Estudio de biocompatibilidad de matrices poliméricas combinadas, responsivas al pH, con aplicación en Ingeniería de Tejido Óseo / Biocompatibility study of combined pH-responsive polymeric matrices with application in Bone Tissue Engineering

El presente trabajo muestra la obtención de un material a partir de un polímero sintético (TerP) y otro natural, mediante entrecruzamiento físico y su caracterización fisicoquímica y biológica, con el fin de emplearlos para regeneración de tejido óseo. Las membranas fueron obtenidas por la técnica de evaporación del solvente y caracterizadas por espectroscopia FTIR, ensayos de hinchamiento, medidas de ángulo de contacto y microscopia electrónica de barrido (SEM). Se encontró que la compatibilidad entre los polímeros que la constituyen es estable a pH fisiológico y que, al incorporar mayor cantidad del TerP a la matriz, esta se vuelve más hidrofóbica y porosa. Además, teniendo en cuenta la aplicación prevista para dichos materiales, se realizaron estudios de biocompatibilidad y citotoxicidad con células progenitoras de médula ósea (CPMO) y células RAW264.7, respectivamente. Se evaluó la proliferación celular, la producción y liberación de óxido nítrico (NO) al medio de cultivo durante 24 y 48 horas y la expresión de citoquinas proinflamatorias IL-1ß y TNF-α de las células crecidas sobre los biomateriales variando la cantidad del polímero sintético. Se encontró mayor proliferación celular y menor producción de NO sobre las matrices que contienen menos proporción del TerP, además de poseer una mejor biocompatibilidad. Los resultados de este estudio muestran que el terpolímero obtenido y su combinación con un polímero natural es una estrategia muy interesante para obtener un biomaterial con posibles aplicaciones en medicina regenerativa y que podría extenderse a otros sistemas estructuralmente relacionados. (AU)

In the present work, the preparation of a biomaterial from a synthetic terpolymer (TerP) and a natural polymer, physically crosslinked, is shown. In order to evaluate the new material for bone tissue regeneration, physicochemical and biological characterizations were performed. The membranes were obtained by solvent casting and characterized using FTIR spectroscopy, swelling tests, contact angle measurements, and scanning electron microscopy (SEM). It was found that the compatibility between the polymers is stable at physiological pH and the incorporation of a higher amount of TerP into the matrix increases hydrophobicity and porosity.Furthermore, considering the intended application of these materials, studies of biocompatibility and cytotoxicity were conducted with Bone Marrow Progenitor Cells (BMPCs) and RAW264.7 cells, respectively. Cell proliferation, NO production and release into the culture medium for 24 and 48 hours, and proinflammatory cytokine expression of IL-1ß and TNF-α from cells grown on the biomaterials while varying the amount of the synthetic polymer were evaluated. Greater cell proliferation and lower NO production were found on matrices containing a lower proportion of TerP, in addition to better biocompatibility. The results of this study demonstrate that the obtained terpolymer and its combination with a natural polymer is a highly interesting strategy for biomaterial preparation with potential applications in regenerative medicine. This approach could be extended to other structurally related systems. (AU)

Muscle strength training is better than the use of growth hormone (GH) in bone health of Wistar rats / Entrenamiento de la fuerza muscular es mejor que el uso de la hormona del crecimiento (GH) en salud ósea del ratas Wistar

SUMMARY: The objective of this study was to evaluate the effects of growth hormone (GH) and muscle strength training (ST) on the composition of bone tissue of Wistar rats through Raman spectroscopy. In total, 40 male rats were randomly distributed into four groups: (N = 10) control (C), control with the application of GH (GHC), strength training (T), and strength training with the application of GH (GHT). The training consisted of four series of 10 water jumps, performed three times a week, with an overload corresponding to 50 % of body weight and duration of four weeks. GH was applied at a dose of 0.2 IU / kg in each animal three times a week and every other day. After four weeks, the animals were euthanized and the right femurs collected for analysis of the bone structure. Raman spectroscopy (ER) was used to observe the following compounds from their respective bands: Calcium Carbonate-Triglycerides (fatty acids) 1073 cm-1, Collagen type I 509 cm-1, Bone-DNA Phosphate (Protein) 589 cm-1, Phosphate Phospholipids 1078 cm-1. For the statistical analysis, the Shapiro-Wilk and ANOVA One-Way variance analysis normality tests were performed, followed by the Tukey post-test. The results showed an increase in the concentrations of calcium carbonate-triglycerides (fatty acids), type I collagen, bone phosphate-DNA (protein), and phosphate phospholipids in all experimental groups, with or without ST and/or GH , But only the isolated training group differed significantly from the control group (P <0.05). It was concluded that all treatments could promote bone tissue gain, however, only the T group demonstrated a significant difference in the mineral compounds analyzed.

