Metabolic effect of low fluoride levels in the islets of NOD mice: integrative morphological, immunohistochemical, and proteomic analyses

Abstract Fluoride (F) has been widely used to control dental caries, and studies suggest beneficial effects against diabetes when a low dose of F is added to the drinking water (10 mgF/L). Objectives This study evaluated metabolic changes in pancreatic islets of NOD mice exposed to low doses of F and the main pathways altered by the treatment. Methodology In total, 42 female NOD mice were randomly divided into two groups, considering the concentration of F administered in the drinking water for 14 weeks: 0 or 10 mgF/L. After the experimental period, the pancreas was collected for morphological and immunohistochemical analysis, and the islets for proteomic analysis. Results In the morphological and immunohistochemical analysis, no significant differences were found in the percentage of cells labelled for insulin, glucagon, and acetylated histone H3, although the treated group had higher percentages than the control group. Moreover, no significant differences were found for the mean percentages of pancreatic areas occupied by islets and for the pancreatic inflammatory infiltrate between the control and treated groups. Proteomic analysis showed large increases in histones H3 and, to a lesser extent, in histone acetyltransferases, concomitant with a decrease in enzymes involved in the formation of acetyl-CoA, besides many changes in proteins involved in several metabolic pathways, especially energy metabolism. The conjunction analysis of these data showed an attempt by the organism to maintain protein synthesis in the islets, even with the dramatic changes in energy metabolism. Conclusion Our data suggests epigenetic alterations in the islets of NOD mice exposed to F levels comparable to those found in public supply water consumed by humans.

Acinar autolysis and mucous extravasation in human sublingual glands: a microscopic postmortem study

Although some morphological investigations on aged human sublingual glands (HSG) found eventual phenomena identified as autolysis and mucous extravasation, the exact meaning of these findings has not been elucidated.Objective The aim of this work is to investigate whether acinar autolysis and mucous extravasation are related to the aging process in human sublingual glands. We also speculate if autolytic changes may assist forensic pathologists in determining time of death.Material and Methods 186 cadavers’ glands were allocated to age groups: I (0–30 years); II (31–60), and III (61–90). Time and mode of death were also recorded. Acinar autolysis and mucous extravasation were classified as present or absent. Ultrastructural analysis was performed using transmission electron microscopy (TEM). Data were compared using Mann-Whitney U, Spearman’s correlation coefficient, Kruskal-Wallis, and Dunn tests (p<0.05).Results There was correlation between age and acinar autolysis (r=0.38; p=0.0001). However, there was no correlation between autolysis and time of death. No differences were observed between genders. TEM showed mucous and serous cells presenting nuclear and membrane alterations and mucous cells were more susceptible to autolysis.Conclusion Acinar autolysis occurred in all age groups and increased with age while mucous extravasation was rarely found. Both findings are independent. Autolysis degrees in HSG could not be used to determine time of death.

Assessment of new bone formation around titanium surface treated implants in diabetic rats / Neoformação óssea ao redor de implantes de titânio com superfície tratada inseridos em ratos diabéticos

Objective: This study aimed to compare the bone formation around titanium implants with machined and acid-etched surfaces, inserted in induceddiabetic rats and in non-diabetic rats, in an attempt to investigate whether there are differences in bone formation between this metaboliccondition and the use of different implant surfaces. Methods: Custom fabricated commercially pure solid cylinder titanium implants, machined and acid-etched surface were inserted in the femora of streptozotocin-induced diabetic rats (n=10) and non-diabetic rats (n=10). Morphometrical bone-implant contact percentage and bone area within the limits of the implant threads (BD) were performed at 21 days of healing. Results: Peri-implant tissue in machined implant showed intense new bone formation within all threads of the implants of the non-diabetic group (BIC= 82.8 ± 9.23 e BD = 38.7 ± 4.27) while diabetic group (BIC = 35.3 ± 9.4 and BD = 20.0 ± 3.8) exhibited small and immature bone formationwithin threads of the implants with thickness fibrous connective tissue interposition between bone-implant interface. In the acid-etchedsurface implants in both, diabetic and non-diabetic groups, the peri-implant tissue showed intense new bone formation within all threads ofthe implants with BIC = 74.4 ± 14.7 e BD = 35.4 ± 3.48 in non-diabetic group and BIC = 63.1 ± 12.9 e BD = 29.6 ± 4.9 in diabetic group.Conclusion: In machined surface implants the diabetes interfere negatively in osseointegration while acid-etched surface promoted major BIC and BD index, indicating its selective use in diabetic patients.

Objetivo: Comparar a formação óssea ao redor de implantes de superfície lisa e tratada, instalados em ratos diabético-induzidos e não-diabéticos,investigando se há diferenças na formação óssea entre os dois quadros metabólicos, melhora no padrão de osteogênese entre as diferentessuperfícies e, sua relação com o diabetes. Métodos: Foram instalados implantes de titânio de superfícies lisa e tratada, no fêmur de ratos diabético-induzidos com estreptozotocina (n=10) e não diabéticos (n=10).A Análise morfométrica da porcentagem de contato osso-implante (COI) foi realizada 21 dias após a cirurgia. Resultados: A neoformação óssea foi intensa ao redor dos implantes de superfície lisa nos ratos não-diabéticos (COI = 82.8 ± 9.23), enquanto que o grupo diabético exibiu pequena e imatura formação óssea (COI=35.3 ± 9.4), com interposição de tecido conjuntivo na interface osso-implante. Ao redor dos implantes com superfície tratada, ocorreu intensa neoformação óssea, tanto nos animais diabéticos (COI = 63.1 ± 12.9) como nos não-diabéticos, (COI = 74.4 ± 14.7). Conclusão: Nos implantes de superfície lisa, o diabetes interfere negativamente na osseointegração, enquanto que as superfícies tratadas com ácido promoveram maior contato osso-implante, indicando seu uso seletivo em pacientes diabéticos.

