Polyhydroxybutyrate-based osteoinductive mineralized electrospun structures that mimic components and tissue interfaces of the osteon for bone tissue engineering.

Scaffolds for bone tissue engineering should enable regeneration of bone tissues with its native hierarchically organized extracellular matrix (ECM) and multiple tissue interfaces. To achieve this, inspired by the structure and properties of bone osteon, we fabricated polyhydroxybutyrate (PHB)-based mineralized electrospun fibrous scaffolds. After studying multiple PHB-based fibers, we chose 7%PHB/1%Gelatin fibers (PG) to fabricate mineralized fibers that mimic mineralized collagen fibers in bone. The mineralized PG (mPG) surface had a rough, hydrophilic layer of low crystalline calcium phosphate which was biocompatible to bone marrow stromal cells (BMSCs), induced their proliferation and was osteoinductive. Subsequently, by modulating the electrospinning process, we fabricated mPG-based novel higher order fibrous scaffolds that mimic the macroscale geometries of osteons of bone ECM. Inspired by the aligned collagen fibers in bone lamellae, we fabricated mPG scaffolds with aligned fibers that could direct anisotropic elongation of mouse BMSC (mBMSCs). Further, we fabricated electrospun mPG-based osteoinductive tubular constructs which can mimic cylindrical bone components like osteons or lamellae or be used as long bone analogues based on their dimensions. Finally, to regenerate tissue interfaces in bone, we introduced a novel bi-layered scaffold-based approach. An electrospun bi-layered tubular construct that had PG in the outer layer and 7%PHB/0.5%Polypyrrole fibers (PPy) in the inner layer was fabricated. The bi-layered tubular construct underwent preferential surface mineralization only on its outer layer. This outer mineralized layer supported osteogenesis while the inner PPy layer could support neural cell growth. Thus, the bi-layered tubular construct may be used to regenerate haversian canal in the osteons which hosts nerve fibers. Overall, the study introduced novel techniques to fabricate biomimetic structures that can regenerate components of bone osteon and its multiple tissue interfaces. The study lays foundation for the fabrication of a modular scaffold that can regenerate bone with its hierarchical structure and complex tissue interfaces.

Microstructural changes in the irradiated and osteoradionecrotic bone: a SEM study.

Radiation exposure is a major health concern due to bone involvement including mandible, causing deleterious effects on bone metabolism, and healing with an increasing risk of infection and osteoradionecrosis. This study aims to investigate the radiotherapy-induced microstructural changes in the human mandible by scanning electron microscopy (SEM). Mandibular cortical bone biopsies were obtained from control, irradiated, and patients with osteoradionecrosis (ORN). Bone samples were prepared for light microscopy and SEM. The SEM images were analyzed for the number of osteons, number of Haversian canal (HC), diameter of osteon (D.O), the diameter of HC (D.HC), osteonal wall thickness (O.W.Th), number of osteocytes, and number of osteocytic dendrites. The number of osteons, D.O, D.HC, O.W.Th, the number of osteocytes, and osteocytic dendrites were significantly decreased in both irradiated and ORN compared to controls (p < .05). The number of HCs decreased in irradiated and ORN bone compared to the control group. However, this was statistically not significant. The deleterious effect of radiation continues gradually altering the bone quality, structure, cellularity, and vascularity in the long term (>5 years mean radiation biopsy interval). The underlying microscopic damage in bone increases its susceptibility and contributes further to radiation-induced bone changes or even ORN.

