Correlation between ultrasound velocity and densitometry in fresh and demineralized cortical bone

OBJECTIVE: To compare ultrasound propagation velocity with densitometry in the diaphyseal compact cortical bone of whole sheep metatarsals. METHODS: The transverse ultrasound velocity and bone mineral density of 5-cm-long diaphyseal bone segments were first measured. The bone segments were then divided into four groups of 15 segments each and demineralized in an aqueous 0.5 N hydrochloric acid solution for 6, 12, 24 or 36 hours. All measurements were repeated after demineralization for each time duration and the values measured before and after demineralization were compared. RESULTS: Ultrasound velocity and bone mineral density decreased with demineralization time, and most differences in the pre- and post-demineralization values within each group and between groups were significant: A moderate correlation coefficient (r=0.75956) together with a moderate agreement was determined between both post-demineralization parameters, detected by the Bland-Altman method. CONCLUSION: We conclude that both ultrasound velocity and bone mineral density decrease as a result of demineralization, thus indicating that bone mineral content is of great importance for maintaining the acoustic parameters of cortical bone, as observed for cancellous bone. Ultrasound velocity can be used to evaluate both compact cortical bone quality and bone mineral density.

Efeito da desmineralização óssea sobre parâmetros de superfície e sobre o comportamento de pré-osteoblastos em cultura: estudo em microscopia eletrônica de varredura e confocal / Effect of bone demineralization on surface parameters and on behavior of pre-osteoblasts in culture: study in scanning electron microscopy and confocal microscopy

A desmineralização óssea superficial tem se demonstrado favorável à consolidação de enxertos e ao comportamento celular, entretanto os mecanismos envolvidos ainda não estão esclarecidos. Os subsídios para o embasamento biológico da desmineralização, proporcionado por publicações anteriores, sugeriram que modificações na superfície óssea teriam influenciado o comportamento de pré-osteoblastos em cultura. Assim, este estudo objetivou comparar o efeito de duas concentrações de ácido cítrico na desmineralização de superfícies ósseas onde foram cultivadas células pré-osteoblásticas (MC3T3-E1), e analisar parâmetros de superfície comparando superfícies desmineralizadas a não desmineralizadas. Setenta amostras ósseas bicorticais foram removidas das calvárias de 35 ratos e divididas em grupos para as análises: 1) Microscopia Eletrônica de Varredura (MEV) para avaliação da área de recobrimento e espessura da camada de células sobre as amostras (n = 15) durante 24, 48 e 72 horas: Grupo AC.10 – amostras desmineralizadas por 30 segundos com ácido cítrico 10 %; Grupo AC.50 –amostras desmineralizadas por 30 segundos com ácido cítrico 50 %; e Grupo C (controle) – amostras não desmineralizadas; 2) Microscopia Confocal para análise da área de expressão e intensidade de fluorescência das BMP-2, -4 e -7: AC.10 – seis amostras desmineralizadas conforme item 1); AC.50 – seis amostras desmineralizadas conforme item 1); C – três amostras não desmineralizadas; 3) Microscopia Confocal para análise da rugosidade superficial média (Ra e Sa): Grupos AC.10 e AC.50 com cinco amostras cada, desmineralizadas conforme o item 1), sendo cada amostra seu próprio controle (análises antes e depois da desmineralização). Também foram avaliadas as distâncias entre picos (P-P) e entre picos e vales (P-V) antes e depois da desmineralização; 4) Microscopia Eletrônica de Varredura / Espectroscopia de Energia Dispersiva (MEV / EDS) para análise da composição superficial: mesmas...

The superficial bone demineralization has proved to be a favorable procedure for bone grafts consolidation and cell behavior, however the underlying mechanisms have not been clarified yet. Therefore, this study aimed to compare the effect of two concentrations of citric acid on demineralization of bone surfaces where pre-osteoblastic cells (MC3T3-E1) were cultivated, and analyze surface parameters comparing demineralized bone surfaces with non-demineralized surfaces. Seventy bicortical bone samples were harvested from the calvaria of 35 rats and divided into groups as follows: 1) Scanning Electron Microscopy (SEM) to evaluate the coating area and thickness of cells layers cultured on the samples (n = 15) for 24, 48, and 72 hours: Group CA.10 – samples demineralized for 30 seconds with 10 % citric acid; Group CA.50 – samples demineralized for 30 seconds with 50 % citric acid, and Group C (control) – non-demineralized samples; 2) Confocal Microscopy for analysis of expression area and intensity of fluorescence of BMP-2, -4, and -7: CA.10 – six samples demineralized as item 1); CA.50 – six samples demineralized as item 1); Group C – three non-demineralized samples; 3) Confocal Microscopy for surface mean roughness analysis (Ra and Sa): Groups CA.10 and CA.50 made up of five samples each and demineralized according to item 1), each sample was its own control (analysis before and after demineralization). The distances between peaks (P-P) and between peaks and valleys (P-V) were also evaluated before and after demineralization; 4) Scanning Electron Microscopy / Energy Dispersive Spectroscopy (SEM / EDS) to analyze the surface composition: the same samples of item 3) were evaluated before and after demineralization for atomic percentage (%A) of carbon, oxygen, magnesium, phosphorus, sulfur and calcium. Statistical test was made by adopting the 95 % significance level. Demineralized samples showed cells with morphology in the later stages of differentiation...

Efeito da desmineralização óssea sobre parâmetros de superfície e sobre o comportamento de pré-osteoblastos em cultura: estudo em microscopia eletrônica de varredura e confocal / Effect of bone demineralization on surface parameters and on behavior of pre-osteoblasts in culture: study in scanning electron microscopy and confocal microscopy