Fluoride effect on the process of alveolar bone repair in rats: evaluation of activity of MMP-2 and 9

Objective: The aim of this study was to evaluate comparatively the effect of fluoride (F) on the activity of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) involved in process of alveolar bone repair. Material and methods: This study used 4 groups of Wistar rats with 80 days of life (n = 160) which received drinking water containing different doses of fluoride (NaF): 5, 15, 50 ppm and deionized water (control) throughout the experiment. These animals had their right upper incisors extracted. After extraction, the animals were euthanized at 7, 14, 21 and 30 days and the hemi-maxillae were collected for microscopic analysis (Hematoxylin and Eosin and immunohistochemistry for MMP-9) and zymography (MMP-2 and 9).Results: Microscopically the process of bone repair was similar in all groups, being noted only a delay of the blood clot resorption and bone formation in the group of 50 ppm F. The expression for MMP-9 showed differences betweengroups only during the initial repair (7 days). However, the zymography showed no significant differences between treated and control groups. Conclusion: Ours results suggest an effect of fluoride on the activity of MMPs 2 and 9 at the initial period of alveolar repair which could be associated to the process of blood clot remission and delay in bone repair. Further studies are needed to establish the relationship between the initial process of resorption of the blood clot, and the involvement of MMPs 2 and 9 and its regulators/tissue inhibitors.

Experimental dry socket: microscopic and molecular evaluation of two treatment modalities / Alveolite experimental: análise microscópica e molecular de duas modalidades de tratamento

PURPOSE: To evaluate two treatment modalities of dry socket in rats and to discuss the first findings of the molecular analysis in this experimental model. METHODS: 84 rats underwent a tooth extraction were divided in 4 groups: I-uninfected socket (control), II-infected socket without any treatment, III-infected socket treated with irrigation of 2 percent sodium iodide and 3 percent hydrogen peroxide solution, IV-infected socket submitted to curettage, irrigation with physiological saline solution and fulfilled with metronidazole paste as base. The groups were subdivided in postoperative sacrifice periods: 6/15/28 days. A quantitative and a qualitative microscopic analysis was performed. Also, a quantitative analysis was performed using a RealTimePCR to evaluate the genes expression in the wound healing: Collagen Type I/COL-I, vascular endothelial growth factor/VEGF, osteocalcin/OCN, alkaline phosphatase/ALP, runt-related transcription factor 2/RUNX2 and tumor necrosis factor alpha/TNF-α. RESULTS: The group I showed higher bone formation, followed by groups IV, III, II respectively. The group II presented higher inflammatory infiltrate and the wound healing was delayed compared with other groups. It was obtained a significant positive correlation between bone neoformation and the expression of OCN and RUNX2, inflammatory infiltrate with TNF-α and a negative correlation between bone neoformation and TNF-α. CONCLUSION: No significant difference was found between the treatments.

OBJETIVO: Avaliar duas modalidades de tratamento da alveolite em ratos e discutir os primeiros achados de uma análise molecular neste modelo experimental. MÉTODOS: 84 ratos foram submetidos a uma extração dentária e foram divididos em quatro grupos: I- alvéolo não infectado (controle), II- alvéolo infectado sem tratamento, III- alvéolo infectado tratado com irrigação de iodeto de sódio a 2 por cento e solução de peróxido de hidrogênio a 3 por cento, IV- alvéolo infectado submetido à curetagem, irrigação com solução salina fisiológica e preenchimento com pasta a base de metronidazol. Os grupos foram subdivididos em períodos de sacrifício pós-operatório: 6/15/28 dias. Uma análise quantitativa e qualitativa microscópica foi realizada. Além disso, uma análise quantitativa foi realizada utilizando RealTimePCR para avaliar a expressão de genes no reparo alveolar: o colágeno tipo I / COL-I, o fator de crescimento endotelial vascular / VEGF, osteocalcina / OCN, fosfatase alcalina / ALP, fator de transcrição runt relacionados 2 / RUNX2 e fator de necrose tumoral alfa / TNF-α. RESULTADOS: O grupo I mostrou maior formação óssea, seguido pelos grupos IV, III, II, respectivamente. O grupo II apresentou maior infiltrado inflamatório e a cicatrização foi atrasada em comparação com outros grupos. Foi obtida uma correlação positiva entre a neoformação óssea e a expressão de OCN e RUNX2, infiltrado inflamatório com TNF-α e uma correlação negativa entre a neoformação óssea e TNF-α. CONCLUSÃO: Nenhuma diferença significativa foi encontrada entre os tratamentos.

Radiological and histopathological evaluation of experimentally-induced periapical lesion in rats

OBJECTIVE: This study evaluated experimentally-induced periapical bone loss sites using digital radiographic and histopathologic parameters. MATERIAL AND METHODS: Twenty-seven Wistar rats were submitted to coronal opening of their mandibular right first molars. They were radiographed at 2, 15 and 30 days after the operative procedure by two digital radiographic storage phosphor plates (Digora®). The images were analyzed by creating a region of interest at the periapical region of each tooth (ImageJ) and registering the corresponding pixel values. After the sacrifice, the specimens were submitted to microscopic analysis in order to confirm the pulpal and periapical status of the tooth. RESULTS: There was significant statistically difference between the control and test sides in all the experimental periods regarding the pixel values (two-way ANOVA; p<0.05). CONCLUSIONS: The microscopic analysis proved that a periapical disease development occurred during the experimental periods with an evolution from pulpal necrosis to periapical bone resorption.