Cortical bone modifications after radiotherapy: cortex porosity and osteonal changes evaluated over time

Abstract Aiming to evaluate cortical bone microarchitecture and osteonal morphology after irradiation, twelve male New Zealand rabbits were used. The animals were divided: control group (no radiation-NIr); and 3 irradiated groups, sacrificed after: 7 (Ir7d); 14 (Ir14d) and 21 (Ir21d) days. A single radiation dose of 30 Gy was used. Computed microtomography analyzed the cortical microarchitecture: cortical thickness (CtTh), bone volume (BV), total porosity (Ct.Po), intracortical porosity (CtPo-cl), channel/pore number (Po.N), fractal dimension (FD) and degree of anisotropy (Ct.DA). After scan, osteonal morphology was histologically assessed by means: area and perimeter of the osteons (O.Ar; O.p) and of the Haversian canals (C.Ar; C.p). Microtomographic analysis were performed by ANOVA, followed by Tukey and Dunnet tests. Osteon morphology analyses were performed by Kruskal-Wallis, and test Dunn's. Cortical thickness was significant difference (p<0.010) between the NIr and irradiated groups, with thicker cortex at Ir7d (1.15±0.09). The intracortical porosity revealed significant difference (p<0.001) between irradiated groups and NIr, with lower value for Ir7d (0.29±0.09). Bone volume was lower in Ir14d compared to control. Area and perimeter of the osteons were statistically different (p<0.0001) between NIr and Ir7d. Haversian canals also revealed lower values (p<0.0001) in Ir7d (80.57±9.3; 31.63±6.5) compared to NIr and irradiated groups. Cortical microarchitecture was affected by radiation, and the effects appear to be time-dependent, mostly regarding the osteons morphology at the initial days. Cortex structure in Ir21d revealed similarities to control suggesting that microarchitecture resembles normal condition after a period.

Resumo Com o objetivo de avaliar a microarquitetura óssea cortical e a morfologia dos osteons após irradiação, foram utilizados doze coelhos machos da Nova Zelândia. Os animais foram divididos: grupo controle (sem radiação-NIr); e 3 grupos irradiados, sacrificados após: 7 (Ir7d); 14 (Ir14d) e 21 (Ir21d) dias. Foi utilizada uma dose única de radiação de 30 Gy. A microtomografia computadorizada analisou a microarquitetura cortical: espessura cortical (CtTh), volume ósseo (BV), porosidade total (Ct.Po), porosidade intracortical (CtPo-cl), número de canal/ poro (Po.N), dimensão fractal (DF) e grau de anisotropia (Ct.DA). Após a varredura, a morfologia dos osteosn foi avaliada histologicamente por meio de: Área e perímetro do osteon (O.Ar; O.p) e dos canais de Havers (C.Ar; C.p). A análise microtomográfica foi realizada por ANOVA, seguida pelos testes de Tukey e Dunnet. As análises morfológicas do osteon foram realizadas por Kruskal-Wallis e testadas por Dunn. A espessura cortical foi diferente (p<0,010) entre os grupos controle e irradiados, com córtex mais espesso no Ir7d (1,15±0,09). A porosidade intracortical revelou diferenças significativas (p<0,001) entre os grupos irradiados e o controle, com menor valor para Ir7d (0,29±0,09). O volume ósseo foi menor no Ir14d em relação ao controle. Área e perímetro do osteon foi diferente (p<0,0001) entre o controle e Ir7d. Os canais haversianos também revelaram valores mais baixos (p<0,0001) em Ir7d (80.57±9.3; 31.63±6.5) em relação ao controle e demais grupos irradiados. A microarquitetura cortical é afetada pela radiação e os efeitos parecem ser dependentes do tempo, principalmente em relação à morfologia dos osteons nos dias iniciais. A estrutura cortical em Ir21d revelou semelhanças com o controle, sugerindo que a microarquitetura se assemelha à condição normal após um período.

Age determination in subadults using histological analysis of bones / Determinación de la edad en subadultos usando análisis histológico de los huesos

Abstract Introduction: According to the literature, the amount of osteons has been suggested as a good proxy to determine the age of death in adults. However in subadults research has not been carried out yet. Objective: To determine the accuracy of the histomorphometric technique predicting the age at death in subadults using bone remains. Methodology: The information of static histomorphometric parameters from about 120 iliac bones retrieved from the exhumed remains of subadults whose age at death was known was taken from the Granada collection. In order to predict the age at death we performed a step by step linear regression to estimate the fittest model. Results: The most closely and significantly associated biopsy findings with age were: the osteon count, the internal cortical width, and the trabecular bone volume. Pearson's correlation index indicated a weak linear association among these variables. To assess the accuracy of the model we used a coefficient of determination with a 0.32 value. 32% of the age variation in the subadults was explained by the three variables. Conclusion: This regression model explains a percentage of the total age variation in the subadult population. However this model is not enough to determine the age at death.

