Oral implant osseointegration model in C57Bl/6 mice: microtomographic, histological, histomorphometric and molecular characterization

Abstract Despite the successful clinical application of titanium (Ti) as a biomaterial, the exact cellular and molecular mechanisms responsible for Ti osseointegration remains unclear, especially because of the limited methodological tools available in this field. Objective: In this study, we present a microscopic and molecular characterization of an oral implant osseointegration model using C57Bl/6 mice. Material and Methods: Forty-eight male wild-type mice received a Ti implant on the edentulous alveolar crest and the peri-implant sites were evaluated through microscopic (μCT, histological and birefringence) and molecular (RealTimePCRarray) analysis in different points in time after surgery (3, 7, 14 and 21 days). Results: The early stages of osseointegration were marked by an increased expression of growth factors and MSC markers. Subsequently, a provisional granulation tissue was formed, with high expression of VEGFb and earlier osteogenic markers (BMPs, ALP and Runx2). The immune/inflammatory phase was evidenced by an increased density of inflammatory cells, and high expression of cytokines (TNF, IL6, IL1) chemokines (CXCL3, CCL2, CCL5 and CXC3CL1) and chemokine receptors (CCR2 and CCR5). Also, iNOS expression remained low, while ARG1 was upregulated, indicating predominance of a M2-type response. At later points in time, the bone matrix density and volume were increased, in agreement with a high expression of Col1a1 and Col21a2. The remodelling process was marked by peaks of MMPs, RANKL and OPG expression at 14 days, and an increased density of osteoclasts. At 21 days, intimate Ti/bone contact was observed, with expression of final osteoblast differentiation markers (PHEX, SOST), as well as red spectrum collagen fibers. Conclusions: This study demonstrated a unique molecular view of oral osseointegration kinetics in C57Bl/6 mice, evidencing potential elements responsible for orchestrating cell migration, proliferation, ECM deposition and maturation, angiogenesis, bone formation and remodeling at the bone-implant interface in parallel with a novel microscopic analysis.

Marcadores de remodelamiento óseo en pacientes con enfermedad periodontal / Bone remodeling biomarkers in patients with periodontal disease

La Fosfatasa Alcalina Ósea (FAO) es una isoforma de la Fosfatasa Alcalina (FAL). La medición de su actividad en saliva es una medida indirecta del proceso de formación ósea, más sensible y específica que la FAL. La catepsina K es la principal colagenasa del proceso de resorción ósea, es capaz de degradar al colágeno tipo I en varios sitios dando lugar a pequeños péptidos N- y C- terminales. El telopéptido C-terminal (CTx) es el marcador más sensible y específico en el aumento de la resorción ósea, ya que el colágeno tipo I constituye más del 90 por ciento de la matriz orgánica del hueso...

Avaliação das citocinas TNF-α, RANKL e OPG e do número de osteoclastos no reparo de defeito ósseo em calvária de ratos diabéticos tratados com matriz óssea desmineralizada / Evaluation of cytokines TNF-α, RANKL and OPG and the number of osteoclasts on repair of bone defects in skulls of diabetic rats treated with demineralized bone matrix

Neste trabalho, foi avaliado a participação dos osteoclastos bem como a ação das citocinas RANKL, OPG e TNF-α durante a formação e remodelação óssea em defeitos ósseos de tamanho crítico em ratos normoglicêmicos e diabéticos tratados ou não com a MAOD. Para isso, foram utilizados 250 ratos machos Wistar. Trinta ratos foram utilizados para coleta dos fêmures e tíbias, os quais foram processados para obtenção da MAOD. Os demais 220 ratos foram divididos em Grupo Não Diabétido (CTL, n=110) e Grupo Diabético (DIAB, n= 110) induzido pela aplicação de uma dose única de 47 mg/Kg de massa corporal de estreptozotocina. Um defeito transósseo de 8 mm de diâmetro foi realizado nos ossos parietais dos ratos, sendo que, nos subgrupos CTL MAOD e DIAB MAOD, os defeitos foram preenchidos com MAOD e nos grupos CTL COAG e DIAB COAG apenas com coágulo sanguíneo. Após 0, 7, 14, 21 e 42 dias, as calotas cranianas foram coletadas para determinação da densidade de volume, número de osteoclastos/mm2 na área do defeito, quantificação por imunoistoquimica e expressão do RNAm para as proteínas RANKL, OPG e TNF-α. Os resultados para volume do tecido ósseo neoformado foi maior nos grupos CTL COAG e CTL MAOD, bem como no grupo DIAB MAOD quando comparado com DIAB COAG (CTL MAOD > CTL COAG e DIAB MAOD > DIAB COAG). O número de osteoclastos nos grupos CTL aumentaram significantemente (3,69 osteoclasto/mm2), enquanto que nos grupos MAOD aumentaram gradualmente até os 42 dias (2,8 osteoclasto/mm2). Os resultados para imunomarcação mostraram que a MAOD promove 1,28 vezes maior expressão de OPG, bem como de TNF-α tanto no grupo CTL (1,59 vezes) como no DIAB (1,76 vezes). Os resultados para expressão do RNAm para OPG mostrou que a média dos valores do grupo COAG comparado com a do grupo MAOD foi 1,91 vezes maior no grupo COAG. Já os valores para expressão de RANKL permaneceram constantes no grupo DIAB MAOD, com aumento significativo de 2,57 vezes aos 42 dias, sendo 4,3 vezes maior, quando comparado com a média dos outros grupos no mesmo período. Conclui-se que nos animais normoglicemicos, o tratamento com a MAOD aumenta a expressão de OPG, RANKL e TNF-α, assim como a atividade osteoclástica, promovendo reabsorção da MAOD e formação de tecido ósseo, enquanto que nos animais diabéticos, a atividade osteoclástica foi reduzida, sem alteração nos níveis de OPG e RANKL, reduzindo a reabsorção da MAOD e consequentemente da formação óssea.(AU)