Experimental alveolitis in rats: microbiological, acute phase response and histometric characterization of delayed alveolar healing

The pathogenesis of alveolitis is not well known and therefore experimental situations that mimic some features of this disease should be developed. OBJECTIVE: In this study, the evolution of the experimentally induced infection in rat sockets is characterized, which leads to clinical signs of suppurative alveolitis with remarkable wound healing disturbs. MATERIAL AND METHODS: Non-infected (Group I) and experimentally infected sockets in Rattus novergicus (Group II) were histometrically evaluated regarding the kinetics of alveolar healing. In addition, the characterization of the present bacteria in inoculation material and the serum levels of C-reactive protein (CRP) were performed. The detected species were Capnocytophaga ochracea, Fusobacterium nucleatum ss nucleatum, Prevotella melaninogenica, Streptococcus anginosus, Treponema socranskii and Streptococcus sanguis. RESULTS: All experimentally infected rats developed suppurative alveolitis, showing higher levels of CRP in comparison to those non-infected ones. Furthermore, infected rats presented a significant delayed wound healing as measured by the histometric analysis (higher persistent polymorphonuclear infiltrate and lower density of newly formed bone). CONCLUSION: These findings indicate that rat sockets with experimentally induced infection produced higher levels of serum CRP, showing the potential of disseminated infection and a disturb in the alveolar repair process in an interesting experimental model for alveolitis studies.

Matriz colagênica de tendão bovino como potencial biomaterial para bioengenharia de tecidos / Collagenic matrix from bovine tendon as a potential biomaterial for tissue bioengineering

A Engenharia de Tecidos é um campo interdisciplinar que busca preservar, restaurar ou criar um tecido funcional, apoiando- se em três elementos fundamentais: células, fatores tróficos e carreadores. Um desses elementos, que ainda permanece sob intensa investigação, é o carreador. O objetivo do trabalho foi avaliar o comportamento tecidual ao implante de membrana colagênica derivada de tendão bovino em subcutâneo de camundongos. Nos animais do grupo controle foi feita apenas a incisão, divulsão e sutura. Depois de 3, 7, 15, 30 e 60 dias, os camundongos foram eutanasiados por dose excessiva de anestésico, sendo os tecidos reacionais coletados para análise histológica. Foram observados os seguintes parâmetros: biodegradação em relação ao tempo, vascularização, integração tecidual e reação de corpo estranho. O tecido adjacente ao material implantado apresentou infiltrado inflamatório nos períodos iniciais, com angiogênese e proliferação fibroblástica. No grupo experimental constatamos uma moderada reabsorção da membrana nos períodos de 15 e 30 dias e absorção completa aos 60 dias. A absorção foi mediada por células tipo macrófagos, sem a necessidade de células gigantes. Concomitantemente, houve a regeneração tecidual. No grupo controle observamos resultados compatíveis com o procedimento operatório, mostrando formação de coágulo e rede de fibrina nos primeiros períodos, proliferação angioblástica e fibroblástica nos períodos seguintes e regeneração tecidual nos 2 últimos períodos analisados. Diante dos resultados obtidos, podemos concluir que a membrana de tendão bovino é biocompatível e reabsorvível, posicionando- se como um promissor material a ser explorado pela medicina regenerativa.

Tissue Engineering is an interdisciplinary field that seeks to preserve, restore or create a functional tissue, relying on three key elements: cells, growth factors and carriers. Thus, our objective was to evaluate the reactional tissue induced by collagenic matrices derived from bovine tendon in the subcutaneous tissue of mice. Thereafter, the animals were killed at 3, 7, 15, 30 and 60 days post-surgery of implantation and tissues collected for histological analysis for analyzing: biodegradation, angiogenesis, tissue integration and foreign body reaction. The reactional tissue showed a moderate inflammatory infiltrate, with angiogenesis and fibroblast- like cells proliferation, while a moderate resorption of the membrane was found at 15 and 30 days and it being complete at 60 days. Our results suggest that the absorption was mediated by mononuclear cells such as macrophages, without giant cells involvement. Based on these results, we conclude that the membrane of bovine tendon is biocompatible and absorbable, it being a promising material to be exploited for regenerative medicine.

Análise morfométrica da resposta tecidual ao osso bovino misto medular implantado em defeito ósseo na calvária de rato / Morphometric analysis of tissue response to mixed bovine bone marrow implanted in bone defects in rat calvaria

O propósito deste trabalho foi avaliar o reparo ósseo de defeito de tamanho crítico em crânio de ratos tratados com disco de osso bovino misto (OBM) medular poroso. Foram utilizados 30 ratos Wistar machos adultos submetidos à cirurgia para confecção de um defeito circular de 8 mm de diâmetro removendo toda a díploe da calvaria. Os defeitos foram preenchidos com um disco de OBM de 8 mm de diâmetro por 3 mm de espessura, enquanto no grupo controle o defeito ósseo foi preenchido com coágulo sanguíneo do próprio animal. Os animais foram mortos após 30, 60 e 120 dias. As peças foram removidas, fixadas em formol 10% tamponado, desmineralizadas e processadas pela técnica histotécnica padrão para coloração em hematoxilina e eosina. Os cortes foram submetidos à análise histológica descritiva e morfometria (densidade de volume). Os resultados demonstraram uma pequena diminuição do material implantado com o decorrer dos períodos experimentais, sugerindo uma reabsorção lenta e gradual do OBM. Em contrapartida foi observado uma pequena neoformação óssea nos dois grupos (teste e controle) e formação de tecido conjuntivo denso nas áreas do defeito ósseo aos 120 dias também nos dois grupos. O tecido conjuntivo, no grupo teste, foi capaz de penetrar nos poros do material e ocupar esses espaços com o passar dos períodos. Dentro dos limites desse estudo, pode-se afirmar que o OBM é biocompativel, no entanto, não apresenta capacidade osteocondutora ou osteoindutora em defeitos críticos na calvária de ratos Wistar.