RESUMEN: El objetivo del estudio fue avaluar el efecto de la aplicación de la hormona del crecimiento (GH) y entrenamiento de la fuerza muscular (EF) en la composición del tejido óseo de ratas Wistar a partir de la espectroscopía Raman. Fueron utilizadas 40 ratas machos distribuidas de forma aleatoria en cuatro grupos (n=10): control (C), control y aplicación de GH (GHC), entrenamiento de la fuerza muscular (EF) y entrenamiento de la fuerza muscular y aplicación del GH (GHE). El entrenamiento fue consistió en cuatro series de 10 saltos acuáticos, realizados tres veces en la semana, con sobrecarga correspondiente a 50 % de la masa corporal y durante cuatro semanas. El GH fue aplicado en la dosificación de 0,2 UI/kg en cada animal, tres veces en la semana y en días alternados. Después de cuatro semanas, los animales fueran eutanasiados y retirados los fémures derechos para un análisis de la estructura ósea. La espectroscopía Raman fue utilizada para observar los siguientes compuestos a partir de las respectivas bandas: Carbonato de Calcio-Triglicéridos (ácidos grasos) 1073 cm-1, Colágeno Tipo I 509 cm-1, Fosfato Óseo-DNA (Proteína) 589 cm1, Fosfato Fosfolípidos 1078 cm-1. Para el análisis estadístico, fueron realizadas las pruebas Shapiro-Wilk y el análisis de variancia ANOVA One-Way, seguida de test post hoc de Tukey. Los resultados revelaran aumento de la concentración de Carbonato de Calcio-Triglicéridos (ácidos grasos), Colágeno Tipo I, Fosfato Óseo- DNA (Proteína), Fosfato Fosfolípidos en todos los grupos experimentales, asociados o no a la realización del EF y/o aplicación del GH. Además, solamente el grupo EF mostró diferencia significativa del grupo C (p<0,05). Es posible concluir que todos los tratamientos mostraran aumentos en el tejido óseo, sin embargo, solamente el grupo T demostró una diferencia significativa en los compuestos minerales analizados.

Diversidad del ADN mitocondrial en restos óseos prehispánicos / Mitochondrial DNA diversity in prehispanic bone remains on the eastern Colombian Andes

Resumen Introducción. El ADN antiguo que se extrae de los restos óseos humanos permite analizar la composición genética de las poblaciones precolombinas y determinar las dinámicas poblacionales que dieron origen a la diversidad de las poblaciones contemporáneas. Objetivo. Determinar la diversidad genética y la relación con otras comunidades contemporáneas y antiguas de América, de los restos óseos asociados al Templo del Sol en Sogamoso, Colombia. Materiales y métodos. Se analizaron 13 individuos pertenecientes al periodo precolombino muisca (siglos IX-XVI d. C.), provenientes de los alrededores del Templo del Sol en Sogamoso, Boyacá, Andes orientales colombianos. Se amplificó el ADN mitocondrial (ADNmt) y se determinaron los polimorfismos de la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism, RFLP) para los cuatro haplogrupos amerindios (A, B, C y D). Además, se amplificaron y analizaron los marcadores autosómicos, incluida la amelogenina, y los marcadores de los polimorfismos de repeticiones cortas en tándem (Short Tandem Repeat, STR) del cromosoma Y. Resultados. El haplogrupo A fue el linaje mitocondrial más frecuente en esta población, seguido de los haplogrupos B y C; no se detectó el haplogrupo D. Los análisis de variación genética indicaron una diversidad semejante a la de las poblaciones pertenecientes a la familia lingüística chibcha, contemporánea en Colombia y Centroamérica. Se logró hacer la determinación molecular del sexo de los individuos estudiados y compararla con los datos osteológicos. Con una sola excepción, los datos bioantropológicos y moleculares concordaron. Conclusiones. Estos resultados aportan nuevos elementos a la hipótesis del origen centroamericano de los grupos chibchas del altiplano cundiboyacense con base en marcadores genéticos, y permitieron establecer el sexo y las relaciones de parentesco.

Abstract Introduction: DNA extracted from ancient human bones allows to analyze the genetic makeup of preColumbian populations and to determine the dynamics that gave rise to the diversity of contemporary populations. Objective: To determine the genetic diversity of skeletal remains associated with the Templo del Sol (Sun Temple) and their relationship with other contemporary and ancient communities of America. Materials and methods: We analyzed 13 individuals belonging to the pre-Columbian Muisca Period (IX-XVI centuries AD) from the vicinities of the Templo del Sol (Sun Temple) (Sogamoso, Boyacá) in the eastern Colombian Andes. Mitochondrial DNA was amplified and RFLPs were performed in order to type the four traditional Amerindian haplogroups (A, B, C and D). In addition, autosomal markers including amelogenin and Y-chromosome STRs were amplified. Results: Among the observed mitochondrial lineages, haplogroup A was the most frequent, followed by haplogroups B and C; no evidence of haplogroup D was found. The genetic variation analysis indicated a similar diversity of pre-Columbian Muiscas to that of contemporary populations belonging to the Chibcha linguistic family from Colombia and Central America. Molecular sexing was accomplished and it was compared to osteological data. With only one exception, anthropological and molecular data were consistent. Conclusions: Our results contribute new genetic elements supporting the hypothesis of Central American origin of the Chibcha groups of the Cundiboyacense plateau, and allowed sex typing and kinship evaluations.

Matrices de tercera generación en la ingeniería de tejidos ósea / Third-generation matrices in bone tissue engineering

Existen numerosas patologías que generan situaciones invalidantes debido a problemas asociados a nivel de defectos óseos. Esto genera, en muchas oportunidades, cuestiones sanitarias de alto impacto. La ingeniería de tejidos óseos pretende generar propuestas novedosas para reparar pérdidas o fracturas óseas, promoviendo regenerar el tejido mediante el implante de matrices biodegradables que puedan actuar como estructuras para la adhesión celular, favoreciendo el crecimiento y la diferenciación hasta formar hueso de novo. El incremento notable de los conocimientos en las áreas biotecnológicas, de síntesis química, así como de biomedicina, permiten el desarrollo de numerosos tipos de matrices de tercera generación, biodegradables y no tóxicas, con características que proponen sean consideradas en la regeneración tisular ósea. Este trabajo intenta resumir los tipos de matrices que mayor impacto han tenido hasta el momento en la medicina regenerativa ósea, mostrando los casos más relevantes de resultados experimentales y clínicos, y propone algunas perspectivas que se deberían considerar para poder aplicarlas a la práctica clínica. Esta es un área que invita a los investigadores a posicionarse en un pensamiento complejo desde el punto de vista científico-filosófico. (AU)