Participation of osteoclasts was evaluated in reabsorption process of demineralized allogenic bone matrix (DABM) as well as the activity of cytokines RANKL, OPG and TNF- α during formation and bone remodeling in critial size defect of normoglycemic and diabetic rats treated or not with DABM. Therefore, 250 male Wistar rats were used. Thirty rats had femurs and tibias collected and processed to obtain DABM. 220 rats were divided into control group (CTL, n=110) and diabetic group (DIAB, n= 110) injected by a single dose of 47 mg/Kg of body weight streptozotocin. Were made 8mm bone defect on skulls of rats, in subgroups CTL DABM and DIAB DABM, defects were filled with DABM and subgroups CTL CLOT and DIAB CLOT were filled with blood clot. After 0, 7, 14, 21 and 42 days, the skulls were collected to determine the volume density, number of osteoclasts/mm2 into defects area, quantification by immunohistochemistry and RNAm expression of RANKL, OPG and TNF-α cytokines. The results of volume density of newly formed bone was higher in CTL CLOT and CTL DABM, as well as in DIAB DABM compared to DIAB CLOT (CTL DABM > CTL CLOT and DIAB DABM > DIAB CLOT). The number of osteoclasts in CTL groups increased to 3,69 osteoclasts/mm2, while in subgroups treated with DABM gradually increased up until 42 days (2,8 osteoclasts/mm2). Immunohistochemistry showed that DABM promotes an increase of 1.28-fold of OPG expression, as well as TNF-a expression in CTL group (1.59-fold) and DIAB group (1.76-fold). The results of RNAm expression of OPG showed that the average values of the CLOT subgroup compared to the average values of DABM subgroup was 1.91- fold higher in CLOT subgroup. The values of RANKL RNAm expression increase 2.57-fold at 42 days, being 4.3-fold higher than the average os the other groups in the same period. In conclusion, in the normoglicemic animals (CTL group), the treatment with DABM increase the expression of OPG, RANKL and TNF-α as the activity of osteoclasts, leading to DABM resorption and bone tissue formation, while in diabetic animals, the osteoclast activity was reduced, without changes in the leves of OPG and RANKL, decreasing DABM resorption and bone formation.(AU)

Mini-implants and miniplates generate sub-absolute and absolute anchorage

The functional demand imposed on bone promotes changes in the spatial properties of osteocytes as well as in their extensions uniformly distributed throughout the mineralized surface. Once spatial deformation is established, osteocytes create the need for structural adaptations that result in bone formation and resorption that happen to meet the functional demands. The endosteum and the periosteum are the effectors responsible for stimulating adaptive osteocytes in the inner and outer surfaces.Changes in shape, volume and position of the jaws as a result of skeletal correction of the maxilla and mandible require anchorage to allow bone remodeling to redefine morphology, esthetics and function as a result of spatial deformation conducted by orthodontic appliances. Examining the degree of changes in shape, volume and structural relationship of areas where mini-implants and miniplates are placed allows us to classify mini-implants as devices of subabsolute anchorage and miniplates as devices of absolute anchorage.

Uma demanda funcional sobre o osso promove alterações na forma espacial da rede de osteócitos e seus prolongamentos, distribuídos uniformemente na estrutura mineralizada. A partir da deformação espacial captada, os osteócitos comandam a necessidade de adaptações estruturais, formando osso em novas áreas e reabsorvendo em outras, para que sejam atendidas as demandas funcionais. O endósteo e o periósteo são os verdadeiros efetores desses estímulos osteocíticos adaptativos, nas superfícies internas e externas. As alterações de forma, volume e posição dos ossos maxilares, nas correções esqueléticas da maxila e mandíbula, requerem uma ancoragem para que a remodelação óssea redefina a morfologia, a estética e as funções, a partir de deformações espaciais dirigidas por aparelhos. Verificar o grau de alterações na forma, volume e relações estruturais das áreas onde se fixaram os mini-implantes e as miniplacas poderá levar à classificação dos mini-implantes como dispositivos de ancoragem subabsoluta e as miniplacas, como de ancoragem absoluta.