The purpose of this study was to evaluate bone repair of critical size defects in the calvarias of rats treated with bovine bone mixed disk (OBM) porous medullary. We used 30 adult male Wistar rats that underwent surgery to confection a bone defect of 8 mm diameter circular removing diploe calvaria completely. The defects were filled with a disc of OBM 8 mm diameter and 3 mm thick, while the control group the bone defect was filled with blood clot. The animals were killed after 30, 60 and 120 days. The pieces were removed, fixed in 10% buffered formalin, demineralized and processed by standard technique to staining with hematoxylin and eosin. The sections were submitted to descriptive histology and morphometry (volume density). The results showed a small decrease of the implanted material in the course of the experimental periods, suggesting a slow and gradual resorption of the OBM. On the other hand, there was a small new bone formation observed in both groups (test and control) and formation of dense connective tissue areas of the bone defect at 120 days also in the two groups. The connective tissue in the test group was able to penetrate the pores of the material and occupy these spaces over the periods. Within the limits of this study, it can be said that the OBM is biocompatible, however, does not have osteoinductive or osteoconductive capacity in critical defects in the calvaria of rats.

Postmortem acinar autolysis in rat sublingual gland: a morphometric study

ABSTRACT OBJECTIVE: To analyze and to quantify morphological acinar postmortem changes in rat sublingual glands (SLG). MATERIAL AND METHODSs: Fifty rats were divided into two groups of 25 animals each. Group I was used for morphological and morphometric evaluations and group II for the determination of gland density and processed gland volume. Acinar autolytic changes were studied at 0 (control group), 3, 6, 12 and 24 h postmortem periods. The morphometric analysis of the volume density (Vv) and total volume (Vt) of intact (ia) and autolyzed (aa) acini was performed under light microscopy using a Zeiss II integration grid with 100 symmetrically distributed points. RESULTS: Morphologically, temporal progressive nuclear alterations and gradual loss of the structural architecture of acinar cells were found. Regarding quantitative results, both the Vvaa and the Vvia showed statistically significant differences among all postmortem periods (p<0.05). Vvaa increased from 0.42 percent at 0 h to 75.84 percent at 24 h postmortem and Vvia decreased from 71.16 percent to 0 percent over the same period. For Vtaa and Vtia, no statistically significant differences occurred between 12-24 h and 0-3 h (p>0.05), respectively. Vtaa increased from 0.18 mm³ at 0 h to 38.17 mm³ at 12 h, while Vtia showed a decrease from 33.47 mm³ to 0 mm³ between 3-24 h postmortem. Data concerning Vtaa were adjusted by two-variable linear regression, obtaining the equation: y=-3.54 + 3.38x (r²=0.90). The Vtaa growth rate calculated by this equation was 3.38 mm³/h between 0-12 h. CONCLUSION: Acinar autolysis on rat SLG demonstrated the most significant signs during the first 6 h postmortem and was widely spread through the gland at 12 h.

Biocompatibility evaluation of a new bioresorbable pin for membrane fixation

The aim of this study was to morphometrically analyze the tissue response to a customized pin obtained from devitalized bovine cortical bone (DBCB-pin) implanted in the subcutaneous tissue of rats, as well as to assess its microstructural aspect by scanning electron microscopy (SEM). The pins were implanted in the subcutaneous tissue of 20 rats, which were killed at 7, 14, 28 and 60 days (5 rats/period) after implantation. In the subcutaneous tissue, DBCB-pin promoted the formation of a fibrous capsule. At 7 days, capsule showed thickness of 70 ± 3.2 µm with higher density of newly formed capillaries and smaller density of collagen fibers. Between 14 and 60 days, more organized fibrous capsule exhibited smaller thickness (53 ± 5.5 µm) with higher density of fibroblasts and collagen fibers. In this period, a small and slow bioresorption of the DBCB-pin by macrophages and rare multinucleated giant cells without tissue damage was observed. The thickness of DBCB-pin resorbed was in mean only of 9.3 µm. During all experimental periods not occurred presence of immune reaction cells as lymphocytes and plasma cells. It was concluded that the pin derived from cortical bovine bone was well tolerated by subcutaneous tissue of rats and slowly resorbed could be an alternative material for membrane fixation in the guided tissue regeneration procedures.

O objetivo deste estudo foi analisar morfometricamente a resposta tecidual a um pino obtido a partir de osso bovino desvitalizado cortical (DBCB pinos) implantado no tecido subcutâneo de ratos, bem como para avaliar o seu aspecto microestrutural por microscopia eletrônica de varredura (MEV). Os pinos foram implantados no tecido subcutâneo de 20 ratos, que foram sacrificados aos 7, 14, 28 e 60 dias (5 animais / período) após a implantação. No tecido subcutâneo, o pino DBCB promoveu a formação de uma cápsula fibrosa. Aos 7 dias, a cápsula apresentou espessura de 70 ± 3,2 μm com maior densidade de capilares neoformados e menor densidade de fibras colágenas. Entre 14 e 60 dias, a cápsula fibrosa apresentava-se mais organizada e exibiram menor espessura (53 ± 5,5 μm) com maior densidade de fibroblastos e fibras colágenas. Nesse período, foi observada uma bioreabsorção pequena e lenta dos pinos DBCB por macrófagos e raras células gigantes multinucleadas, sem dano tecidual. A espessura dos pinos DBCB reabsorvidos foi em média de apenas 9,3 µm. Durante todos os períodos experimentais não ocorreu presença de células como linfócitos e células plasmáticas. Concluiu-se que o pino derivado de osso bovino cortical foi bem tolerado pelo tecido subcutâneo de ratos e reabsorvido lentamente, sendo um potencial material alternativo para fixação da membrana nos procedimentos de regeneração tecidual guiada.