There are several pathologies that generate disability due to complications associated with bone defects. This often generates high impact health troubles. Bone tissue engineering aims to generate novel means to repair bone loss or bone fractures, promoting tissue regeneration through the implantation biodegradables scaffolds, which can act as structures for cell adhesion, that promts cell growth and differentiation for the novo bone formation. The remarkable for the novo bone formation in biotechnology, chemical synthesis, and biomedical knowledge allows the development of numerous types of third generation scaffolds, applied to promote bone tissue regeneration. This brief report aims to review the scaffolds that have had more impact in bone regenerative medicine so far, describing the most relevant experimental and clinical results. This is an area that invites researchers to situate themselves in a complex thought of scientific-philosophical point of view. (AU)

Biomecánica de las fracturas por stress / Biomechanical features of stress fractures

Se define como estrés (stress) tanto la fuerza que una carga externa ejerce sobre un cuerpo sólido como la fuerza reactiva que acompaña a la primera (Ley de Newton), por unidad de área imaginaria transversal a su dirección. Las cargas internas reactivas inducen deformaciones proporcionales del cuerpo. La resistencia del cuerpo a deformarse se llama rigidez. La deformación puede resquebrajar el cuerpo y, eventualmente, producir una fractura por confluencia de trazos. La resistencia del cuerpo a separarse en fragmentos por esa causa se llama tenacidad. La resistencia del cuerpo a la fractura es proporcional al stress que puede soportar sin separarse en fragmentos por deformación (no hay fractura sin deformación y sin stress previo). El stress máximo que un cuerpo puede soportar sin fracturarse resulta de una combinación de ambas propiedades: rigidez y tenacidad, cada una con distintos determinantes biológicos. Una o varias deformaciones del cuerpo pueden provocarle resquebrajaduras sin fracturarlo. La acumulación de resquebrajaduras determina la "fatiga" del material constitutivo del cuerpo, que reduce su rigidez, tenacidad y resistencia a la fractura para la próxima ocasión ("fragilidad por fatiga"). En el caso de los huesos, en general, los términos stress y fatiga tienen las connotaciones amplias referidas, respecto de todas las fracturas posibles. La fatiga predispone a fracturas a cargas bajas, que se denominan (correctamente) "fracturas por fatiga" y también (incorrectamente) "fracturas por stress", para distinguirlas de las que ocurren corrientemente, sin resquebrajaduras previas al trauma, que se denominan (incorrectamente) "fracturas por fragilidad, o por insuficiencia". En realidad, todas las fracturas se producen por stress y por fragilidad o insuficiencia (en conjunto); pero la distinción grosera entre fracturas "por fatiga, o por stress", por un lado, y "por fragilidad" o "por insuficiencia", por otro, aceptando las amplias connotaciones referidas antes, tiene valor en la práctica clínica. Este artículo intenta explicar esas particularidades biomecánicas y describir las distintas condiciones que predisponen a las fracturas "por fatiga o por stress" en la clínica, distinguiéndolas de las fracturas "por fragilidad o por insuficiencia" (manteniendo estas denominaciones) y detallando las características de interés directo para su diagnóstico y tratamiento. (AU)

The term "stress" expresses the force exerted by an external load on a solid body and the accompanying, opposed force (Newton's Law), expressed per unit of an imaginary area perpendicular to the loading direction. The internal loads generated this way deform (strain) proportionally the body's structure. The resistance of the body to strain expresses its stiffness. Critical strain magnitudes may induce micro-fractures (microdamage), the confluence of which may fracture the body. The body's resistance to separation into fragments determines its toughness. Hence, the body's resistance to fracture is proportional to the stress the body can support (or give back) while it is not fractured by the loadinduced strain (no stress, no strain -> no fracture). Therefore, the maximal stress the body can stand prior to fracture is determined by a combination of both, its stiffness and its toughness; and each of those properties is differently determined biologically. One or more deformations of the body may induce some microdamage but not a fracture. Microdamage accumulation determines the fatigue of the material constitutive of the body and reduces body's toughness, leading to a "fatigue-induced fragility". In case of bones, in general, both stress and fatigue have the referred, wide connotations, regarding any kind of fractures. In particular, bone fatigue predisposes to low-stress fractures, which are named (correctly) "fatigue fractures" and also misnamed "stress fractures", to distinguish them from the current fractures that occur without any excess of microdamage, that are named (wrongly) "fragility" or "insufficiency" fractures. In fact, all fractures result from all stress and fragility or insufficiency as a whole; however, the gross distinction between "fatigue or stress fractures", on one side, and "fragility or insufficiency fractures", on the other, accepting the wide connotations of the corresponding terminology, is relevant to clinical practice. This article aims to explain the above biomechanical features and describe the different instances that predispose to "fatigue or stress fractures" in clinical practice, as a different entity from "insufficiency or fragility fractures" (maintaining this nomenclature), and describe their relevant features to their diagnosis and therapy. (AU)

Bioacumulación de níquel en encía, saliva y hueso alveolar de paciente con aparatología ortodóncica fija: reporte de un caso / Bioaccumulation of nickel in gingiva, saliva and alveolar bone in a patient with a fixed orthodontic appliance: a case report