A desmineralização óssea superficial tem se demonstrado favorável à consolidação de enxertos e ao comportamento celular, entretanto os mecanismos envolvidos ainda não estão esclarecidos. Os subsídios para o embasamento biológico da desmineralização, proporcionado por publicações anteriores, sugeriram que modificações na superfície óssea teriam influenciado o comportamento de pré-osteoblastos em cultura. Assim, este estudo objetivou comparar o efeito de duas concentrações de ácido cítrico na desmineralização de superfícies ósseas onde foram cultivadas células pré-osteoblásticas (MC3T3-E1), e analisar parâmetros de superfície comparando superfícies desmineralizadas a não desmineralizadas. Setenta amostras ósseas bicorticais foram removidas das calvárias de 35 ratos e divididas em grupos para as análises: 1) Microscopia Eletrônica de Varredura (MEV) para avaliação da área de recobrimento e espessura da camada de células sobre as amostras (n = 15) durante 24, 48 e 72 horas: Grupo AC.10 – amostras desmineralizadas por 30 segundos com ácido cítrico 10 %; Grupo AC.50 –amostras desmineralizadas por 30 segundos com ácido cítrico 50 %; e Grupo C (controle) – amostras não desmineralizadas; 2) Microscopia Confocal para análise da área de expressão e intensidade de fluorescência das BMP-2, -4 e -7: AC.10 – seis amostras desmineralizadas conforme item 1); AC.50 – seis amostras desmineralizadas conforme item 1); C – três amostras não desmineralizadas; 3) Microscopia Confocal para análise da rugosidade superficial média (Ra e Sa): Grupos AC.10 e AC.50 com cinco amostras cada, desmineralizadas conforme o item 1), sendo cada amostra seu próprio controle (análises antes e depois da desmineralização). Também foram avaliadas as distâncias entre picos (P-P) e entre picos e vales (P-V) antes e depois da desmineralização; 4) Microscopia Eletrônica de Varredura / Espectroscopia de Energia Dispersiva (MEV / EDS) para análise da composição superficial: mesmas...

The superficial bone demineralization has proved to be a favorable procedure for bone grafts consolidation and cell behavior, however the underlying mechanisms have not been clarified yet. Therefore, this study aimed to compare the effect of two concentrations of citric acid on demineralization of bone surfaces where pre-osteoblastic cells (MC3T3-E1) were cultivated, and analyze surface parameters comparing demineralized bone surfaces with non-demineralized surfaces. Seventy bicortical bone samples were harvested from the calvaria of 35 rats and divided into groups as follows: 1) Scanning Electron Microscopy (SEM) to evaluate the coating area and thickness of cells layers cultured on the samples (n = 15) for 24, 48, and 72 hours: Group CA.10 – samples demineralized for 30 seconds with 10 % citric acid; Group CA.50 – samples demineralized for 30 seconds with 50 % citric acid, and Group C (control) – non-demineralized samples; 2) Confocal Microscopy for analysis of expression area and intensity of fluorescence of BMP-2, -4, and -7: CA.10 – six samples demineralized as item 1); CA.50 – six samples demineralized as item 1); Group C – three non-demineralized samples; 3) Confocal Microscopy for surface mean roughness analysis (Ra and Sa): Groups CA.10 and CA.50 made up of five samples each and demineralized according to item 1), each sample was its own control (analysis before and after demineralization). The distances between peaks (P-P) and between peaks and valleys (P-V) were also evaluated before and after demineralization; 4) Scanning Electron Microscopy / Energy Dispersive Spectroscopy (SEM / EDS) to analyze the surface composition: the same samples of item 3) were evaluated before and after demineralization for atomic percentage (%A) of carbon, oxygen, magnesium, phosphorus, sulfur and calcium. Statistical test was made by adopting the 95 % significance level. Demineralized samples showed cells with morphology in the later stages of differentiation...

Osteogenic potential of mesenchymal cells derived from canine umbilical cord matrix co-cultured with platelet-rich plasma and demineralized bone matrix

Canine mesenchymal cells (MSCs) derived from Wharton's jelly were co-cultured, then supplemented or not supplemented with platelet rich plasma (PRP) and demineralized bone matrix (DBM) to verify osteogenic differentiation. Osteoblastic differentiation followed by mineralized bone matrix production was found to be significantly higher (p < 0.05) when MSCs were associated with PRP/DBM in culture after 14-21-days of induction. Osteopontin and osteocalcin gene expression were significantly superior (p < 0.05) under the same culture conditions after 21 days of observation. In conclusion, addition of PRP to DBM co-cultured with MSCs successfully induced osteogenesis in vitro.

Alterações Ósseas e do Sistema Estomatognático associadas a Doenças Endócrino Metabólicas / Cambios en los huesos y del sistema estomatognático asociadas con las enfermedades endocrino-metabólicas / Diseases Bone Changes and changes in the Stomatognathic System Associated with Metabolic Endocrine Diseases

Introdução: a prevalência de doenças endócrino-metabólicas tem aumentado nos últimos anos com a melhoria da expectativa de vida da população. Diabete Melittus, hipotireoidismo, hipertireoidismo, hipoparatireoidismo e hiperparatireoidismo podem levar as alterações no sistema estomatognático, contudo, a literatura ainda é limitada nesta área. Objetivo: Abordar as características e o processo fisiopatológico de doenças endócrino-metabólicas, com destaque para as alterações ósseas e do sistema estomatognático. Metodologia: Os dados foram coletados através da seleção de artigos sobre alterações ósseas e do sistema estomatognático associadas ao hipotireoidismo, hipertireoidismo, hipoparatireoidismo, hiperparatireoidismo e diabete mellitus, publicados nos bancos de dados, Bireme, MEDLINE e Scielo. Resultados: Foram coletados 73 artigos publicados na literatura entre os anos 1995 e 2012, dos quais 40 foram selecionados por se enquadrar no tema proposto. A literatura consultada demonstrou que alterações dos níveis séricos dos hormônios tireoidianos e do paratormônio, assim como diabete mellitus, influenciam no metabolismo ósseo. Atrasos na esfoliação e erupção dentária, no crescimento e desenvolvimento ósseo são observados no hipotireoidismo, enquanto o hipertireoidismo apresenta densidade óssea mineral diminuída, osteoporose dos ossos maxilares, esfoliação acelerada dos dentes decíduos e erupção prematura dos permanentes. No hipoparatireoidismo, observa-se osteoesclerose, dentição hipoplásica e reduzida remodelação óssea. Intensa atividade osteoclástica, ausência de lâmina dura e tumor marrom podem estar presentes no hiperparatireoidismo. Diabete mellitus pode ocasionar oesteoporose, doença periodontal e hipoplasia de esmalte. Embora conhecidas, a magnitude e as causas destas alterações no sistema estomatognático ainda são pouco elucidadas. Conclusões: É imprescindível que o cirurgião dentista tenha conhecimento sobre as manifestações locais ou sistêmicas associadas a estas desordens endócrino-metabólicas que podem acometer os pacientes, para que possa reconhecê-las e contribuir com o seu diagnóstico e tratamento precoces(AU)