The Effect of Poloxamer 407-Based Hydrogel on the Osteoinductivity of Demineralized Bone Matrix

BACKGROUND: Demineralized bone matrix (DBM) is used for bone healing due to its osteoinductivity, but it requires a carrier for clinical application. Here, we report the effects on the osteoinductivity of DBM by use of a poloxamer 407-based hydrogel as the carrier, compared to sterile water. METHODS: DBM-W and DBM-H represent 27 wt% of DBM with sterile water and DBM with a poloxamer 407-based hydrogel, respectively. Both of the compositions were applied to human mesenchymal stem cell (MSC) cultures, and monitored for alkaline phosphatase (ALP) staining and ALP activity. Six 10-week-old athymic nude rats were used for abdominal muscle grafting with either DBM-W or DBM-H, and were tested by plane radiography, microfocus X-ray computed tomography (CT), and decalcified histology to evaluate ectopic bone formation. RESULTS: The DBM-W group showed stronger ALP staining at 7, 14, and 21 days of treatment, and significantly higher ALP activity at 7 and 14 days of treatment, compared to the DBM-H group. Plane radiography could not confirm the radio-opaque lesions in the rat ectopic bone formulation model. However, ectopic bone formation was observed in both groups by micro-CT. Compared to the DBM-H group, the DBM-W group showed higher bone volume, percent bone volume and trabecular number, and the difference in percent bone volume was statistically significant. Decalcified histology found bony tissue with lamellation in both groups. CONCLUSIONS: Our results suggest that poloxamer 407-based hydrogel has efficacy as a DBM carrier since it shows ectopic bone formation, but its effects on the quality and quantity of osteoblastic differentiation in rat abdominal ectopic bone and MSC are considered negative.

Comparable bone healing capacity of different bone graft matrices in a rabbit segmental defect model

We compared the bone healing capacity of three different demineralized bone matrix (DBM) products applied using different carrier molecules (hyaluronic acid [HA] vs. carboxymethylcellulose [CMC]) or bone compositions (cortical bone vs. cortical bone and cancellous bone) in a rabbit segmental defect model. Overall, 15-mm segmental defects in the left and right radiuses were created in 36 New Zealand White rabbits and filled with HA-based demineralized cortical bone matrix (DBX), CMC-based demineralized cortical bone matrix (DB) or CMC-based demineralized cortical bone with cancellous bone (NDDB), and the wound area was evaluated at 4, 8, and 12 weeks post-implantation. DBX showed significantly lower radiopacity, bone volume fraction, and bone mineral density than DB and NDDB before implantation. However, bone healing score, bone volume fraction, bone mineral density, and residual bone area at 4, 8, and 12 weeks post-implantation revealed no significant differences in bone healing capacity. Overall, three DBM products with different carrier molecules or bone compositions showed similar bone healing capacity.

Particulate bone matrix usage for alveolar bone conservation. A histomorphometric study / Particulate bone matrix usage for alveolar bone conservation. A histomorphometric study.

UNLABELLED: Different filling materials have been used in an attempt to repair bone loss situations. OBJECTIVE: The present study aimed to examine the effect of a bone matrix in post - extraction remodelling of the alveolar bone, and to perform a histomorphometric analysis of the residual alveolar ridges in Wistar rats. MATERIAL AND METHODS: Both rat first lower molars were extracted and the right alveoli were filled with particles of a bone matrix with mineral components (MO - UNC) (experimental group, EG). The left alveoli were used as a control group (CG). The animals were sacrificed at 0 hr., 15, 30 and 60 days after extraction, and the samples were processed. Histological sections were made at the level of the mesial alveolus of the first lower molar. Repair of the alveoli was histologically evaluated and a histomorphometric study of total alveolar volume (TAV), height of the buccal plate (Bh), height of the lingual plate (Lh) and percentage of osseointegration (OI) of the particles was performed to compare the residual ridges of CG with those of the EG. Statistical analysis of the data was performed. RESULTS: In the cases of the experimental group, newly - formed bone tissue was identified around the MO - UNC particles (osseointegration). Histomorphometric data indicate that, at 60 days post - extraction, TAV was significantly greater for EG when compared with CG (p <0.05) and the percentage of osseointegration of the particles increased as a function of time (57.6

, for EG at 15, 30 y 60 days respectively). CONCLUSIONS: The bone matrix (MO - UNC) evaluated in this study is an osteoconductive material that prevents the collapse of post - extraction alveolar bone.