Se presenta el caso de una paciente de 28 años, con tratamiento ortodóncico activo, que acudió a consulta odontológica por motivos estéticos. Luego del examen intraoral se le diagnosticó agrandamiento gingival y exostosis maxilar. El plan de tratamiento consistió en cirugía estética periodontal y exéresis de la exostosis. Durante el tratamiento quirúrgico se tomaron muestras de encía, saliva y hueso alveolar, previo consentimiento informado. Una vez procesadas las muestras se midieron las concentraciones de níquel en ng/ml mediante espectrofotometría de absorción atómica; el promedio de dichas concentraciones en cada una de las muestras fueron: 986,4 ng/ml en saliva, 779,5 ng/ml en hueso y 620,5 ng/ml para el caso de la encía. La acumulación de níquel evidenciada en cada una de las muestras resulta de especial interés, pues la exposición a metales contenidos en la aparatología ortodóncica constituye actualmente un importante tema de investigación.

A case is reported of a 28-year-old female patient, with active orthodontic treatment, who was treated in the dental practice for aesthetic reasons. The diagnosis was gingival overgrowth, induced by orthodontic treatment, and maxillary exostosis. The treatment consisted of periodontal cosmetic surgery and removing the exostosis. During surgery, samples of gingiva, saliva and alveolar bone were obtained. Before take the samples, the patient signed an informed consent. Nickel concentrations in ng/mL were measured by atomic absorption spectrophotometry. The mean nickel concentration in the samples were: 986.4 ng/ml in saliva, 779.5 ng/ml in alveolar bone, and 620.5 ng/ml in the case of gingiva. The high accumulation of nickel observed in each sample type is of particular interest because exposure to metals contained in orthodontic appliances is currently an important public health problem in dentistry.

Cytokine expression patterns and mesenchymal stem cell karyotypes from the bone marrow microenvironment of patients with myelodysplastic syndromes

The purpose of this study was to explore cytokine expression patterns and cytogenetic abnormalities of mesenchymal stem cells (MSCs) from the bone marrow microenvironment of Chinese patients with myelodysplastic syndromes (MDS). Bone marrow samples were obtained from 30 cases of MDS (MDS group) and 30 healthy donors (control group). The expression pattern of cytokines was detected by customized protein array. The karyotypes of MSCs were analyzed using fluorescence in situ hybridization. Compared with the control group, leukemia inhibitory factor, stem cell factor (SCF), stromal cell-derived factor (SDF-1), bone morphogenetic protein 4, hematopoietic stem cell (HSC) stimulating factor, and transforming growth factor-β in the MDS group were significantly downregulated (P<0.05), while interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and programmed death ligand (B7-H1) were significantly upregulated (P<0.05). For chromosome abnormality analysis, the detection rate of abnormal karyotypes (+8, -8, -20, 20q-, -Y, -7, 5q-) was 30% in the MDS group and 0% in the control group. In conclusion, the up- and downregulated expression of these cytokines might play a key role in the pathogenesis of MDS. Among them, SCF and SDF-1 may play roles in the apoptosis of HSCs in MDS; and IFN-γ, TNF-α, and B7-H1 may be associated with apoptosis of bone marrow cells in MDS. In addition, the abnormal karyotypes might be actively involved in the pathogenesis of MDS. Further studies are required to determine the role of abnormal karyotypes in the occurrence and development of MDS.

Efecto biológico de la hipertermia hídrica controlada sobre hueso sano de conejos / Biological effect of controlled fluid hyperthermia on rabbit healthy bones

La hipertermia hídrica se ha utilizado para tratamientos de diversas formas de cáncer, en hueso aún no se ha determinado cuál es su efecto; se realizó este estudio prospectivo para demostrar los cambios estructurales y metabólicos y los efectos en la consolidación de hueso expuesto a hipertermia hídrica. Se utilizaron 30 conejos de raza New-Zealand. Con peso entre 2.8 y 3.2 kg divididos en 9 grupos y 3 subgrupos, se realizó osteotomía en el tercio medio del fémur derecho; este segmento fue expuesto a hipertermia a 15, 20 y 25 grados centígrados, durante 50, 60 y 70 minutos, gammagrafía ósea cada cuatro semanas y radiográficos cada semana hasta la semana 13. Para el análisis estadístico se utilizó la prueba de post-hoc y test de Tukey, encontrando diferencias en las concentraciones de elementos químicos a temperaturas superiores a 60 grados durante 20 minutos con significancia estadística, retardo en la consolidación y evidencia de actividad metabólica. Se concluye que con la exposición a temperaturas de 60 grados durante 20 minutos se presentaron alteraciones morfológicas en potasio, magnesio, azufre y fósforo y retardo en la consolidación ósea. Estos resultados son usados como parámetros para el tratamiento con hipertermia hídrica controlada en tumores óseos.

Fluid hyperthermia has been used to treat various types of cancers, but its effects on bone have not been determined. The purpose of this prospective study was to show the structural and metabolic changes of bone exposed to fluid hyperthermia and the effects of the latter on bone healing. Thirty New Zealand rabbits were used, weighting 2.8-3.2 kg; they were divided into 9 groups and 3 subgroups. An osteotomy was performed in the mid third of the right femur and this segment was exposed to hyperthermia at 15, 20 and 25 degrees centigrade for 50, 60 and 70 minutes. A bone scan was performed every 4 weeks and X-rays were taken every week up to week 13. The post-hoc Tukey test was used for the statistical analysis. Differences were found in the concentration of chemicals at temperatures above 60 degrees for 20 minutes with statistical significance; bone healing was delayed and there was evidence of metabolic activity. We conclude that exposure to temperatures above 60 degrees for 20 minutes resulted in morphologic alterations in potassium, magnesium, sulfur and phosphorus, and delayed bone healing. These results are used as parameters for the treatment of bone tumors with fluid hyperthermia.