Introducción: La prevalencia de las enfermedades endocrino-metabólicas se ha incrementado en los últimos años por el incremento de la esperanza de vida de la población. La diabetes mellitus, hipotiroidismo, hipertiroidismo, hipoparatiroidismo e hiperparatiroidismo pueden presentar alteraciones en el sistema estomatognático, sin embargo, los estudios encontrados en la literatura son todavía limitados en esta área. Objetivo: abordar las características y procesos fisiopatológicos de estas enfermedades y los cambios especialmente en los huesos y del sistema estomatognático. Métodos: los datos fueron recolectados a través de la selección de artículos sobre las alteraciones óseas del sistema estomatognático en pacientes afectados con el hipotiroidismo, el hipertiroidismo, el hipoparatiroidismo, el hiperparatiroidismo y la diabetes mellitus, publicada en las bases de datos, BIREME, SciELO y MEDLINE. Resultados: se recolectaron 73 artículos publicados entre los años 1995 y 2012, de los cuales 40 fueron seleccionados por la relación con el tema de interés para nuestro propósito. La literatura ha demostrado que los cambios en los niveles séricos de la hormona tiroidea y paratiroidea, y la diabetes mellitus afectan el metabolismo óseo. En el hipotiroidismo se observan retrasos en la exfoliación y erupción de los dientes, en el crecimiento y desarrollo óseo. El hipertiroidismo, hay disminución de la densidad mineral ósea, osteoporosis de los huesos de la mandíbula, exfoliación acelerada de los dientes temporales y erupción prematura de los permanentes. En el hipoparatiroidismo, se observan osteosclerosis, dientes hipoplásicos, y remodelación ósea reducida. Puede estar presente intensa actividad de los osteoclastos, ausencia de la lámina dura y tumor pardo en el hiperparatiroidismo. La diabete mellitus puede causar oesteoporosis, enfermedad periodontal e hipoplasia del esmalte. Aunque conocidas, la magnitud y las causas de estos cambios en el sistema estomatognático aún no están claros.Conclusiones: es esencial que el dentista tenga conocimiento de las manifestaciones locales o sistémicas asociadas con estos trastornos endocrinos y metabólicos para que pueda reconocerlos y ayudar en el diagnóstico precoz y el tratamiento(AU)

Introduction: the prevalence of endocrine-metabolic diseases has increased in recent years due to improvement of the life expectancy of the population. Diabetes mellitus, hypothyroidism, hyperthyroidism, hypoparathyroidism and hyperparathyroidism may present alterations in the stomatognathic system; however, some studies found in literature are still limited in this area. Objective: to address the characteristics and pathophysiological process of these diseases, especially bone changes and in the stomatognathic system. Methods: data were collected through the selection of articles about bone alterations of the stomatognathic system associated with hypothyroidism, hyperthyroidism, hypoparathyroidism, hyperparathyroidism, and diabetes, published in databases, BIREME, MEDLINE and SciELO. Results: seventy three articles published between 1995 y 2012 were revised, and 40 were selected on the basis of their association with the topic of interest. It has shown that changes in serum levels of thyroid and parathyroid hormones, and diabetes mellitus have an impact on bone metabolism. Delays in tooth eruption and exfoliation, bone growth and development are observed in hypothyroidism, whereas decreased bone mineral density, jawbones osteoporosis, accelerated exfoliation of deciduous teeth and premature eruption of permanent teeth were observed in hyperthyroidism. In hypoparathyroidism, there were osteosclerosis, hypoplastic teeth, and reduced bone remodeling. Intense osteoclastic activity, brown tumors and the absence of lamina dura in hyperparathyroidism may be observed. Diabetes mellitus can cause osteoporosis, periodontal disease and enamel hypoplasia. Although they are known, the magnitude and causes of these changes in the stomatognathic system are still unclear. Conclusions: it is essential that dentist can recognize the local or systemic manifestations associated with these endocrine and metabolic disorders and help to early diagnosis and treatment(AU)

Stabilizing effects of a particulate demineralized bone matrix in the L4 interbody space with and without PEEK Cage: a literature review and preliminary results of a cadaveric biomechanical study / Estabilización de los efectos de una matriz osea desmineralizada Particulada en el espacio intersomático a nivel L4 con o sin cajetín PEEK: revisión de la literatura y resultados preliminares de un estudio biomecánico cadavérico

We reviewed the biological elements supporting the usefulness of a specifically designed particulate form of demineralized bone matrix (DBM) in spinal fusion, and report some limitations of its use described in the medical literature and in the interbody space using a cadaveric biomechanical model. A literature review and description of the techniques used to augment spinal fusion are presented, including a more thorough review of recent findings of cadaveric biomechanical flexibility studies using DBM alone at different percentage fills of the existing disc space and DBM with a polyetheretherketone (PEEK) interbody cage. The need for DBM was established by reviewing limitations of autografts and allografts in spinal fusion. Demineralized bone matrix used alone did not increase stability post discectomy at L4-L5, but was demonstrated to exhibit satisfactory stability when used with a PEEK interbody cage. There may be a future role for DBM that hardens and fills disc space more rigidly, overcoming this limitation to its use.

Examinamos los elementos biológicos que respaldan la utilidad de una forma particulada específicamente diseñada de matriz ósea desmineralizada (MOD) con fusión espinal. Asimismo reportamos algunas limitaciones de su uso en el espacio intersomático descritas en la literatura médica, mediante un modelo biomecánico cadavérico. Se presenta una revisión de la literatura, acompañada de una descripción de las técnicas utilizadas para aumentar la fusión espinal, incluyendo una revisión más exhaustiva de los hallazgos recientes de los estudios de flexibilidad biomecánico cadavérica, utilizando sólo MOD en diferentes rellenos de porcentajes del espacio discal existente, y MOD con cajetines intervertebrales de polieteretercetona (PEEK). La necesidad de MOD se estableció examinando las limitaciones de los autoinjertos y los aloinjertos en la fusión espinal. El uso de la matriz ósea desmineralizada sola, no aumentaba la estabilidad post-disectómica a nivel L4-L5, pero se demostró que presentaba una estabilidad satisfactoria cuando se utilizaba con un cajetín intervertebral PEEK. Puede haber un papel futuro para un MOD que se endurezca y rellene el espacio del disco más rígidamente, venciendo así esta limitación a su uso.