Resumen Introducción: La capacidad de predicción de las osteonas para determinar la edad de muerte de los individuos ha sido descrito en la literatura científica. No obstante, no se ha determinado dicha capacidad en individuos subadultos. Objetivo: Determinar la eficacia de lo parámetros histomorfometricos en población subadulta. Metodología: Se realizaron biopsias de hueso ilíaco en los restos de 120 subadultos, de la Colección Osteológica de Granada, con edad conocida en el momento de la muerte. Para establecer la capacidad de predicción se utilizó el R2 obtenido a partir de regresión lineal múltiple. Resultados: Las variables con mayor nivel predictivo y significativo para la estimación de la edad fueron: recuento de osteonas tipo 2 de la cortical interna y externa, y el volumen óseo trabecular; En la evaluación del modelo, se obtuvo un coeficiente de determinación de 0.32, es decir, el 32% de la variación en la edad de los subadultos se explica por el modelo. Sin embargo, se evidenció diferencias en la capacidad de predicción por sexo. Conclusión: Este modelo de regresión explica un porcentaje sustancial de la varianza de la edad de los individuos en la muestra. No obstante, no es suficiente para garantizar una adecuada predicción de la edad al momento de muerte de los individuos subdultos.

Changes in cortical bone channels network and osteocyte organization after the use of zoledronic acid

Objective The aim of this study was to evaluate the effects of zoledronic acid (ZA) on the cortical bone channels network (CBCN) and osteocyte organization in relation to the bone channels. Materials and methods Eighteen male Wistar rats were divided into control (CG) and test groups (TG). Twelve animals from TG received 3 ZA doses (7.5 µg/kg), and 6 animals from CG did not receive any medication. TG animals were euthanized at 14 (n = 6) and 75 (n = 6) dadys after drug injection. CBCN was analyzed in mandibles and tibias using computational routines. The osteocyte organization was qualitatively evaluated in tibias using a three-dimensional reconstruction of images from serial histological sections. Results Significant differences in CBCN of tibia were found between the treated and untreated rats, with a wider range of sizes and shapes of the channels after the use of ZA (channels area p = 0.0063, channels area SD p = 0.0276) and less bone matrix (bone volume p = 0.0388). The alterations in the channels’ morphology were more evident at 75 days after the drug injection (channels perimeter p = 0.0286). No differences were found in mandibles CBCN. The osteocyte distribution revealed more variable patterns of cell distribution in ZA groups, with non-homogeneous distribution of cells in relation to the bone channels. Conclusion Zoledronic acid induces structural changes in CBCN and modifies the osteocyte arrangement in cortical bone in the tibia; also, the variability in the morphology of bone channels became more evident after a certain time of the use of the drug.

Familial osteopetrosis in Agouti paca: report of nine cases / Osteopetrose familiar em Agouti paca: relato de nove casos

Nine cases of familial osteopetrosis were studied in Agouti paca rodents maintained in captivity. Animals were distributed in three groups depending on the severity of their skeletal lesions. Based upon clinical, radiological, and microscopic findings, it was concluded that one animal had level I lesions, three animals had level II lesions, and five animals had level III osteopetrosis and osteonecrosis. Throughout the entire axial and appendicular skeleton, there was an increased amount of both trabecular and cortical bone tissue. All analyzed bones showed thickened cortex and reduced medullary canals. Bone trabeculae were thick and confluent. Cortex showed a narrowing of Haversian canals. Numerous cementing lines resulted in typical mosaic patterns. Osteocytes were pycnotic. Osteonecrosis was characterized by the disappearance of osteocytes and bone matrix decomposition.