Níveis de fósforo disponível em dietas para suínos dos 15 aos 30kg / Available phosphorus levels in diets for pigs from 15 to 30kg

Avaliou-se a suplementação de dietas com fósforo disponível (Pd) para suínos selecionados para deposição de carne, utilizando-se 100 leitões com média de peso inicial de 15,03±0,33kg, distribuídos em delineamento de blocos ao acaso, com cinco tratamentos, cinco repetições e quatro animais por unidade experimental. Os tratamentos foram dieta basal sem suplementação de fosfato bicálcico e outras quatro dietas obtidas pela suplementação da dieta basal com fosfato bicálcico, resultando em cinco concentrações de Pd, 0,106; 0,221; 0,310; 0,406; e 0,493%. As porcentagens de Pd influenciaram o consumo diário de ração, que aumentou de forma linear. O ganho de peso diário aumentou e a conversão alimentar melhorou de forma quadrática até a suplementação máxima de 0,450 e 0,390% de Pd, respectivamente. Não houve efeito da suplementação das dietas sobre a resistência óssea. As porcentagens de Pd influenciaram de forma quadrática os teores de cálcio e de fósforo nos ossos, que aumentaram até a adição máxima de 0,388 e 0,369% de Pd, respectivamente. Não houve efeito da porcentagem de Pd sobre o teor de cinza óssea. Suínos dos 15 aos 30kg exigem maior concentração de Pd para máximo ganho de peso (0,450% ou 5,33g/dia) e melhor conversão alimentar (0,390% ou 4,52g/dia) em relação às recomendações atuais.

The supplementation of diets with available phosphorus (aP) for pigs selected for meat deposition was evaluated, using 100 commercial hybrid piglets, 50 castrated males and 50 females, with average initial weight of 15.03±0.33kg allotted in a randomized block design with five treatments, five replicates, and four pigs, 2 males and 2 females per experimental unit. The treatments were a basal diet and other four diets obtained through basal diet supplementation with dicalcium phosphate to obtain five aP concentrations, 0.106, 0.221, 0.310, 0,406, and 0.493%. The percentages of aP influenced daily feed intake, which increased linearly. Daily weight gain increased and feed conversion improved quadratically up to 0.450 and 0.390% maximum responses. There was no effect of diet supplementation on bone strength. The percentages of aP influenced the calcium and phosphorus contents in the bones, which increased quadratically up to 0.388 and 0.369% maximum responses, respectively. There was no effect of aP percentage in the bone ashe content. Pigs from 15 to 30kg require greater amount of aP in the diet for maximal weight gain (0.450% or 5.33g/d) and greatest feed conversion (0.390% or 4.52g/d) compared to current requirements.

Desempenho ponderal de bovinos Nelore suplementados com fontes alternativas de fósforo / Weight gain of Nelore cattle supplemented with different phosphorus sources

O desempenho produtivo e a possível interferência do flúor sobre a saúde dos animais foram investigados em bovinos Nelore suplementados, por 866 dias, com distintas fontes alternativas de fósforo com diferentes relações fósforo:fluor (P:F). Os tratamentos experimentais foram: Controle negativo (CONTNEG, sem qualquer suplementação com P), fosfato bicálcico (FB 120:1, FB 30:1 e FB 10:1), fosfato monobicálcico (FMBC 60:1), superfosfato triplo (SFT 30:1) e fosfato de rocha de Cajati (FR 10:1). Foram utilizados 49 novilhos, desmamados aos oito meses de idade, castrados e com 230 kg de peso médio, distribuídos em sete piquetes com água e mistura mineral formulada sem P. A dieta padrão foi feita com bagaço de cana (0,03% de P) como volumoso e um concentrado contendo 0,239 % de P oferecido na base de 1% do peso dos animais para permitir um ganho de peso aproximado de 0,50 kg/dia. Até o dia 134, não houve diferença estatística entre os diversos lotes, inclusive para o tratamento CONTNEG, que não recebeu fósforo suplementar na dieta e ganhou 71,6 kg de peso ou 0,633 kg/dia. Após 866 dias de confinamento (2,37 anos), os animais suplementados com o fosfato bicálcico padrão (120:1) ganharam menos peso que os suplementados com as fontes FMCB 60:1, FB 30:1 e SFT 30:1. Até um ano de suplementação fosfórica com fosfato bicálcico padrão (120:1) artificialmente fluoretado com NaF ou com o fosfato de rocha não se detectou danos à saúde ou ao ganho de peso dos animais. As análises de fósforo nos ossos mostraram diferença estatística apenas entre o tratamento CONTNEG e os que tinham fosfato bicálcico. As concentrações de flúor nos ossos se mostraram intimamente associadas à quantidade de flúor disponível nas fontes utilizadas. Conforme a proporção P:F na dieta foi diminuindo, características relacionadas à fluorose dentária ficaram mais evidentes, sendo que os animais que receberam fontes com relação 10:1, apresentaram, ao final do experimento, dentes incisivos permanentes mal formados, quebradiços e com manchas esbranquiçadas.