Cadmium chloride toxicity suppresses osteogenic potential of rat bone marrow mesenchymal stem cells through reducing cell viability and bone matrix mineralization.

Background and Objectives: Cadmium an environmental pollutant, exert several risks to human health. In this study we investigated the effect of cadmium chloride (CdCl 2 ) on Viability, morphology and bone Matrix Miniralization of Rat Bone Marrow Mesenchymal Stem Cells (rMSCs). Materials and Methods: rMSCs were cultured in DMEM containing 15% FBS and pen-strep. After 21 days of treatment with the selected doses of 750 and 2000 nM of CdCl 2 viability, colony forming unit, population doubling number, DAN breakage and the morphology of the cells were studied. Also to study the effects of CdCl2 on differentiation property, the morphology and bone matrix mineralization via estimation of intracellular calcium concentration and quantitative alizarin red were also evaluated in the cells using Hoechst, Acridine orange and Alizarin red staining. Data was analyzed using one-way ANOVA and Tukey ' s test and the means difference was considered significant at P<0.05. Results: The mean viability, colony forming unit, population doubling number and also the mean bone matrix mineralization of the rMSCs treated with CdCl 2 significantly reduced in a dose dependent manner. Nuclear fragmentation and cytoplasm shrinkage was also seen in the treated cells. Conclusion: CdCl 2 can reduce the viability and bone matrix mineralization of rMSCs even at low doses.

Influence of the association between simvastatin and demineralized bovine bone matrix on bone repair in rats

Simvastatin, a drug used to lower blood cholesterol, has been reported to have an anabolic effect on bone. The purpose of this study was to evaluate the influence of simvastatin and demineralized bovine bone matrix (DBBM) on the repair of rat calvarial defects. Defects of 5 mm were created in 64 rats, divided into four groups: no local treatment (control); treatment with DBBM (DBBM); treatment with a combination of simvastatin solution (2.2 mg/50 μl) and DBBM (DBBMSIM-1); and treatment with simvastatin solution (0.5 mg/50 μl) and DBBM (DBBMSIM-2). Animals were sacrificed on postoperative day 30 or 60, after which the calvariae were X-rayed and prepared for histomorphometric evaluation. The data were submitted to statistical analysis (p < 0.05). X-rays revealed that, on postoperative day 30, animals treated with a lower dose of simvastatin presented the lowest bone density, whereas on postoperative day 60 the use of simvastatin, regardless of the dose, resulted in lower density than that observed in control and DBBM group samples. Histomorphometric analysis revealed that, on postoperative day 30, both DBBM and DBBMSIM-1 had a negative impact on bone formation. On postoperative day 60, none of the combinations tested impaired bone repair. These results showed that the association between DBBM and simvastatin had a negative impact on bone repair.

Avaliação do reparo de defeito ósseo em calvária de ratos diabéticos tratados com matriz óssea desmineralizada / Evaluation of repair of bone defects in skulls of diabetic rats treated with demineralized bone matrix

O objetivo deste trabalho foi avaliar as atividades osteoindutoras e osteocondutoras da matriz alogênica óssea desmineralizada (MAOD) frente à diabetes no reparo de defeito de tamanho crítico em calvárias de ratos diabéticos. Para isso, 100 ratos machos Wistar foram divididos em 2 grupos: no grupo diabético (DIAB, n=50) foi injetado 47 mg/Kg de massa corporal de estreptozotocina, enquanto que no grupo controle (CTL, n=50) foi injetado solução fisiológica a 0,9%. A MAOD foi obtida de 50 ratos, cujo fêmur e tíbia foram retirados, desmineralizados com HCl a 0,6M por 24 horas, particulados em 1-2mm³, neutralizados com soro fisiológico e armazenados em álcool. Após a anestesia, foram realizados defeitos ósseos de 8 mm nas calvárias dos animais, sendo os grupos CTL COAG (n=25) e DIAB COAG (n=25) preenchidos com coágulo e os grupos CTL MAOD (n=25) e DIAB MAOD (n=25) preenchidos com MAOD. Após os períodos de 0, 7, 14, 21 e 42 dias, as calvárias foram coletadas. A análise radiográfica mostrou que houve formação de ilhas radiodensas no interior dos defeitos nos grupos CTL e DIAB tratados com MAOD, enquanto que nos grupos tratados com coágulo houve formação de áreas mais radiodensas somente nas bordas do defeito, corroborando com os resultados morfológicos, que mostraram nos grupos tratados com coágulo que o reparo ósseo teve início nas bordas do defeito, enquanto que nos grupos tratados com MAOD, a neoformação óssea ocorreu também nas áreas de reabsorção nas partículas de MAOD. De acordo com os resultados morfométricos, o volume de tecido ósseo aumentou gradativamente em todos os grupos, porém, esse aumento foi maior nos grupos CTL em relação aos seus respectivos tratamentos nos grupos DIAB (CTL COAG > DIAB COAG e CTL MAOD > DIAB MAOD) e maior quando comparados os grupos tratados com MAOD versus os respectivos grupos tratados com COAG (CTL MAOD > CTL COAG e DIAB MAOD > DIAB COAG). Assim, ao término de 42 dias, o volume de tecido ósseo no grupo CTL MAOD foi...