Acompanhamento histomorfométrico e radiográfico do reparo de defeito crítico em crânio de ratos preenchidos com associação de osso medular bovino e BMP / Histomorphometric and radiographic monitoring the repair of critical defects in skulls of rats completed with combination of medular bovine bone plus BMP

O objetivo deste trabalho foi avaliar histomorfometricamente o processo de reparo de defeito ósseo perene na calvária de ratos preenchidos com grânulos de osso medular bovino desproteinizado, associado a um pool de proteínas morfogenéticas ósseas (BMPs) bovinas. Após 1, 3 e 6 meses, as calotas cranianas foram coletadas, processadas e as sessões obtidas coradas em Hematoxilina e Eosina. A análise histomorfométrica mostrou que o xenoenxerto não foi completamente degradado durante os períodos estudados, com a densidade de volume do material implantado permanecendo próximo de 50% em todos os períodos (p > 0,05). No grupo controle a densidade de volume do tecido conjuntivo era de 62,5% ± 8,81% (1° mês), diminuindo significantemente para 40,2% ± 7,5% após 6 meses (p < 0,05), enquanto que o volume de tecido ósseo neoformado passou de 37,5% ± 8,81% no 1° mês para 59,8% ± 7,5% aos 6 meses (p < 0.05). No grupo experimental, o volume de tecido conjuntivo diminuiu de 45,8% ± 7,8% (1° mês) para 31,8% ± 8,38% (6° mês), ao passo que o volume do tecido ósseo neoformado aumentou de 7,0% ± 2,45% (1° mês) para 19,6% ± 11,04% (6° mês) (p < 0,05). Conclui-se que, a despeito da biocompatibilidade e propriedade osteocondutora do xenoenxerto, o composto estudado retardou o reparo ósseo em defeito críticos no crânio de ratos.

The objective of this study was to evaluate the healing process of perennial bone defect in the skull of rats promoted by treating the defects with inorganic granules of bovine bone plus a pool of bone morphogenetic proteins (BMPs). After 1, 3 and 6 months from surgery, the whole skulls were carefully collected and sections obtained were stained with Hematoxylin & Eosin. We showed that implanted material was not completely resorbed during the periods studied, remaining around 50% in all periods (p > 0.05). In the control group the volume of connective tissue decreased significantly from 62.5% ± 8.81% (1 month) to 40.2% ± 7.5% after 6 months (p < 0.05), while that the amount of neoformed bone increased from 37.5% to ± 8.81% (1 month) to 59.8% ± 7.5% (6 months) (p < 0.05). On the other hand, in the experimental group, the volume of connective tissue decreased from 45.8% ± 7.8% (1 month) to 31.8% ± 8.38% (6 months), while the amount of neoformed bone tissue increased from 7.0% ± 2.45% (1 month) to 19.6% ± 11.04% (6 months) (p < 0.05). Based on results, we concluded that, despite the biocompatibility and osteoconductive property of signaling molecules such as BMPs, their combination with bovine bone particles delayed the new bone formation in critical defects rats.

Regeneração óssea em Odontologia com a utilização do substituto ósseo composto xenogênico Gen Mix / Bone regeneration in Dentistry with use of composed bone substitute Gen Mix

O presente estudo avaliou quatro casos clínicos onde um xenoenxerto bovino composto osteossubstituto foi usado para regeneração da perda óssea. Três casos de alvéolos dentais pós-extração atraumática e um caso de procedimento de elevação de seio maxilar usando técnica de reposição lateral, objetivando a colocação de implante, foram tratados com xenoenxerto composto. Quatro a oito meses, previamente à colocação de implante, as áreas enxertadas foram reoperadas e biópsias ósseas removidas por brocas trefinas. Após seis meses da colocação dos implantes, os elementos protéticos estiveram em função e mostrando sucesso clínico. A análise histológica das biópsias mostrou ausência de infiltrado inflamatório e presença de tecido ósseo neoformado fora e dentro de poros e fendas do biomaterial. O xenoenxerto foi biocompatível e permitiu a aposição de novo osso, indicando seu uso antes da colocação de implantes osseointegrados.

The present study evaluated four clinical cases where a xenograft bovine compound bone substitute was used for regeneration of bone loss. Three cases of dental sockets after non-traumatic tooth extractions and one case of maxillary sinus augmentation procedure using the lateral approach technique, aiming further implant placement, were treated with xenograft compound. Four to eight months before implant placement, the grafted areas were reopened and bone biopsies carried out by trephine burs. Six months after implant placement, the prosthetic elements were in function showing clinical success. Histological analysis of biopsies showed absence of inflammatory infiltrate and the presence of new bone tissue over and inside pores and grooves of the biomaterial as well. The xenograft was biocompatible and allowed the apposition of new bone, supporting its use before dental implant placement.

Aspectos celulares, teciduais e moleculares da osteogênese ectópica e ortotópica induzida pela matriz alogênica óssea e dentinária / Cellular, tissue and molecular aspects of the ectopic and orthotopic osteogenese induced by bone and dentine allogenic matrix

O objetivo do atual trabalho, foi correlacionar os eventos celulares e teciduais com a expressão das proteínas VEGF, BMP-7, RANKL e OPG durante a osteogênese ectópica e ortotópica, induzida pela matriz óssea (MO) e dentinária (MD) alogênica. Matrizes alogênicas desmineralizada em HCl a 0,6N, obtidas de fêmur e incisivo de ratos, fori implantada entre as fáscias musculares da coxa e em defeito trans-ósseo de 8mm de diâmetro nos ossos parietais. As análises radiográfica e histomorfométrica da neoformação óssea e, a imunohistoquímica e o western blotting para as proteínas VEGF, BMP, RANKL e OPG, mostraram que: a) o volume da região do enxerto nos sítios ortotópicos reduziu 19% em 42 dias; b) em ambos tipos de enxerto e locais de implantação, ocorreu formação de tecido cartilaginoso e ósseo; c) nos sítios intramusculares, a reabsorção da matriz alogênica e a remodelação do tecido cartilaginoso, ósseo e medular foi mais acelerado, em relação a implantação ortotópico; d) o aumento na densidade de volume dos vasos sanguíneos e no número de osteoblastos/osteócitos e osteoclastos ocorreu simultaneamente e estava associado à maior reabsorção da matriz alogênica e à formação do tecido medular (hematopoiético); e) as proteínas VEGF, BMP-7, RANKL, OPG foram expressas em condrócitos, osteoblastos ativos, osteócitos recém aprisionados na matriz e em células estromais próximas aos osteoblastos ou às áreas da matriz alogênica reabsorvida; e f) a expressão das proteínas VEGF, BMP-7, RANKL e OPG foi maior no grupo MO. O pico de expressão dessas proteínas ocorreu nos períodos de 14 aos 21 dias no grupo da MO e 21 e 28 dias no grupo da MD.