Weight gain and possible interference of fluoride on animal health was investigated in Nellore cattle supplemented during 866 days with alternative sources of phosphorus with different phosphorus:fluor (P:F) ratios. The five treatments were: (1) Negative control (NC, without any supplemental P), (2) dicalcium phosphate (DCP 120:1, DCP 30:1 and DCP 10:1), (3) monodicalcium phosphate (MDCP 60:1), (4) triple superphosphate (TSF 30:1), and (5) cajati rock phosphate (RP 10:1). We used 49 oxen weaned with 8 months of age and an average weight of 230 kg, distributed into seven paddocks with water and mineral mixture formulated without P. A standard diet consisting of sugar cane bagasse (0.03% P) as roughage and a concentrate containing 0.239% P was provided on 1% of live weight to allow a weight gain of about 0.50 kg/day. Until day 134, there was no statistical difference between the various groups, including the treatment NC which received no supplemental phosphorus in the diet and gained weight of 71.6 kg or 0.633 kg/day. After 866 days of confinement (2.37 years), the oxen supplemented with dicalcium phosphate standard (120:1) gained less weight than those supplemented with sources MDCP 60:1, DCP 30:1 and TSF 30:1. Up to one year of supplementation with dicalcium phosphate artificially fluoridated with NaF or with rock phosphate did not result in damage to health or in weight gain of the animals. Analyses of phosphorus in bones showed statistical difference between treatments, and group NC that did not receive any supplemental P showed the lowest values. The concentration of fluoride in bones proved to be closely related to the amount of fluoride available in the sources used. As the ratio P:F in the diet decreased, characteristic signs related to dental fluorosis became more evident, and the animals that received P supplies with 10:1 ratio showed at the end of the experiment permanent malformed incisor teeth, brittle and whitish stained.

DNA extraction from bones and teeth using AutoMate Express forensic DNA extraction system / 法医学杂志

OBJECTIVE@#To explore a new method in order to extract DNA from bones and teeth automatically.@*METHODS@#Samples of 33 bones and 15 teeth were acquired by freeze-mill method and manual method, respectively. DNA materials were extracted and quantified from the triturated samples by AutoMate Express forensic DNA extraction system.@*RESULTS@#DNA extraction from bones and teeth were completed in 3 hours using the AutoMate Express forensic DNA extraction system. There was no statistical difference between the two methods in the DNA concentration of bones. Both bones and teeth got the good STR typing by freeze-mill method, and the DNA concentration of teeth was higher than those by manual method.@*CONCLUSION@#AutoMate Express forensic DNA extraction system is a new method to extract DNA from bones and teeth, which can be applied in forensic practice.

Progress in application of microbeam X-ray fluorescence spectroscopy in forensic science / 法医学杂志

Microbeam X-ray fluorescence (micro-XRF) spectrometry has been raised as an analytical technique of microbeam during the recent years. With its advantages of high sensitivity, small sample requirement, high testing accuracy and non-destruction, the technique is widely utilized in forensic science. This review bases on recent researches at home and abroad, describes its applications including identification of gunshot residue, visualization of fingerprints, discrimination of drug source, production process, and other material evidences of analysis in crime scene. Thanks to the advances in technology, intelligent and portable micro-XRF equipment has appeared to be applied. It is believed that it may be more popular and frequent in administration of forensic science in the near future.

Effect of Combined Sex Hormone Replacement on Bone/Cartilage Turnover in a Murine Model of Osteoarthritis

BACKGROUND: Estrogens act on estrogen receptors distributed in articular cartilages, synovial membrane, and ligaments, which are thought to be related with degenerative changes. Meanwhile, progesterone is known to have a weak anabolic action on bone formation This study evaluates the effects of estrogen and progesterone hormone on bone/cartilage turnover in ovariectomized (OVX) rats. METHODS: Thirty-five 7-month-old female Sprague-Dawley rats were randomly divided into 5 groups and then ovariectomized bilaterally except the sham control group. The first and the second group acting as controls did not receive hormonal therapy, the third group received estrogen, the fourth group received progesterone, and the fifth group received combination of both hormones 10 weeks after surgery. Evaluations were done using the serum levels of cartilage oligomeric matrix protein (COMP) for cartilage turnover, collagen type I C-telopeptide (CTX-1) and osteocalcin (OC) for bone turnover at 11, 15, 19 weeks after OVX and histology using the Osteoarthritis Research Society International (OARSI) osteoarthritis (OA) cartilage histopathology assessment system. RESULTS: Significantly less cartilage degradation (decreased levels of COMP) was found in the combined hormone treated group in comparison with OVX group. Similarly, both hormonal treatment resulted in increased bone formation and decreased bone resorption i.e., a low overall bone turnover status (decrease in the serum OC and CTX-1 levels). CONCLUSIONS: Combined estrogen and progesterone therapy was found to be convincing in terms of reducing the severity of OA in this experimental model.

Evidence for estrogen receptor expression during medullary bone formation and resorption in estrogen-treated male Japanese quails (Coturnix coturnix japonica)

The temporal expression of estrogen receptor (ER)-alpha and ER-beta mRNA was examined in male Japanese quails. Femurs of quails receiving 17beta-estradiol underwent RTPCR and histochemical analysis 1 to 15 days after treatment. Untreated quails were used as controls (day 0). Between days 0 and 5, cells lining the bone endosteal surface differentiated into osteoblasts, which in turn formed medullary bone. Expression of ER-alpha was already observed on day 0 and increased slightly during bone formation whereas ER-beta was hardly detected throughout this process. After osteoclasts appeared on the medullary bone surface, this type of bone disappeared from the bone marrow cavity (days 7~15). ER-alpha expression simultaneously decreased slightly and ER-beta levels remained very low. These results suggest that estrogen activity mediated by ER-alpha not only affects medullary bone formation but also bone resorption.