The aim of this work was to evaluate the osteoinductive and osteoconductive activities of demineralized allogeneic bone matrix (DABM) against diabetes in repairing critical size defects in diabetic rats skulls. Therefore, 100 male Wistar rats were shered into two groups: in the diabetic group (DIAB, n=50) was 47 mg/Kg of body weight streptozotocin, while in the control group (CTL, n=50) was injected saline 0.9%. The DABM was obteined using 50 rats which were removed their femur and tibia bones, demineralized in 0.6 N HCl during 24 hours, cut into 1-2mm³ pieces, neutralized in saline and stored in alcohol. After anesthesia, were made 8 mm bone defects on skulls of rats, being the CTL CLOT group (n=25) and DIAB CLOT group (n=25) filled with blood clot and the CTL DABM group (n=25) and DIAB DABM group (n=25) filled with DABM. After 0, 7, 14, 21 and 42 days, the skulls were collected. The radiographic analysis showed radiodense islets inside the defects filled with DABM in CTL and DIAB groups, while groups filled with blood clot showed radiodense areas near the defect border, which is in agreement to the morphologic results, that had showed the begining of bone healing was near the defects border in groups filled with blood clot, while groups filled with DABM showed new bone formation also in resorption DABM areas. According to morphometric results, the volume of bone tissue had increased in all groups, however, this increase was more accentuated in CTL groups when compared to DIAB groups with respected treatments (CTL CLOT > DIAB CLOT and CTL DABM > DIAB DABM) and bigger when groups treated with DABM are compared to respestive groups treated with CLOT (CTL DABM > CTL CLOT e DIAB DABM > DIAB CLOT). Thereby, at the end of 42 days, the CTL DABM bone tissue volume was 3.24 greater than the other groups, the CTL CLOT and DIAB DABM groups didnt show any significant differenceand the DIAB DABM was 1,81 greater than DIAB CLOT. From these results, the conclusion...

Ação da matriz óssea bovina desmineralizada na neoformação óssea em ratos submetidos ao alcoolismo experimental: avaliação histológica e histométrica / Action of demineralized bovine bone matrix on bone neoformation in rats submitted to experimental alcoholism: histological and histometric evaluation

O etanol inibe a proliferação de células osteoblásticas, gerando baixa massa óssea e aumento na prevalência de fraturas na população alcoólatra. A quantidade de defeitos ósseos criados cirurgicamente, e pelos vários tipos de acidentes, tem aumentado atualmente e existe uma preocupação muito grande na descoberta de substâncias que acelerem a neoformação óssea que preencham essas cavidades. Baseado no exposto anteriormente resolveu-se realizar este trabalho com o objetivo de observar se a matriz óssea bovina desmineralizada (Genox ®) altera a neoformação óssea em ratos submetidos ao alcoolismo experimental, usando para isso análise histológica e histométrica. Para isso foram utilizados 40 ratos machos (Rattus norvegicus), separados em 2 grupos de 20 animais cada, assim distribuídos: Grupo E1, que recebeu álcool etílico a 25%, diluído em água de torneira, e cavidade cirúrgica preenchida somente por coágulo sanguíneo, e Grupo E2, que recebeu álcool etílico a 25%, diluído em água de torneira, e cavidade cirúrgica preenchida por Gen-ox®. Após um período de 3 semanas de adaptação gradativa ao álcool, os animais receberam dieta alcoólica de 25% por um período de 90 dias. Decorrido esse período, a tíbia esquerda de todos os animais foi submetida a uma cirurgia onde se produziu uma cavidade cirúrgica experimental, que no Grupo E1 ficou preenchida por coágulo sanguíneo, e no Grupo E2 preenchida por Gen-ox®. Cinco animais de cada grupo foram sacrificados em períodos de 10, 20, 40 e 60 dias contados a partir do dia da cirurgia experimental, para retirada de parte da tíbia, onde a cavidade cirúrgica foi realizada. Os blocos retirados foram processados histologicamente e submetidos à coloração por Tricrômico de Masson, para estudo histomorfológico e histométrico da área total do defeito, quantidade de tecido conjuntivo presente e quantidade de tecido ósseo neoformado. Os resultados mostraram que a reorganização da medula óssea e reparação total da...