Concluímos que, a capacidade osteoindutora da matriz alogênica desmineralizada está relacionado a origem da matriz e ao sítio de implantação e que, as proteínas VEGF, BMP-7, RANKL e OPG estão associadas a maior reabsorção da matriz implantada, promovendo uma rápida e contínua liberação dos morfógenos contidos em seu interior que, induzem temporal e espacialmente a formação óssea/medular.

The aim of the present work was to correlate the cellular and tissue events with the expression of VEGF, BMP-7, RANKL and OPG during ectopic and orthotopic osteogenesis, induced by bone and dentin allogeneic matrix. Allogenic matrices obtained from femur and incisor of rats and demineralized in 0.6 N HCl were implanted into a intramuscular pocket and a 8mm-diameter bone defect in the skull. The radiographic and histomorphometric analysis of new bone formation, and immunohistochemistry and western blotting for VEGF, BMP, RANKL and OPG proteins, showed that: a) the total volume of the graft region in orthotopic site decreased 19% at 42 days b) in both graft types and implantation sites occurred formation of cartilaginous and bone tissues, c) in intramuscular sites, the resorption of allogenic matrix and remodeling of the new formed cartilage and bone were faster, in relation to orthotopic implantation sites; d) the increase in the volume density of blood vessels and in the number of osteoblasts/osteocytes and osteoclasts occurred simultaneously and was associated with greater reabsorption of the allogenic matrix and hematopoietic bone marrow formation; e) VEGF, BMP-7, RANKL, OPG proteins were expressed in chondrocytes, active osteoblasts, newly osteocytes confined and stromal cells located near the osteoblasts or in the surface of the reabsorbed matrix; and f) the VEGF, BMP-7, RANKL and OPG expression was higher in MO grafts than in the MD. The peak of expression of these proteins each occurred at 14 and 21 days in MO and 21 and 28 days in MD. We concluded that, the osteoinductive capacity of allogeneic demineralized matrix is related to matrix origin and implantation site and that the VEGF, BMP-7, RANKL and OPG proteins are associated with greater reabsorption of the implanted matrix, promoting rapid and continuous matrix-release morphogens that induces spatially and temporally the bone and bone marrow formation.

Marcadores bioquímicos e microscópicos como ferramentas investigativas da resposta tecidual envolvidas com a associação de osso cortical bovino/colágeno em subcutâneo de ratos / Biochemical and microscopic analyses as investigative tool of tissue response involved with the association of bovine cortical bone/collagen in subcutaneous of rats

O objetivo deste trabalho foi avaliar a reposta tecidual à associação de osso bovino inorgânico e colágeno bovino liofilizado, implantados em subcutâneo de ratos. O composto (osso bovino + colágeno) foi acondicionado em cápsulas de colágeno transparente e posteriormente implantado no tecido conjuntivo subcutâneo de ratos. Os animais foram mortos após 10, 20, 30 e 60 dias da cirurgia e as peças recolhidas para análise microscópica e enzimática. Foram analisados os aspectos microscópicos da resposta tecidual, bem como a atividade específica da fosfatase ácida total, fosfatase ácida lisossomal, fosfatase ácida de baixa massa molecular, fosfatase ácida resistente ao tartarato (marcador de osteoclastos e alguns tipos de células gigantes multinucleadas) e da fosfatase alcalina. A análise enzimática mostrou que a atividade da fosfatase ácida lisossomal coincidiu com os períodos de maior presença de células gigantes multinucleadas inflamatórias. A análise microscópica revelou um moderado infiltrado inflamatório apenas no período de 10 dias, desaparecendo nos períodos seguintes. A presença de linfócitos e plasmócitos foi leve, sendo notada apenas no período de 10 dias. O grau de fibrosamento tendeu de moderado a intenso até o final do experimento. Dentro dos limites deste trabalho pode-se concluir que a combinação de osso cortical bovino + colágeno bovino liofilizado é biocompatível não exibindo sinais de resposta imunogênica, além disso, a atividade das fosfatases ácidas pode ser modulada em função do tempo, durante a resposta tecidual ao composto implantado em subcutâneo de ratos. O exato papel de cada uma dessas enzimas nesta complexa cadeia de eventos deve, ainda, ser investigado.

The objective of this work was to evaluate the rat tissue response under association of inorganic bovine bone and bovine collagen. Before experimentation, the association (bovine bone + collagen) was conditioned in collagen capsules and after implanted in rats subcutaneous tissue. Afterwards, the animals were killed as follows: 10, 20, 30 and 60 days after surgery. Immediately, the biopsies were collected and appropriately conditioned for both microscopic and biochemical analysis. The biochemical parameters evaluated were: specific activity of total acid phosphatase, lisossomal acid phosphatase, low molecular weight phosphatase, tartarateresistant acid phosphatase and of alkaline phosphatase. These results showed that lisossomal acid phosphatase activity coincided with the periods of bigger incidence of inflammatory multinucleated giant cells. Results from microscopic analysis showed a moderate infiltrated inflammatory at 10 days, decreasing at the following periods. Also, the presence of lymphocytes and plasmocytes was scored as light at 10 days. We found that fibrosis degrees were between moderate to intense at the final periods. Taken together, our results showed that association of cortical bovine bone plus bovine collagen is biocompatible by not showing signals of immune response. On the other hand, both acid and alkaline and phosphatase activities were modulated along the periods evaluated as responsive events.