Efeito da desmineralização ácida da interface enxerto-leito na consolidação de enxertos ósseos autógenos em bloco / Influence of acid demineralization of contacting osseous surfaces on the consolidation of autogenous onlay bone grafts

Para testar a hipótese de que a desmineralização in situ das superfícies de contato enxerto-leito, e a forma como o enxerto é estabilizado ao leito, podem influenciar os mecanismos envolvidos na consolidação do enxerto, fragmentos ósseos de 10 mm de diâmetro foram removidos das metáfises proximais tibiais de 36 coelhos (Oryctolagus Cuniculus) e transplantados para uma área adjacente. Na tíbia esquerda dos animais, as superfícies de contato do enxerto e do leito hospedeiro foram desmineralizadas com ácido cítrico pH 1,0 por 3 minutos antes dos enxertos serem fixados ao leito. Na tíbia direita, o transplante do bloco ósseo não foi precedido de desmineralização. Metade dos enxertos foi imobilizada sobre o leito pela superposição de uma membrana não reabsorvível de politetrafluoretileno colada com cianoacrilato ao leito à distância da interface enxerto-leito. A outra metade dos enxertos foi fixada por um parafuso de titânio no centro do enxerto. Assim, foram formados 4 grupos de estudo: membrana (M), membrana + ácido (MA), parafuso (P) e parafuso + ácido (PA). Três animais de cada grupo forneceram espécimes para análise microscópica quantitativa e qualitativa aos 15, 30 e 45 dias de pós-operatório. A análise qualitativa demonstrou que não houve formação óssea na interface em nenhum espécime aos 15 dias e que nos demais períodos, em todos os grupos, a quantidade de tecido ósseo neoformado na interface e seu estágio de maturação aumentaram com o tempo. Ambos os métodos de fixação empregados foram eficientes em manter os enxertos em posição, porém a membrana promoveu menor reabsorção da estrutura do enxerto. A análise quantitativa computadorizada revelou que, aos 30 dias, os grupos MA e PA apresentaram maior área de formação óssea na interface (71,34 ± 12,03%; 56,74 ± 2,15% respectivamente) em relação aos grupos M e P (51,75 ± 11,02%; 43,95 ± 4,05% respectivamente) e superfícies de consolidação óssea mais extensas (93,41 ± 5,95%; 93,73 ± 4,96%...

In order to test the hypothesis that the demineralization "in situ" of contacting surfaces of bone graft/bone bed and the fixation method used for graft stabilization can influence the mechanisms involved in the consolidation of the graft, bone fragments of 10 mm in diameter were removed from the proximal tibial metaphysis of thirty-six male rabbits (Oryctolagus Cuniculus) and transplanted to an adjacent area. In the left tibia of the animals, the contacting surfaces of the graft and host bed were demineralized with citric acid pH 1.0 for 3 minutes before the grafts were fixed to the receptor bed. In the right tibia, the bone block transplantation was not preceded by demineralization. Half of the grafts were immobilized on the bone bed by a nonresorbable polytetrafluoroethylene membrane glued with cyanoacrylate adhesive to the host bed distant from the bone graft-bone bed interface. The other half of the grafts were fixed by a titanium screw in the center of the graft. Thus, four groups were formed: membrane (M), membrane + acid (MA), screw (P) and screw + acid (PA). Three animals from each group provided specimens for quantitative and qualitative microscopic analysis at 15, 30 and 45 days postoperatively. Qualitative analysis showed no significant bone formation at the interface in any specimen of the groups at 15 days and on the other periods in all groups, the amount of newly formed bone at the interface as well as the stage of bone maturation increased with time. Both fixation methods were effective in maintaining the graft in position, but the membrane resulted in less resorption of the graft. Quantitative analysis, performed by means of a computer program for image analysis, showed that at day 30, groups MA and PA, showed greater area of bone formation at the interface (71.34 ± 12.03%; 56.74 ± 2 15%) than groups M and P (51.75 ± 11.02%, 43.95 ± 4.05%) and more osseointegrated bone surfaces (93.41 ± 5.95%, 93.73 ± 4.96%) than those...

Efeito da desmineralização ácida da interface enxerto-leito na consolidação de enxertos ósseos autógenos em bloco / Influence of acid demineralization of contacting osseous surfaces on the consolidation of autogenous onlay bone grafts

Para testar a hipótese de que a desmineralização in situ das superfícies de contato enxerto-leito, e a forma como o enxerto é estabilizado ao leito, podem influenciar os mecanismos envolvidos na consolidação do enxerto, fragmentos ósseos de 10 mm de diâmetro foram removidos das metáfises proximais tibiais de 36 coelhos (Oryctolagus Cuniculus) e transplantados para uma área adjacente. Na tíbia esquerda dos animais, as superfícies de contato do enxerto e do leito hospedeiro foram desmineralizadas com ácido cítrico pH 1,0 por 3 minutos antes dos enxertos serem fixados ao leito. Na tíbia direita, o transplante do bloco ósseo não foi precedido de desmineralização. Metade dos enxertos foi imobilizada sobre o leito pela superposição de uma membrana não reabsorvível de politetrafluoretileno colada com cianoacrilato ao leito à distância da interface enxerto-leito. A outra metade dos enxertos foi fixada por um parafuso de titânio no centro do enxerto. Assim, foram formados 4 grupos de estudo: membrana (M), membrana + ácido (MA), parafuso (P) e parafuso + ácido (PA). Três animais de cada grupo forneceram espécimes para análise microscópica quantitativa e qualitativa aos 15, 30 e 45 dias de pós-operatório. A análise qualitativa demonstrou que não houve formação óssea na interface em nenhum espécime aos 15 dias e que nos demais períodos, em todos os grupos, a quantidade de tecido ósseo neoformado na interface e seu estágio de maturação aumentaram com o tempo. Ambos os métodos de fixação empregados foram eficientes em manter os enxertos em posição, porém a membrana promoveu menor reabsorção da estrutura do enxerto. A análise quantitativa computadorizada revelou que, aos 30 dias, os grupos MA e PA apresentaram maior área de formação óssea na interface (71,34 ± 12,03%; 56,74 ± 2,15% respectivamente) em relação aos grupos M e P (51,75 ± 11,02%; 43,95 ± 4,05% respectivamente) e superfícies de consolidação óssea mais extensas (93,41 ± 5,95%; 93,73 ± 4,96%...

