagen type III expression. 112 male Wistar rats weighing 250–300 g had their maxillary second molar extracted. Sockets filled with blood clot (BC) or treated with L (empty liposome), P (PBS), BP (BMP-4 in PBS) and TP (TGF-β1 in PBS), as well as with BL (BMP-4 in liposome) and TL (TGF-β1 in liposome) administered isolated or in association (BTL) were obtained. The animals were sacrificed at 3, 7, 14 and 21 days after surgery. Results An increased percentage of bone trabeculae, and a higher number of blood vessels were observed in groups BL or TL administered isolated or in association when compared to groups BC, L, P, BP and TP. Fibronectin and collagen type III analysis revealed enhanced expression firstly detected at 3 days followed by a peak at 7 days. Lower levels of immunoreactivity were observed in the sockets filled with blood clot, and treated with L, P, BP and TP when compared with sockets from groups BL, TL and BTL. Conclusion The present study indicates growth factors carried by liposomes, either in isolated or associated forms, as successful enhancers of the healing process in rat tooth sockets. We also conclude that the expression of fibronectin and collagen type III increases during the early phases of bone repair.

Tissue engineering has emerged as a novel treatment for replacement of lost bone tissue. This study evaluated the effects of a chitosan–gelatin scaffold seeded with bone marrow mesenchymal stem cells (BMMSCs) in the healing process of tooth sockets in rats. BMMSCs isolated from transgenic rats expressing enhanced green fluorescent protein (eGFP) were expanded and seeded on a chitosan–gelatin scaffold. These constructs were cultured for three days and characterized by scanning electronic microscopy (SEM) and energy dispersion spectroscopy (EDS). Receptor rats received the implant in the left sockets, after upper first-molar extraction. Right alveoli served as control. Animals were sacrificed at days 5, 21, and 35 post-graft for examination. Morphometry demonstrated increased bone mineralization after 21 and 35 days in transplanted sockets. Migration, differentiation, and fate of eGFP-labeled BMMSCs were monitored by immunohistochemistry. Tartrate-resistant acid phosphatase staining (TRAP) was carried out at 21 days, to identify the involvement of osteoclastic cells in the scaffold resorption. The biomaterial was resorbed by TRAP-negative giant cells in a typical foreign body reaction. Immunohistochemical findings showed that BMMSCs contributed to bone, epithelial, and vascular repair. Together, results indicate that BMMSCs loaded in the chitosan–gelatin scaffold is a strategy for tissue development in bone engineering. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A:2775–2786, 2012.

This work evaluated the bone-forming potential of the platelet-derived growth factor isoform BB (PDGF-BB), insulin-like growth factor I (IGF-I), and mixed PDGF-BB/IGF-I delivered in liposomes compared with phosphate buffered saline (PBS), in the healing process of rat tooth sockets. One hundred and twelve Wistar rats were randomized into 7 groups of 16 animals each and were evaluated at 3, 7, 14 and 21 days after extraction of the maxillary second molars. The left sockets were treated with PBS (P), empty liposome (L), IGF-I in PBS (IP), IGF-I in liposome (IL), PDGF-BB in PBS (PDP), PDGF-BB in liposome (PDL) and both growth factors (GFs) together within liposomes (PDIL). The right sockets were filled with blood clot (BC). Histological and histomorphometric analyses were used to evaluate the formation of new bone and blood vessels. Immunohistochemistry was performed to evaluate the expression of osteocalcin and vascular endothelial growth factor (VEGF) during bone repair. Data were tested statistically using a Tukey's test according to a Dunn's analysis and Mann-Whitney U test followed by Kruskal-Wallis one-way analysis. Results were considered significant when p