Osteoporose em bubalinos em relação à idade, à condição clínica animal e ao estado físico-químico ósseo e do cobre hepático / Osteoporosis in water buffalo, in relation to age, clinical condition of the animal and physical-chemical bone and hepatic copper status

O presente trabalho faz parte de um programa de pesquisa de doenças metabólicas nutricionais em bubalinos no Trópico Úmido Amazônico e, nesse contexto, objetivou-se estudar os processos osteoporóticos em bubalinos criados em sistema de pastejo na Ilha de Marajó e a relação da enfermidade com a faixa etária e a condição físico-química óssea e do cobre hepático. Foram utilizados 110 animais: 39 jovens e 71 adultos, apresentando Índice de Condição Corporal entre 1 e 2. Avaliou-se um grupo composto por animais jovens e outro por animais adultos. Análises anátomoclínica e da qualidade óssea possibilitaram subdividir cada grupo em dois subgrupos, segundo a presença ou não de manifestações osteopáticas aparentes. Do terço médio da 11ª costela direita analisou-se o cálcio, fósforo, cinza e densidade óssea e do tecido hepático determinou-se a concentração do cobre. No exame anatomopatológico, respectivamente 98,44% dos animais jovens e 96,16% dos animais adultos apresentavam algum grau de osteoporose (+, ++, +++). Foram demonstrados baixos percentuais na média do fósforo (10,69%), nas cinzas (60,24%) e na densidade (1,46 g/ml). O cobre hepático apresentou valores médios igualmente baixos (19,51 mg/kg). Os resultados mostraram que a osteoporose apresenta um quadro mais severo nos animais jovens, sendo que o processo pode estar relacionado com as baixas reservas de fósforo no tecido ósseo e do cobre no tecido hepático.

This paper is part of a research program of metabolic nutritional diseases in water buffalo in the Humid Tropical Amazon. In that context, the objective was to study osteoporotic processes in buffalo raised in a grazing system on Marajó Island and the relation of the disease with the age as well as physical-chemical bone and hepatic copper status. One hundred and ten animals were evaluated: 39 young or juvenile and 71 adults that presented Body Condition Index from 1 to 2. One group was composed by young buffalo and another by adult animals. Based on anatomical-clinical and bone quality analyses, the animals were subdivided in two sub-groups, according to presence or absence of clinical signs of apparent osteopathic. Analysis of calcium, phosphorus, ash and bone density was performed on the middle third of the 11th right rib, and the copper concentration was determined from liver tissue. Pathological anatomical findings showed that 98.44% of juvenile animals and 96.16% of adult animals, respectively, presented some degree of osteoporosis (+, ++, +++). Low average percentages were demonstrated for phosphorus (10.69%), in ashes (60.24%) and in density (1.46 g/ml). Hepatic copper presented similar low values (19.51 mg/kg). The results showed that juvenile animals presented more severe clinical sign of osteoporosis, and the pathogenesis may be related to low reserves of phosphorus in bony tissue and copper in liver tissue.

DNA-based personal identification in disaster / 法医学杂志

The disaster is a sudden unexpected event that causes serious human injuries and deaths as well as missing persons. The main tasks of forensic DNA laboratories are to identify victim in the disaster. After reviewed the previous disasters and related studies, we proposed a new procedure of DNA identification for the use of disaster in the future, which includes preparation works, samples collection, samples storage, DNA extraction, typing, data analysis and interpretation of results. Some experiences and problems about the DNA identification are also discussed.

[Removal of methylene blue dye from synthetic wastewater with bone char]

Dyes are organic materials with complex structures, toxic, carcinogenic, teratogenic, nonbiodegredable properties and the most important pollutants of textile industrial wastewaters. The goal of this study was to survey the feasibility application of bone char [BC] as a sorbent for the removal of methylene blue [MB] from synthetic wastewater. The sub goals of the research were to determine the adsorption isotherm, effects of primary concentration of dye, adsorbent dose, contact time, and pH for the adsorption of MB with BC. BC was prepared under laboratory conditions by using of electrical furnace at 400°C for 2h. The prepared BC was crushed and pulverized by standard ASTM sieves with range of 10-16 mesh [1.18-2 mm]. The chemical composition and solid structure of BC was analyzed using X-ray diffraction [XRD] and scanning electronic microscopy [SEM]. Measurement of the surface area was carried out by N2 gas via BET isotherm and Belsorb software. The concentration of dye was measured by photometric method [663nm]. Predominant composition of BC is calcium hydroxyl apatite [Ca5 [PO4]3OH with 14m2/g surface area. The results of this study showed that increasing of primary concentration of dye, adsorbent dose and pH [5 to12] would lead to increasing of adsorption/removal of MB dye. Equilibration of dye adsorption was reached at lapse of 2h and optimum pH for adsorption of MB with BC found in the rage of 8.5-12. Adsorption of MB with BC complies with freundlich isotherm [R2: 0.99]. Conclusion: Bone char is a cheap component that can be used as an adsorbent in water and wastewater treatment. Based on optimum pH of 8.5-12 found for the removal of MB and the fact that many of textile industrial wastewaters have an alkaline pH, this adsorbent can be used for the removal of dyes from these wastewaters

Metabolismo e distribuição do flúor em ovinos jovens tratados cronicamente com fluoreto de sódio / Metabolism and distribution of fluor in lambs chronically treated with sodium fluoride