In order to test the hypothesis that the demineralization "in situ" of contacting surfaces of bone graft/bone bed and the fixation method used for graft stabilization can influence the mechanisms involved in the consolidation of the graft, bone fragments of 10 mm in diameter were removed from the proximal tibial metaphysis of thirty-six male rabbits (Oryctolagus Cuniculus) and transplanted to an adjacent area. In the left tibia of the animals, the contacting surfaces of the graft and host bed were demineralized with citric acid pH 1.0 for 3 minutes before the grafts were fixed to the receptor bed. In the right tibia, the bone block transplantation was not preceded by demineralization. Half of the grafts were immobilized on the bone bed by a nonresorbable polytetrafluoroethylene membrane glued with cyanoacrylate adhesive to the host bed distant from the bone graft-bone bed interface. The other half of the grafts were fixed by a titanium screw in the center of the graft. Thus, four groups were formed: membrane (M), membrane + acid (MA), screw (P) and screw + acid (PA). Three animals from each group provided specimens for quantitative and qualitative microscopic analysis at 15, 30 and 45 days postoperatively. Qualitative analysis showed no significant bone formation at the interface in any specimen of the groups at 15 days and on the other periods in all groups, the amount of newly formed bone at the interface as well as the stage of bone maturation increased with time. Both fixation methods were effective in maintaining the graft in position, but the membrane resulted in less resorption of the graft. Quantitative analysis, performed by means of a computer program for image analysis, showed that at day 30, groups MA and PA, showed greater area of bone formation at the interface (71.34 ± 12.03%; 56.74 ± 2 15%) than groups M and P (51.75 ± 11.02%, 43.95 ± 4.05%) and more osseointegrated bone surfaces (93.41 ± 5.95%, 93.73 ± 4.96%) than those...

Análisis geométrico de la arquitectura trabecular de un hueso de bovino durante el proceso inducido de desmineralización / Geometric analysis of the trabecular architecture in a bovine bone during the process of induced demineralization

La masificación de la radiología digital ha hecho posible el estudio de diferentes patologías mediante imágenes de alta calidad diagnóstica. Existen diferentes patologías que afectan al tejido óseo, y que producen pérdida del mineral(1). Aquellas patologías se caracterizan por la pérdida de la arquitectura trabecular y un adelgazamiento de la cortical(7), visibles en la radiología. Aquellos cambios llevan al paciente a caer en el riesgo de sufrir futuras fracturas(8), por lo que se considera importante realizar un análisis de la geometría de las trabéculas ante este tipo de patología, con el fin de prever riesgos de fractura. Material y Métodos. Para realizar este estudio, se escogió un fémur de bovino(19). Este fue sumergido en ácido acético al 4 por ciento, con el fin de producir su desmineralización. Se le realizó una medición cada 24 horas mediante la adquisición de imágenes radiológicas, que fueron obtenidas con un equipo digital directo hasta observar cambios radiológicos evidentes en la población trabecular. Las imágenes fueron evaluadas mediante un software de libre acceso llamado ImageJ®(23), realizando mediciones trabeculares mediante la herramienta ROI, y se adquirieron los valores de área, perímetro y circularidad. Resultados. En las 10 trabéculas estudiadas se observaron y cuantificaron cambios en la arquitectura trabecular, aumentando el área en un 124 por ciento, el perímetro en un 53 por ciento y la circularidad se mantuvo en promedio constante. Conclusiones: Mediante la radiología digital, es posible evaluar la arquitectura trabecular mediante parámetros geométricos, los cuales nos indican que existen cambios muy pequeños a lo largo del tiempo. Se observó un aumento de tamaño en las trabéculas, pero sin pérdida de su forma.

Introduction. The mass use of digital radiology has made possible the study of different pathologies through high quality diagnostic images. There are different diseases that affect bone tissue and which produce mineral loss (1). Those diseases are characterized by loss of trabecular architecture and cortical thinning (7), visible in radiology. Those changes lead the patient to suffer the risk of future fractures (8), therefore it is considered important to analyze the geometry of the trabeculae in this kind of pathology in order to anticipate fracture risk. Material and Methods. For this study, a bovine's femur was chosen(19). This was immersed in 4 percent acetic acid to produce demineralization. Measurement was performed (in Clinica Alemana Santiago) every 24 hours by radiological imagings, which were obtained with digital radiology (DR) to observe obvious radiological changes in trabecular population. The images were evaluated by a freely available software called ImageJ® (23), by performing Trabecular measurements using the ROI tool, acquiring the values of area, perimeter and circularity. Results. In the 10 trabeculae studied, we observed and quantified changes in trabecular architecture, increasing the value of average area in 124 percent, perimeter in 53 percent and no change in circularity during the demineralization process. Conclusions. With digital radiography, it is possible to evaluate the trabecular architecture using geometric parameters, which indicate that there are very small changes over time. An increase in size of the trabeculae was observed, trabeculae was observed, but without loss of shape.

Efeito da desmineralização óssea superficial na migração, adesão e diferenciação de pré-osteoblastos / The effect of surface bone demineralization on the migration, adhesion and differentiation of pre-osteoblasts