Descreveram-se nove casos de osteopetrose familiar em Agouti paca mantidas em cativeiro. Os animais foram distribuídos em três grupos de acordo com a gravidade das lesões do esqueleto. Com base nos exames clínico, radiológico e microscópico, foi concluído que um animal apresentou lesões de nível I, três animais tiveram lesões de nível II e cinco animais tiveram osteopetrose de nível III. Por todo o esqueleto axial e apendicular, a quantidade de osso trabecular e osteônico estava aumentada. Todos os ossos analisados mostraram córtex espesso e canais medulares reduzidos. As trabéculas ósseas eram espessas e confluentes. No córtex, verificou-se um estreitamento de canais de Havers. Numerosas linhas de cimentação resultaram em um padrão de mosaico típico. Osteócitos estavam picnóticos e a osteonecrose foi caracterizada pela morte dos osteócitos, com desintegração da matriz óssea.

La importancia pericial del estudio comparativo histomorfológico del hueso humano y de otros géneros / Forensic importance of the comparative histomorphometric study of the human bone and bones from other genders

Es un hecho notable que la identificación de huesos o fragmentos de éstos contribuye sobremanera en el esclarecimiento de problemas judiciales. Cuando no se dispone del esqueleto o de huesos íntegros, el análisis de características histomorfológicas óseas se torna un factor decisivo en la elucidación de ciertos casos de identificación. El propósito del presente estudio fue ampliar o buscar nuevas posibilidades en la identificación de fragmentos óseos de seres humanos y de otros 19 mamíferos. Así, analizamos los sistemas de Havers, los canales de Havers, los osteocitos y los canalículos de los mismos, del hombre, anta, aracambé, buey, cabra, perro montés, perro, capivara, carnero, cateto, caballo, conejo, cutia, gato, mono-clavo, jaguar, puerco espín, cerdo, oso hormiguero y venado. Fueron analizados cortes histológicos por desgaste del tejido óseo en sentido longitudinal y transversal y analizados con microscopio de luz. Los resultados demuestran con claridad y precisión que el análisis histomorfológico de las estructuras citadas posibilita la identificación y diferenciación de los géneros estudiados o excluyen la posibilidad de que fragmentos óseos examinados pertenezcan al género Homo sapiens

Descripción histológica de la substancia cortical dorsal en la parte media de la diáfisis del metacarpiano III en yegua mestiza / Histological description of the dorsal cortical bone in the midle diaphysis of the metacarpial III in the mixed mare

El hueso es el unicó tejido capaz de adaptarse estructural y geométricamente ante presiones impuestas sobre él. Durante la vida, el hueso es afectado por procesos externos e internos. Los externos producen cambios en sus medidas y forma. Los internos remodelan su arquitectura interna. Además, los huesos son afectados por factores que incluyen edad, sexo, especie, origen y contenido de minerales. Las variaciones estructurales en la organización microscópica del tejido óseo ocurren de manera constante y consistente mediante el crecimiento y la remodelación por parte de cada especie animal. El objetivo del presente trabajo fue estudiar la estructura microscópica inorgánica del cuadrante dorsal del metacarpiano III en la parte media de la diáfisis en distintos grupos etarios. Se estudió la substancia cortical dorsal de los huesos metarcapianos III izquierdos provenientes de 30 yeguas mestizas, con edades entre 2 y 5 años. Extraídos los huesos se los liberó de los tejidos blandos. El cuadrante dorsal fue muestreado, en la parte media de la diáfisis. Posteriormente, se realizó el pulido en forma manual. Las muestras fueron observadas al microscopio de luz polarizada. Se observaron diferencias histológica en los diferentes grupos etarios. Se concluyó que se producen variaciones microscópicas de la subtancia cortical dorsal del metarcapiano III en la parte media de la diáfisis, en el transcurso de 4 años