Considering the potential use of growth factors carried by liposomes for bone repair, this study aimed to assess the progress of bone healing process in injured alveoli of rats after administering EGF within liposomes. For this assessment we used 48 male Wistar rats that had their maxillary second molar extracted and separated into 5 groups: sockets filled with blood clot (BC), treated with empty liposome (L), PBS (P), EGF in PBS (EGF-P) and EGF in liposome (EGF-L). The animals were sacrificed after 3, 7, 14 and 21 days after surgery. Histological, histomorphometric and immunohistochemistry analysis were performed to evaluate new bone and blood vessels formation as well as the expression of fibronectin and collagen type III, two determinant proteins for early wound regeneration. Our analysis showed a continuous transformation of sockets during all stages of wound healing. Nevertheless, groups BC, L, P and EGF-P followed a regular time for regeneration significantly different from the EGF-L group, which showed faster recovering. A higher expression of fibronectin and type III collagen in the group EGF-L after 3 and 7 days of surgery was observed and might be explained by the ability of the liposome to deliver EGF in a controlled manner, stimulating mesenchymal cells migration and osteoblast differentiation. As liposome efficiently regulated the availability of EGF without risks for its function and protected the factor from early absorption and degradation, the present work indicates that liposomes can be successful used as carriers for controlled delivery of growth factors in bone healing.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico A utilização de xenoenxertos derivados de osso bovino tem se configurado como uma alternativa aos autoenxertos na substituição de perdas óssea. Entretanto, sua propriedade de osteoindução e potencial osteogênico ainda são limitados. Por essa razão, algumas estratégias como a desmineralização de fragmentos ósseos, bem como a utilização de células tronco mesenquimais tem sido proposta para a otimização do reparo ósseo. Nesse contexto, este estudo avaliou os efeitos da associação de uma matriz óssea de origem bovina, marca comercial- Criteria®, desmineralizada em EDTA 10%, às CTPD’s (modificadas para eGFP - enhancedgreenfluorescentprotein) no reparo de um defeito ósseo intrabucal em ratos. Os defeitos foram preenchidos por: 1. Matriz óssea desmineralizada (MOD); 2. CTPD’s em suspensão; 3. CTPD+MOD; 4. Coágulo sanguíneo (controle). Os animais foram sacrificados após 7, 14 e 49 dias para avaliações histomorfométricas e imunohistoquímica para GFP (visando o monitoramento das células tronco transplantadas); e após 3 e 7 dias para análise de expressão de genes indicativos de diferenciação osteoblástica (Runx2, Osx e iBSP) por RT-PCRq. Aos 7 dias, o fechamento epitelial foi incompleto em todos os grupos. A morfometria demonstrou que a deposição óssea na área do defeito cirúrgico foi maior no grupo que recebeu a associação proposta, seguido pela enxertia com as CTPD’s isoladas. Este resultado está em concordância com a análise molecular, que demonstrou a expressão do marcador de diferenciação osteoblástica iBSP, 64 vezes maior no mesmo período. Esse padrão de deposição óssea na análise morfométrica se manteve nos períodos de 14 e 49 dias, evidenciando a associação benéfica entre CTPD+MOD para terapias de regeneração tecidual óssea. O nível de maturação da matriz foi avaliado por coloração de PicroSirius Red e revelou a maior maturação no grupo CTPD+MOD. Resumidamente, os resultados demonstraram otimização do processo de regeneração óssea com a associação CTPD+MOD em todos os períodos. O estudo traz esclarecimentos sobre o xenoenxerto de fabricação nacional e apresenta uma proposta de otimização para o seu uso no desenvolvimento de terapias de regeneração tecidual. The use of xenografts derived from bovine bone has been configured as an alternative to autografts in the replacement of bone loss. However, its property of osteoinduction and osteogenic potential are still limited. For this reason, some strategies such as demineralization of bone fragments as well as the use of mesenchymal stem cells have been proposed for the optimization of bone repair. In this context, this study evaluated the effects of the association of a bone matrix of bovine origin, commercial brand - Criteria ®, demineralized in EDTA 10%, to the CTPDs (modified for eGFP - enhancedgreenfluorescentprotein) in the repair of an intrabuccal bone defect in rats. The defects were filled by: 1. Demineralized