Ethanol inhibits the proliferation of osteoblastic cells, leading to low bone mass and increased prevalence of fractures in the alcoholic population. The amount of bone defects surgically created, and various types of accidents has increased and there is currently a great concern in the discovery of substances that accelerate new bone formation to fill those cavities. Based on the foregoing it was decided to undertake this work in order to see whether demineralized bovine bone (Gen-ox®) alters bone formation in rats subjected to experimental alcoholism, using it for histological and histometric analysis. For this we used 40 male rats (Rattus norvegicus) separated in two groups of 20 animals each one, distributed as follows: Group E1, which received 25% ethanol, diluted in tap water, and the surgical cavity filled only by a blood clot, and Group E2, which received 25% ethanol, diluted in tap water, and the surgical cavity filled with Gen-ox®. After a period of three weeks of gradual adaptation to alcohol, the animals received 25% alcohol diet for a period of 90 days. After this period, the left tibia of all animals underwent a surgery where it produced an experimental surgical cavity, which in Group E1 was filled by blood clot, and in Group E2 filled with Gen-ox®. Five animals from each group were sacrificed on days 10, 20, 40 and 60 days from the day of experimental surgery to remove part of the tibia, where the sinus surgery was done. The blocks were removed and processed histologically stained by Masson trichrome, for histomorphological and histometric study of the total area of the defect, amount of connective tissue and amount of new bone. The results showed that the reorganization of the bone marrow and full repair of the surgical cavity in Group E1 had occurred in a shorter time than in Group E2. It was also noted that in the final period, the animals in Group E2 showed areas of connective tissue and thick bone...

Ação da matriz óssea bovina desmineralizada na neoformação óssea em ratos submetidos ao alcoolismo experimental: avaliação histológica e histométrica / Action of demineralized bovine bone matrix on bone neoformation in rats submitted to experimental alcoholism: histological and histometric evaluation

O etanol inibe a proliferação de células osteoblásticas, gerando baixa massa óssea e aumento na prevalência de fraturas na população alcoólatra. A quantidade de defeitos ósseos criados cirurgicamente, e pelos vários tipos de acidentes, tem aumentado atualmente e existe uma preocupação muito grande na descoberta de substâncias que acelerem a neoformação óssea que preencham essas cavidades. Baseado no exposto anteriormente resolveu-se realizar este trabalho com o objetivo de observar se a matriz óssea bovina desmineralizada (Genox ®) altera a neoformação óssea em ratos submetidos ao alcoolismo experimental, usando para isso análise histológica e histométrica. Para isso foram utilizados 40 ratos machos (Rattus norvegicus), separados em 2 grupos de 20 animais cada, assim distribuídos: Grupo E1, que recebeu álcool etílico a 25%, diluído em água de torneira, e cavidade cirúrgica preenchida somente por coágulo sanguíneo, e Grupo E2, que recebeu álcool etílico a 25%, diluído em água de torneira, e cavidade cirúrgica preenchida por Gen-ox®. Após um período de 3 semanas de adaptação gradativa ao álcool, os animais receberam dieta alcoólica de 25% por um período de 90 dias. Decorrido esse período, a tíbia esquerda de todos os animais foi submetida a uma cirurgia onde se produziu uma cavidade cirúrgica experimental, que no Grupo E1 ficou preenchida por coágulo sanguíneo, e no Grupo E2 preenchida por Gen-ox®. Cinco animais de cada grupo foram sacrificados em períodos de 10, 20, 40 e 60 dias contados a partir do dia da cirurgia experimental, para retirada de parte da tíbia, onde a cavidade cirúrgica foi realizada. Os blocos retirados foram processados histologicamente e submetidos à coloração por Tricrômico de Masson, para estudo histomorfológico e histométrico da área total do defeito, quantidade de tecido conjuntivo presente e quantidade de tecido ósseo neoformado. Os resultados mostraram que a reorganização da medula óssea e reparação total da...

Ethanol inhibits the proliferation of osteoblastic cells, leading to low bone mass and increased prevalence of fractures in the alcoholic population. The amount of bone defects surgically created, and various types of accidents has increased and there is currently a great concern in the discovery of substances that accelerate new bone formation to fill those cavities. Based on the foregoing it was decided to undertake this work in order to see whether demineralized bovine bone (Gen-ox®) alters bone formation in rats subjected to experimental alcoholism, using it for histological and histometric analysis. For this we used 40 male rats (Rattus norvegicus) separated in two groups of 20 animals each one, distributed as follows: Group E1, which received 25% ethanol, diluted in tap water, and the surgical cavity filled only by a blood clot, and Group E2, which received 25% ethanol, diluted in tap water, and the surgical cavity filled with Gen-ox®. After a period of three weeks of gradual adaptation to alcohol, the animals received 25% alcohol diet for a period of 90 days. After this period, the left tibia of all animals underwent a surgery where it produced an experimental surgical cavity, which in Group E1 was filled by blood clot, and in Group E2 filled with Gen-ox®. Five animals from each group were sacrificed on days 10, 20, 40 and 60 days from the day of experimental surgery to remove part of the tibia, where the sinus surgery was done. The blocks were removed and processed histologically stained by Masson trichrome, for histomorphological and histometric study of the total area of the defect, amount of connective tissue and amount of new bone. The results showed that the reorganization of the bone marrow and full repair of the surgical cavity in Group E1 had occurred in a shorter time than in Group E2. It was also noted that in the final period, the animals in Group E2 showed areas of connective tissue and thick bone...