Resultados de pesquisas prévias tem encontrado potencial aumentado para a consolidação de enxertos ósseos mediante desmineralização do material enxertado e/ou das superfícies de consolidação. Entretanto há carência de embasamento apoiado em evidências biológicas do benefício de tal procedimento. Para testar esta hipótese, o tecido ósseo da calvária de cobaias (Cavia porcellus) foi exposto ao condicionamento por ácido cítrico durante 15, 30, 90 e 180 segundos (grupos teste). Quarenta e cinco discos ósseos de três milímetros de diâmetro foram removidos dos animais, dos quais 36 foram condicionados com ácido cítrico pH 1 a 50% e nove não receberam condicionamento (grupo controle). Sobre nove discos de cada grupo foram cultivados pré-osteoblastos MC3T3-E1 durante 24, 48 e 72 horas (três discos de cada grupo em cada tempo). Análises da morfologia celular, do número de células aderidas sobre as superfícies e da área de cobertura destas superfícies por préosteoblastos foram realizadas à microscopia eletrônica de varredura. Observou-se aumento do número de células aderidas às superfícies com o tempo, independentemente de haver condicionamento ou de seu tempo de aplicação. Entretanto, essa diferença só foi estatisticamente significante intragrupos (p<0,05) e quando comparados os períodos de 24 e 72 horas de incubação. A área de cobertura das superfícies por células aumentou significantemente com o tempo somente nos grupos teste, também entre os períodos de incubação de 24 e 72 horas (p<0,01). O grupo controle apresentou-se com 50% ou menos de área de cobertura superficial em relação aos demais. A duração de aplicação do ácido não interferiu significantemente nesse parâmetro de avaliação, mas nos grupos 15 e 30, a área de recobrimento ósseo mais do que triplicou às 72 horas em relação às 24 horas (p<0,01), com cerca de 70% das superfícies cobertas por células, contra 30% no grupo controle. Conclui-se que a desmineralização óssea...

Results of previous research has found increased potential for the consolidation of bone grafts by demineralization of the graft material and / or areas of consolidation. However there is a lack of foundation supported by biological evidence of benefits from such procedures. To test this hypothesis, the bone tissue of the calvaria of guinea pigs (Cavia porcellus) were exposed to conditioning by citric acid for 15, 30, 90 and 180 seconds (test group). Forty-five bone disks measuring three millimeters in diameter were removed from the animals, of which 36 were conditioned with citric acid pH 1 to 50% and nine did not receive conditioning (control group). About nine disks in each group were pre-cultured with MC3T3- E1 osteoblasts for 24, 48 and 72 hours (three discs of each group at each time point). Analysis of cell morphology, number of cells attached on the surface and the coverage area of these surfaces by pre-osteoblasts were performed on scanning electron microscopy. There was na increase in the number of cells attached to surfaces over time, regardless of conditioning or application time. However, this difference was not statistically significant intra-group (p <0.05) when comparing the periods of 24 and 72 hours of incubation. The coverage area of the surfaces of cells increased significantly with time only in the test groups, also among the incubation periods of 24 and 72 hours (p <0.01). The control group presented with 50% or less of surface area coverage compared to the other. The duration of application of the acid did not affect significantly this parameter of evaluation, but in groups 15 and 30, the bonearea covered more than tripled from 24 to 72 hours (p <0.01), with about 70 % of the area covered by cells, versus 30% in the control group. It was concluded that bone demineralization in the studied conditioning times provides a substrate on which cells acquire pre-osteoblastic morphology compatible with...

Investigate of DNA extraction of os cervi / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a convenient, practical and high efficient method of DNA extraction of os cervi, and lay the foundation of identification of animal bones.</p><p><b>METHOD</b>The bones of sika deer, red deer, cattle, dog and pig were used to extract DNA under different decalcification time (24,48,72 h) and decalcification temperature (4,25,37,56,70 degrees C), and extract method.</p><p><b>RESULT</b>It proved by experiments that demineralization process promotes the cracking of osteocyte. In a broad of decalcification time and temperature, DNA could be extracted from all bone samples successfully while the quantity varied slightly.</p><p><b>CONCLUSION</b>Samples (about 0.1 g) decalcify with 0. mol x L(-1) EDTA at 4 degrees C for 24 h, then water-bath for 1 h after lysis buffer added, DNA extracted via the method above is of high quality and can be used for PCR.</p>

Grafting Using Injectable Calcium Sulfate in Bone Tumor Surgery: Comparison with Demineralized Bone Matrix-based Grafting

BACKGROUND: Injectable calcium sulfate is a clinically proven osteoconductive biomaterial, and it is an injectable, resorbable and semi-structural bone graft material. The purpose of this study was to validate the clinical outcomes of injectable calcium sulfate (ICS) grafts as compared with those of a demineralized bone matrix (DBM)-based graft for filling in contained bony defects created by tumor surgery. METHODS: Fifty-six patients (41 males and 15 females) with various bone tumors and who were surgically treated between September 2003 and October 2007 were included for this study. The patients were randomly allocated into two groups, and either an ICS graft (28 patients) or a DBM-based graft (28 patients) was implanted into each contained defect that was developed by the surgery. The radiographic outcomes were compared between the two groups and various clinical factors were included for the statistical analysis. RESULTS: When one case with early postoperative pathologic fracture in the DBM group was excluded, the overall success rates of the ICS and DBM grafting were 85.7% (24/28) and 88.9% (24/27) (p > 0.05), respectively. The average time to complete healing was 17.3 weeks in the ICS group and 14.9 weeks in the DBM group (p > 0.05). Additionally, the ICS was completely resorbed within 3 months, except for one case. CONCLUSIONS: Although the rate of resorption of ICS is a concern, the injectable calcium sulfate appears to be a comparable bone graft substitute for a DBM-based graft, with a lower cost, for the treatment of the bone defects created during surgery for various bone tumors.

Treatment of gingival recession with collagen membrane and DFDBA: a histometric study in dogs

In a previous study, we evaluated the findings related to the use of resorbable collagen membranes in humans along with DFDBA (demineralized freeze-dried bone allograft). The aim of this subsequent study was to histometrically evaluate in dogs, the healing response of gingival recessions treated with collagen membrane + DFDBA (Guided Tissue Regeneration, GTR) compared to a coronally positioned flap (CPF). Two types of treatment were randomly carried out in a split-mouth study. Group 1 was considered as test (GTR: collagen membrane + DFDBA), whereas Group 2 stood for the control (only CPF). The dogs were given chemical bacterial plaque control with 0.2 percent chlorhexidine digluconate during a 90-day repair period. Afterwards, the animals were killed to obtain biopsies and histometric evaluation of the process of cementum and bone formation, epithelial migration and gingival level. A statistically significant difference was found between groups with a larger extension of neoformed cementum (GTR = 32.72 percent; CPF = 18.82 percent; p = 0.0004), new bone (GTR = 23.20 percent; CPF = 09.90 percent; p = 0.0401) and with a smaller area of residual gingival recession in the test group (GTR = 50.69 percent; CPF = 59.73 percent; p = 0.0055) compared to the control group. The only item assessed that showed no statistical difference was epithelial proliferation on the root surface, with means of 15.14 percent for the GTR group and 20.34 percent for the CPF group (p = 0.0890). Within the limits of this study we concluded that the treatment of gingival recession defects with GTR, associating collagen membrane with DFDBA, showed better outcomes in terms of a larger extension of neoformed cementum and bone, as well as in terms of a smaller proportion of residual recessions.