bone matrix (MOD); 2. TDPs in suspension; 3. CTPD + MOD; 4. Blood clot (control). The animals were sacrificed after 7, 14 and 49 days for histomorphometric and immunohistochemical evaluations for GFP (aiming at the monitoring of transplanted stem cells); and after 3 and 7 days for expression analysis of genes indicative of osteoblastic differentiation (Runx2, Osx and iBSP) by RT-PCRq. At 7 days, epithelial closure was incomplete in all groups. Morphometry demonstrated that bone deposition in the area of the surgical defect was higher in the group that received the proposed association, followed by grafting with the isolated TCDC's. This result is in agreement with the molecular analysis, which demonstrated the expression of the osteoblast differentiation marker iBSP, 64 times higher in the same period. This pattern of bone deposition in the morphometric analysis was maintained in the periods of 14 and 49 days, evidencing the beneficial association between CTPD + MOD for tissue regeneration therapies. The maturation level of the matrix was evaluated by staining of PicroSirius Red and revealed the highest maturation in the CTPD + MOD group. Briefly, the results demonstrated optimization of the bone regeneration process with the association CTPD + MOD in all periods. The study brings clarifications about the xenograft of national manufacture and presents an optimization proposal for its use in the development of tissue regeneration therapies.

Apesar de sua capacidade de reparo, o tecido ósseo pode ser submetido a alguns tipos de fraturas, cirurgias ou patologias que podem levar a grandes defeitos ósseos. As principais estratégias de tratamento de defeitos ósseos são baseados em osteoindução ou osteocondução. Matriz dentinária desmineralizada humana (MDDH) é uma alternativa biocompatível para preencher defeitos ósseos, melhorando a qualidade e quantidade de osso produzido. 24 Ratos Wistar foram selecionados, submetidos à extração de ambos os segundos molares superiores (direito e esquerdo). Os alvéolos foram separados em dois grupos: controle (direita) preenchido com coágulo sanguíneo e experimental (esquerda) preenchido com MDDH. Os animais foram sacrificados aos 5, 10 e 21 dias. Foram realizadas análises histológicas, histomorfométricas (análise de variância - ANOVA e teste de Tukey) e imunohistoquímica para osteopontina (OPN) como indicador de osteogênese. Aos 5 dias MDDH foi incorporada pelas novas trabéculasósseas. Aos 10 dias observou-se organização do tecido conjuntivo etrabéculas no grupo experimental. Detectou-se coloração intensa para OPN em área adjacente à MDDH no grupo experimental. Aos 21 dias no grupo experimental verificou-se trabéculas maduras. Houve diferençaestatísticamente significatva (p

O processo de odontogênese é guiado por uma cascata de sinalização na qual inúmeras moléculas, como os fatores de crescimento fibroblástico 2, 3 e 4 (FGF2, FGF3 e FGF4), garantem o desenvolvimento dos grupos dentários. A maioria dos dados sobre a odontogênese advém de roedores, que não possuem todos os grupos dentários. O gambá Didelphis albiventris é um mamífero marsupial com dentição mais próxima à humana. Além disso, a odontogênese nesta espécie ocorre nos neonatos já no marsúpio, facilitando o acesso e a coleta de amostras para o estudo da formação dos dentes. O objetivo deste estudo foi determinar o padrão de expressão de FGF2, FGF3 e FGF4 durante a odontogênese de D. albiventris. Para isso, neonatos de D. albiventris foram coletados e processados para seguintes análises: (1) histológica, para caracterizar morfologicamente a odontogênese de molares de D. albiventris; (2) de imunolocalização, para caracterizar o padrão de expressão de FGF2, FGF3 e FGF4 durante a odontogênese nesta espécie de gambá; e (3) de expressão gênica, para clonar e sequenciar essas moléculas de interesse no gambá, além de permitir a caracterização do padrão de expressão dos RNAm para FGFs, por hibridização in situ. A análise histológica foi realizada por coloração HE; e a imunolocalização dos FGFs foi realizada por imunoperoxidase indireta. Para a análise de expressão gênica, oligonucleotídeos foram construídos com base na sequência conhecida de Monodelphis domestica, o marsupial mais próximo com