Proteoliposomes as matrix vesicles' biomimetics to study the initiation of skeletal mineralization

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite (HA) seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Ion transporters control the availability of phosphate and calcium needed for HA deposition. The lipidic microenvironment in which MV-associated enzymes and transporters function plays a crucial physiological role and must be taken into account when attempting to elucidate their interplay during the initiation of biomineralization. In this short mini-review, we discuss the potential use of proteoliposome systems as chondrocyte- and osteoblast-derived MVs biomimetics, as a means of reconstituting a phospholipid microenvironment in a manner that recapitulates the native functional MV microenvironment. Such a system can be used to elucidate the interplay of MV enzymes during catalysis of biomineralization substrates and in modulating in vitro calcification. As such, the enzymatic defects associated with disease-causing mutations in MV enzymes could be studied in an artificial vesicular environment that better mimics their in vivo biological milieu. These artificial systems could also be used for the screening of small molecule compounds able to modulate the activity of MV enzymes for potential therapeutic uses. Such a nanovesicular system could also prove useful for the repair/treatment of craniofacial and other skeletal defects and to facilitate the mineralization of titanium-based tooth implants.

effect of leptin on the osteoinductive activity of recombinant human bone morphogenetic protein-2 in nude mice

To investigate the effect of leptin in the model of ectopic bone formation that utilizes subcutaneously implanted recombinant human bone morphogenetic protein-2 [rhBMP-2]-containing type I collagen discs. This study was performed in the Clinical Research Center, Southern Medical University, Guangzhou, China from November 2008 to June 2009. A single dose of 20 micro l saline [group A], 20 micro g leptin [group B], 2 micro g rhBMP-2 [group C], 2 micro g rhBMP-2 + 20 micro g leptin [group D], and type I collagen disks as a carrier were mixed, and subcutaneouly implanted into the back of nude mice [n=12]. The effect of ectopic bone formation was evaluated by radiography, dual energy X absorptiometry, biochemical examination of alkaline phosphatase [ALP] activity, histological observation, and semi-quantitative evaluation 4 and 8 weeks after surgery. At 4 and 8 weeks after operation the radiographs, bone mass density, ALP, and histology values showed significant intragroup and intergroup differences, with those at 8 weeks being higher than those at 4 weeks. Our results indicated that the sustainedly released material with collagen as a carrier combined treatment with leptin and rhBMP-2 has a very good osteoinductive activity. Leptin is a positive modulator for the osteoinductive efficacy of BMPs, they have synergistic effect. Its mechanisms are probably related to promote the formation of new-vessels and proliferation/ differentiation of many kinds of cells

El periostio: una alternativa más para obtener buena calidad de matriz ósea orgánica / The periosteum: another alternative to obtain good quality organic bone matrix

Se estudiaron las propiedades osteogénicas del periostio y de los osteoblastos colocados a modo de injerto en cavidades óseas. Se evaluaron aspectos experimentales y sus resultados transferidos a la clínica. En la faz experimental, se emplearon conejos machos de 4.5 kg y de un año de edad, a los que se lesinjertó periostio en cavidades óseas artificiales talladas a tal fin, con su respectivos controles en cada una de las extremidades traseras. El seguimiento radiográfico, la histología convencional y el análisis computarizado de las densidades óseas corroboraron la mayor formación en cantidad de tejido óseo en las cavidades problemas. La investigación clínica permitió establecer la evolución de las cavidades control y problema sin que se experimentara rechazo del injerto aplicado a modo de rollo y a cielo cerrado. Los resultados clínicos y radiográficos demostraron, a través de la comparación de las radiodensidades de las cavidades controles y problemas, que la curación de las heridas óseas ocurre mucho antes que se hagan visibles en las radiografías, ya que la densidad del hueso maduro no es la misma que el hueso osteoide que se está formando.