Osteoinductive activity of demineralized bone matrix and deprotenized bone derived from human avascular necrotic femoral head / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To observe the osteoinductive activity of demineralized bone matrix (DBM) and deproteinized bone (DPB) made from human avascular necrotic femoral head.</p><p><b>METHODS</b>The femoral head was cut into pieces with the size of 3 mm x 3 mm x 5 mm, which were made into DBM and DPB. These two kinds of biomaterials were cocultured with human bone mesenchymal stem cells (hBMSCs). Monolayer cells without biomaterials were cultured as control. Proliferative activity of hBMSCs was evaluated on days 1, 3, 5, 7 and 14. The concentration of alkaline phosphatase (ALP), osteocalcin (OC), and Ca(2+) were detected on days 1, 7, 14 and 21.</p><p><b>RESULTS</b>Cells cultured in DBM showed higher proliferative activity than did in DPB and monolayer cells (F =39.773, P <0.01). DBM and DPB also had osteoinductive activity. The concentrations of ALP (F=93.162, P <0.01), OC (F =236.852, P < 0.01), Ca(2+)(F =80.711, P <0.01) of DBM group were significantly higher than that of DPB and control groups.</p><p><b>CONCLUSIONS</b>In vitro, DBM and DPB made from avascular necrotic femoral head have osteoinductive activity when cocultured with hBMSCs, and the former is stronger than the latter.</p>

Dinámica de la descalcificación de tejido mineralizado de perros a través de microondas / Decalcification dynamic of dog mineralized tissue by microwaves

The aim of this article is to present the decalcification process dynamic of mineralized tissue in dogs, teeth and jaw, comparing the traditional decalcification method, immersion, and microwave, immersion followed by irradiation using a domestic microwave oven, accompanying the liberation of calcium through spectrophotometer of atomic absorption. It was used as decalcified agent, EDTA solution or nitric acid. The results showed that with the use of nitric acid (5 percent), after 15 days, the irradiated fragments could be processed for histological analysis, otherwise the tooth not irradiated need to be submerged for 65 days. The EDTA decalcified action was slower than the nitric acid. The histological observations of the irradiated samples showed an excellent preservation of the morphological characteristics, independently of the decalcified agent used.

El objetivo de este artículo fue presentar, un método de descalcificación dinámico de tejido mineralizado de perros, dientes y mandíbula, comparando el método de descalcificación tradicional e irradiación en horno de microondas, analizando la liberación de calcio en espectro fotómetro de absorción atómica. Usamos como agente descalcificante, solución de EDTA y acido nítrico. Los resultados mostraron que los fragmentos descalcificados con ácido nítrico después de 15 días, ya podían ser preparados para análisis histológico, el diente al ser irradiado tardó 65 días para ser descalcificado. El EDTA descalcificó lentamente, en relación al ácido nítrico. Las observaciones histológicas de las muestras irradiadas mostraron una excelente preservación de las características morfológicas independiente del agente descacificador usado.

Evaluation of the cytocompatibility of mixed bovine bone

Treatment of bovine bone with peroxides and chaotropic agents aims to obtain an acellular bone matrix that is able to maintain the collagen-apatite complex and a higher mechanical resistance, a mixed biomaterial hereby named mixed bovine bone (MBB). The purpose of this study was to evaluate the cytocompatibility of MBB and cell-MBB interaction. Cell morphology, number of viable cells, ability to reduce methyltetrazolium and to incorporate neutral red upon exposure to different concentrations of the hydrosoluble extract of MBB were assessed in Balb-c 3T3 cells according to ISO 10993-5 standard. The interaction between cells and MBB surface was evaluated by scanning electron microscopy. The water-soluble MBB extracts were cytotoxic and led to cell death possibly due to its effect on mitochondrial function and membrane permeability. Cells plated directly onto the MBB did not survive, although after dialysis and material conditioning in DMEM + 10 percent FCS, the cells adhered and proliferated onto the material. It may be concluded that, in vitro, water-soluble MBB extracts were cytotoxic. Nevertheless, MBB cytotoxic effect was reverted by dialysis resulting in a material that is suitable for cell based-therapy in the bioengineering field.

O tratamento do osso bovino com peróxidos e agentes caotrópicos visa à obtenção de uma matriz óssea acelular, com manutenção do complexo colágeno-apatita e maior resistência mecânica, denominada osso bovino misto (OBM). O objetivo desse estudo foi avaliar a citocompatitiblidade do osso bovino misto e a interação célula-OBM. Células Balb-c 3T3 foram tratadas com diferentes concentrações do extrato hidrossolúvel do OBM (0,1g por mililitro de meio de cultura sem soro). Foram avaliadas a morfologia e número de células, a capacidade de redução do metil tetrazol e a incorporação de vermelho neutro, de acordo com o padrão ISO 10993-5. A interação células-OBM foi estudada por microscopia eletrônica de varredura. Os extratos hidrosolúveis do OBM levaram à morte celular, possivelmente devido à redução da função mitocondrial e aumento da permeabilidade de membrana. Células plaqueadas diretamente sobre o OBM não sobreviveram. Entretanto, após diálise e condicionamento com DMEM +10 por cento de soro fetal bovino, as células aderiram e proliferaram sobre o material. Com base nos resultados obtidos, concluiu-se que o OBM é citotóxico. A citotoxicidade foi revertida por diálise resultando em um material adequado para terapia celular no campo da bioengenharia.