genoma sequenciado. Oligos foram desenhados para amplificar fragmentos de FGF3 e FGF4 (uma vez que FGF2 já havia sido sequenciado anteriormente). A sonda de RNA antisenso foi sintetizada in vitro, com marcação para digoxigenina. A análise histológica das fases da odontogênese mostrou que o gambá D. albiventris possui as mesmas estruturas presentes em roedores, nos mesmos estágios da odontogênese, além de apresentar uma lâmina dentária secundária vestigial. A imunohistoquímica permitiu a observação de FGF2, FGF3 e FGF4 em um maior número de estruturas dentárias em relação à literatura referente a roedores, sugerindo um papel mais amplo dessas moléculas no gambá. Não foi possível amplificar FGF3 de D. albiventris, sugerindo diferenças nas sequências entre as espécies de gambás. O fragmento de FGF4 foi clonado e sequenciado. O clone também serviu como molde para a síntese da sonda de RNA antisenso, utilizada na hibridização in situ. O padrão de expressão indicou a presença de FGF4 no germe dentário, tecido conjuntivo, musculatura esquelética, cartilagem, epitélio, fígado e tecido nervoso. O conhecimento da sinalização que determina a odontogênese em um modelo animal com dentição completa pode contribuir para o desenvolvimento de terapias para a reposição de dentes perdidos. Este estudo ainda representa mais um passo no conhecimento dessa espécie de gambá e na sua implementação para estudos em Biologia do Desenvolvimento Odontogenesis is guided by a complex signaling cascade in which several molecules, including the fibroblastic growth factors 2, 3 and 4 (FGF2, FGF3 and FGF4), ensure dental groups development and specificity. Most of the data on odontogenesis derives from rodents, which does not have all dental groups. Didelphis albiventris is a marsupial mammal with the closest dentition to humans. Besides, odontogenesis in this species occurs when the newborns are already in the pouch, favouring samples harvesting for the study of tooth development. This study aimed to determinate the expression pattern of FGF2, FGF3 and FGF4 during the D. albiventris odontogenesis. For this, D. albiventris newborns were harvested and processed for the following analysis: (1) histological, to allow the morphological characterization of the D. albiventris molars odontogenesis; (2) immunolozalization, to establish FGF2, FGF3, and FGF4 expression pattern during odontogenesis; and (3) of gene expression, to allow the cloning and sequencing of these molecules in this opossum species, providing tools for the localtemporal characterization of the FGFs mRNA expression patterns, by in situ hybridization. Histological analysis was performed using HE; and FGFs immunolocalization was performed by indirect immunoperoxidase. For gene expression analysis, oligos were obtained based on sequences available for Monodelphis domestica, the closest marsupial with genome sequenced. Oligos were obtained to amplify D. albiventris FGF3 and FGF4 (as FGF2 was cloned and sequenced before). Antisense RNA probe were in vitro synthesized, using digoxigenin for labelling. Histological analysis of the odontogenesis stages showed similarity of dental structures between D. albiventris and rodents, at the same odontogenesis stages, in addition to the presence of a vestigial secondary dental lamina in opossum. Immunolocalization allowed the observation of FGF2, FGF3 and FGF4 in a larger number of dental structures than related data for rodents, suggesting broader functions for these molecules in this opossum species. It was not possible to amplify D. albiventris FGF3, suggesting differences in the sequence level between these two opossum species. A FGF4 fragment was cloned and sequenced. This clone was also used to allow the synthesis of an antisense RNA probe, for in situ hybridization. FGF4 trancripts were detected in the dental germ, connective tissue, skeletal muscle, cartilage, epithelium, liver, and nervous tissue. The knowledge of the signaling that determinates odontogenesis in an animal model with complete dentition may contribute to the development of therapies for the replacement of lost teeth. The present study represents one step further in knowing this opossum species and in its implementation for Developmental Biology studies.