Hacia el conocimiento molecular del metabolismo óseo: una revisión del actual estado del arte / Toward the molecular understanding of bone metabolism: a review of the state-of-the-art

El tejido óseo posee características propias, tanto histológicas como fisológicas, que lo diferencian como un tejido altamente complejo, por sus propiedades de regulación mediante el mecanismo de formación-reabsorción; está comandado por hormonas como la paratiroidea, el péptido relacionado con la hormona paratiroidea, la vitamina D3, estrógenos, andrógenos, glucocorticoides, hormonas tiroideas y del crecimiento, entre otras. La matriz ósea está compuesta por dos constituyentes principales: la matriz orgánica y las sales inorgánicas. La matriz orgánica tiene como componente principal el colágeno tipo I, así como los tipos II y III, y por lo menos 12 proteínas no colágenas, a cuyas mutaciones se les atribuye la causa de algunos sindromes. Se han estudiado la glicoproteína acídica ósea, la osteocalcina, osteopontina, osteonectina, sialoproteína ósea, con propiedades y formaciones específicas para la síntesis y metabolismo del tejido óseo. La matriz inorgánica se compone de cantidades de citrato, carbonato e hidroxiapatita. La interacción molecular de estos componentes conduce a la mineralización del tejido con el concurso de componentes celulares como el osteoblasto y el osteoclasto. La proliferación y la maduración de la matriz preceden a la etapa de mineralización que se regula por actividades esencialmente hormonales. Los procesos de osteoclastogénesis y osteoblastogénesis se modulan molecularmente por la osteoprotegerina, que inhibe el desarrollo de osteoclastos bloqueando la molécula RANKL, factor de diferenciación osteoclástica con un importante papel en la reabsorción ósea. El aspecto molecular de la osteoblastogénesis es menos conocido, y hasta el momento sólo se conocen las proteínas morfogenéticas que participan en la diferenciación osteoblástica

Sensibilidad de los marcadores del remodelamiento óseo: su modificación en la menopausia, ante la terapia estrogénica de reemplazo y ante una enfermedad metabólica generalizada / Markers of bone turnover, their sensibility to bone remodeling: changes in menopause, in hormone replacement therapy and in a metabolic bone disease

Evalua una batería de marcadores bioquímicos específicos y sensibles para predecir cambios en la velocidad del remodelamiento óseo. Se estudió a mujeres sanas pre y postmenospáusicas y en estas últimas a su vez se evaluó los cambios producidos después de 90 días de una terapia hormonal de reemplazo. Respecto de los marcadores bioquímicos de formación, la FA total aumentó en las postmenospáusicas respecto del grupo premenospáusico (P < 0,0001), al igual que la BGP (1 < 0,01); en cambio, la FA ósea no registró cambios significativos. Luego de la terapia hormonal de reemplazo ninguno de estos marcadores registraron cambios significativos. Todos los marcadores de resorción aumentaron en als mujeres postmenospáusicas. Los convencionales tales como el calcio urinario/creatinina y la hidroxiprolina con un p < 0,01 y p < 0,006 respectivamente. Los nuevos marcadores de resorción presentaron los siguientes cambios: Pyr p < 0,001; 72 por ciento; D-Pyr p < 0,003, 27 por ciento y Crosslaps p < 0,003, 70 por ciento de aumento respectivamente. Ante la terapia estrogénica, si bien los marcadores convencionales no mostraron diferencias significativas respecto del nivel basal, la Pyr disminuyó significativamente en un 15 por ciento (p < 0,03), la D-Pyr en un 15 por ciento (p < 0,04) y los Crosslaps en un 39 por ciento (p < 0,001). También se investigó la precisión diagnóstica de los marcadores bioquímicos en pacientes con un remodelamiento óseo aumentado, como es el caso de enfermedades celíacas. Estas se compararon con los normales que presentaban igual estado estrogénico observándose distintos comportamientos entre pre y postmenospáusicas. La BGP aumentó sólo en las celíacas premenospáusicas (p < 0,003). La FA total en los dos grupos estrogénicos (p < 0,0001 y p < 0,005 respectivamente), al igual que la FA ósea (p < 0,0003 y p< 0,0004, respectivamente). El marcador de resorción D-Pyr no presentó diferencias significativas en ninguno de los dos grupos, la Pyr sílo en las premenopáusicas (p < 0,01). La hidroxiprolina aumentó en ambos (p < 0,04 y p < 0,004, respectivamente), al igual que los Crosslaps (p < 0,001 para los dos grupos estrogénicos)

Bone homeostasis in type II diabetes mellitus

This study was conducted on 80 cases of adult onset diabetes mellitus [type II DM]. There was significant increase in serum calcium, alkaline phosphatase, albumin, urea and creatinine. This was associated with high serum insulin level and low T3 levels which were measured by radioimmunoassay. The study showed that DM causes changes in mineral and calcium homeostasis but these changes were not related to serum glucose level, serum insulin level or the duration of DM. The decreased serum T3 and elevated serum reverse T3 [r T3] were found to progress towards normalization upon achievement of good metabolic control