Avaliação da biocompatibilidade de uma membrana de pericárdio bovino acelular e seu potencial como carreador de osteoblastos / Evaluation of the biocompatibility of an acellular bovine pericardium membrane and its potential as an osteoblast scaffold

A biocompatibilidade de uma membrana de pericárdio bovino foi avaliada em tecido subcutâneo de camundongos 3,7, 15, 30 e 60 dias após a implantação. Os componentes celulares da resposta inflamatória, a degradação da membranae as características do colágeno foram analisadas em cortes histológicos corados pela hematoxilina-eosina, tricrômico de Masson e Picro-Sírius, respectivamente. Para verificar seu potencial como carreador celular, osteoblastos humanos(hFOB1.19, ATCC) foram semeados sobre a membrana e mantidos em DMEM/F12 por 7 dias. Os resultados in vitro mostraram que os osteoblastos proliferaram em monocamada na superfície da membrana, mas sem penetrar em seu interior. A análise dos cortes histológicos demonstrou 3 dias após a implantação apenas a formação da rede de fibrina. Aos 7 dias, o material implantado estava circundado por células inflamatórias mononucleares, com pouca penetração celular no seu interior. Após 15 dias foi observado um intenso infiltrado inflamatório em contato e dentro do material,bem como sinais de degradação interna e externa. No período de 30 dias, o material, em processo bastante avançado de absorção, estava totalmente tomado por fibroblastos e macrófagos. Aos 60 dias pós-implantação, o material não foi maisdetectado em quaisquer dos animais e a tecido subcutâneo apresentava-se normal. Os cortes corados com Picro-Sírius e observados sob luz polarizada mostraram o remodelamento tecidual. Em conclusão, a membrana de pericárdio é bioabsorvívele biocompatível, porém, in vitro, não proporciona uma adequada matriz tridimensional para osteoblastos.

The biocompatibility of a pericardium membrane was evaluated in the subcutaneous tissue of mouse killed 3, 7, 15, 30 and 60 days post implantation. The cellular components of inflammatory infiltrate, the membrane degradation, and the collagen characteristic were analyzed in histological sections stained with hematoxilyn and eosin, Tricromic of Masson and Sirius Red, respectively. The potential features as a tissue engineering scaffold was tested in vitro using human osteoblasts (h.Fob 1.19, ATCC) seeded over the membrane and maintained for 7 days in DMEM/F12. We observed in vitro the monolayer proliferation of osteoblasts, but without penetrating in the membrane. The histological sections showed after 3 days of implantation only the presence of a fibrin net. At the 7-day period, mononuclear inflammatory cells were observed around the implant, but a few one were observed inside the membrane. After 15 days the inflammatory infiltrate was more intense than in the previous period and the cells were inside and in close contact to the material showing evident signs of internal and external degradation. The implant degradation was intense after 30 days and theresidual material was fulfilled of fibroblasts and macrophages. No signs of membrane were observed after 60 days in any animals and the subcutaneous tissue presented normal aspect. Sirius Red staining at polarized light had evidenced the tissue remodeling throughout the experimental periods. In conclusion, the pericardium membrane is bioabsorbable and biocompatible, but, in vitro, do not fulfill the requirements as a tridimensional scaffold to osteoblast.

Evaluation of clinical periodontal conditions in smokers and non-smokers

Given that tobacco smoking habit is a risk factor for periodontal diseases, the aim of this study was to compare clinical periodontal aspects between smokers and non-smokers. The clinical status were assessed in 55 patients, 29 smokers and 26 non-smokers, aged 30 to 50 years, with mean age of 40. The clinical parameters used were: probing depth (PD), plaque index (PI), gingival index (GI), clinical attachment level (CAL), gingival recession (GR) and gingival bleeding index (GBI) for arches (upper and lower ) and teeth (anterior and posterior). Tooth loss was also evaluated in both groups. Multiple regression analysis showed: tendency of greater probing depth and clinical attachment level means for smokers; greater amount of plaque in smokers in all regions; greater gingival index means for non-smokers with clinical significance (p<0.05) in all regions. Although, without statistical significance, the analysis showed greater gingival bleeding index means almost always for non-smokers; similar gingival recession means in both groups and tendency of upper tooth loss in smokers and lower tooth loss in non-smokers. The findings of this study showed that clinical periodontal parameters may be different in smokers when compared to non-smokers and that masking of some periodontal signs can be a result of nicotine's vasoconstrictor effect.

Ultrastructural and immunohistochemical study of the influence of fluoride excess on the development of rat incisor tooth buds

Little information is available on the pathogenesis of fluorosis during the fetal and initial postnatal period. In the present study, female rats received 0 (control), 7 or 100 ppm of sodium fluoride in drinking water, one week before breeding and throughout gestation and nursing periods. The hemimandibles of the offspring were collected at 0, 7 and 14 days of postnatal life (n = 5) and processed for morphological analyses by light and electron microscopy, immunohistochemical analysis for amelogenin and morphometric study of enamel matrix and ameloblasts of incisors. The results showed a decrease in matrix production at the secretory phase at all study periods for the 100 ppm group. In this same group, the secretory ameloblasts showed reduction of enamel matrix secretion, disorganization of mitochondrial crests, large vacuoles at the apical portion of the cytoplasm, retention of intracisternal material and dilatation of some cisterns in the rough endoplasmic reticulum. In the groups of animals aged 7 and 14 days, analysis of variance showed significant reduction (p<0.05) in cytoplasmic volume of 23.80 percent and 24.75 percent, respectively, in relation to the control group. The smooth-ended maturation ameloblasts exhibited a large number of vacuoles with electron-dense endocytic matrix, suggesting a delay in the resorption process. Immunohistochemical analysis showed no difference in the intensity and labeling pattern of the enamel matrix in any study group. Interestingly, in offspring at the age of 14 days for the 7 ppm group, there was an increase in the matrix length at the secretory phase. Therefore, part of the excessive dose of sodium fluoride given to the mother in drinking water can reach the offspring through the placenta and mother's milk, causing morphological changes in ameloblasts and suggesting a reduction in secretion and a delay in matrix resorption.