Este estudo teve por objetivo avaliar o metabolismo do flúor (F) em ovinos. Para tanto, utilizaram-se 12 animais, com cinco meses de idade, os quais receberam como dieta base 3 por cento do peso vivo de feno de alfafa e água ad libitum. Os animais foram divididos e constituíram um grupo Controle, que recebeu apenas sal iodado (5g de NaCl/animal + 0,2mg I/kg matéria seca) e, um grupo Tratado, que recebeu sal iodado adicionado de fluoreto de sódio (4,7mg F/kg de peso corporal). Esses sais foram administrados via sonda oro-esofágica, diariamente por um período de 150 dias. Para análise de F, coletaram-se amostras de sangue, urina e fezes e, ao fim do período experimental, após a eutanásia dos animais, coletou-se a glândula pineal e amostras de osso. Também nesta ocasião, coletou-se uma amostra de rim para exame histopatológico. Analisando-se os teores séricos, urinários e ósseos de F, verificou-se que foram significativamente superiores nos animais Tratados em relação aos Controles. Quanto ao F contido na glândula pineal, não houve diferença significativa entre os grupos. Na análise histológica do rim, não foram observadas alterações. Conclui-se que a administração crônica de flúor induz ao acúmulo desse elemento nos ossos, mesmo havendo um alto teor de cálcio na alimentação e esse acúmulo parece não ser nocivo aos animais. Em ovinos, a capacidade orgânica de acúmulo ósseo e excreção urinária do flúor é diferente de outras espécies animais.

The objective of the present study was to evaluate fluorine metabolism in growing lambs. Twelve 5-month-old male lambs maintained on alfalfa hay (3 percent BW) and non-fluorinated water ad libitum were used. Animals were allocated into Control, receiving 5g NaCl/animal/day + 0.2mg I/kg dry matter) and Treated group, receiving the same treatment plus sodium fluoride (4.7mg F/kg body weight). Mineral treatment was given by gavage, daily for 150 days. Blood, urine and fecal samples were collected during and the end of the experiment. At the end of treatment period animals were euthanized and kidney, pineal and bone samples were collected. Urine F was higher in treated animals throughout the experiment. Bone F levels were also increased in treated animals; pineal F content however, was not different between groups. Kidney histology revealed no differences. It is concluded that chronic F administration induces accumulation of the element in the skeleton. However such fact appears not to be detrimental to animals. Rates of F accumulation in bone and urine excretion obtained in other species can not be used in growing lambs.

Morphological and chemical analysis of bone substitutes by scanning electron microscopy and microanalysis by spectroscopy of dispersion energy

This study evaluated the morphological and chemical composition of the following bone substitutes: cancellous and cortical organic bovine bone with macro and microparticle size ranging from 1.0 to 2.0 mm and 0.25 to 1.0 mm, respectively; inorganic bovine bone with particle size ranging from 0.25 to 1.0 mm; hydroxyapatite with particle size ranging from 0.75 to 1.0 mm; and demineralized freeze-dried bone allograft with particle size ranging from 0.25 to 0.5 mm. The samples were sputter-coated with gold in an ion coater, the morphology was observed and particle size was measured under vacuum by scanning electron microscopy (SEM). The chemical composition was evaluated by spectroscopy of dispersion energy (EDS) microanalysis using samples without coating. SEM analysis provided visual evidence that all examined materials have irregular shape and particle sizes larger than those informed by the manufacturer. EDS microanalysis detected the presence of sodium, calcium and phosphorus that are usual elements of the bone tissue. However, mineral elements were detected in all analyzed particles of organic bovine bone except for macro cancellous organic bovine bone. These results suggest that the examined organic bovine bone cannot be considered as a pure organic material.

Neste estudo foram avaliados a morfologia, o tamanho e a composição química dos seguintes substitutos ósseos: osso bovino orgânico cortical e esponjoso com micropartículas medindo entre 0,25 e 1,0 mm e macropartículas medindo entre 1,0 e 2,0 mm; osso bovino cortical inorgânico com partículas medindo entre 0,25 e 1,0 mm; hidroxiapatita com partículas medindo entre 0,75 e 1,0 mm; e osso humano descalcificado, congelado e seco medindo entre 0,25 a 0,5 mm. Para a analise da morfologia e tamanho das partículas, as amostras foram preparadas em porta-espécime, metalizadas em ouro e analisadas a vácuo em microscopia eletrônico de varredura (MEV). Para a análise da composição química, as partículas não foram metalizadas e foram analisadas por microanálise por espectroscopia por dispersão de energia (EDS). A análise em MEV, demonstrou que as partículas substitutos ossos apresentaram formato irregular e tamanho variável, maior do que o mencionado pelo fabricante. A microanálise por EDS detectou a presença de elementos como sódio, cálcio e fósforo, que são comuns à composição do tecido ósseo, porém revelaram a presença de elementos químicos nas partículas de osso bovino orgânico, exceto para a macropartícula de osso bovino orgânico esponjoso. Esses resultados sugerem que o osso bovino orgânico não pode ser considerado um material orgânico puro.

Morphogenetic protein rhBMP-2 and new bone formation / Proteína Morfogenética rhBMP-2 y Tejido Ósteoneoformado

Esta revisión de la literatura tiene como objetvo mostrar las principales características morfogenéticas, de la proteína rhBMP-2, de mayor prpiedad osteoinductiva, estudiados desde su descubrimiento hasta la actualidad, señalando las diferentes utilizaciones y aplicaciones de esta proteína.

This work aim to show by literature review the principal characteristics of morphogenetic proteins, in special of the rhBMP-2, with the major osteoinductive properties, presented in the prime works count from it discovery until actually, showing the most varieties and applications of this protein.