[Effect of demineralized bone matrix on induction and conduction of osteogenesis in alveolar bone of type I diabetic rats]

There are few studies on application of Demineralized Bone Matrix [DBM] in presence of a disease, such as type I diabetes. The aim of this study was to determine the osteoinductive and osteoconductive effects of DBM on the repair of alveolar bone in diabetic rats treated with insulin. This experimental study was carried out on 50 adult male [8 weeks old] rats [200-250g body weight]. The animals were divided into four groups as follow: first group, containing 8 animals, as control group and second, third and fourth groups, containing 14 animals [in each group], were diabetic groups. Diabetes was induced by alloxan. Among the diabetic groups, only group 4 received 1 unit of Insulin NPH daily. After 10 days, the upper right incisor tooth was extracted and the socket was filled with DBM in groups 3 and 4. At the end of the first and second weeks, half of the rats in each group were decapitated. The specimens were prepared and stained with H and E [Hematoxylin-Eozine] method. The histological changes around the DBM particle showing osteoblastic activity were studied. In group 4, at the end of first week, osteoblastic activity and formation of bone trabecula and collagen fibers[5 from 7 animals], and at the end of second week, osteogenesis with formation of more and thicker bone trabecula in vicinity of DBM was observed. In group 3, at the end of first week, osteoinductivity and osteoconductive effects were seen in some area around DBM Particles [3 from 7 animales], meanwhile degeneration of bone matrix were seen by osteoclastic activity. At the end of second week, osteogenesis was dispersly seen. In group 2, hematom and inflammation were the dominant features at the end of first week and at the end of second week, osteogenesis was seen weakly. In group 1, histological findings at the end of first and second weeks, were osteogenesis around periodental ligament [PDL] and osteogenesis with formation of bone trabecula respectively. Our results indicated that DBM particles can stimulate undifferentiated cells to start differentiate to pro-osteoblast and osteoblast in control diabetic rats, so osteogenesis in this group, was better in comparison with other diabetic groups and it is a good mater for graft

Assessment of bovine biomaterials containing bone morphogenetic proteins bound to absorbable hydroxyapatite in rabbit segmental bone defects

PURPOSE: To evaluate the osteo-regenerative capacity of two proprietary bone grafting materials, using a segmental defect model in both radial diaphyses of rabbits. METHODS: The right defect was filled with pooled bone morphogenetic proteins (pBMPs) bound to absorbable ultrathin powdered hydroxyapatite (HA) mixed with inorganic and demineralized bone matrix and bone-derived collagen, derived from bovine bone (Group A). The left defect was filled with bovine demineralized bone matrix and pBMPs bound to absorbable ultrathin powdered HA (Group B). In both groups, an absorbable membrane of demineralized bovine cortical was used to retain the biomaterials in the bone defects, and to guide the tissue regeneration. The rabbits were euthanized 30, 90 and 150 days after surgery. Radiographic, tomographic and histologic evaluations were carried out on all specimens. RESULTS: At 30 days, the demineralized cortical bone cover was totally resorbed in both groups. HA was totally resorbed from Group A defects, whereas HA persisted in Group B defects. A prominent foreign body reaction was evident with both products, more pronounced in sections from Group B. At 90 days, the defects in Group B exhibited more new bone than Group A. However, at 150 days after surgery, neither treatment had stimulated complete repair of the defect. CONCLUSION: The partial bone healing of the segmental defect occurred with low or none performance of the biomaterials tested.

OBJETIVO: Avaliar a capacidade osteo-regenerativa de dois biomateriais utilizando um modelo de defeito segmentar efetuado nas diáfises do rádio de coelhos. MÉTODOS: O defeito direito foi preenchido com pool de proteínas morfogenéticas ósseas (pBMPs) e hidroxiapatita em pó ultrafina absorvível (HA) combinada com matriz óssea inorgânica desmineralizada e colágeno, derivados do osso bovino (Grupo A). O defeito esquerdo foi preenchido com matriz óssea desmineralizada bovina com pBMPs e hidroxiapatita em pó ultrafina absorvível (Grupo B). Em ambos os defeitos utilizou-se membrana reabsorvível de cortical bovina desmineralizada para reter os biomateriais no defeito ósseo e guiar a regeneração tecidual. Os coelhos foram submetidos à eutanásia aos 30, 90 e 150 dias após a cirurgia. Foram efetuados exames radiográficos, tomográficos e histológicos em todos os espécimes. RESULTADOS: Aos 30 dias de pós-cirúrgico, o osso cortical desmineralizado foi totalmente reabsorvido em ambos os grupos. A HA tinha reabsorvido nos defeitos do Grupo A, mas persistiu nos do Grupo B. Uma reação de corpo estranho foi evidente com ambos os produtos, porém mais pronunciada no Grupo B. Aos 90 dias os defeitos do grupo B tinham mais formação óssea que os do Grupo A. Entretanto, aos 150 dias após a cirurgia, nenhum tratamento havia promovido o completo reparo do defeito. CONCLUSÃO: Os biomateriais testados contribuíram pouco ou quase nada para a reconstituição do defeito segmentar.

Effects of demineralized bone matrix on proliferation and osteogenic differentiation of mesenchymal stem cells from human umbilical cord.

BACKGROUND: Mesenchymal stem cells or mesenchymal progenitor cells are defined as self-renewable, multipotent progenitor cells with the unlimited capacity to differentiate into multiple lineage-specific cells that form bone, cartilage, fat, and muscle tissues. Demineralized bone matrix (DBM) has been extensively utilized in orthopaedic, periodontal, and maxillofacial applications and widely investigated as a biomaterial to promote new bone formation. OBJECTIVE: To isolate and characterize umbilical cord mesenchymal stem (UCMS) cells and examine the biological activity of DBM in the UCMS cells MATERIAL AND METHOD: UCMS cells were obtained from human umbilical cord culture. Cells were treated with or without DBM over 7 days of culture. Cell proliferation was examined by direct cell counting. Osteogenic differentiation of the UCMS cells was analysed with alkaline phosphatase staining assay. RESULTS: Phenotypic characteristics ofhuman UCMS cells were spindle and stellate shapes with fine homogenous cytoplasm, typically associated with fibroblast-like cells. The control cells (without DBM treatment) exhibited a spindle shape with little extracellular matrix, whereas the DBM treated cells appeared shortened and flattened, and they were surrounded by extracellular matrix. DBM inhibited the growth of the UCMS cells by 50%, as determined by direct cell counting. Morphologic and histochemical studies confirmed that DBM had a strong stimulatory effect on the alkaline phosphatase activities of UCMS cells, a very early marker of cell differentiation into the osteogenic lineage. CONCLUSION: Mesenchymal progenitor cells derived from umbilical cord could differentiate along an osteogenic lineage and thus provide an alternative source for cell-based therapies and tissue engineering strategies.