Avaliação da resposta tecidual ao implante de osso bovino misto medular em subcutâneo de ratos / Evaluation of tissue repair, in case of bovine bone implantation mixed with bone marrow graft, in subcutaneous region of rats

O objetivo deste trabalho foi avaliar a resposta tecidual ao implante do osso bovino misto (OBM) acelular em subcutâneo de ratos. Blocos de OBM (10x10x5mm) foram implantados no tecido subcutâneo de 25 ratos machos Wistar, os quais foram mortos após 3, 7, 14, 30 e 60 dias (n = 5/período) da cirurgia. O tecido reacional foi coletado, fixado em formol 10% tamponado, lavado, desidratado, diafanizado em xilol e incluído em parafina. Cortes com 6µm de espessura foram corados com Hematoxilina e Eosina. A análise microscópica subjetiva indicou que o OBM foi circundado por tecido de granulação (composto de células mononucleares semelhantes a fibroblastos e polimorfonucleares) aos 3 dias. Entre 7 e 30 dias observou-se a invasão gradual dos poros do material implantado pelo tecido de granulação apresentando um discreto infiltrado inflamatório mononuclear com poucas células gigantes multinucleadas (CGM). Concomitantemente à angiogênese observa-se aparecimento de adipócitos no interior do material confirmando porosidade interconectada dos poros. Após 60 dias os espaços medulares estavam completamente ocupados por tecido de granulação composto por células semelhantes a fibroblastos e raras células inflamatórias mononucleares e CGM. Não se observou indícios de degradação ou absorção do material até o período de 60 dias. Considerando a ausência de necrose ou outros sinais de destruição tecidual, a presença de proliferação celular e angiogênese para o interior do material e que não se observou absorção do material, concluiu-se que o OBM é biocompatível e não absorvível, tendo potencial aplicação na engenharia de tecidos.

The aim of this study was to evaluate the tissue response to acellular mixed bovine bone graft (MBG) implanted in the subcutaneous tissue of rats. MBG in block (10x10x5mm) was implanted in 25 adult male Wistar rats, killed 3, 7, 14, 30 e 60 days after surgery (n=5/period). The granulation tissue was fixed in 10% buffered formalin, washed, dehydrated in ethanol, diafanized in xylene, and included in paraffin according to standard technique. Histological sections measuring 6µm in thickness were stained with haematoxylin and eosin. Histological analysis 3 days after surgery showed a granulation tissue around the MGB block presenting mononuclear cells like fibroblasts and neutrophils. From 7 to 30 days, chronic inflammatory infiltrate consisting mainly of macrophages and few infl ammatory multinucleated giant cells (MGC) were observed invading the porous of MGB. Parallel to angiogenesis, adipocytes were present in the interior of material, corroborating the presence of interconnected porous. After 60 days, medullar spaces were fulfilled with granulation tissue presenting fibroblast-like cells and rare mononuclear inflammatory cells and MGC. No signs of material degradation or absorption were noted throughout the experimental periods. Considering the absence of necrosis or other signs of tissue destruction, cell growth and angiogenesis inside the material and the absence of material absorption, it was concluded that MGB was biocompatible and non absorbable being potentially adequate for tissue engineering.

Avaliação histológica do implante do osso fetal bovino acelular e desmineralizado em subcutâneo de ratos / Histological evaluation of acellular and demineralized fetal bovine bone in the subcutaneous of rats

Objetivo: Avaliar a resposta tecidual ao implante do osso fetal bovino (FBB) acelular e desmineralizado em subcutâneo de ratos. Métodos: O FBB foi obtido em frigorífico local, transportado em gelo para o laboratório, onde foi lavado, tratado mecânica e quimicamente (álcalis e solventes orgânicos) para remoção de débris, células e sangue; a desmineralização do material foi obtida com ácido clorídrico 0,6M a 4°C e neutralização com tampão fosfato em pH 7,4). Foram utilizados 40 ratos machos Wistar divididos em dois grupos: teste e controle (n = 5). No grupo teste um bloco de FBB (1 x 1 x 1cm) foi implantado no tecido subcutâneo de cada animal. No grupo controle os procedimentos cirúrgicos foram idênticos aos do grupo teste, mas sem a colocação do material. Após 10, 20, 30 e 60 dias os animais foram mortos e as peças coletadas, fixadas em formol a 10 por cento tamponado, lavadas, desidratadas, diafanizadas em xilol e incluídas em parafina. Cortes com 6µm de espessura foram corados com hematoxilina-cosina. Resultado: A análise microscópica indicou que o osso fetal bovino foi circundado por tecido conjuntivo aos 10 dias. Entre 20 e 30 dias observou-se a substituição gradual do tecido conjuntivo em contato com o material implantado por tecido adiposo. No período de 60 dias notou-se acentuada diminuição das trabéculas ósseas do material implantado, sugerindo discreta absorção do material implantado e a substituição do tecido conjuntivo frouxo que circundava as partículas por tecido adiposo. Em nenhum dos períodos experimentais foram observados linfócitos ou plasmócitos no tecido reacional ou destruição tecidual. Raras áreas apresentaram células gigantes multinucleadas. A análise semiquantitativa das lâminas não demonstrou diferença significativa (p>0,05) entre os grupos controle e FBB com relação ao infiltrado inflamatório, presença de vasos e grau de fibrosamento. Conclusão: Considerando que o material não induziu destruição tecidual ou recrutamento de células do sistema imunológico, foi parcialmente absorvido e bem tolerado pelo tecido, os autores concluem que o osso fetal bovino foi biocompatível